Modified antigen-binding molecules with changed cell signal activity

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 dwg

 

Background of the invention

The technical field to which the invention relates

The present invention relates to antigen-binding molecules (AFM). In some embodiments, the present invention relates to recombinant monoclonal antibodies or their fragments, including chimeric, primaryservername or humanised antibodies or their fragments with altered ability to mediate cell signaling activity of the antigen-targeted and/or altered ability to mediate cross-linking of one or more antigens-targets. In addition, the present invention relates to nucleic acid molecules encoding such AFM, as well as vectors and cells-owners, including such nucleic acid molecules. The present invention also relates to methods for producing the AFM of the present invention and to methods of using the AFM for the treatment of diseases. In addition, the present invention relates to the AFM with a modified glycosylation, having improved therapeutic properties, including antibodies with increased binding to Fc-receptor and increased effector function.

The level of technology

Antibodies, also called immunoglobulins, have a structure which consists of four polypeptide chains: two identical �azelie chain (heavy N), paired with two identical light chains (light - L). Each heavy and light chain includes a variable region (variable region VH and VL, respectively) and constant region (constant region CH and CL, respectively). Area SN contains 3 domains (CH1, CH2 and CH3), although less of the CL contains only one domain (which is simply denoted by "CL"). Each VH and VL region comprises 3 complementary deterministic region (complementarity determining region, CDR), flanked by four frame sections in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Complementary deterministic region (CDR) are the most variable part of the field V, and determine the specificity of the antibody. Together paired VH and VL form the binding site of the antigen, and bivalent antibodies have two such antigenspecific site. It should be noted that the basic structure of the antibody can be modified in different ways (for example, the generation of structural fragments), although it maintains or even improves the desired functions and/or the antigen-binding activity.

On the border between the domains VH and CH1 contains conservative amino acids. This contact area can be described under the title "molecular spherical joint" or "articulation". Such articulation determines the movement of the elbow and is therefore called the "elbow angle" regions VH and VL relative�individual regions CH1 and CL and prevents hard contact between regions V and C (Lesk, Chothia, Nature 335, 1988, cc.188-190). "An improved spherical joint" is formed by the residues of amino acids in skeleton plot VH, especially in positions 11, 110 and 112 (according to the Kabat numbering system, and others in kN. "Sequences of Proteins of Immunological Interest", 1987, 4th ed., publishing house of Public Health Services, NIH, Washington, D.C.). (Cm. Lesk, Chothia, Nature, 335, 1988, cc.188-190). "Spherical projection" of such a "spherical joint" is located in the CH1 domain and is formed mainly by two amino acids at positions 148 and 149 (see Landolfi, etc., J. Immunol. 166, 2001, cc.1748-1754, Lesk, Chothia, Nature 335, 1988, cc.188-190) (where CH1 residues forming the "spherical projection", numbered 149 and 150, respectively). Differences in amino acids in these positions may dictate the elbow angle, which is formed between regions V and C, and therefore the orientation of the dimer VH-VL (see Lesk, Chothia, Nature 335, 1988, cc.188-190). Amino acid residues occupying these positions in VH, are highly conservative for immunoglobulin sequences (see, e.g., Lesk, Chothia, Nature 335, 1988, cc.188-190). All amino acid residues involved in the articulation (for instance, regulations 11, 110, 112, 148, and 149), are located in the textured regions (e.g., residues 11, 110 and 112) or in a constant domain (e.g., 148 and 149 according to Landolfi, etc., or 149 and 150 according to Lesk and Chothia), and, apparently, not involved directly in binding to the antigen. Landolfi, etc., J. Immunol. 166, 001, cc.1748-1754.

In addition to mediating effector functions such as antibody dependent cell-induced cytotoxicity (antibody dependent cell-also been other ideas where cytotoxicity - ADCC) and complement-dependent cytotoxicity (complement dependent cytotoxicity CDC), monoclonal antibodies can modulate cellular functions by induction or suppression of cellular signaling pathways. For example, it was found that monoclonal antibodies for mediating antigenic cross-stitching activate the death receptors (e.g., facilitating the oligomerization of receptors or mimikriya ligand binding) and block ligand-mediated transfer of cellular signal during the growth and differentiation of cells and/or metabolic pathway of cell proliferation (see, e.g., Ludwig, etc., Oncogene 22, 2003, cc.9097-9106).

Apoptosis, or programmed cell death can be induced by several different mechanisms. For example, activation of signaling pathways associated with using a cellular membrane "death receptors", for example, representatives of the superfamily of receptors of tumor necrosis factor (tumor necrosis factor receptor - TNFR), can lead to induction of apoptosis. Similarly, the dimerization or cross-linking of the surface antigen may lead to the induction of apoptosis. So dimerization or cross-linking surface antigen, e.g., anti�ena CD20, can also induce apoptosis (see, e.g., Ludwig, etc., Oncogene 22, 2003, cc.9097-9106).

Still remains a need for improved therapeutic approaches to the targeting of antigens associated with the transmission of cellular signals, including but not limited to, induction of apoptosis, for the treatment of the disease in primates, including humans, but not limited to them.

Brief description of the invention

Recognition of the enormous therapeutic potential of modified antigen-binding molecules (AFM) that have altered ability to mediate cellular signaling by antigen-targeted and/or altered ability to mediate cross-linking of one or more antigens-targets, the present invention is designed such AFM and method for producing such ACM. In addition, the present method involves the preparation of recombinant chimeric antibodies or chimeric fragments. The effectiveness of such modified AFM can be further enhanced by designing the profile of glycosylation of the Fc region of the antibody.

Thus, one object of the present invention is a modified antigen-binding molecule comprising the variable region of the heavy or light chain comprising at least one substitution of an amino acid residue in at least one frame section of the decree�the x variable regions of the heavy or light chain, compared with the variable region of the heavy or light chain of the original antigen-binding molecule, where the said changes lead to altered cell signaling activity of the antigen target, if the modified antigen-binding molecule is combined with a specified antigen target. In one embodiment of the present invention, a modified activity for the transmission of the cellular signal is apoptosis. In one of the embodiments of the present invention is a modified antigen-binding molecule has an increased ability to induce apoptosis. In another embodiment of the present invention is a modified antigen-binding molecule has a decreased ability to induce apoptosis.

Another object of the present invention relates to a modified antigen-binding molecule comprising the variable region of the heavy or light chain, in which at least one amino acid substituted by at least one frame area of the specified variable regions of the heavy or light chain, compared with the variable region of the heavy or light chain of the original antigen-binding molecules, and specified a modified antigen-binding molecule has altered ability to mediate cross-shivani� one or more antigens-targets as a result of any such replacement.

In one of the embodiments of the present invention is a modified antigen-binding molecule of the present invention includes a replacement plot in frame FR1 variable regions of the heavy chain. In another embodiment of the present invention, the substitution is a substitution selected from the group consisting of at least 2 at least 3 or at least 4 amino acids.

In yet another variant implementation of the present invention, the replacement is a complete replacement frame section FR1 variable regions of the heavy chain. In another embodiment of the present invention FR1 completely replaced on VH FR1 cells of the germ line. In yet another embodiment of the present invention, the VH FR1 cells of the germ line includes 8-13 amino acid sequence according to Kabat numbering selected from the group consisting of sequences SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105.

In one of the embodiments of the present invention is a modified AFM includes replacement plot in frame FR1 variable regions of the heavy chain in the form of a replacement amino acid residue at one or more positions 8, 9, 10, 11, 12 or 13 on numbering Kbat.

In one of the embodiments of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 8 on Kabat numbering. In a more specific embodiment of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 8 according to Kabat numbering for the amino acid residue selected from the group consisting of arginine and glycine.

In another specific embodiment of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 9 according to the Kabat numbering. In a more specific embodiment of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 9 according to the numbering of Kabat the amino acid residue selected from the group consisting of alanine, Proline, glycine, serine, and histidine.

In one of specific embodiments of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 10 according to the Kabat numbering. In a more specific embodiment of the present invention, the substitution is a replacement of the amino acid residue in position 10 in the numbering of Kabat the amino acid residue is selected from the group consisting of glutamate, threonine, glycine, alanine and valine.

In another embodiment of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 11 according to the Kabat numbering. In a more specific embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 11 to Kabat numbering for the remainder of any amino acids except leucine. In yet another embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 11 to the Kabat numbering on non-polar amino acid. In another embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 11 to the numbering of Kabat the amino acid residue selected from the group comprising valine, leucine, isoleucine, serine and phenylalanine. In one of the embodiments of the present invention, the substitution is a replacement of the amino acid residue at position 11 to the Kabat numbering on leucine.

In another embodiment of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue in position 12 according to the Kabat numbering. In a more specific embodiment of the present invention, the substitution is the replacement of amino acids�th residue in position 12 by Kabat numbering in the residue of any amino acid, selected from the group comprising lysine, valine, leucine and isoleucine.

In one of the embodiments of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residues at positions 11 and 12 on Kabat numbering. In a specific embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 11 to Kabat numbering for valine and at position 12 to Kabat numbering for lysine; a substitution of amino acid residue in position 11 according to the Kabat numbering on leucine and at position 12 to Kabat numbering for valine; replacement of amino acid residue in position 11 and Kabat numbering for valine and at position 12 to Kabat numbering for isoleucine; or a substitution of amino acid residue in position 11 and Kabat numbering for valine and at position 12 to Kabat numbering for valine.

In another embodiment of the present invention, the substitution in FR1 of the variable region of the heavy chain is a replacement amino acid residue at position 13 on Kabat numbering. In a specific embodiment of the present invention, the substitution is a replacement of the amino acid residue at the position 13 to the numbering of Kabat the amino acid residue selected from the group comprising lysine, arginine, glutamine and glutamate.

In yet another embodiment of the present invention is the change in the AFM is replaced�have at least one amino acid residue in FR4 of the variable region of the heavy chain. In a specific embodiment of the present invention, the substitution in FR4 of the variable region of the heavy chain is a replacement amino acid residues at either or both of the provisions 110 or 112 according to the Kabat numbering.

In one of specific embodiments of the present invention, the replacement amino acid residue at position 110 according to Kabat numbering for amino acid selected from the group comprising leucine, isoleucine, threonine or series. In a more specific embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 110 by Kabat numbering in isoleucine.

In another embodiment of the present invention, the replacement amino acid residue at position 112 according to Kabat numbering for amino acid selected from the group comprising valine, leucine, isoleucine, or threonine. In a more specific embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 112 by Kabat numbering in isoleucine.

One of the objects of the present invention is associated with a modified antigen-binding molecule comprising the CH1 domain that contains at least one substitution of an amino acid residue compared to the CH1 domain of the original polypeptide, and the substitution leads to altered cell signaling activity of the antigen target, if modified�ƈ antigen-binding molecule is combined with the antigen target.

Another object of the present invention also relates to a modified antigen-binding molecule comprising the CH1 domain that contains at least one substitution of an amino acid residue compared to the CH1 domain of the original polypeptide, and antigen-binding molecule as a result of such replacement has altered ability to mediate cross-linking of one or more antigens-targets.

In one of the embodiments of the present invention, the change in CH1 is the replacement amino acid residue at one or more of the provisions 148, 149 or 150. In a specific embodiment of the present invention, the substitution is a replacement of the amino acid residue at position 149 on leucine. In another embodiment of the present invention, the substitution is a replacement of a CH1 domain. In yet another embodiment of the present invention, the substitution is the replacement of the CH1 domain of IgG on CH1 domain of IgM.

Another object of the present invention is a modified antigen-binding molecule comprising at least one substitution of an amino acid, where the specified substitution is the replacement of amino acid residues in the light chain in a boundary region between the variable and constant regions, and the substitution leads to an altered activity on transfer glue�full-time signal of the antigen-target binding molecules, if the modified antigen-binding molecule is combined with a specified antigen target.

Another object of the present invention is associated with a modified antigen-binding molecule comprising at least one substitution of an amino acid, where the specified substitution is the replacement of amino acid residues in the light chain in a boundary region between the variable and constant regions, and specified a modified antigen-binding molecule has altered as a result of this replacement the ability to mediate cross-linking of one or more antigens-targets.

In one of the embodiments of the present invention, the substitution in a variable region light chain AFM is a replacement amino acid at one or more of the provisions 10, 12, 39, 40, 41, 80, 81, 83, 84, 103, 105, 106 and 108 according to the Kabat numbering. In a specific embodiment of the present invention, the substitution in a variable region light chain AFM is the replacement of amino acid residues in one or more of the provisions of 40, 80, 83, 105 or 106 according to the Kabat numbering.

In another embodiment of the present invention, the substitution in the light chain of the AFM is the replacement of amino acid residues in one or more of the provisions of 40, 80, 83, 105 or 106 according to the Kabat numbering on non-polar amino acid.

� another specific embodiment of the present invention, the substitution in the light chain of the AFM is the replacement amino acid residue in position 40 on Kabat numbering by alanine.

In another specific embodiment of the present invention, the substitution in the light chain of the AFM is the replacement amino acid residue in position 80 according to Kabat numbering on Proline.

In another specific embodiment of the present invention, the substitution in the light chain of the AFM is the replacement amino acid residue at position 83 according to Kabat numbering on phenylalanine.

In another specific embodiment of the present invention, the substitution in the light chain of the AFM is the replacement amino acid residue in position 105 according to the Kabat numbering by alanine.

In another specific embodiment of the present invention, the substitution in the light chain of the AFM is the replacement amino acid residue in position 106 according to the Kabat numbering by alanine. In another specific embodiment of the present invention, the substitution in the light chain of the AFM is the replacement amino acid residue in position 106 according to the numbering of Kabat, wherein the antigen-binding molecules of the reduced ability to induce apoptosis.

In some embodiments of the present invention, the replacement according to the present invention is a combination of substitutions of any of the amino acid residues in variable and/or constant regions of the heavy and/or light chain as described in the present invention.

One of the areas of research�tion of the present invention is the amino acid replacement (substitution) in modified AFM of the present invention, which leads to altered cell signaling activity of the antigen target, if modified AFM is combined with the antigen target.

In one of the embodiments of the present invention altered cell signaling activity is increased agonistic activity. In one of specific embodiments of the present invention increased agonistic activity selected from the group including induction of apoptosis and induction of cell differentiation.

In one of the embodiments of the present invention altered cell signaling activity is increased antagonistic activity. In a specific embodiment of the present invention antagonistic activity selected from the group including the survival of cells, cell growth, cell proliferation and angiogenesis.

Another object of the present invention is a modified antigen-binding molecule of the present invention, which specifically binds to the antigen CD20 human. Another object of the present invention is a modified antigen-binding molecule that specifically binds with a representative of the superfamily of TNF receptor human. In one of the embodiments of the present invention, the representative of the superfamily of �of receptor human TNF is selected from the group including TNFR1, CD95, TRAILR1, TRAILR2, EDAR and p75NGFR.

Another object of the present invention is a modified antigen-binding molecule that specifically binds to receptor receptor. In one of specific embodiments of the present invention receptor tyrosinekinase selected from the group including HER1 (EGFR1), HER2/neu, HER3, HER4, IGF-1R, FGFR, PDGFR, VEGFR1, VEGFR2 and VEGFR3. In one of specific embodiments of the present invention receptor receptor is HER1 (EGFR1).

Another object of the present invention is also a modified antigen-binding molecule (AFM), which can be selected from the group including a whole antibody, a Fab fragment or a fusion protein, fragment F(ab')2 or fusion protein, Manantial, double antibody, triple antibody and Quaternary antibody, but their list is not limited. In one specific embodiment of the present invention, the AFM is a chimeric molecule or a molecule that is completely derived from a person. In another more specific embodiment of the present invention is a modified AFM is humanized. In another embodiment of the present invention is a modified AFM is multispecific. In a more specific embodiment of the present image�etenia modified AFM is bespecifically.

Another object of the present invention is a source of antigen-binding molecule comprising a variable region heavy chain selected from the group comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62.

Another object of the present invention is a source of antigen-binding molecule comprising a light chain selected from the group comprising SEQ ID NO:48, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133 and SEQ ID NO:134.

Another object of the present invention is a modified AFM also includes the Fc region of a human. According to one embodiment of the present invention, the Fc region of the modified AFM modified in such a way that includes the modified oligosaccharides. In one specific embodiment of the present invention, the Fc region has been modified to reduce the proportion of fucose residues compared to the unmodified Fc region. In another specific embodiment of the present invention, the Fc region contains a higher proportion of branched oligosaccharides compared with the unmodified Fc region. In yet another specific embodiment of the present invention, the modified oligosaccharides are branched complex. In another embodiment of the present invention are modified together by manipulating� oligosaccharides have an increased proportion of branched nefokusirana oligosaccharides in the Fc region as compared with the unmodified Fc region. In one specific embodiment of the present invention, the Fc region contains a higher proportion of residues of N-acetylglucosamine (GlcNAc) residues of fucose in a modified Fc region as compared with the unmodified Fc region. In a specific embodiment of the present invention branched nefokusirana oligosaccharides oligosaccharides are hybrid. In another specific embodiment of the present invention branched nefokusirana oligosaccharides are complex.

Another object of the present invention is the receptor on the cell surface, selected from the group consisting of membrane transport receptor, G-protein-receptor binding and enzyme-binding receptor. In one specific embodiment of the present invention, membrane transport receptor is a channel-linked receptor. In another specific embodiment of the present invention fermentopathy receptor selected from the group consisting of receptor huaylillas, receptor tyrosine kinases, tyrosinekinase-coupled receptor, the receptor tyrosinosis and receptor serine/threonine kinases.

Another object of the present invention relates to pharmaceutical composition comprising a modified And�M of the present invention. It is envisaged that the pharmaceutical composition may also include pharmaceutically acceptable carrier, adjuvant or a combination thereof.

The present invention also relates to a method of treating a disease that is treatable through altered cell signaling activity in a patient, comprising the introduction to the patient a therapeutically effective amount of a pharmaceutical composition comprising a modified AFM according to the present invention and a pharmaceutically acceptable carrier.

Another object of the present invention is associated with the selected polynucleotide that encodes a polypeptide comprising the variable region of the heavy or light chain and the variable region of the heavy or light chain includes at least one substitution of amino acid residue in at least one frame section, compared to the original variable region of the heavy or light chain, in which a change occurs in the cellular signaling activity of the antigen target, if the polypeptide is combined with the antigen target.

Another object of the present invention is associated with the selected polynucleotide that encodes a polypeptide comprising the variable region of the heavy or light chain and the variable region of the heavy or light chain includes at least one substitution of amino�kislotnogo residue in at least one frame section, compared to the original variable region of the heavy or light chain, wherein the polypeptide has an altered ability to mediate cross-linking of one or more antigens-targets as a result of such replacement.

In one embodiment of the present invention, the polynucleotide of the present invention encodes a polypeptide comprising light or heavy chain of the antibody. In another embodiment of the present invention, the polynucleotide of the present invention encodes a polypeptide, wherein the polypeptide is a fusion protein. The present invention also relates to polypeptides encoded by the polynucleotides of the present invention.

The present invention also relates to a vector comprising the polynucleotide according to the present invention and a host cell comprising this vector.

The present invention also relates to polynucleotide, codereuse polypeptide comprising the variable region of the heavy or light chain, which contains at least one amino acid residue in at least one wireframe plot of the variable region of the heavy or light chain, compared with the variable region of the heavy or light source of the antigen-binding molecule, wherein the polypeptide is a modified AFM according to the present invention./p>

The present invention also relates to a cage-owner, is so constructed that it is capable of expressing at least one nucleic acid encoding a polypeptide having an activity of β(1,4)-N-acetylglucosaminyltransferase III, in an amount sufficient to modify the oligosaccharides in the Fc region of a polypeptide produced by a host cell, wherein the polypeptide is a modified AFM according to the present invention. In one embodiment of the present invention, the polypeptide having the activity of β(1,4)-N-acetylglucosaminyltransferase III, is a hybrid polypeptide. In one embodiment of the present invention, the hybrid polypeptide is the catalytic domain of β(1,4)-N-acetylglucosaminyltransferase III. In another embodiment of the present invention, the hybrid polypeptide also includes domain localization Golgi heterological resident polypeptide Golgi. Domain Golgi localization can be selected from the group including domain mannosidase II, the localization domain of β(1,2)-N - acetylglucosaminyltransferase I, the localization domain of β(1,2)-N - acetylglucosaminyltransferase II, the localization domain of mannosidase I and domain localization and 1-6 capsid fucosyltransferase, but their list is not limited.

In another embodiment osushestvlyaetsya the invention of the AFM includes the area equivalent to the Fc region of human IgG.

In another embodiment of the present invention AFM secreted by the host cell of the present invention, exhibits an increased binding affinity of the Fc and/or increased effector function in connection with a modification of the oligosaccharide chains. According to the present invention increased effector function is selected from the group comprising: increased Fc-mediated cellular cytotoxicity, increased binding to natural killer cells (GAC), increased binding to macrophages, increased binding to the PAC polymorphisim cells, increased binding to monocytes, increased directional signal-induced apoptosis, increased the maturation dendrocygna cells and increased premirovanii T cells. In one embodiment of the present invention, the Fc receptor is an Fcγ activating receptor. In another embodiment of the present invention, the receptor Fc receptor is FcγRIIIA receptor.

According to the present invention, the host may be selected from the group that includes cells: Cho, HEK293-EBNA, BHK, NSO, SP2/0, cells, YO myeloma cells of mouse myeloma RH, PER, PER.C6 or cells of hybridomas.

Another embodiment of the present invention relates to a method for producing modified AFM comprising a variable region �agelou or light chain, comprising at least one substitution of amino acid residue in at least one wireframe plot of the variable region of the heavy or light chain compared with the variable region of the heavy or light chain of the original AFM, and the substitution leads to changes in cellular signaling activity of the antigen target, if modified AFM is combined with the antigen target; said method includes: (i) culturing the host cell of the present invention under conditions permitting expression of the polynucleotide, and (ii) the allocation of the modified AFM from the culture medium.

Another object of the present invention relates to a method for producing modified AFM comprising the variable region of the heavy or light chain comprising at least one substitution of amino acid residue in at least one wireframe plot of the variable region of the heavy or light chain compared with the variable region of the heavy or light chain of the original antigen-binding molecule in which a modified antigen-binding molecule has altered ability to mediate cross-stitching as a result of replacement, which includes: (i) culturing the host cell of the present invention under conditions permitting expression of the polynucleotide, and (ii) the allocation of the modified AFM from culture �Reda.

Another object of the present invention relates to a method of modifying the ability of the AFM to facilitate the formation of complexes with the antigen, the target antigen-binding molecules (AFM), which includes: replacing at least one amino acid residue in at least one wireframe plot of the variable region of the heavy or light chain of the original AFM. In one embodiment of the present invention, the AFM promotes the induction of apoptosis in a cell expressing the antigen target. In another embodiment of the present invention AFM enhances the induction of cell differentiation in a cell expressing the antigen target.

The present invention also relates to a method of inducing apoptosis in a cell, the method comprising contacting the cells with a modified AFM, comprising the variable region of the heavy or light chain, which comprises at least one substitution of amino acid residue in at least one frame area of the specified variable regions of the heavy or light chain compared with the variable region of the heavy or light chain of the original AFM, in which a modified AFM has a high ability to induce apoptosis compared with the original polypeptide. In one of specific embodiments of the present invention, the cell is swollen�left cell. In one embodiment of the present invention, the contacting occurs in vivo.

Another object of the present invention also relates to a method of treating a disease or disorder that is treatable altered cell signaling activity of the antigen target, method, representing an introduction to the subject in need, a therapeutically effective amount of a modified AFM, and a modified AFM includes a variable region of the heavy or light chain comprising at least one substitution of an amino acid in at least one wireframe plot of the variable region of the heavy or light chain compared with the variable region of the heavy or light chain of the original AFM, and the substitution leads to altered activity of transmission of the cellular signal is the antigen target if the modified AFM combined with the antigen target.

Another object of the present invention also relates to a method of treating a disease or disorder that is treatable with an altered ability to mediate cross-linking of one or more antigens of the target, the method comprising administering to a subject in need, a therapeutically effective amount of a modified AFM, in which a modified AFM includes a variable region of a heavy or �egcoa chain containing at least one amino acid substitution in at least one wireframe plot of the variable region of the heavy or light chain compared with the variable region of the heavy or light chain of the original ACM, and a modified AFM has the ability to mediate cross-linking of one or more antigens-targets as a result of such replacement.

In one of specific embodiments of the present invention is a modified AFM imposed according to the present invention, includes a variable region heavy chain selected from the group comprising the sequences: SEQ ID NO:4, SEQ ID NO:36 and SEQ ID NO:38.

In one embodiment of the present invention, the disease or disorder susceptible to treatment with a modified AFM according to the present invention, is a disorder associated with cell proliferation. In another variant implementation of the present invention a disorder associated with cell proliferation is cancer. In yet another embodiment of the present invention, the disease or disorder susceptible to treatment with a modified AFM according to the present invention, is a disorder of b-cells. In a specific embodiment of the present invention, the disorder of b-cells is a b-cell lymphoma.

Infusion�the invention also relates to the use of a modified AFM of the present invention to obtain drugs for treatment or prevention of cancer.

In one of specific embodiments the present invention relates to the use of a modified AFM of the present invention to obtain drugs for treatment or prevention of a cancer selected from the group comprising: b-cell lymphoma, breast cancer, bladder cancer, head and neck cancer, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and brain cancer.

In another specific embodiment of the present invention involves the use of a modified AFM of the present invention to obtain drugs for treatment or prevention of cancer, where the specified antigen-binding molecule is used in therapeutically effective amounts, lag of about 1.0 mg/kg to about 15 mg/kg In another embodiment of the present invention, therapeutically effective amount is from about 1.5 mg/kg to about 12 mg/kg In yet another variant implementation of the present invention, a therapeutically effective amount is from about 1.5 mg/kg to about 4.5 mg/kg In another embodiment of the present invention, therapeutically effective amount is from about 4.5 mg/kg to about 12 mg/kg In yet another variant implementation of the present and�gaining a therapeutically effective amount is about 1.5 mg/kg. In another embodiment of the present invention, therapeutically effective amount is approximately 4.5 mg/kg. In yet another embodiment of the present invention, therapeutically effective amount is about 12 mg/kg.

The present invention also relates to a method of treatment or prevention of cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a patient in need of it. In one of specific embodiments of the present invention the cancer is selected from the group including: b-cell lymphoma, breast cancer, bladder cancer, head and neck cancer, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and brain cancer.

The present invention also relates to a method of treatment or prophylaxis of a precancerous condition or disorders, comprising administering a therapeutically effective amount of the pharmaceutical composition under item 158 85 or patient in need of it. In one specific embodiment of the present invention, a precancerous condition or violation is selected from the group including leukoplakia of the oral cavity, actinic keratosis (senile warts), precancerous polyps of the colon or rectum, gastric, epithelial�th dysplasia, adenomatous dysplasia syndrome hereditary nonpolyposis colon cancer (SNRD), ulcers Barrett's esophagus, bladder dysplasia, and precancerous cervical conditions.

The present invention also relates to a modified antigen-binding molecule of the present invention for the treatment or prevention of cancer. In one of the embodiments of the present invention the cancer is selected from the group including b-cell lymphoma, breast cancer, bladder cancer, head and neck cancer, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and brain cancer.

The present invention also relates to a modified antigen-binding molecule of the present invention for use in the treatment or prophylaxis of a precancerous condition or violation. In one embodiment of the present invention, a precancerous condition or violation is selected from the group including leukoplakia of the oral cavity, actinic keratosis (senile warts), precancerous polyps of the colon or rectum, gastric epithelial dysplasia, adenomatous dysplasia syndrome hereditary nonpolyposis colon cancer (SNRD), ulcers Barrett's esophagus, bladder dysplasia, and precancerous cervical conditions.

The present invention also linked�GSI to a modified antigen-binding molecule of the present invention for use in treating disorders associated with altered signaling activity and/or altered by crosslinking one or more antigens-targets.

Brief description of figures

Fig.1. Alignment of amino acid sequences of the various structures of the variable regions of the heavy chain of anti-CD20 antibodies. Amino acid sequence of the variable regions of the heavy chain of monoclonal antibody 1F5 used as a control sequence. The differences of amino acids when compared with 1F5 shaded.

Fig.2. The binding of various humanized anti-CD20 antibodies from b-cells one. Source (chimeric) antibody B-ly1 compared with two humanized heavy chain variants for which it was established that they induce strong apoptosis (VNN and VNN), as well as with those derived (humanised does not cause apoptosis) of B-HL8, which supposedly restores this effect (variants b-HL11 to In-HL 17). All humanized variants of the heavy chain pair with the same humanized variant of light chain BKV1.

Fig.3. Binding of rituximab (O) and chB-ly1 (Δ) with CD20 on b-cells lymphoma one.

Fig.4. Comparison of the induction of antibody-dependent apoptosis in three anti-CD20 antibodies. Antibody chB-ly1 wt is the design of the chimeric antibody B-ly1, comprising the variable region of the rodent and the constant region h�person. Antibody BHH2-BKV1 represents a humanized variant comprising the CDRs of the rodent antibody B-ly1 and frame areas of a person derived from class VH1 V genes of the cells of the germline of the person for the heavy chain, and coupled with BKV1 light chain is a humanized antibody B-ly1. Antibody BHL8-BKV1wt represents a humanized variant comprising CDR of the murine antibody B-ly1 and frame areas of a person derived from V genes from cells of two different germ lines of a person, paired with a light chain BKV1 humanized antibody B-ly1.

Fig.5. Comparison of the induction of antibody-dependent apoptosis of five humanized variants of the anti-CD20 antibody B-ly1. Variant antibodies BHH2-BKV1 represents a humanized variant comprising the CDRs of the rodent antibody B-ly1 and frame areas of a person derived from class for VH1 heavy chain (VN) and paired with BKV1 light chain humanized antibody B-ly1. Variant antibodies BHL8-BKV1wt represents a humanized variant comprising the CDRs of the rodent antibody B-ly1 and frame areas of a person derived from V genes of cells of two different germ lines of human and paired with BKV1 light chain humanized antibody B-ly1. Variant antibodies BHL14-BKV1 is derived BHL8 with the replacement of valine, the lysine at position 12 to the numbering of Kabat and with the replacement of valine by methionine at position 48 by Kabat numbering in variabe�encourages creativity region of the heavy chain, coupled with the design BKV1 light chain. Variant antibodies BHL15-BKV1 W1 is also derived BHL8 with the replacement of glycine by serine at position 16 to the numbering of Kabat, and valine by methionine at position 48 by Kabat numbering in the variable regions of the heavy chain, coupled with the design BKV1 light chain. Variant antibodies BHL16-BKV1 W1 is derived antibodies BHL8 replacement of leucine with valine in position 20 according to Kabat numbering and valine by methionine at position 48 by Kabat numbering in the variable regions of the heavy chain, coupled with the design of the light chain of the antibody BKV1. Variant antibodies BHL17-BKV1 W1 is derived antibodies BHL8 with the replacement of valine by methionine at position 48 by Kabat numbering in the variable regions of the heavy chain and paired with the design of the light chain of the antibody BKV1.

Fig.6. Comparison of induction of apoptosis in cells Z-138 caused by SV anti-CD20 monoclonal antibody and two humanized versions of the antibody B-ly1, BHH2-BKV1 and BHL13-BKV1. Variant antibodies BHH2-BKV1 represents a humanized variant comprising CDR of the antibody B-ly1 rodents and frame areas of a person derived from class VH1 V genes of the germline of the human heavy chain paired with BKV1 light chain humanized antibody B-ly1. Variant antibodies BHL13-BKV1 is derived antibodies BHL8 (see Fig.5 above) with the substitution of leucine for valine at position 11 to the numbering of Kabat and�ene of valine by methionine at position 48 by Kabat numbering in the variable regions of the heavy chain and paired with a light chain BKV1.

Fig.7. Depletion of b cells by rituximab (◇) and chB-ly1 (■) in the whole blood of three different classes of genotype of FcγRIIIa-158V/F: (A) whole blood donor F/F homozygous for the receptor with low affinity, (B) whole blood donor F/V heterozygous for the receptor affinity, and (B) whole blood donor V/V homozygous for the receptor with high affinity.

Fig.8. Profile MALDI-TOF glycoengineered chimeric antibody B-ly1. (A) table showing the percentage content of specific peaks, (B) Spectrum glycoengineered chimeric antibody B-ly1, (B) Spectrum glycoengineered chimeric antibody B-Ly1, treated with Endo-H.

Fig.9. The binding of various humanized anti-CD20 antibodies from b-cells one. Differences design IN-N from designs B-HL8 and B-HL11 localized in frame portions 1 and 2 with all three CDRs that are identical. Design B-HL8 and B-HL11 have a sequence of frame sections that are derived from VH3 class people, although full frame plot-NN is derived from a human VH1. B-HL11 is a derivative of B-HL8 single mutation Glu1Gln, and Gln is an amino acid residue in the design of IN-NN. This means that the replacement Glu1Gln not alter the binding affinity or intensity. Other differences between IN-N and B-HL8 balances are 14 FR, of which one or more can affect antigens�binding property of the antibody.

Fig.10. Binding of humanized anti-CD20 antibody BHL4-BKV1 on target cells one. Design B-HL4 antibodies obtained from IN-N replacement frame section in FR1 IN-NN on a wireframe plot of the sequence IGHV1-45 (room H) germline of the person. This design shows reduced binding ability, despite the presence of different amino acids only four provisions of the plot in frame FR1. These residues are localized in positions 2, 14, 28 and 30 on numeralia Kabat. It is assumed that among these are important provisions 28 and 30 form part of CDR1 Chothia numbering.

Fig.11. A comparison of the sequestration capacity between IN-N, IN-N, IN-N (all paired with BKV1 light chain gumanitarnogo antibody B-ly1), and the initial antibody B-ly1. These data show that all antibodies show similar values of the EU50, but the design IN-N binds with a lower intensity/ stoichiometry than options IN-N and IN-N. In-N can be distinguished from IN-N and IN-N by partially humanised regions CDR1 and CDR2 (Kabat definition) and polymorphism, Ala/Thr at position 28 (Kabat numbering). This means that either position 28 or a whole region CDR1 and/or CDR2 region important for the interaction of an antibody with the antigen.

Fig.12. Comparison of the binding activity between the B-HL1, IN-N source and the antibody B-ly1. Submitted�Lenno data show the absence of any binding activity design B-HL1 and about two times less active on the intensity of binding/stoichiometry IN-N compared with the antibody B-ly1. And B-HL1, and IN-N designed on the basis of the acceptor skeleton plots, derivatives VH1 man. Among other differences, the most significant is the difference in position 71 (according to Kabat numbering) design B-HL1, which is important for binding antigen.

Fig.13. Comparison of binding structures B-HL2 and B-HL3 heavy chain of anti-CD20 antibodies with their antigen. In both cases, the sequence of VL of rodents combined with humanized heavy chains. These data indicate that B-HL2 and B-HL3 not show binding activity to the antigen CD-20.

Fig.14. Appticable the action of anti-CD20 antibodies on the cells tuckaleechee leukemia Z-138 MCL.

Fig.15. Apoptosis caused by anti-CD20 antibodies. Details of the research: 5×105cells/well was seeded into 24-well plates (5×105cells/ml) in culture medium. In the wells is added to a final concentration of 10 μg/ml of the respective antibody, FSB for a negative control or 5 mm camptothecin for positive control. Samples were incubated overnight (16 h), stained with AnnV-FITC and analyzed FACS. The study was conducted in three replicates. (*): Signal for one of the FSB subtract (one FSB gives 8% and 2% AnnV+ cells for PR-1 and Z-138, respectively. Using antibody: C2 B8 (chimeric, not subjected to glycoengineering); BHH2-BKV1 (a humanized, not subjected Glyco�kienerii). Note: this study does not include any additional effector cells, only the target + antibody or controls.

Fig.16. The destruction of target cells anti-CD20 antibodies with immune effector cells. Details of the research: the depletion of b-cells in normal whole blood determined by incubation over night and analyzed by FACS method on CD19+/CD3+. ADCC is determined, using as effectors of mononuclear cells of peripheral blood (PBMC) after incubation for 4 h at a ratio of 25:1 effector to the target. The destruction of target cells is measured by the delay of calcein compared to detergent lysis (100%) and a lysis without the antibody (0%). Using the following antibodies: C2 B8 (chimeric, not subjected to glycoengineering), BHH2-BKV1-dt (humanitariannet, not subjected to glycoengineered form BHH2-BKV1), BHH2-BKV1-GE (humanitariannet subjected to glycoengineered form BHH2-BKV1).

Fig.17. Profile MALDI/TOF-MS PNGaseF-released Fc-oligosaccharides unmodified uncircumcised glycoengineering BHH2-BKV1 humanized IgG1 B-ly1 antibody to human CD20 antibody.

Fig.18. Profile MALDI/TOF-MS PNGaseF-released Fc-oligosaccharides been glycoengineered BHH2-BKV1 humanized IgG1 B-ly1 antibody to human CD20 antibody. Glycoengineering conduct joint expression in the cells of the host�x of antibody genes and gene encoding an enzyme with catalytic activity of β-1,4-N-acetylglucosaminyltransferase III (GnT-III).

Fig.19. Profile MALDI/TOF-MS PNGaseF-released Fc-oligosaccharides glycoengineered BHH2-BKV1g2 humanized IgG1 B-ly1 anti-human CD20 antibody. Glycoengineering conduct joint expression in the cells-the owners of antibody genes and genes encoding an enzyme with catalytic activity of β-1,4-N-acetylglucosaminyltransferase III (GnT-III) and encoding an enzyme with catalytic activity of α-mannosidase II Golgi.

Fig.20. Binding of antibodies that have been glycoengineered and not subjected her (g2 version; glycosylation profiles, see Fig.17-19), with the receptor FcγRIIIa person shown on the surface of Cho cells expressing recombinant CD16.

Fig.21. Appticable the action of anti-CD20 antibodies, subjected and not subjected to glycoengineering in the field Fc, cells tuckaleechee leukemia Z-138. Details of the research: 5×105cells/well was seeded into 24-well plates (5×105cells/ml) in culture medium. In the wells is added to a final concentration of 10 μg/ml of the respective antibody or PBS for negative control. Samples were incubated overnight (16 h), stained with AnnV-FITC and analyzed by FACS. The study was conducted in three replicates. Using antibody: SW=rituximab (chimeric, not subjected�Otoe glycoengineered antibody), BHH2-BKV1 (a humanized, not subjected to glycoengineering - glycosylation profiles, see Fig.17-19), BHH2-BKV1g1 (a humanized subjected to glycoengineering), BHH2-BKV1g2 (a humanized subjected to glycoengineering). Note: this study does not include any effector cells, only the target + antibody or control. (*): Signal for FSK deducted.

Fig.22. Linking different humanisierung anti-CD20 antibodies from b-cells one. Design VNN humanized heavy chain compared to its derivatives VN and VNN. In addition, the options shown are options that determine the impact of regulations 28 and 30 according to the Kabat numbering (BHH8 and VNN).

Fig.23. The impact of single amino acid substitutions in apoptosis of anti-CD20 antibodies cells tuckaleechee leukemia Z-138. Details of the research: 5×105cells/well was seeded into 24-well plates (5×105cells/ml) in culture medium. In the wells is added to a final concentration of 10 μg/ml of the respective antibody or PBS for negative control (no antibody). Samples were incubated overnight (16 h), stained with AnnV-FITC and analyzed by FACS. The study was conducted in three replicates. Using antibody: C2 B8 (chimeric, not subjected to glycoengineered antibody), BHH2-BKV1 (a humanized, not subjected to glycoengineering), VN-A (derived VNN with Deputy�Noah valine by leucine at position 11 to Kabat numbering) and VN-IN (derived VNN with the replacement of lysine valine at position 12 by Kabat numbering), the last three antibody paired with a light chain BKV1. KDbinding of antigen by replacing remains unchanged. Note: this study does not include any effector cells, only the target + antibody or control. (*): Signal for FSK deducted.

Fig.24. Impact of single amino acid substitutions on apoptosis before an inactive anti-CD20 antibodies on the cells tuckaleechee leukemia Z-138 MCL. Details of the research: 5×105cells/well was seeded into 24-well plates (5×105cells/ml) in culture medium. In the wells is added to a final concentration of 10 μg/ml of the respective antibody or PBS for negative control. Samples were incubated overnight (16 h), stained with AnnV-FITC and analyzed by FACS. The study was conducted in three replicates. Using antibody: SV (chimeric, not subjected to glycoengineered antibody), BHL8 (a humanized, not subjected to glycoengineering), BHL13 (derived BHL8 replacement of leucine with valine at position 11 and valine by methionine at position 48 by Kabat numbering) and BHL14 (derived BHL8 with the replacement of valine with lysine in position 12 and valine by methionine at position 48 by Kabat numbering), the last three antibodies paired with a light chain BKV1. Note: this study does not include any effector cells, only the target + antibody or control.

Fig.25. The influence of W�exchange of a single amino acid in the light chain on apoptosis, caused by anti-CD20 antibodies in the cells of Z-138 MCL. Research: 5×105cells/well was seeded into 24-well plates (5×105cells/ml) in culture medium. The final concentration of 10 μg/ml of the respective antibody, PBS as negative control (no antibody) or 5 mm of camptothecin for positive control to make the hole. Samples were incubated overnight (16 h), stained with AnnV-FITC and analyzed FACS. The study was conducted in three replicates. Using the following antibodies: antibody VN-A (derived VNN with the substitution of valine for leucine at position 11 to Kabat numbering), paired with a light chain BKV1, antibody VN (derived VNN with the substitution of methionine for isoleucine at position 34 by Kabat numbering), paired with a light chain BKV1, and antibody VN, paired with a light chain BKV14 (derived BKV1 with the replacement of isoleucine by alanine at position 106 according to Kabat numbering).

Fig.26. A three-dimensional image of the molecular "spherical joint" on the border of the domains VH and CH1.

Detailed description of the invention

The terms used in the present invention are basically common, otherwise here is the explanation.

In the context of the present invention, the term "antigen-binding molecule (AFM)" refers in the broadest sense to the molecule, that specifically binds antigenic d�terminant. The term "specifically binds" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific relationships. In the context of the present invention, the term "modified antigen-binding molecule (or a modified AFM)" refers to the AFM, including the replacement of at least one amino acid residue in the variable regions of the heavy chain and/or region CH1, and/or replacing at least one amino acid residue in the variable region of light chain, and/or the field of CL.

Used in the present invention the term "antibody" refers to whole antibody molecules, including monoclonal, polyclonal and multispecific (e.g. bispecific) antibodies and fragments of antibodies having an Fc region, retains the binding specificity, and hybrid proteins that include a region equivalent to the Fc region of immunoglobulin, and maintain the binding specificity. The present concept also refers to fragments of antibodies, which kept the binding specificity, for example, fragments of VHfragments VL, Fab fragments, fragments F(ab')2the scFv fragments, Fv fragments, Manantial, double antibody, triple antibody and Quaternary antibodies, but their list is not limited to (see, e.g., Hudson and Souriau, Nature Md. 9, 2003, cc.129-134, the essence of which is given in the present invention by reference). The present concept also relates to humanized, primaryservername and chimeric antibodies. In the context of the present invention, the concept of a whole antibody refers to an immunoglobulin molecule comprising two heavy chains and two light chains, each of which includes a variable and a constant region.

In the context of the present invention, the term "variable region" refers to the N-terminal domain of the heavy or light chain of immunoglobulin. According to one embodiment of the present invention, a modified AFM may include a functional fragment of the variable region.

In the context of the present invention, the term "variable region of the heavy chain" refers to N-terminal domain of the heavy chain of immunoglobulin. In one example, the variable region of the heavy chain expressed by Kabat provisions 1-113 (with possible insertion at specific residues according to Kabat, etc., in proc.: 'Sequence of Proteins of Immunological Interest", 1983, publishing house of the Department of health and human services USA). In one of the embodiments of the present invention is a modified AFM may include a functional fragment of the variable region of the heavy chain.

In the context of the present invention, the term "constant region of the heavy chain" on�worn to the C-terminal domain of the heavy chain of immunoglobulin. There are five natural classes of constant regions of heavy chains: IgA, IgG, IgE, IgD and IgM. In one example of the constant region of the heavy chain comprises domains CH1, CH2 and CH3.

In the context of the present invention, the term "region CH1" refers to the domain of the heavy chain of immunoglobulin, which is C-terminal to the variable region and N-terminal to the hinge region. Immunoglobulin type IgG CH1 region according to Kabat expressed in normal provisions 114-228.

In the context of the present invention, the term "apoptosis" refers to programmed cell death that is characterized by certain cellular changes, such as fragmentation of the nucleus and/or the formation of apofaticheski bodies by condensation of cytoplasm, plasma membranes and/or organelles.

In the context of the present invention, the term "agonist activity" refers to the activity of the agent (e.g., antigen-binding molecules) interacting (e.g., binding) with a molecule associated with the surface of the cells, and the initiation or induction of the reaction.

In the context of the present invention, the term "antagonistic activity" refers to the activity of the agent (e.g., antigen-binding molecules) interacting (e.g., binding) with the molecule on the cell surface and as a result prevents initial�Yu or induction of response or reaction stops.

In the context of the present invention the concept of "hybrid" and "chimeric" as applied to polypeptides, e.g., AFM, are polypeptides comprising amino acid sequences derived from two or more heterologous peptides, for example, parts of antibodies from different species. To obtain chimeric AFM components that do not have antigen-binding properties, for example, can be obtained from a variety of species, including primates, e.g. chimpanzees and humans. Constant region chimeric AFM is most preferably essentially identical to the constant region of a natural human antibody and the variable region of the chimeric antibody is most preferably derived antigen-binding molecules (derived not from man, but from another donor), which specifically binds the investigated antigen. Chimeric AFM may include full variable region of the donor, in another embodiment, the chimeric antibody can include a humanized or primaryservername antibody. Humanized antibodies are the most preferred forms of hybrid or chimeric antibodies.

In the context of the present invention, the term "humanized" refers to antigen-binding molecule, which is derived antigen-binding molecules derived not from man,�reamer, murine antibody, that retains or substantially retains the antigen-binding properties of the original molecule, but to a lesser extent immunogene for people. This can be achieved in various ways, including (a) transplantation of entire variable domains derived not from man, in the constant region of a person to obtain chimeric antibodies, (b) transplant only CDR received from a man in a skeleton of the plot and the constant region of the person keeping or not keeping fundamentally important frame sites (i.e., those that are important for maintaining good antigen-binding affinity or functions of the antibody), or (b) transplantation of entire variable domains derived not from man, but "masking" their land, such humanitarianlaw, by replacement of surface residues. Such methods are described by Jones and others, Morrison, etc., Proc. Natl. Acad. Sci., 81, 1984, cc.6851-6855, Morrison and Oi, Adv. Immunol., 44, 1988, cc.65-92, Verhoeyen et, Science 239, 1988, cc.1534-1536, Padlan, Molec. Immun., 28, 1991, cc.489-498, Padlan, Molec. Immun., 31, 1994, cc.169-217; all these works are included in the present description in the form of links to their essence.

Usually, there are three complementary deterministic region (complementarity determining regions CDR), CDR1, CDR2 and CDR3, each of the variable domains of the heavy and light chains of the antibody, which are flanked by four skeleton subcatname (i.e., FR1, FR2, FR3 and FR4) in each of the variable� domains of the heavy and light chains of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. With the discussion of the humanized antibodies along with other works can be found in the patent US 6632927 and published application US No. 2003/0175269, the essence of which is included in the present description as references.

In a similar way in the context of the present invention, the term "primaryservername" refers to antigen-binding molecule, which is derived antigen-binding molecules are not from the body of a Primate, e.g., a rodent antibody, that retains or substantially retains the antigen-binding properties of the original molecule, but to a lesser extent immunogen in the body of primates.

In the case when the concept is used and/or referred to in the art, may have two or more interpretations, in the context of the present invention apply to him - all the concepts, except those that obviously have a different meaning. A specific example in the context of the present invention is the concept of "complementary predetermined region" ("complementarity determining region CDR") used to describe the non-contiguous antigen combining sites that are defined in the variable region polypeptides and heavy and light chain. This area is described by Kabat and others in kN.: "Sequences of Proteins of Immunological Interest", the Department of health and human services USA, 1983, and Chothia and d�. in J. Mol. Biol. 196, 1987, cc.901-917, and both works are mentioned in the present invention and the included links, where the definitions include overlapping or subsets of amino acid residues in comparison with each other. However, the use of any of the definitions for CDR of the antibody or its variants refers to this notion used in the present invention. Relevant amino acid residues which are part of the CDR according to the descriptions of the two publications cited above, are shown for comparison below in table.1, the Numbers of amino acid residues constituting a specific predetermined complementary region (CDR), can vary depending on the sequence and size of the CDR. The person skilled in the art can easily determine which residues contain a specific CDR, obtaining the amino acid sequence of the variable regions of the antibody.

Table 1.
Denote CDR1
According to KabatBy ChothiaOxAbM2
VHCDR131-3526-3226-35
VHCDR250-6552-5850-58
VHCDR395-10295-10295-102
VLCDR124-3426-3224-34
VLCDR250-5650-5250-56
VLCDR389-9791-9689-97

1In table.1 shows the different symbols all CDR and compliance of these symbols to the numbering of Kabat and others (see below).
2"OxAbM" refers to the designation CDR according to the software for modeling antibody Oxford Molecular's "AbM".

Kabat and others also described the numbering system for a variable domain sequences, which is applicable to any antibody. Specialist in this field can definitely be applied a system of "Kabat numbering" to any sequence �arabellege domain regardless of any experimental data beyond the sequence itself. In the context of the present invention "Kabat numbering" refers to a counting system that is installed Kabat and others in kN.: "Sequences of Proteins of Immunological Interest", the Department of health and human services USA, 1983. Unless otherwise stated, references to specific amino acid residues in the composition of the AFM correspond to the Kabat numbering system. The sequence in the sequence listing not numbered according to the Kabat system.

In the context of the present invention is related to the polypeptide the term "GnTIII activity" means polypeptides that can catalyze the addition of N-acetylglucosamine residue (GlcNAc) β-1-4 bonds to β-linked mannoside timenational core N-linked oligosaccharides. These include hybrid polypeptides exhibiting enzymatic activity similar (but not identical) with the activity of β(1,4)-N-acetylglucosaminyltransferase III, also known as β-1,4-mannosyl-glycoprotein 4-beta-M-acetylglucosaminyltransferase (EU 2.4.1.144) according to the numbering of the Committee on nomenclature of the International Union of biochemistry and molecular biology, Nomenclature Committee of the International Union of Biochemistry and Molecular Biology - NC-IUBMB), as measured in a particular biological study and dependent or independent of dose. In cases where there is a dose-dependence, it should not be identical dose-dependence GnTIII, but preferably in Zn�siderable extent, similar to the dose-dependence of this activity in comparison with GnTIII (i.e., the investigated polypeptide may show increased activity, or activity that is less than about 25 times, preferably not reduced by more than about 10 times, and most preferably, reduced by not more than about 3 times the GnTIII activity.)

In the context of the present invention, the term "variant" or "analog" refers to a polypeptide that differs from a specific polypeptide of the present invention by insertion, graduations or substitutions of amino acids, produced through, for example, methods of DNA recombination. Options AFM of the present invention include chimeric, primaryservername or humanized antigen-binding molecules in which one or several amino acid residues are modified by replacing, adding and/or division in such a way that would not substantially affect antigen-binding affinity. The approach to determining which amino acid residues can be substituted, added or delegated without loss of activity can be established by comparing the sequence of a particular polypeptide with sequences homologous peptides and minimizing the number of changes in amino acid sequence highly homologous (conservative) or by replacing amino acids on concensus of posledovatel�ness.

Using another approach, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by "excess" in the genetic code. Different replacement codons, for example, silent changes, which cause the formation of various restriction sites, may be introduced to optimize cloning into plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, change properties, for example, ligand-binding affinities, miaocheng affinity or the rate of loss/turnover.

In the context of the present invention, the term "replacement" amino acid residue" means the replacement of one or several amino acids in a reference sequence (for example, in the original molecule, for example, antigen-binding molecule). In one of the embodiments of the present invention, the replacement amino acid residue may be, for example, in the form of point mutations in nucleic acid sequence that encodes a polypeptide that distinguishes it from the original peptide. In another embodiment of the present Fig�plants are the replacement of amino acid residues can be achieved by the replacement of the whole skeleton of the plot of the original polypeptide, for example, a wireframe plot of the sequence VH germ line, which includes the desired amino acid in the position, which will be replaced relative to the source sequence.

In the context of the present invention, the term "conservative amino acid sequence" refers to sequences that can be obtained by replacing one amino acid with another having similar structure and/or chemical properties, i.e., obtained as a result of conservative amino acid substitutions, and the basis may be the similarity in polarity, solubility, hydrophobicity, hydrophilicity and/or amphipathicity nature. For example, nonpolar (hydrophobic) amino acids include glycine, alanine, leucine, isoleucine, valine, Proline, phenylalamine, tryptophan and methionine, to the polar neutral amino acids include series, threonine, cysteine, tyrosine, asparagine and glutamine, to a positively charged (basic) amino acids include arginine, lysine, and histidine, and to negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The size of insertions and deletions is preferably 1-20 amino acids, more preferably 1-10 amino acids. The allowable variation can be experimentally determined by systematically produce inserti�, deletions or substitutions of amino acids in the polypeptide molecule produced by the methods of DNA recombination and subsequent evaluation of the activity of recombinant variants.

In the context of the present invention, the term "original antigen-binding molecule" or "original molecule" means a polypeptide with a specific amino acid sequence encoded by the polynucleotide sequence. The sequence of the original molecule (i.e. the original sequence) serves as a control sequence when making substitutions of amino acid residues which could change the ability of the formed molecule (e.g., a modified antigen-binding molecules) to induce or block the activity of transmission of the cellular signal and/or cross-linking antigen. In addition, the original molecule is control in determining whether to replace the antigen effect on the transmission of cellular signals and/or cross-linking antigen, and, if necessary, the degree of manifestation of this action. A sequence containing one or more substitutions of amino acids compared to the original sequence (for example, a modified AFM) can in turn serve as the initial sequence for further substitutions.

In the context� present invention, the term "altered cellular signaling activity" refers to increasing or decreasing the ability of the AFM to induce or inhibit cellular signaling activity of the antigen target.

In the context of the present invention, the term "altered cross-linking of one or more antigens-targets" means the increase or decrease of the ability of the AFM to contact in close proximity to each other and/or in close proximity with other membrane-associated molecules, and/or in a more favorable conformation for interacting antigens-targets that are able to form complexes (e.g., through cross-linkage of proteins or oligomerization of membrane-associated receptors) to initiate cellular signaling metabolic pathways.

In the context of the present invention, the term "transmission mechanism of cell signal" or "cellular signaling activity" refers to the complete metabolic pathway of signal transmission (i.e., signal transduction), which leads to a specific change in the cell or to a specific biological effect, as well as any signal stages on this metabolic pathway.

In the context of the present invention, if it is stated that a nucleic acid or nucleotide sequence, for example at least 95% "identical" reference nucleotide sequence of the present invention, this means that the nucleotide sequence of the polynucleotide is identical to the control �posledovatelnosti except the circumstances that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the control nucleotide sequence. In other words, to obtain nucleotide sequences at least 95% identical to a control nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be delegated or substituted with other nucleotides, or up to 5% of all nucleotides in the reference sequence may be insertion in the control sequence.

In practice, in order to determine whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence or polypeptide sequence of the present invention can be applied a conventional method with the use of computer programs. The preferred method of determining the most General coincidence between the studied sequence (sequence of the present invention) and a subject sequence, also called a common sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et, Comp. The RDAs. Biosci. 6, 1990, cc.237-245. For the alignment of the sequences under study and subject are both pic�egovernance DNA. The RNA sequence can be mapped in the conversion of Y in T. the Result of the reconciliation specified shared sequence identity is expressed in percents. Preferred parameters used in the FASTDB alignment of DNA sequences and calculation of percent identity are: matrix = unitary, k-ratio = 4, penalty for the incorrect pairing of bases = 1, penalty colspan = 30, the length of a randomised group = 0, the boundary cut-off = 1, gap penalty = 5, penalty size of the gap = 0.05, and window size = 500 or the length of the subject nucleotide sequence, as it was shorter.

If the subject sequence is shorter than the studied sequence because of 5' or 3' deletions, not because of internal deletions, can be made by manual correction of the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at 5'- or 3'-ends as compared with the studied sequence, the percent identity is correct by counting the number of bases of the studied sequence, which represents the 5' and 3' of the subject sequence, which are not compatible/not aligned, as a percent of the total number of bases studied in the pic�egovernance. Whether the nucleotide compatible/aligned is determined by results of sequence alignment as a result of using the FASTDB program. This percentage is then subtracted from the percent identity, calculated by using the above FASTDB program using the specified parameters, to achieve the totals percent identity. Received the adjusted value is used for the purposes of the present invention. Only bases outside the 5' and 3'bases of the subject sequence, as shown in the reconciliation of using the FASTDB program which is not compatible/not aligned with the target sequence, count for manually adjusting the percent identity.

For example, a 90 base pairs of subject sequence is compared with 100 bases of the studied sequence to determine percent identity. Deletions are located from the 5'-end of the subject sequence and therefore, the reconciliation method FASTDB doesn't show the error/the coincidence of the first 10 bases from the 5' end. the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3'ends does not match the total number of bases in the studied sequence) so 10% is subtracted from estimates of the percent identity calculated by FASTDB program. If the remaining 90 bases full�STU correspond, the final percent identity might be 90%. In another example, a subject sequence length 90 bases is compared with the test sequence length of 100 bases. This time the deletions are internal divisions because there are no bases on the 5'- or 3'-end of the subject sequence, which are not compatible/not vyravneny with the control sequence. In this case the percent identity score using the FASTDB and do not adjust manually. Once again, only bases 5' and 3'end of the subject sequence, which are not compatible/not vyravneny with the studied sequence, correct manually. For the purposes of the present invention, no other manual adjustment is not made.

With the help of a polypeptide having an amino acid sequence at least 95% "identical" to the studied amino acid sequence of the present invention, it is meant that the amino acid sequence of the subject polypeptide is identical to the studied sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the studied amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence�alnost at least 95% identical to the investigated amino acid sequence, up to 5% of amino acid residues in the subject sequence may be insertion, delegated, or replaced by other amino acid residues. Such change of control sequences can be amino - or carboxy-end of the control amino acid sequence or anywhere between those terminal positions, distributed or half individually among the amino acid residues in the reference sequence or in one or more neighboring groups in the control sequence.

For example, in order to determine whether a particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference polypeptide, can be used known to the control computer program. The preferred method of determining the greatest common overlap between the studied sequence (sequence of the present invention) and a subject sequence, also referred to as a method of determining alignment of a considered overall sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et, Comp. The RDAs. Biosci. 6, 1990, cc.237-245. During reconciliation reference and subject sequences they can be either nucleotide or amino acid sequences. The result of the reconciliation of the considered � whole sequence is the percentage identity. Preferred parameters used in the alignment of amino acids using the FASTDB program are: matrix = PAM 0, k-multiplicity = 2, penalty for the incorrect pairing of bases = 1, penalty colspan = 20, the length of a randomised group = 0, the boundary cut-off = 1, window size = sequence length, gap penalty = 5, penalty size of the gap = 0.05, and window size = 500 or the length of the subject amino acid sequence, as she was shorter.

Preferred parameters used in the FASTDB alignment of DNA sequences and calculation of percent identity are: matrix = unitary, k-ratio = 4, penalty for the incorrect pairing of bases = 1, penalty colspan = 30, the length of a randomised group = 0, the boundary cut-off = 1, gap penalty = 5, penalty size of the gap = 0.05, and window size = 500 or the length of the subject nucleotide sequence, as it was shorter.

If the subject sequence is shorter than the analyzed sequence due to N - or C-terminal rather than internal, deletions, to obtain the result should be produced by manual correction. This is because the program ASTDB does not account for N - and C-terminal truncations of the subject sequence when calculating the total percentage of identity. For subject sequences truncated � N - and C-Termini, when comparing with the studied sequence percentage identity is correct by counting the number of residues of the investigated sequences that are N - and C-terminal residues of the subject sequence which are not matched/not well aligned with a corresponding subject residue in the form of a percentage of the total number of bases of the studied sequence. Whether a residue is matched/reconciled determined by the results of sequence alignment using the FASTDB program. The resulting percentage is then subtracted from the percent identity, calculated by using the above FASTDB program using the specified parameters, to obtain the final estimates of percent identity. The final estimate of the percentage of identity is used for the purposes of the present invention. Only residues to the N - and C-ends of the subject sequence which are not matched/not reconciled with the studied sequence, take into account when manually adjusting the percent identity. It is only the provisions of the investigated residues outside the N - and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with the target sequence of 100 residues to determine the percentage identity. Deletion is from N-Terminus of the subject sequence�activities and, consequently, the use of the program for FASTDB alignment does not show a match/alignment of the first 10 residues from the N-end. the 10 unpaired residues represent 10% of the sequence (number of residues with N - and C-end does not coincide with the total number of residues in the examined sequence) so 10% is subtracted from the magnitude of the estimate of the percent identity calculated by FASTDB program. If the remaining 90 residues are fully compatible, the final percent identity might be 90%. In another example, the subjective sequence of 90 residues is compared with the target sequence of 100 residues.

In this case, the deletions are internal, so there are no residues at the N - or C-ends of the subject sequence, which are not the same/not reconciled with the studied sequence. In this case the percent identity score using the FASTDB program without manual correction. Thus, only the residues outside the N - and C-Termini of the subject sequence, as was shown by the combination of the FASTDB program, do not match/are not verified with the test sequence, and correction is carried manually. Other correction for the purposes of the present invention are not conducted.

In the context of the present invention a nucleic acid that "hybridizing under stringent conditions" with serial�the activities of the nucleic acids of the present invention, means a polynucleotide, which hybridizers over night at 42°C in a solution comprising 50% formamide, 5x SSC (750 mm NaCl, 75 mm sodium citrate), 50 mm sodium phosphate (pH 7.6), 5x solution Denhardt, 10% of dextransucrase and 20 µg/ml denatured cut DNA salmon sperm, followed by washing the filters in 0.1 x SSC at about 65°C.

In the context of the present invention, the term "Fc region" refers to the C-terminal region of the heavy chain of IgG. Although the boundaries of the Fc region in IgG heavy chain can vary slightly, the Fc region of the heavy chain of human IgG is usually determined in the form of a length of the amino acid residue at position Cys226 to the carboxy end of the sequence.

In the context of the present invention, the term "region equivalent to the Fc region of immunoglobulin" includes natural allelic variants of the Fc region of immunoglobulin, as well as variants having alterations caused by substitutions, additions or deletions, but does not significantly reduce the ability of the immunoglobulin to mediate effector functions (e.g. antibody-dependent cellular cytotoxicity). For example, there may be one or more deletions of amino acids from the N-end or C-end of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected by observing a few rules, famous�x in this field to identify minimal impact on the activity. (See, for example, Bowie J. U., etc., Science 247, 1990, cc.1306-1310). In one embodiment of the present invention, the region equivalent to the Fc region" may also form part of heterological hybrid protein. In some embodiments of the present invention, the region equivalent to the Fc region" also includes the corresponding region of the heavy chain from another class of immunoglobulin (e.g., IgA, IgE, IgD and IgM).

In the context of the present invention, the term "domain Golgi localization" refers to the amino acid sequence of a resident polypeptide Golgi, which is responsible for zakalivanie polypeptide localization in the Golgi complex. Typically, the localization domains contain amino-terminal "tail" of the enzyme.

In the context of the present invention, the term "effector function" refers to those biological activities that are associated with the Fc region (a native sequence Fc region or amino acid sequence region of the variant Fc) antibodies. Examples of effector functions of antibodies include, but is not limited to, binding affinity Fc-receptor, antibody-dependent cellular cytotoxicity (antibody-dependent cellular cytotoxicity - ADCC), antibody-dependent cellular phagocytosis (antibody-dependent cellular phagocytosis - ADCP), the secretion of cyto�ina, mediated by immune complex consumption antigen antigen-presenting cells, loss of regulation of cell surface receptors, etc.

In the context of the present invention the concept of "engineering, engineering, glycoengineering, glycoengineered and glycosylation engineering" includes any manipulation of the glycosylated sample of natural or recombinant polypeptide, for example, antigen-binding molecule (AFM) or its fragment. Glycosylation involves the metabolic engineering of the glycosylation mechanism of the cell, including genetic manipulation of metabolic pathways for the synthesis of oligosaccharides to achieve altered glycosylation of a murine cells glycoproteins. In one of the embodiments of the present invention is directed to the glycosylation engineering is the activity of glycosyltransferases. In one of specific embodiments of the present invention engineering leads to altered activity glucosaminidase and/or activity fucosyltransferase.

In the context of the present invention, the term "host" refers to any type of cellular system which can be designed to obtain polypeptides and antigen-binding molecules of the present invention. To cells-owners from�Osada cultivated cells, for example, cultured mammalian cells, such as Cho cells, BHK, HEK293-EBNA, NS0, SP2/0, cells, YO myeloma cells of mouse myeloma RH, cells PER, PER.C6, or cell hybridomas, yeast cells, insect cells and plant cells, including the cells of the transgenic animal, transgenic plant or cultured plant tissue or animal. In one of the embodiments of the present invention designs a host cell to obtain the antigen-binding molecule with modified glycoforms. In a preferred embodiment of the present invention the antigen-binding activity is an antibody, antibody fragment or hybrid protein. In some embodiments of the present invention the host cell are further manipulated to Express increased levels of one or more polypeptides having GnTIII activity. In other embodiments of the present invention are engineering the host cell to acquire eliminated, reduced or suppressed activity of the cow α1,6-fucosyltransferase. The concept of "cow activity of α1,6-fucosyltransferase" extends to the capsid gene expression of α1,6-fucosyltransferase, and crustal interaction of the enzyme α1,6-fucosyltransferase and a suitable substrate.

In con�Exte present invention, the term "Fc-mediated cellular cytotoxicity" includes antibody-dependent cytotoxicity and cytotoxicity, mediated by soluble Fc-hybrid protein containing the Fc region of a human. This is an immune mechanism leading to the lysis of the target cells for the antibody" immune effector cells", and:

In the context of the present invention, the term "immune effector cells" are a population of cells on which surface Fc receptors appear on the cell surface, through which they bind to the Fc region of the antibodies or Fc-hybrid proteins and perform effectornyi functions. Such population may include but it not limited to, mononuclear cells of peripheral blood (PBMC) and/or natural killer cells (GAC).

In the context of the present invention, the term "target cells antibodies" means cells associated with antibodies or Fc-hybrid proteins. The antibodies or Fc-hybrid proteins bind target cells via protein portion of the N-Terminus of the Fc region.

In the context of the present invention the term "increased Fc-mediated cellular cytotoxicity" means either the increase of the number of target cells antibodies that lyse at a given time at a given antibody concentration or the concentration of Fc-hybrid protein, in the medium surrounding the target cells, by the mechanism described above Fc-mediated cellular cytotoxicity, and/or a decrease in the concentration of anti�La or Fc-hybrid protein in the environment, surrounding the target cells, required to achieve the lysis of a given number of target cells antibodies" at this time, by the mechanism of Fc-mediated cellular cytotoxicity. Increased Fc-mediated cellular cytotoxicity relative cell cytotoxicity mediated by the same antibody or Fc-hybrid protein formed by the same type of host cells, using the same standard methods of receiving, cleaning, processing and storage, known to specialists in this field, but not produced by the cells-the hosts created for the expression of glycosyltransferases GnTIII, the methods described in the present invention.

By "antibody with increased antibody dependent cellular citotoxicity (ADCC) in the context of the present invention means an antibody with enhanced ADCC, which is determined by appropriate methods known to experts in this field. One of the methods of research ADCC described below in the example. Other reasonable means of determining in vitro ADCC is the following method:

1) in this study use target cells, known to Express the antigen is a target recognized by the antigen-binding region of the antibody,

2) in a study using mononuclear cells of peripheral blood (PBMC) isolated from blood about random�once selected donor as effector cells,

3) the study was conducted according to the following Protocol:

(i) PBMC isolated using centrifugation in standard density, and suspended to a density of 5×106cells/ml of culture medium RPMI,

(ii) the target cells are grown using standard methods of tissue culture, cells harvested in the exponential phase of growth with viability more than 90%, washed in RPMI medium for cell culture, put the label in the amount of 100 McCurry51Cr, washed twice with culture medium for cells and was resuspended in the medium for cell cultures at a density of 105cells/ml,

(iii) 100 μl of the above suspension of cells contributing to each well of 96-hole tablet for micrometrology,

(iv) the antibody is serially diluted from 4000 ng/ml to 0.04 ng/ml in the medium for cell culture and 50 μl of the obtained solutions make antibodies to target cells in 96-well plates for micrometrology testing in three replicates each concentration of antibody across the range of concentrations specified above,

(v) controls for the maximum release (MB) in three additional wells in the plate containing the labeled target cells, contribute 50 ál of 2 vol.% aqueous solution of a nonionic detergent (product Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above)

(vi) controls for spontaneous vysvobozhdeny� (SV) in three additional wells in the plate, containing the labeled target cells, contribute 50 ál of culture medium RPMI instead of the antibody solution (point iv above)

(vii) then 96-well plates for micrometrology centrifuged in a mode of 50 g for 1 min and incubated for 1 h at 4°C.

viii) 50 microliters of the PBMC suspension (point i above) are making to each well to obtain the ratio of effector: target cells was 25:1 and the plates were incubated in an atmosphere of 5% CO2at 37°C for 4 h,

(ix) the supernatant without cells from each well were collected and quantify the released radioactivity (BP) using the count of gamma-rays,

x) the percentage of specific lysis counts for each antibody concentration according to the formula: (BP-MB)/(MB-CB)×100, where BP denotes the calculated average radioactivity (see point ix above) for that antibody concentration, MB denotes the calculated average radioactivity (see point ix above) for controls MB (see point v above) and ST denotes the average radioactivity (see point ix above) for controls (see point vi above)

4) the concept of "antibody-dependent cellular cytotoxicity - ADCC" means or increasing the maximum percentage of specific lysis observed within the range of concentrations of the antibodies tested above, and/or a decrease in the antibody concentration required to achieve ½ the maximum percentage of the specific soil�ski lysis, observed within the above the tested concentration range of the antibody. The increase in ADCC relative to the magnitude of the ADCC, measured in the above study, performed with the same antibody produced by the same type of host cells, using the same methods standard production, purification, processing and storage, known to specialists in this field, but not by the cells of the host, designed for sverkhekspressiya GnTIII.

Antigen-binding molecules with substitutions of amino acids in the heavy and/or light chain

One of the objects of the present invention is associated with antigen-binding molecules (AFM), including modified V region and/or heavy and/or light chain, as well as the discovery of the fact that the ability of such AFM to induce cellular signaling activity of the antigen target and/or to mediate cross-linking of the antigen target can be increased (i.e., induced or increased) or reduced (i.e. suppressed or reduced) as a result of such modifications. Thus, the present invention provides polypeptides, including AFM, having a modified V regions and/or areas With heavy and/or light chain nucleic acid sequences (e.g., vectors) that encode such polypeptides, methods for obtaining poly�of aptidon, with modified V and/or areas With heavy and/or light chain, and methods of their use for the treatment of various diseases and disorders.

There are several mechanisms involved in therapeutic efficacy of antibodies, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (complement-dependent cytotoxicity CDC), induction cessation of growth or apoptosis, blocking or inhibition of cell growth, cell proliferation, cell survival, and/or other cellular processes. For example, described the case of induction of cell death and other cellular signaling events agonistic monoclonal antibodies. Cerisano and others have described the induction of caspase-independent cell death, characterized apoptosome signs (including the exposure of phosphatidyl-serine (PS), morphological changes and/or reflects the consumption of the iodide (PI)) and gomotopicheskoi aggregation of Ewing's sarcoma cells, stimulation of the agonistic antibodies against the transmembrane glycoprotein CD99 (for example, anti-S 013 MAb and O662 MAb) (Cerisano, etc., Oncogene 23, 2003, cc.5664-5674). In addition. Likewise, Hahn, etc. installed activation of MAPK signaling pathways through engagement of CD99 with anti-S monoclonal antibodies (e.g., DN16 and YG32), which leads to gomotopicheskoi aggregation of cells (Hahn et, FEBS Letters 470, 2000, cc.50-354). Pettersen and others identified a new functional domain of CD99, which can be activated using anti-S monoclonal antibodies, Ad20, activation of which induces apoptosis in transformed T-cells (Pettersen et, J. Immunol. 166, 2001, cc.4931-4942). Monoclonal antibody against the antigen CD47 (e.g., VN) can also induce caspase-dependent cell death, which is associated with metabolic signaling pathways reorganization of the cytoskeleton (Mateo, etc., Blood 100, 2002, cc.2882-2890). The essence of each of the above cited works included in this description by reference.

In other examples, it is shown that some antibodies against the antigen CD20 (e.g., rituximab and it) and CD52 (SAMRAT-1H) directly induce apoptosis in cancer cells. Cm. Ludwig, etc., Oncogene 22, 2003, cc.9097-9106. For rituximab and some other monoclonal antibodies with small signal activity or without signalling activity (anti-CD19, CD21, CD22 and Neg) the ability to induce apoptosis or to stop growth is enhanced by chemical conversion of antibodies in homodimeric IgG-IgG. Ghetie, etc., Proc. Natl. Acad. Sci. 94, 1997, cc.7509-7514). It was suggested that the increase was due to increased negative signal transmission and/or increased cross-stitching tetravalent homodimeric antibodies. Ghetie, etc., Proc. Natl. Acad. Sci. 94, 1997, cc.7509-7514. Cross shivanie increased apoptosis have also been achieved through the use of secondary antibodies or Fc-receptor-bearing helper cells. Cm. Jazhirehi and Bonavida, Oncogene 24, 2005, cc.2121-2143. The essence of each of the above cited works included in this description by reference.

Not relying on a theoretical basis, the present invention determines that modification of amino acid residues in the region of the hinge antigen-binding molecules can affect the ability of the AFM to induce or inhibit the signaling activity and/or cross-linking of the antigen target. The angle of the hinge region controls the orientation of V With respect to the immunoglobulin and thus facilitates the interaction of antibodies with antigen and effector proteins. Cm. Lesk and Chothia, Nature 335, 1988, cc.188-190. Lesk and Chothia mounted residues that support the "molecular ball-and-socket joint" in the "elbow angle" in antibodies, namely in positions 11, 110 and 112 in the VH region and at positions 149 and 150 in the CH1 region according to Kabat numbering, and found that there is a high degree of conservativeness of antibodies, namely amino acid residues that support the joint. Lesk and Chothia, Nature 335, 1988, cc.188-190 (the essence of this work is given in the present description by reference). However, they did not receive modifications of residues spherical articulation or residues that are close. Landolfi and others showed that modifications in the provisions 10-13 by Kabat numbering in the AF2 antibody that neutralizes IFN-γ cel�century lead to a substantial loss of neutralizing activity of the antibody, but do not affect the binding of an antibody with its antigen target. Landolfi, etc., J. Immunol. 166, 2001, cc.1748-1754 (the essence of this work is given in the present description by reference). However, Landolfi and others found no effect on the ability of antibodies to induce cell signal or to mediate cross-linking antigen.

The multivalent AFM the ability to change orientationselective sites allows you to change the proximity associated antigenic units when combining multiple antigen-binding sites. The closeness of the antigenic units to each other facilitates high or low interaction (e.g., cross-stitching, dimerization, etc.) between antigenic units. For example, if the bending angle of each pair of VH/VL-CH1/CL in the AFM is oriented in such a way that the antigen-binding sites are in closer contact with each other, the unit of binding to the antigen (e.g., cell surface receptor molecules) can also come into contact tightly with each other or acquire a conformation that is more favorable for interaction. This proximity or conformational changes can mediate the interaction, for example, cross-linking or oligomerization, communicating between antig�us. On the other hand the orientation of the antigen-binding sites is that they are farther apart or are less favorable conformation that can prevent their interaction.

The amino acid residues at the boundary of VL-CL can also be modified to influence the orientation of the binding site of the antigen. For example, residues at positions 40, 80, 83, 105 and 106 by Kabat numbering in the frame sections of variable region light chain is located on the border of the VL/CL.

The activity of any cellular signaling mechanisms may be altered (i.e., induced or suppressed) of antigen-binding molecules (AFM) of the present invention. The present invention also relates to cellular signaling mechanisms, including those that are initiated via cell surface receptor proteins, including associated with ion channels, G-protein and parentclassname of cell surface receptor proteins. Cm. kN.: "Molecular Biology of the Cell", 1994, ed. by Alberts and others, 3rd ed., Chapter 5 included in the present invention by reference. Thus, for example, of the present invention to a cellular signaling activity includes, but is not limited to, the activities that cause apoptosis, cell differentiation, cell growth, cell proliferation and survival of cells, as well as any stage gear� signal by a metabolic pathway. In one embodiment of the present invention, a cellular signaling activity is manifested through fermentopathy receptor; in one of specific embodiments of the present invention fermentopathy receptor is a receptor receptor. In another embodiment of the present invention, a cellular signaling activity through the receptor's associated ion channel.

Modified V region and/or the heavy or light chains of the AFM of the present invention differ from the corresponding unmodified areas of the original polypeptide (for example, the source of antigen-binding molecules)at least one amino acid substitution. The concept of "original", "primary" or "non-modified" polypeptide preferably includes at least a portion of the heavy or light chain of an antibody and may be prepared by methods known in this field to obtain polypeptides comprising the V region of the heavy chain, or the CH1 region or part, and/or V region light chain or the area or part thereof. In one specific embodiment of the present invention, the original polypeptide is an antigen-binding molecule and includes at least a portion of the field of VH or VL. In some embodiments of the present invention modification�carovana V region of the heavy and/or light chain can be obtained (for example, described in the present invention methods) and can be hybridisierung with a heterologous polypeptide such as an antibody Fc. In one of the embodiments of the present invention is a modified AFM or include a snippet of the hybrid protein in which the modified V region of the heavy chain or its fragment hybridisierung constant region of the heavy chain selected from the group including IgA, IgG, IgE, IgD and IgM, fragment or derivative. In one specific embodiment of the present invention, the constant region of the heavy chain is an IgG. In another embodiment of the present invention is a modified AFM or include a snippet of the hybrid protein in which a modified V region light chain or a fragment hybridizing with a constant region light chain selected from the group including IgA, IgG, IgE, IgD or IgM, or a fragment, or derivative. In one specific embodiment of the present invention, the constant region of the light chain is an IgG. In specific embodiments of the present invention the polypeptides of the present invention include whole antibodies (e.g. IgG) containing a light chain and a heavy chain having a modified V region of the heavy and/or light chain.

Polynucleotides that encode a polypeptide containing modifici�created V region or the CH1 region of the heavy chain or a modified V region or CL region of light chain, can be obtained by methods known in this field according to the present description for certain sequences. These methods include, but are not limited to, the preparation method of site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis previously obtained nucleic acid that encodes a polypeptide. Site-directed mutagenesis is the preferred method of obtaining substituted variants. This method is well known in the art (see, for example. Carter, etc. Nucleic Acids Res. 13, 1985, cc.4431-4443 and Kunkel, etc., Proc. Natl. Acad. Sci. USA 82, 1987, p. 488, both publications included in the present invention in the form of links). In short, when performing site-directed mutagenesis of the original DNA is modified first by hybridization of an oligonucleotide encoding the desired mutation with a single chain of such starting DNA. After hybridization, DNA polymerase is used to synthesize an entire second circuit, using the hybridized oligonucleotide as a primer and using one of the primary chain DNA as template. Thus, the oligonucleotide encoding the desired mutation, incorporate in formed Dunaeva DNA.

PCR mutagenesis is also suitable for the production of variants unmodified amino acid sequence of the starting polypeptide (see, for example, the publication Vallette, etc., Nuc. Acids Res. 17, 1989, cc.723-733 included in the present invention by reference). Briefly, when small amounts of matrix DNA as starting material for PCR primers, a slightly different sequence from the corresponding region of matrix DNA, can be used to obtain relatively large quantities of a specific DNA fragment that differs from the matrix sequence only at the positions where the primers differ from the matrix.

Cassette mutagenesis is another way to obtain variants of the AFM, based on the method described by Wells and others in the publication of Gene 34, 1985, cc.315-323 included in the present invention by reference. The starting material is the plasmid (or other vector) comprising the starting polypeptide DNA that will be modified. Identify the codons (codons) in the starting DNA, which then cause mutation. Must be unique site restriction enzymes on each side of the identified site (s) mutations. If no such restriction sites are absent they can be obtained using the method described oligonucleotide-mediated mutagenesis for their introduction in accordance with the relevant localization in the starting polypeptide DNA. Plasmid DNA is cut at these sites, resulting in it b�Xia linear. Double-oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation (mutation) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridizing using standard techniques. This double-nucleotide is called a cluster. Such a cassette is created to obtain the 5' and 3' ends that are compatible with the ends of the linearized plasmid, and it can be directly Legerova in the plasmid. This plasmid now contains the mutated DNA sequence.

Alternatively or additionally, the desired amino acid sequence encoding a polypeptide variant can be determined, and a nucleic acid sequence encoding such variant amino acid sequences may be obtained synthetically.

The amino acid sequence of the original polypeptide can be modified to obtain the AFM with a modified V region and/or a modified region CH1 of the heavy chain and/or a modified V region and/or a modified region CL light chain, with an altered ability to induce cellular signaling activity of the antigen target, if modified AFM is combined (e.g., binds) with the antigen target. Cle�full-time signaling activity can be agonistic activity or antagonistic activity. According to one object of the present invention agonistic activity is induced by a modified antigen-binding molecule if it binds to cellular membraneassociated receptor and initiates cellular signaling metabolic pathway. In one of the embodiments of the present invention cellular signaling metabolic pathway is apoptosis metabolic path. In another embodiment of the present invention cellular signaling metabolic pathway is the metabolic process by differentiation. According to another object of the present invention antagonistic activity of the modified antigen-binding molecules is shown, for example, in the case where the AFM binds membrane-associated receptor and prevents the induction of cellular signaling metabolic pathway or interrupts the current signal. Antagonistic activity can be achieved, for example, blocking the binding and subsequent signal transduction of endogenous ligand and/or prevention of cross-stitching or oligomerization of receptors or other molecules that may be necessary for the induction of cellular signaling metabolic pathway. In one embodiment of the present invention, a cellular signal�iny metabolic pathway, which is suppressed or interrupted, is the metabolic path of cellular growth. In another embodiment of the present invention cellular signaling metabolic route, which is suppressed or interrupted, is the metabolic pathway of cell division. In yet another variant implementation of the present invention cellular signaling metabolic route, which is suppressed or interrupted, is the metabolic pathway of cell survival.

The amino acid sequence of the original polypeptide can also be modified to obtain the AFM with a modified V region or the modified region With the heavy chain (for example, a modified region CH1), and/or a modified V region and/or a modified CL region light chain with an altered ability to mediate cross-linking of one or more antigens-targets, if modified AFM is combined (e.g., binds) with the antigen-targeted antigens-targets). In one of the embodiments of the present invention associated antigens-targets (e.g., cell surface receptor molecules) come in closer contact with each other and/or in a more favorable conformation for interaction compared with the corresponding original nimodipine�agreed AFM, that leads to increased cross-stitching and oligomerization between related antigens. In another embodiment of the present invention associated antigens-targets (e.g., cell surface receptor molecules) remain remote from each other and/or are in a less favorable conformation for interaction compared to the corresponding unmodified original AFM, reducing or preventing cross-linking or oligomerization between related antigens. In one of specific embodiments of the present invention, the increased cross-linking or oligomerization leads to increased apoptosis. In another embodiment of the present invention, the increased cross-linking or oligomerization results in increased cell differentiation. In one embodiment of the present invention, the reduced cross-linking or oligomerization lead to reduced cell growth, reduced cell division or decreased cell survival.

A substantial modification of the biological properties of the region V or the CH1 region of the heavy chain or area V or area CL light chain can be accomplished by selecting substitutions that differ significantly in their impact on receiving (a) structureproperties framework substitutions, for example, flat or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (C) the magnitude of the side chain. Naturally existing amino acid residues are divided into classes based on common properties of the side chain:

(1) vibraphone: met, ala, val, leu, ile,

(2) neutral hydrophobic: cys, ser, thr,

(3) acidic: asp, glu,

(4) basic: asn, gln, his, lys, arg,

(5) residues that influence chain orientation: gly, pro, and

(6) aromatic: trp, tyr, phe.

Substitutions, non-conservative, can cause the replacement of the representative of one of these classes to the representative of another class. Conservative substitutions can cause the replacement of the representative of these classes for another representative of the same class.

Examples of polypeptides. including modified AFM

The present invention also relates to antigen-binding molecules with amino acid modifications that alter the ability of the AFM to induce cellular signaling activity and/or postraduate cross-linking of antigens. In one of the embodiments of the present invention, the modification of the AFM includes replacement of at least one wireframe plot of the variable region of the heavy or light chain compared to the original molecule. In a specific embodiment of the present invention by replacing PR�comes the replacement amino acid residue plot in frame FR1 of the heavy chain. In a preferred embodiment of the present invention, modification of the AFM includes replacement of amino acid residues at one or several positions 8, 9, 10, 11, 12 or 13 by Kabat numbering in the variable regions of the heavy chain. In another embodiment of the present invention, modification of the AFM is the replacement of amino acid residues in frame section FR4 of the heavy chain. In one of specific embodiments of the present invention, the modification of the AFM is the replacement amino acid residues at one or several positions 110 or 112 by Kabat numbering in the variable regions of the heavy chain. In another embodiment of the present invention, modification of the AFM is replacing at least one amino acid residue in the light chain of V and S. In one particular embodiment of the present invention, modification of the AFM is the replacement amino acid residue at one or more provisions of 40, 80, 83, 105 or 106 according to the Kabat numbering.

In one of the embodiments of the present invention, the amino acid may be substituted by a point mutation in the source sequence, which leads to the desired substitution of amino acid residue (residues). In another embodiment, the modification of the AFM may represent a replacement of the whole skeleton of the plot in the original m�lekule on a wireframe plot which includes the desired amino acid residue in a certain position. In one of the embodiments of the present invention, the modification of the AFM is the replacement frame section FR1 original molecule plot on frame FR1 encoded variable gene sequence of the germ line.

In another embodiment of the present invention, the AFM includes at least the CH1 region, and the modification of the AFM consists of replacing at least one amino acid residue compared to the original polypeptide. In one of specific embodiments of the present invention, the modification of the AFM consists of replacing one or more amino acid residues at positions 148, 149 and/or 150 in the constant region of the heavy chain.

The present invention also relates to a modified antigen-binding molecules comprising one or more complement-predetermined region (CDR) of the original antigen-binding molecules. Such truncated CDR can contain at least amino acid residues that determine the specificity of this CDR. The concept of "balance that determines specificity" indicates the residues that are directly involved in the interaction with the antigen. In General terms, only about one-fifth to one-third of the residues in a particular CDR is involved in binding and�of Tigana. Define the specificity residues in a particular CDR can be identified, for example, the calculation of interatomic contacts from three-dimensional modeling and determination of the sequence variability at a given residue position in accordance with the methods described in Padlan et, FASEB J. 9, 1995, cc.133-139, the essence of which is included in the present invention by reference.

Thus, the present invention also relates to selected polynucleotide, comprising at least one complementary determined region of the original molecules, or variants or truncated forms containing at least residues that determine the specificity for a given complementary determined region, and specified the selected polynucleotide encodes a hybrid polypeptide. Preferably the selected polynucleotides encode a hybrid polypeptide, which is a modified antigen-binding molecule. In one of the embodiments of the present invention the polynucleotide comprises three complementary deterministic source molecules, or variants or truncated forms containing at least residues determine the specificity for each of the three complementary deterministic fields. In another embodiment of the present image�etenia the polynucleotide encodes the entire variable region of a light or heavy chain of a chimeric (e.g., humanized) antibodies. The present invention also relates to polypeptides, encoding such polynucleotides.

In another embodiment of the present invention is related to a modified antigen-binding molecule comprising at least one complementary predetermined area of the original molecules, or variants or truncated forms containing at least residues that determine the specificity for a given complementary predetermined region, and comprising a sequence derived from heterological polypeptide. In one of the embodiments of the present invention is a modified antigen-binding molecule comprises three complementary predetermined region of the original molecules, or variants of its truncated forms containing at least residues that determine the specificity for each of the three complementary predetermined areas. The object of the present invention is also a modified antigen-binding molecule comprising a variable region of an antibody light or heavy chain. In one particularly preferred embodiment of the present invention the antigen-binding molecule is chimeric, for example, humanized antibody. The present invention also relates to methods of obtaining such m�modified antigen-binding molecules, and also to their use for the treatment of diseases, including those associated with cell proliferation.

It is known that some of the mechanisms associated with therapeutic efficacy of antibodies, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and the induction of cardiac growth, cell differentiation or apoptosis.

The present invention is associated with modified AFM, which have an enhanced ability to induce apoptosis compared to the corresponding unmodified original AFM. For example, the original AFM that weakly induces apoptosis or not it induces, can be modified according to the present invention for generating a modified AFM, which has no ability to induce apoptosis or has an increased ability to induce apoptosis. The present invention also relates to a modified AFM, which has a higher ability to induce cardiac cell growth or differentiation in comparison with the corresponding unmodified original AFM. For example, the original AFM, which does not cause the cessation of cell growth or differentiation, or raises them slightly, may be modified according to the present invention for generating a modified AFM, which does not cause terminated�I growth or differentiation, or which has a high ability to induce cessation of growth or differentiation.

In particular, such as anti-CD20 antibodies, a large number of experimental data shows that rituximab acts through conventional effector mechanisms, as measured by research CDC and ADCC. Similarly, it was shown that the stability of the different lymphoma cells to rituximab in vivo is a function of their sensitivity to CDC in vitro. On the contrary, the type of action in vivo of another anti-CD20 antibody approved for medical use, B1, requires neither complement nor natural killer cell activity. More precisely the efficiency of B1 in vivo is related to its ability to induce strong apoptosis. Typically anti-CD20 monoclonal antibodies are divided into two different categories based on their mechanism of action for the elimination of lymphoma cells. Anti-CD20 antibodies type I mainly utilized the complement for the destruction of target cells, while antibodies type II are great mechanisms, mainly through apoptosis. Rituximab and 1F5 are examples of anti-CD20 antibodies type I, a B1 is an example of a type II antibody. See, for example, Cragg, M. S., Glennie, M. J., Blood 103, 2004, cc.2738-2743, Teeling, J. L. et al., Blood 104, 2004, cc.1793-1800, the contents of which are incorporated into this description by reference.

In the patent application US 2005/0123546 (�, which is included in the present invention by reference) first constructed an anti-CD20 antibody for type I increased effector functions, for example, ADCC and induction of powerful abilities to apoptosis, effectively modifying anti-CD20 antibody type I anti-CD20 antibody type II. In one of the embodiments of the present invention is associated with a modified anti-CD20 antibody comprising the substitution in variable regions of the heavy or light chain, compared with the original anti-CD20 antibody type I leads to increased induction of apoptosis modified anti-CD20 antibody. In another embodiment of the present invention is related to anti-CD20 antibodies type II, having as a result of engineering increased effector function, and without loss of pronounced ability to induce apoptosis. In one of the embodiments of the present invention anti-CD20 antibodies type II include replacing one or more amino acids in the variable regions of the heavy chain or light chain compared to the original molecule. In another embodiment of the present invention, the engineered anti-CD20 antibodies type I and/or type II for changes in patterns of glycosylation in the Fc region. In one specific embodiment of the present invention, the altered glycosylation of the modified AFM contains elevated levels of branched complex of amino acid residues in the Fc region. In another embodiment of the present invention, isentropically modified AFM leads to low fokusnik residues in the Fc region. Cm. patent application US 2004 0093621, the contents of which are included in the present invention by reference. In another embodiment of the present invention, the anti-CD20 antibodies type I or type II were subjected to a polypeptide engineered according to the method described in US 6737056, or patent application US 2004 0185045 (firm Macrogenics), or US patent application 2004 0132101 (firm Xencor), the essence of which is included in the present invention by reference. The present invention also relates to methods of producing such design antibodies type I or type II, and to methods of using such antibodies for treatment of various disorders involving the b-cells, including b-cell lymphomas. Chimeric and humanized modified AFM Known chimeric antibody mouse/human. See, for example, Morrison, S. L., etc., PNAS 1, 1984, cc.6851-6854, European patent publication 173494, Boulianna G. L etc., Nature 312, 1984, p. 642, Neubeiger M. S., etc., Nature 314, 1985, p. 268, European patent publication 125023; Tan, etc., J. Immunol. 135, 1985, p 8564, Sun L. K, etc., Hybridoma 5, 1986, p. 517, Sahagan, etc., J. Immunol. 137, 1986, pp. 1066-1074. See, for example, the publication Muron, Nature 312, 1984, p. 597, Dickson, Genetic Engineering News 5(3), 1985, Marx, Science 229, 1985, p. 455, Morrison, Science 229, 1985, cc.1202-1207. Robinson and others in PCT WO/88104936 described chimeric antibody with a constant region of a human and the variable region of the rodent, with specificity to the CD20 epitope, part of the chimeric antibody derived from rodent�, according to Robinson is derived monoclonal mouse antibody N (gamma 2b, Kappa). Although in the above paper the authors note that the described chimeric antibody is a "leading candidate" for the treatment of disorders involving the b-cells, the specialists in this field can consider this statement in relation to the specified antibody only as assumptions, mainly because in the link there was no indication that therapeutic efficacy, as well as data received on a highly developed mammals, such as primates or humans.

Methodological approaches for obtaining chimeric antibodies known to specialists in this field. For example, light and heavy chains can be expressed separately using, for example, light chain immunoglobulin and heavy chain immunoglobulin in different plasmids or in one vector (e.g., polycistron). They can then be purified and assembled in vitro in the complete antibodies, and methodological approaches for performing such Assembly is described. See, for example, Scharff, M., Harvey Lectures 69) 1974, p. 125. Also were are the parameters of the formation in vitro of IgG antibodies from a shortened heavy and light chains. See, for example. Sears and others, Biochem. 16, 1977, cc.2016-2025.

In one of preferred embodiments of the present invention chimeric AFM real�of the invention is a humanized antibody. Ways to humanitarian antibodies not belonging to the man known in the art. For example, humanized AFM of the present invention can be prepared by methods described in US 5225539, US 6180370, US 6632927, US No. 2003/0039649, US No. 2004/0044187, US No. 2005/0033028, the contents of which are included in the present invention in the form of links. Preferably, a humanized antibody has one or more amino acid residues introduced into its composition from the source, which is not. Such amino acid residues are not taken from the person, often referred to as "import" residues and are typically taken from an "import" variable domain. Humanitarian can be produced by the method of Winter and others (Jones et, Nature, 321, 1986, cc.522-525, Riechmann et, Nature, 332, 1988, cc.323 to 327, Verhoeyen et, Science 239, 1988, cc.1534-1536) by replacing sequences of the hypervariable regions for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US 4816567), which is substantially less than an intact human variable domain substituted with the corresponding sequence from another species (not human). In practice, humanized antibodies are typically human antibodies in which some hypervariable residues region and possibly some�haunted remains of the skeleton of the plot is substituted by residues from analogous sites of antibodies rodents. Humanized anti-CD20 antibodies of the subject can include a constant region of human immunoglobulin.

The choice of the variable domains and light, and heavy, used for obtaining humanized antibodies is very important to reduce antigenicity. Using a method called "optimal fit", carry out the screening sequence of the variable domain of the antibody rodent relative to the entire library of known human sequences with variable domains. The human sequence which is closest to the sequence of the rodent is then considered as a wire-frame plot (FR) for the humanized antibody (Sims and others, J. Immunol., 151, 1993, p. 2296, Chothia et, J. Mol. Biol., 196, 1987, p. 901). Another method of selecting a sequence of frame area of the person is to compare the sequence of each individual subcaste full frame footage rodent (i.e., FR1, FR2, FR3 and FR4) or some combination of individual sabucedo (e.g., FR1 and FR2) against a library of known sequences of variable region of a person corresponding to the frame sabucedo (for example, by Kabat numbering), and in the choice of the sequence of a person for each subcaste or combination closest to sabucedo or combination (US patent application No. 2003/0040606A1). In �the other way apply a certain frame section, derived from consensuses sequence of all human antibodies of a particular subgroup of light or heavy chains. The same wireframe plot can be used for several different humanized antibodies (Carter et, Proc. Natl. Acad. Sci. USA, 89, 1992, p. 4285, Presta et, J. Immunol., 151, 1993, p. 2623, the essence of which is included in the present invention in the form of links).

It is also important that antibodies humanitarium maintaining a high degree of affinity for the antigen and other valuable biological properties. To achieve this goal by using your preferred method, humanized antibodies are obtained by the analysis of the source sequences and various conceptual humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional model of the immunoglobulin can be obtained using computer programs known to specialists in this field (e.g. InsightII, accelrys inc (formerly MSI), or on the website http://swissmodel.expasy.org/, described by Schwede, etc. in Nucleic Acids Res. 13, 2003, cc.3381-3385). The analysis of such models allows to identify the possible role of amino acid residues in the functioning of the studied immunoglobulin sequence, i.e. to identify the amino acid residues that influence the ability of the investigated immunoglobulin to bind the corresponding antigen. In this way, FR residues can� to be selected and combined from the recipient and valuable sequences thus that produces the desired properties of the antibody, for example, maintaining affinity with the antigen-targeted antigens-targets). In General, the amino acid residues hypervariable region are directly and most substantially affect binding to the antigen.

In another embodiment of the present invention, antigen-binding molecules of the present invention are designed to increase binding affinity, for example, by the methods described in the patent application US 2004/0132066, the content of which is provided in the present invention in the form of a link..

In a preferred embodiment of the present invention, the selected polynucleotide comprises a sequence that encodes a polypeptide containing the amino acid sequence shown below in table.3 and/or 5. The present invention also provides for isolated nucleic acid comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence presented below in table.2 and/or 4. In another embodiment of the present invention is directed to isolated nucleic acid comprising a sequence that encodes a polypeptide with an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical Amin�acid sequence, presented below in table.3 and/or 5. The present invention also includes isolated nucleic acid comprising a sequence that encodes a polypeptide containing the amino acid sequence of any of the designs presented in table.3 and/or 5, with conservative amino acid substitutions. In some embodiments of the present invention, any of the polynucleotides or polypeptides of table.2-5 can be rejected. In this regard, for example, in some embodiments of the present invention is a modified AFM and/or the polynucleotide encoding a modified AFM does not contain any or all of the following sequences: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38. In another example, in some embodiments of the present invention, a modified AFM of the present invention does not contain any or all of the following sequences: SEQ ID NO:55-62.

In another embodiment of the present invention is directed to a selected polypeptide comprising the amino acid sequence shown below in table.3 and/or 5. The present invention is also directed to a selected polypeptide comprising a sequence encoded by the nucleotide sequence presented below in table.2 and/or 4. In dragomiresti implementation of the present invention is directed to a selected polypeptide, comprising the amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence presented below in table.3 and/or 5. The present invention also comprises the selected polypeptide containing the amino acid sequence of any of the designs presented in table.3 and/or 5, with conservative amino acid substitutions. In certain embodiments of the present invention, any of the polynucleotides or polypeptides presented in table.2-5, can be excluded. In this regard, for example, in certain embodiments of the present invention, the polypeptide contains the amino acid sequence corresponding to or encode any or all of the following sequences: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38. In another example, some embodiments of the present invention is a modified AFM of the present invention does not contain any or all of the following sequences: SEQ ID NO:55-62.

td align="justify"> CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACACCTTCAGCTATTCT TGGATGAGCTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACGCACAGAAA TTCCAAGGAAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA
Table 2.
DesignNucleotide sequenceSEQ ID NO
In-N1

DesignNucleotide sequenceSEQ ID NO
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACGCCTTCAGCTATTCT TGGATGAACTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA3
In-minority charge carriersCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACGCCTTCAGCTATTCT TGGATGAACTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTAT CTGTGTGCAAGAAATGTCTTTGATGGTTACTG GCTTGTTTACTGGGGCCAGGGAACCCTGGTCA CCGTCTCCTCAGCTAGCACC5
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGAGCTTCAGTGAAGGTCTCCT GCAAGGTCTCCGGATACGCGTTCAGCTATTCT TGGATGAACTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA7
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACGCGTTCAGCTATTCT TGGATGAGCTGGGTGCGGCAGGCGCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA9

DesignNucleotide sequenceSEQ ID NO
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACGCCTTCAGCTATTCT TGGATCAATTGGGTGCGGCAGGCGCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA11
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACGCCTTCAGCTATTCT TGGATCTCGTGGGTGCGGCAGGCGCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA13
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGCGCCTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACACCTTCACATACAGC TGGATGAACTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA 15
In-NCAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGCGCCTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACACCTTCAGCTATTCT TGGATGAACTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACAATGGGAAA TTCAAGGGCAGAGTCACAATTACCGCCGACA AATCCACTAGCACAGCCTATATGGAGCTGAG CAGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA17

DesignNucleotide sequenceSEQ ID NO
B-HL1CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCCGGATACACCTTCACCTATTCT TGGATGCACTGGGTGCGGCAGGCCCCTGGAC AAGGGCTCGAGTGGATGGGACGGATCTTTCC CGGCGATGGGGATACTGACTACGCACAGAAA TTCCAAGGAAGAGTCACAATGACACGGGACA CGTCCACTTCCACCGTCTATATGGAGCTGAGC AGCCTGAGATCTGAGGACACGGCCGTGTATT ACTGTGCAAGAAATGTCTTTGATGGTTACTGG CTTGTTTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA19
B-HL2GAGGTGCAATTGGTGCAGTCTGGCGCTGAAG TTAAGAAGCCTGGGGCCACCGTGAAGATCTC CTGCAAGGTGTCCGGATACACCTTCACCTATT CTTGGATGCACTGGGTGCAGCAGGCCCCTGG AAAGGGGCTCGAGTGGATGGGACGGATCTTT CCCGGCGATGGGGATACTGACTACGCAGAGA AATTCCAAGGAAGAGTCACAATCACAGCCGA CACGTCCACTGACACCGCCTATATGGAGCTGA GCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAACCAATGTCTTTGATGGTTACT GGCTTGTTTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA21
B-HL3GAGGTGCAATTGGTGCAGTCTGGCGCTGAAG TTAAGAAGCCTGGGGCCACCGTGAAGATCTC CTGCAAGGTGTCCGGATACACCTTCACCTATT CTTGGATGAACTGGGTGCAGCAGGCCCCTGG AAAGGGGCTCGAGTGGATGGGACGGATCTTT CCCGGCGATGGGGATACTGACTACAATGGGA AATTCAAGGGAAGAGTCACAATCACAGCCGA CACGTCCACTGACACCGCCTATATGGAGCTGA GCAGCCTGAGATCTGAGACACGGCCGTGTA TTACTGTGCAACCAATGTCTTTGATGGTTACT GGCTTGTTTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA 23
B-HL4CAGATGCAATTGGTGCAGTCTGGCGCTGAAGT TAAGAAGACCGGGAGTTCAGTGAAGGTCTCC TGCAAGGCTTCCGGATACACCTTCACCTATTC TTGGATGAGCTGGGTGCGGCAGGCCCCTGGA CAAGGGCTCGAGTGGATGGGACGGATCTTTC CCGGCGATGGGGATACTGACTACGCACAGAA ATTCCAAGGAAGAGTCACAATTACCGCCGAC AAATCCACTAGCACAGCCTATATGGAGCTGA GCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACT GGCTTGTTTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCAGCTAGCACC25

DesignNucleotide sequenceSEQ ID NO
B-HL8GAAGTGCAGCTGGTGGAGTCTGGAGGAGGCT TGGTCAAGCCTGGCGGGTCCCTGCGGCTCTCC TGTGCAGCCTCTGGATTCACATTTAGCTATTC TTGGATGAACTGGGTGCGGCAGGCTCCTGGA AAGGGCCTCGAGTGGGTGGGACGGATCTTTC CCGGCGATGGGGATACTGACTACAATGGGAA ATTCAAGGGCAGAGTCACAATTACCGCCGAC AAATCCACTAGCACAGCCTATATGGAGCTGA GCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACT GGCTTGTTTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA27
B-HL10CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCCCACTCCGAAGTGCAGCT GGTGGAGTCTGGAGGAGGCTTGGTCAAGCCT GGCGGGTCCCTGCGGCTCTCCTGTGCAGCCTC TGGATTCGCATTCAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA29
B-HL11CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCT TGGTCAAGCCTGGCGGGTCCCTGCGGCTCTCC TGTGCAGCCTCTGGATTCACATTTAGCTATTC TTGGATGAACTGGGTGCGGCAGGCTCCTGGA AAGGGCCTCGAGTGGGTGGGACGGATCTTTC CCGCGATGGGGATACTGACTACAATGGGAA ATTCAAGGGCAGAGTCACAATTACCGCCGAC AAATCCACTAGCACAGCCTATATGGAGCTGA GCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACT GGCTTGTTTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA 31

DesignNucleotide sequenceSEQ ID NO
B-HL12CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCTCACTCCGAAGTGCAGCT CGTGGAGTCTGGAGCAGGCTTGGTCAAGCCT GGCGGGTCCCTGCGGCTCTCCTGCGCAGCCTC TGGATTCACATTTAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA33
B-HL13CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCTCACTCCGAAGTGCAGCT CGTCGAGTCTGGAGGAGGCGTGGTCAAGCCT GGCGGGTCCCTGCGGCTCTCCTGCGCAGCCTC TGGATTCACATTTAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA35
B-HL14CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCTCACTCCGAAGTGCAGCT GGTCGAGTCCGGAGGAGGCTTGAAGAAGCCT GGCGGGTCCCTGCGGCTCTCCTGCGCAGCCTC TGGATTCACATTTAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA37

45
DesignNucleotide sequenceSEQ ID NO
B-HL15CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCCCACTCCGAAGTGCAGCT GGTGGAGTCTGGAGGAGGCTTGGTCAAGCCT GGCTCTTCCCTGCGGCTCTCCTGCGCAGCCTC TGGATTCACATTTAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA39
B-HL16CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCCCACTCCGAAGTGCAGCT GGTGGAGTCTGGAGGAGGCTTGGTCAAGCCT GGCGGGTCCCTGCGGGTCAGCTGCGCAGCCTC TGGATTCACATTTAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA41
B-HL17CGGAATTCGGCCCACCGGTGGCCACCATGGA CTGGACCTGGAGGATCCTCTTCTTGGTGGCAG CAGCCACAGGAGCCCACTCCGAAGTGCAGCT GGTGGAGTCTGGAGGAGGCTTGGTCAAGCCT GGCGGGTCCCTGCGGCTCTCCTGCGCAGCCTC TGGATTCACATTTAGCTATTCTTGGATGAACT GGGTGCGGCAGGCTCCTGGAAAGGGCCTCGA GTGGGTGGGACGGATCTTTCCCGGCGATGGG GATACTGACTACAATGGGAAATTCAAGGGCA GAGTCACAATTACCGCCGACAAATCCACTAG CACAGCCTATATGGAGCTGAGCAGCCTGAGA TCTGAGGACACGGCCGTGTATTACTGTGCAAG AAATGTCTTTGATGGTTACTGGCTTGTTTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GCTAGCGAATTCTCGA43
The signal sequence VHATGGACTGGACCTGGAGGATCCTCTTCTTGGT GGCAGCAGCCACAGGAGCCCACTCC

DesignNucleotide sequenceSEQ ID NO
B-KV1GATATCGTGATGACCCAGACTCCACTCTCCCT GCCCGTCACCCCTGGAGAGCCCGCCAGCATTA GCTGCAGGTCTAGCAAGAGCCTCTTGCACAGC AATGGCATCACTTATTTGTATTGGTACCTGCA AAAGCCAGGGCAGTCTCCACAGCTCCTGATTT ATCAAATGTCCAACCTTGTCTCTGGCGTCCCT GACCGGTTCTCCGGATCCGGGTCAGGCACTGA TTTCACACTGAAAATCAGCAGGGTGGAGGCT GAGGATGTTGGAGTTTATTACTGCGCTCAGAA TCTAGAACTTCCTTACACCTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGTACGGTG47
The signal sequence VLATGGACATGAGGGTCCCCGCTCAGCTCCTGGG CCTCCTGCTGCTCTGGTTCCCAGGTGCCAGGTGT49
Table 3.
DesignNucleotide sequenceSEQ ID NO
In-NQVQLVQSGAEVKKPGSSVKVSCKASGYTFSYSWM SWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS2
In-NQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS4
In-NAQVQLVQSGAELKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS14
In-NWQVQLVQSGAEVVKPGSSVKVSCKASGYAFSYSW MNWVRQAPGQGLEWMGRIFPGDGDTDYNGKFK GRVTITADKSTSTAYMELSSLRSEDTAVYYCARN VFDGYWLVYWGQGTLVTVSS125
In-NSQVQLVQSGGEVKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS126
B-HH2DQVQLVQSGAGVKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS127
In-NNEQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVIVSS128

DesignNucleotide sequenceSEQ ID NO
HH2FQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVIS129
In-NQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYLCARNVFD GYWLVYWGQGTLVTVSS6
In-NQVQLVQSGAEVKKPGASVKVSCKVSGYAFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS
In-NQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM SWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS10
In-NQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIN WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS12
In-NQVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIS WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS14
In-NQVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS16
In-NQVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWM NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS18
B-HL1QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWM HWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGR VTMTRDTSTSTVYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS20
B-HL2EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWM HWVQQAPGKGLEWMGRIFPGDGDTDYAEKFQGR VTITADTSTDTAYMELSSLRSEDTAVYYCATNVFD GYWLVYWGQGTLVTVSS22
B-HL3EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWM NWVQQAPGKGLEWMGRIFPGDGDTDYNGKFKGR VTITADTSTDTAYMELSSLRSEDTAVYYCATNVFD GYWLVYWGQGTLVTVSS24

DesignNucleotide sequenceSEQ ID NO
B-HL4QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYSWM SWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS26
B-HL8EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS28
B-HL10EVQLVESGGGLVKPGGSLRLSCAASGFAFSYSWMN WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS30
B-HL11QVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS32
B-HL12EVQLVESGAGLVKPGGSLRLSCAASGFTFSYSWMN WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS34
B-HL13EVQLVESGGGVVKPGGSLRLSCAASGFTFSYSWMN WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS36
B-HL14EVQLVESGGGLKKPGGSLRLSCAASGFTFSYSWMN WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS38
B-HL15EVQLVESGGGLVKPGSSLRLSCAASGFTFSYSWMN WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS 40
B-HL16EVQLVESGGGLVKPGGSLRVSCAASGFTFSYSWM NWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS42
B-HL17EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWM NWVRQAPGKGLEWMGRIFPGDGDTDYNGKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCARNV FDGYWLVYWGQGTLVTVSS44
The signal sequence VHMDWTWRILFLVAAATGAHS46

DesignNucleotide sequenceSEQ ID NO
B-KV1DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITY LYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSG SGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGG TKVEIKRTV48
B-KV10DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL YWYLQKAGQSPQLLIYQMSNLVSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGT KVEIKRTV130
B-KV11DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL YWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSG TDFTLKISRVEPEDVGVYYCAQNLELPYTFGGGTK VEIKRTV131
B-KV12DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL YWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSSG TDFTLKISRVEAEDFGVYYCAQNLELPYTFGGGTK VEIKRTV 132
B-KV13DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL YWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTK VAIKRTV133
B-KV14DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL YWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTK VEAKRTV134
The signal sequence VLMDMRVPAQLLGLLLLWFPGARC50
Table 4.
DesignNucleotide sequenceSEQ ID NO
I-HHDCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCT GGTTTCACATTCACTGACTACAAGATACACTGGGTGCG ACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGATATT TCAACCCTAACAGCGGTTATAGTACCTACGCACAGAAG TTCCAGGGCAGGGTCACCATTACCGCGGACAAATCCAC GAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTG51

AGGACACGGCCGTGTATTACTGTGCGAGACTATCCC CAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAG GGACCACCGTGACCGTCTCCTCA
M-NPAGAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCAA GCCTGGCGGGTCCCTGCGGCTCTCCTGTGCAGCCTC CGGATTCACATTTAGCAACTATTGGATGAACGGGT GCGGCAGGCTCCTGGAAAGGGCCTCGAGTGGGTGG GAGAGATCAGATTGAAATCCAATAACTTCGGAAGAT ATTACGCTGCAAGCGTGAAGGGCCGGTTCACCATCA GCAGAGATGATTCCAAGAACACGCTGTACCTGCAGA TGAACAGCCTGAAGACCGAGGATACGGCCGTGTATT ACTGTACCACATACGGCAACTACGTTGGGCACTACT TCGACCACTGGGGCCAAGGGACCACCGTCACCGTCTCCAGT 53
Table 5.
DesignNucleotide sequenceSEQ ID NO
I-HHDQVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWV RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADK STSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS52
M-NPAEVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMNWV RQAPGKGLEWVGEIRLKSNNFGRYYAASVKGRFTISRD DSKNTLYLQMNSLKTEDTAVYYCTTYGNYVGHYFDHW GQGTTVTVSS54

In another embodiment of the present invention provides the selected nucleotide comprising a sequence that encodes a polypeptide with an amino acid sequence which is derived from the original sequence, shown in Fig.1 and table.6, and comprising at least one amino acid substitution in at least one frame section of the heavy chain. In another embodiment of the present invention provides a selected polypeptide comprising the amino acid sequence, derived from the original sequence, shown in Fig.1 and table.6, and comprising at least one amino acid substitution at �'ere in a single frame plot heavy chain.

Table 6.
Designation sequenceAmino acid sequenceSEQ ID NO
1F5-VHQVQLRQPGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFK GKATLTADKSSSTAYMQLSSLTSEDSAVYYCAR SHYGSNYVDYFDYWGQGTLVTVST55
1F5-VHQVQLRQPGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFK GKATLTADKSSSTAYMQLSSLTSEDSAVYYCAR SHYGSNYVDYFDYWGQGTLVTVST55
B9E9-VHQVQLVQSGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFK GKATLTADKSSSTAYMQLSSLTSEDSAVYYCAR AQLRPNYWYFDVWGAGTTVTVS56
C2B8-VHQVQLQQPGAELVKPGASVKMSCKASGYTFTSYN MHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFK GKATLTADKSSSTAYMQLSSLTSEDSAVYYCAR STYYGGDWYFNVWGAGTTVTVSA57
2H7-VHQAYLQQSGAELVRPGASVKMSCKASGYTFTSYN MHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFK GKATLTVDKSSSTAYMQLSSLTSEDSAVYFCAR VVYYSNSYWYFDVWGTGTTVTVS58
B-ly1-VHEVKLQQSGPELVKPGASVKISCKASGYAFSYSW MNWVKLRPGQGLEWIGRIFPGDGDTDYNGKFK GKATLTADKSSNTAYMQLTSLTSVDSAVYLCR NVFDGYWLVYWGQGTLVTVSA59
2F2-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYA MHWVRQAPGKGLEWVSTSWNSGSIGYADSVK GRFTISRDNAKKSLYLQMNSLRAEDTALYYCA KDIQYGNYYYGMDVWGQGTTVTVSS 60
7D8-VHEVQLVESGGGLVQPDRSLRLSCAASGFTFHDY AMHWVRQAPGKGLEWVSTISWNSGTIGYADS VKGRFTISRDNAKNSLYLQMNSLRAEDTALY YCAKDIQYGNYYYGMDVWGQGTTVTVSS61
11B8-VHEVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYH AMHWVRQAPGKGLEWVSIIGTGGVTYYADS VKGRFTISRDNVKNSLYLQMNSLRAEDMAVY YCARDYYGAGSFYDGLYGMDVWGQGTTVTVSS62

In one of the embodiments of the present invention modified AFM of the present invention may include the replacement of the whole skeleton of the plot compared to the original AFM. For example, the present invention also relates to the selected polynucleotide comprising a sequence that encodes a polypeptide containing at least one skeleton plot heavy chain derived from a VH sequence of the germline of the person. In a preferred embodiment of the present invention, the sequence of the VH germline of the human plot in frame FR1, or in positions 8-13 on Kabat numbering, received from any of the sequences presented below in table.7. These sequences can be obtained from the IMGT database (http://mighf.cines.fro:8104/texts/IMGTrepertoire}. moreover, each sequence having the identification number included in the present invention by reference.

Table 7.
The number in the database IMGTInventory numberSEQ ID NO (nucleotide sequence)
IMGT_hVH_1_2H82
IMGT_hVH_2_5H83
IMGT_hVH_2_26M84
IMGT_hVH_2_70L2196985
IMGT_hVH_3_7M86
IMGT_hVH_3_11M87
IMGT_hVH_3_19M88
IMGT_hVH_3_20M89
IMGT_hVH_3_33L0661890
IMGT_hVH_3_43M91
IMGT_hVH_3_53MIMGT_hVH_3_dZ1889893
IMGT_hVH_4_4H94
IMGT_hVH_4_30_2L1008995
IMGT_hVH_4_34H96
IMGT_hVH_5_51M97
IMGT_hVH_6_1H98
IMGT_hVH_7_4_1L1005799
IMGT_hVH_7_81Z27509100

In another embodiment of the present invention is associated with the selected polynucleotide comprising a sequence that encodes a polypeptide comprising the amino acid sequence in positions 8-13 Kabat numbering variable regions of the heavy chain or any subgroup (e.g., positions 9-12, the provisions 10-12, etc.) for any of the sequences shown below in table.8. In one of the embodiments of the present invention is connected with a dedicated polypep�house, comprising the amino acid sequence in position 8-13 according to the Kabat numbering or any subgroup (e.g., positions 9-12, the provisions 10-12, etc.) for any of the sequences shown below in table.8.

Table 8.
Amino acid sequenceSEQ ID NO
GAEVKK63
GPTLVK64
GPVLVK65
GPALVK66
GGGLVQ67
GGGLVK68
GGGLVE69
GGGVVR70
GGGVVQ71
GGVVVQ72
GGGLIQ73
RGVLVQ74
GPGLVK75
GSGLVK 76
GAGLLK77
GSELKK78
GHEVKQ79
GAELKK101
GAEVVK102
GGEVKK103
GAGVKK104
GGGVVK105

In another embodiment of the present invention is directed to a selected polynucleotide comprising a sequence that encodes a polypeptide that includes the amino acid in position 108-113 Kabat numbering variable regions of the heavy chain or any subgroup (e.g., position 110 to 112, positions 110 and 112, etc.). In one specific embodiment of the present invention, the sequence at positions 108 to 113 shown in Fig.9 below. In another embodiment of the present invention relates to the selected polypeptide comprising the amino acid sequence at positions 108 to 113 according to the Kabat numbering or any subgroup (e.g., position 110 to 112, positions 110 and 112, etc.) for any of the sequences presented below vtable.9.

Table 9.
Amino acid sequenceSEQ ID NO
LVTVSS106
LVIVSS107
LVTVIS108
LVIVIS109
LVGVSS110
LVTVGS111
LVGVGS112
LVAVSS113
LVTVAS114
LVAVAS115
LVVVSS116
LVTVVS117
LVVVVS118
LVLVSS119
LVTVLS120
LVLVLS121
LVSVSS122
LVTVTS123

In another embodiment of the present invention relates to expression vectors and/or host cell that includes one or more selected polynucleotides of the present invention.

Usually for expression of the AFM according to the present invention can be used any type of cultured cell line. In a preferred embodiment of the present invention, Cho cells, HEK293-EBNA, BHK, NSO, SP2/0, cells, YO myeloma cells of mouse myeloma RH, cell PER cell PER.C6 or cell hybridomas, other mammalian cells, yeast, insect cells and plant cells are used as the starting cell line for receiving the engineered host cells of the present invention.

Modified AFM, further comprising Fc region and options for the Fc regions

In one embodiment of the present invention, the AFM of the present invention comprising one or more amino acid substitutions in the V and/or the CH1 regions of the heavy chain and/or V and/or With areas of light chain, may also include the Fc region of a human. In one specific embodiment of the present invention, the constant region of human IgG1 is shown in SEQ ID NO 80 and 81 below:

The nucleotide sequence of IgG1 (SEQ ID NO:8)

ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Amino acid sequence of IgG1 (SEQ ID NO:81)

TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Variants and isoforms Fc region of a person are also objects of the present invention. For example, variants of the Fc region, applicable in the present invention can be obtained by methods described in US 6737056 (variants of the Fc region with altered effector function through modification of one or more amino acids), or in patent applications US 60/439498, 60/456041, 60/514549 or WO 2004/063351 (variants of the Fc region with enhanced binding affinity due to amino acid modification), or in patent applications US 10/672280 and WO 2004/099249 (Fc variants with modified tie�the burden with FcγR due to amino acid modification), the essence of which is included in the present invention in the form of links.

In another embodiment of the present invention, the AFM comprising one or more amino acid substitutions in the V and/or the CH1 regions of the heavy chain and/or V and/or areas With light chain may further comprise a variant Fc region. Specialist can design an Fc region for obtaining variants with altered binding affinity for one or more Fc regions. For example, it may be modified one or more amino acid residues in the Fc region to change (e.g. increase or decrease) binding to FcR, as described in provisional US patent application 60/678,776, the essence of which is included in the present invention by reference. Can usually be replaced with one or more amino acids in the Fc region, which determine the binding of FcR to obtain such a variant Fc region. In preferred embodiments of the present invention is not more than 1-10 residues in the Fc region may be delegated or substituted. In the present invention, the Fc region comprising one or more amino acid modifications (e.g., substitutions) may preferably comprise at least about 80%, more preferably at least about 90%, and most preferably at least about 95%, POS�egovernance original Fc region or a native sequence Fc region of a human.

Can also be modified Fc region with the insertion of amino acids whose variants have an altered effector function. For example, it is possible to introduce at least one amino acid residue (such as 1-10 amino acid residues and usually not more than 10 residues), which is adjacent to one or more provisions of the Fc region, which, according to the present invention, affects the binding of the FcR, In the context of the present invention under the "junction" means the addition of one or two amino acids in the Fc region. Such variants of the Fc region can be increased or decreased binding to FcR and/or effector function. To access these options with the insertion it is possible to assess the overall crystalline structure of the polypeptide including a binding region FcR (e.g., the extracellular domain of the investigated receptor (FcR)) and Fc region, which insertional amino acid residue (residues) (see, e.g., article Sondermann, etc., Nature 406, 2000, pp. 267, Deisenhofer, Biochemistry 20, 1981, cc.2361-2370, Burmeister, etc., Nature 3442, 1994, cc.379-383 included in the present invention in the form of links) for a rational design of a modified Fc region, which manifests, for example, improved ability to bind to FcR.

Through the introduction of appropriate modifications of the amino acid sequence in the source area� Fc can be obtained by a variant Fc region, which (a) mediates one or more effector functions in the presence of effector cells more or less effectively and/or (b) binds an Fcγ receptor (FcγR) or the neonatal Fc receptor (FcRn) with better affinity compared with the original polypeptide. Such modified Fc region typically may include at least one amino acid modification in the Fc region.

In preferred embodiments of the present invention, the Fc region of the original polypeptide is an Fc region, for example, a native Fc region of human IgG1 (a and not A allotype), IgG2, IgG3, IgG4 and all allotype known or described in any species. The Fc Region. These sequences have, for example, described in provisional US patent application No. 60/678776, which is included in the present invention in reference to its essence.

In some embodiments of the present invention to obtain the AFM comprising one or more amino acid substitutions in regions V and/or CH1 of the heavy chain and/or areas V and/or light chain and further comprising a modified Fc region with enhanced effector function (e.g., ADCC), the original polypeptide preferably has pre-existing ADCC activity (e.g., the original polypeptide comprises a human IgG1 or Fc region of human IgG3) In some embodiments of the present invention is a modified Fc region with improved ADCC mediates ADCC substantially more effectively than an antibody with a native sequence Fc region in IgG1 or IgG3.

In some embodiments of the present invention, the amino acid modification (modification) introduced in the CH2 domain of the source area Fc.

The polypeptides of the present invention, having modified Fc regions may be subjected to one or more additional modifications, depending on the desired or intended use of the polypeptide. Such modifications may include, for example, the additional change in the amino acid sequence (substitution, insertion and/or deletion of amino acid residues), hybridization with heterological a polypeptide (or polypeptides) and/or covalent modifications. Such additional modifications may be performed before, simultaneously or after the amino acid modification (modifications) described above, that changes the binding of the Fc receptor and/or effector function.

Alternatively or additionally, it may be useful to combine amino acid modifications with one or more additional amino acid modifications that result in altered Clq binding and/or function of complement-dependent cytotoxicity Fc region. Associated with this the original polypeptide of particular interest in the present invention the tri�Xia polypeptide, which binds to Clq and exhibits complement-dependent cytotoxicity (CDC). The amino acid substitutions described in the present invention, can result in a change in the ability of the original polypeptide to contact Clq and/or modify its function complement-dependent cytotoxicity (for example, to reduce and preferably to weaken these effector function). However, polypeptides comprising substitutions at one or more of the described positions with improved Clq binding and/or function of complement-dependent cytotoxicity (complement dependent cytotoxicity CDC), discussed in the present invention. For example, it may be that the original polypeptide is unable to bind Clq and/or mediate CDC and may be modified by the described in the present invention methods in such a way that he acquires such additional effector functions. In addition, the polypeptides with pre-existing Clq binding activity, optionally optionally include the ability to mediate CDC and may be modified in such a way that one or both of these activities increase. Amino acid modifications that alter Clq and/or modify its function complement-dependent cytotoxicity, as described, for example, in the patent application WO00/42072 included in the present invention by reference.

As described� above, can be constructed in the Fc region, or parts with an altered effector function, e.g., by modifying Clq binding and/or FcR binding, and thereby changing CDC activity and/or ADCC activity. For example, there can be obtained a modified Fc region with improved Clq binding and improved FcγRIII binding (for example, both improved ADCC activity and improved CDC activity). In another embodiment, if necessary, to effector function has been reduced or removed, can be constructed with a modified Fc region with reduced CDC activity and/or reduced ADCC activity. In other embodiments, the present invention can improve only one of these activities, and optionally also reduce the other activity, for example, to obtain a modified Fc region with improved ADCC activity, but reduced CDC activity and Vice versa.

Another type of amino acid replacements change the type of glycosylation of the polypeptide. This can be achieved, for example, by dividing one or more of a carbohydrate part of the molecule identified in the polypeptide, and/or adding one or more glycosylation sites not found in the polypeptide. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linking refers to the accession of the carbohydrate moiety to the side chain OST�TKA asparagine. Peptide sequences asparagine-X-serine and asparagine-X-threonine, where X denotes any amino acid except Proline, are recognition sequences for enzymatic joining of the carbohydrate moiety to the side chain of asparagine. Thus, the presence of one of these peptide sequences in the polypeptide creates a powerful site of glycosylation. O-linked glycosylation refers to the accession of one of the sugars N-acetylgalactosamine, galactose, or xylose to hydroxynicotinate, usually serina or threonine, although can also be used 5-hydroxyproline or 5-hydroxylysine.

In some embodiments, the present invention provides compositions comprising a modification of the original polypeptide having an Fc region, wherein the modified Fc region comprises at least one modification of surface residue amino acid (see, e.g., Deisenhofer, Biochemistry, 28, 1981, cc.2361-2370, and WO00/42072; both sources are included in the present invention in the form of links). In other embodiments, the present invention provides compositions comprising a modification of the original polypeptide having an Fc region, wherein the modified Fc region comprises at least one modification is not associated with the surface amino acids. In other embodiments implemented�I of the present invention is described variant of the original polypeptide, having a Fc region, wherein the variant comprises at least one modification of surface amino acids and at least one modification is not associated with the surface amino acids.

Therapeutic efficacy of modified AFM of the present invention can be further enhanced by their reception in the host cell that has been affected glycoengineering, for the acquisition of altered expression of at least one glycoproteinsmolecules glycosyltransferases. In one of the embodiments of the present invention subjected to glycoengineering a host additionally expresses one or more of the following molecules: a polynucleotide encoding a polypeptide having GnTIII activity, a polynucleotide encoding a polypeptide having Democracy activity, or a polynucleotide encoding a polypeptide having GalT activity. In one of preferred embodiments of the present invention, the host expresses a polynucleotide encoding a polypeptide having GnTIII activity or Democracy. In another embodiment of the present invention, the host expresses a polynucleotide encoding a polypeptide having GnTIII activity, a polynucleotide encoding a polypeptide having the activity of Democracy. In another preferred variant� the implementation of the present invention, the polypeptide, having GnTIII activity is a hybrid polypeptide comprising a domain of localization of Golgi resident polypeptide Golgi. In one of preferred embodiments of the present invention, the expression modified by the AFM of the present invention in the host cell that expresses a polynucleotide encoding a polypeptide having GnTIII activity, leads to modification of the AFM with an increased binding affinity to Fc receptor and increased effector function. Accordingly, in one embodiment of the present invention, it is directed to a host cell comprising (a) isolated nucleic acid comprising a sequence encoding a polypeptide having GnTIII activity; and (b) allocated to the polynucleotide encoding the AFM of the present invention, for example, chimeric, primaryservername or a humanized antibody that binds to human antigen CD20. In a preferred embodiment of the present invention, the polypeptide having GnTIII activity is a hybrid polypeptide comprising a catalytic domain of GnTIII domain and Golgi localization domain is the localization of mannosidase II. Methods for obtaining such hybrid polypeptides and their use to generate antibodies with enhanced effector functions described in preliminary results�the diesel patent application US 60/495,142 and in the patent application US 2004/0241817, summary of which is included in the present invention in the form of links. In another preferred embodiment of the present invention chimeric AFM is a chimeric antibody or its fragment capable of specifically contacting the antibody B-Ly1 rodents. In a particularly preferred embodiment of the present invention is a chimeric antibody includes a human Fc. In another preferred embodiment of the present invention, the antibody is primaryservername or humanised.

In one embodiment of the present invention, one or more polynucleotides encoding an AVM of the present invention can be expressed under the control of a constitutive promoter or, alternatively, a regulated expression system. To apply regulated expression systems include, but are not limited to, regulated by the tetracycline expression system, ecdyson-inducible expression system, a lac-inducible expression system, a glucocorticoid-inducible expression system, the temperature-inducible promoter system and metallothionein metal-inducible expression system. If several different nucleic acids encoding the AFM of the present invention are contained in the system of the host cell, some of them can expressional�Xia under the control of a constitutive promoter, although others expressed under the control of a regulated promoter. Under the maximum level of expression meaning the highest possible level stable expression of the polypeptide, which does not have a significant adverse effect on the growth rate of cells, and can be determined using conventional experiments. The expression levels determined by methods known in this field, including by the method of Western blotting using antibodies specific for AFM, or antibodies that are specific against peptide label, hybridized with the AFM, and Northern blotting. As a further variant, the polynucleotide may be operatively linked with a reporter gene; the expression levels of the modified AFM with almost the same binding specificity of the original antibody, determined by measurement of a signal correlating with the level of expression of reporter gene. Reporter gene may be transcribed together with the nucleic acid (acids) encoding the indicated hybrid polypeptide as a single mRNA molecule; their respective coding sequences may be linked either IRES site (internal binding site of the ribosome), or cap-independent translational enhancer (cap-independent translation enhancer - CITE). Reporter gene may be translated together�e at least one nucleic acid, encoding a modified AFM, has almost the same binding specificity of the original antibody in such a way that creates a single polypeptide chain. Nucleic acids that encode the AFM of the present invention can be operatively linked to reporter gene under the control of a single promoter such that nucleic acid that encodes the hybrid polypeptide and the reporter gene are transcribed into an RNA molecule, which in another embodiment playerbase in two separate molecules of the messenger RNA (mRNA); one of the formed molecules of mRNA is translated into the specified reporter protein, while others are broadcast on a specified hybrid polypeptide.

The expression modified by the AFM;

Ways, well known to specialists in this field can be used to construct expression vectors containing a sequence encoding a modified AFM, has almost the same binding specificity of the original antibody along with appropriate control signals transcription/translation. These methods include techniques of DNA recombination in vitro, methods of synthesis and recombination in vivo/genetic recombination. See, for example, the methods described Maniatis and others in kN.: "Molecular cloning a laboratory manual", publisher Cold Spring Harbor Laboratory, 1989, new York, � Ausubel, etc., in the book: "Current protocols in molecular biology", publishing house Greene Publishing Associates and Wiley Interscience, 1989, new York.

Various vector systems for the expression in a host can be used for expression of the coding sequence of the AFM of the present invention. Preferably used as systems of host cells mammalian cells subjected to transfection with expression vectors plasmid DNA or kosmidou DNA containing the encoding sequence of the studied protein and the sequence encoding the hybrid polypeptide. Most preferably used as systems of host cells of Cho cells, HEK293-EBNA, BHK, NSO, SP2/0, cells, YO myeloma cells of mouse myeloma RSH, cells PER, PER.C6 or cell hybridomas, other mammalian cells, yeast cells, insect cells and plant cells. Some examples of expression systems and selection methods are described in the following works: Borth, etc., Biotechnol. Bioen. 71, 2000-2001, cc.266-273, Werner, etc., Arzneimittelforschung/Drug Res. 48, 1998, cc.870-880 (1998), Andersen and Krummen, Curr. Op. Biotechnol. 13, 2002, cc.117 to 123, Chadd and Chamow, Curr. Op.Biotechnol. 12, 2001, cc.188-194, Giddings, Curr. Op. Biotechnol. 12, 2001, cc.450-454. In other embodiments, the present invention can be considered other systems of eukaryotic host cells, including yeast cells transformed with recombinant yeast expression vectors containing the sequence encoding the AFM real�th invention, for example, expression systems described in patent applications US 60/344169 and WO 03/056914 (ways of getting type glycoprotein glycoprotein of human rights in eukaryotic the host cell, non-human cell) (the essence of the patent applications included in the present invention in the form of links); system insect cells infected with recombinant virus expression vectors (e.g., baculovirus) carrying a sequence encoding a modified AFM, has almost the same binding specificity as the original antibody; systems of plant cells infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, the tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the sequence encoding the AFM of the present invention, including, but not limited to, the expression systems described in US 6815184 (ways of expression and secretion of biologically active polypeptides from genetically modified duckweed), WO 2004/057002 (formation of glycated proteins by the cells of mosses as a result of the introduction of the glycosyltransferases gene), WO 2004/024927 (ways of generating extracellular heterological non-plant proteins in protoplasts of the moss), patent US 60/365769, 60/368047 and WO 2003078614 (glycoprotein processing in transgenic plants, including functional enzyme of the mammalian GnTIII) (the essence of each work included in the present invention in the form of a link) or system of animal cells infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus) including cell lines engineered to support a large number of copies of DNA that encodes a modified AFM, which has almost the same binding specificity of the original antibiotic or stable amplificatoare (CHO/dhfr) or unstable amplificatoare in double microchromosome (e.g., a rodent cell line). In one embodiment of the present invention, the vector comprising the polynucleotide (polynucleotides) encoding the AFM of the present invention is polycistronic. In addition, in one of the embodiments of the present invention, ACM, discussed above, is an antibody or its fragment. In a preferred embodiment of the present invention, the AFM is a humanized antibody.

For methods of the present invention, stable expression is generally preferable to unstable expression, because it usually gives more reproducible results, and subjected to increasingly large-scale production. Before the use of expression vectors containing the replication mechanism of viral origin, the host cell can be transformed with the appropriate encoding nucleic acids controlled by appropriate control elements of expression (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selective marker. After the introduction of the foreign DNA, engineered cells can be left to grow for 1-2 days in an enriched medium and then transferred to a selective medium. The selective marker in the recombinant plasmid shows resistance to the selection and allows you to select cells that have stably integrated into the chromosome of the plasmid and grow for the formation of foci that in turn can be cloned and be the beginning of cell lines.

Can be applied a number of selective systems, including, but not limited to, timedancing virus herpes simplex (Wigler et, Cell 11, 1977, p. 223), genes hypoxanthine-guanine-phosphoribosyltransferase (Szybalska, Szybalski, Proc. Natl. Acad. Sci. USA 48, 1962, p. 2026), and adenine-phosphoribosyltransferase (Lowy, etc., Cell 22, 1980, p. 817), which can be applied in cells tk-, hgprt-or aprt-respectively. Of antimetabolitnyh sustainability can also be applied as a basis for the selection of genes dhfr, which causes resistance to methotrexate is used (Wigler, etc. Natl. Acad. Sci. USA 77, 1989, p. 3567, O'hare, etc., Proc. Natl. Acad. Sci. USA 78, 1981, p. 1527), gpt, which causes resistance to mycophenolic acid (Mulligan, Berg, Proc. Natl. Acad. Sci. USA 78, 1981, p. 2072), peo, which causes resistance to the aminoglycoside G-418 (Colberre-Garapin, etc., J. Mol. Biol. 150, 1981, p. 1), and hygro, which causes resistance to hygromycin (Santerre, etc., Gene 30, 1984, p. 147). Were previously described selective genes, namely trpB, which allows cells to utilize indole instead of tryptophan, hisD, which allows cells to utilize gastinel instead of histidine (Hartman, Mulligan, Proc. Natl. Acad. Sci. USA 85, 1988, pp. 8047), the system glutaminase, and ODC (intendencias), which causes resistance to the inhibitor emitintermediate, 2-(deformity)-DL-ornithine, DFMO (McConlogue, in kN.: "Current Communications in Molecular Biology", 1987, publishing Cold Spring Harbor Laboratory).

Expression of modified antigen-binding molecules (AFM). comprising a Fc region with altered glycosylation

The present invention also relates to a method of modifying the glycosylation profile of the modified AFM comprising at least one substitution of an amino acid in region V or CH1 formed in the host cell, including expressiona specified in the host cell nucleic acid that encodes a modified AFM of the present invention, and nucleic acids, caliraya polypeptide with the activity� GnTIII, or a vector comprising such nucleic acid. Preferably, the modified polypeptide is IgG or a fragment comprising the Fc region. In one particularly preferred embodiments of the present invention AFM is a humanized antibody or its fragment. In another embodiment of the present invention designs a host cell for co-expression of the AFM of the present invention, GnTIII and mannosidase II (Democracy).

Modified AFM produced by the cells-the hosts of the present invention, exhibit increased Fc receptor binding affinity and/or increased effector function is the result of the modification. In a particularly preferred embodiment of the present invention, the AFM is an humanized antibody or its fragment containing the Fc region. Preferably, the increased Fc receptor binding affinity is increased binding to an activating Fcγ receptor, for example FcγRIIIa receptor. The increased effector function is preferably improve one or more of the following: increased antibody-dependent cellular cytotoxicity, increased antibody-dependent cellular phagocytosis (antibody-dependent cellular phagocytosis - ADCP), increased cytokine secretion, increased mediated immune complexe� consumption antigen antigen-presenting cells, increased Fc-mediated cellular cytotoxicity, increased binding to cells GAC, increased binding to macrophages, increased binding of polynuclear cells, increased binding to monocytes, increased cross-stitching associated with the target antibodies, increased direct signal-induced apoptosis, increased maturation dendrocygna cells and elevated premirovanii with T-cells.

Effector function can be measured and/or determined by various methods known to experts in this field. Various methods of measurement of effector functions, including Fc receptor binding affinity and complement-dependent cytotoxicity, as described in the patent application US 2004/A, the essence of which is included in the present invention by reference. Secretion of the cytokine can be measured, for example, using the method of sandwich ELISA, see, e.g., McRae, etc., J. Immunol. 164, 2000, cc.23-28, and the Protocol for the sandwich ELISA, which can be found on the website www.bdbiosciences.com/pharmingen/protocols or methods described Takahashi, etc., British J. Pharmacol. 137, 2002, cc.315-322, the publications included in the present invention in the form of links. Maturation dendrocygna cells, for example, can be set using the method described Kalergis, Ravetch, J. Exp.Med. 195, 2002, cc.1653-1659, the essence of which is given in the present invention by reference. Approx�ry study of phagocytosis and consumption/antigen presentation proposed in Gresham, etc., J. Exp. Med. 191, 2000, cc.515-528, Krauss, etc., J. Immunol. 153, 1994, cc.1769-1777, Rafiq, etc., J. Clin. Invest. 110, 2002, cc.71-79, Hamano, etc., J. Immunol. 164, 2000, cc.6113-6119, each of which is included in the present invention by reference. Reduced regulation of cell surface receptors can be measured, for example, the methods described Liao, etc., Blood 83, 1994, cc.2294-2304, the essence of which is provided in the present invention is in the form of links. Basic methods, protocols and techniques contained in the book: "Cell Biology: A Laboratory Handbook", 1998, edited by J. E. Celis, which is included in the present invention by reference. The person skilled in the art can adapt the above methods, protocols and techniques for use in the present invention.

The present invention also relates to a method for producing the AFM of the present invention, having modified oligosaccharides into the host cell comprising (a) cultivating a host cell which is designed to Express at least one nucleic acid encoding a polypeptide having GnTIII activity under conditions that allow to develop the AFM according to the present invention, moreover, the specified polypeptide having GnTIII activity, is expressed in an amount sufficient to modify the oligosaccharides in the Fc region of the specified ASM formed of the specified host cell, and (b) the allocation specified� AFM. In a preferred embodiment of the present invention, the polypeptide having GnTIII activity is a hybrid polypeptide containing the catalytic domain of GnTIII. In one particularly preferred embodiments of the present invention, the hybrid polypeptide also includes domain localization Golgi resident polypeptide Golgi.

Preferably the domain of the Golgi localization domain is the localization of mannosidase II or GnTI. In another embodiment, the localization domain of the Golgi selected from the group including: localization domain of mannosidase I, the localization domain GnTII and domain localization and 1-6 cow fucosyltransferase. Obtained by such methods of the present invention AFM have increased Fc receptor binding affinity and/or increased effector function. Preferably, the increased effector function is one or more of the following: increased Fc-mediated cellular cytotoxicity (including increased antibody-dependent cellular cytotoxicity), increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased consumption of antigen-presenting cells is mediated by the immune complex of the antigen, increased binding to natural killer cells (GAC), increased binding to macrophages, POV�weighted binding to monocytes, increased binding to macrophages, increased binding to polymorphisim cells, increased direct signal-induced apoptosis, increased cross-stitching associated with the target antibodies, increased maturation dendrocygna cells and elevated premirovanii T cells. The increased Fc receptor binding affinity is preferably increased binding to Fc activating receptors, such as receptor FcγRIIIa. In a particularly preferred embodiment of the present invention, the AFM is an humanized antibody or its fragment.

In another embodiment of the present invention is directed to a modified AFM, has almost the same binding specificity as the original antibody, and obtained using the methods of the present invention, which contains in the increased proportion of branched oligosaccharides in the Fc region of the specified polypeptide. Assume that such AFM includes antibodies and their fragments containing the Fc region. In a preferred embodiment of the present invention, the AFM is a humanized antibody. In one of the embodiments of the present invention, the percentage content of branched oligosaccharides in the Fc region of the AFM is at least 50%, more preferably at measures� 60% at least 70%, at least 80%, or at least 90%, and most preferably at least 90-95% of the total content of oligosaccharides. In yet another embodiment of the present invention, ACM, obtained by the methods of the present invention, has a higher content of nefokusirana oligosaccharides in the Fc region due to the modification of its oligosaccharides by the methods of the present invention. In another embodiment of the present invention, the percentage content nefokusirana oligosaccharides is at least 50%, preferably at least about 60-70%, most preferably at least 75%. Nefokusirana oligosaccharides can be hybrid or complex. In a particularly preferred embodiment of the present invention AFM by the cells of the host by methods of the present invention contains an increased proportion of branched nefokusirana oligosaccharides in the Fc region. Branched nefokusirana oligosaccharides can be either hybrid, or complex. Specifically, the methods of the present invention can be applied for obtaining the AFM, in which at least 15%, more preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 35% of the oligosaccharides in the field F in the AFM are branched nefokusirana. The methods of the present invention can also be applied for obtaining polypeptides that have at least 15%, more preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 35% of the oligosaccharides in the Fc region polypeptide are branched hybrid nefokusirana.

In another embodiment of the present invention involves a modified AFM, has almost the same binding specificity as the original antibody, which are designed to enhance the effector function and/or enhance Fc receptor binding affinity methods of the present invention. Preferably, the increased effector function is one or more of the following: increased Fc-mediated cellular cytotoxicity (including increased antibody-dependent cellular cytotoxicity), increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased consumption of antigen-presenting cells is mediated by the immune complex of the antigen, increased binding to cells GAC; increased binding to macrophages, increased binding to monocytes, increased binding to polymorphisim cells, increased signal-induced �popes, increased cross-stitching associated with the target antibodies, increased maturation dendrocygna cells or increased premirovanii T cells. In a preferred embodiment of the present invention, the increased Fc receptor binding affinity is increased binding to Fc activating receptor, most preferably FcγRIIIa receptor In one of the embodiments of the present invention is a modified AFM is an antibody, an antibody fragment containing the Fc region, or a hybrid protein that includes a region equivalent to the Fc region of immunoglobulin. In a particularly preferred embodiment of the present invention, the AFM is a humanized antibody.

Pharmaceutical composition comprising a modified antigen-binding molecules (ASM)

The present invention also relates to pharmaceutical compositions comprising the modified AFM of the present invention and a pharmaceutically acceptable carrier.

Can be used with any traditional media. The carrier material may be organic or inorganic, suitable for oral, subcutaneous or parenteral administration. To acceptable carriers include water, gelatin, gum Arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkyl�glycol, vaseline and other. In addition, the pharmaceutical preparations can contain other pharmaceutical agents actions. Additional auxiliary means, for example, flavoring agents, stabilizers, emulsifiers, buffers, etc. may be added in accordance with accepted methods of preparation of pharmaceutical mixtures.

The term "pharmaceutically acceptable" in the context of this invention refers to those compounds, materials, compositions and/or dosed with the forms from the medical point of view suitable for applications involving contact with the tissues of humans or animals, without severe toxicity, irritation, allergic reactions or other complications, weighing the benefit/risk tolerance.

The present invention is also directed to such pharmaceutical compositions for the treatment or prevention of cancer. The present invention is also directed to a method for the treatment or prophylaxis of cancer, comprising introducing a therapeutically effective amount of the pharmaceutical composition of the present invention.

Preferably the cancer is selected from the group consisting of breast cancer, bladder cancer, head and neck cancer, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and brain cancer.

The present invent�s also relates to pharmaceutical compositions for the treatment or prophylaxis of a precancerous condition or damage. The present invention also relates to a method of treatment or prophylaxis of a precancerous condition or damage, comprising introducing a therapeutically effective amount of the pharmaceutical composition of the present invention.

Preferably a pre-cancerous lesion or damage selected from the group consisting of oral leukoplakia, actinic keratosis (senile warts), precancerous polyps of the colon or rectum, epithelial dysplasia of the stomach, adenomatous dysplasia syndrome hereditary nonpolyposis colon cancer, ulcer, Barrett esophagus, bladder dysplasia, and precancerous cervical conditions.

The present invention also relates to methods of obtaining and application of systems of host cells for production of glycoforms of modified AFM of the present invention, which has an increased Fc receptor binding affinity is preferably increased binding to Fc activating receptors, and/or with enhanced effector functions including antibody-dependent cellular cytotoxicity. Methodology glycoengineering applicable for modified AFM of the present invention, described in detail in the patent US 6602684, provisional US patent application 60/441307 and in WO 2004/065540, the contents of which are included in the present invention in the form of a reference�K. Modified AFM of the present invention in another embodiment, its implementation can be subjected to glycoengineering to reduce the content fokusnik residues in the Fc region, made by the methods described in the patent EP 1 176 195 Al, the contents of which are included in the present invention by reference.

Obtaining cell lines for the generation of modified AFM with a modified glycosylation

The present invention provides expression systems in the cells of the host to generate modified by AFM of the present invention having a modified glycosylation. In particular, the present invention provides for a system of host cells for generating modified glycoforms AFM of the present invention having improved therapeutic properties. Thus, the present invention provides a system for expression host cells selected or constructed for expression of the polypeptide having GnTIII activity. In one embodiment of the present invention, the polypeptide having GnTIII activity is a hybrid polypeptide comprising a domain of a Golgi localization heterological resident polypeptide Golgi. Specifically, such systems expression in the cells of the host can be designed to include molecules of recombi�annoy nucleic acid, encodes a polypeptide carrying GnTIII, operatively linked to a constitutive or regulated promoter system.

In one of specific embodiments of the present invention is provided by the host cell, which were constructed for expression of at least one nucleic acid that encodes a hybrid polypeptide having GnTIII activity and comprising the domain of legalization Golgi heterological resident polypeptide Golgi. In one of the embodiments of the present invention in the host cell are introducing a nucleic acid molecule comprising at least one gene encoding a hybrid polypeptide having GnTIII activity and comprising a localization domain of a heterologous Golgi resident polypeptide Golgi.

Usually any type of cultured cell lines, including the lines described above, can be used as the basis for engineering lines of host cells of the present invention. In a preferred embodiment of the present invention the cells of Cho, BHK, NS0, SP2/0, cells, YO myeloma cells of mouse myeloma RH, cells PER, PER.C6 or cell hybridomas, other mammalian cells, yeast cells, insect cells or plant cells are used as the basis for creating the engineered host cells of the present izobreteny�.

The present invention also includes any designed the host cell expressing the polypeptide with GnTIII activity, including hybrid polypeptide comprising a domain localization in heterologous Golgi resident polypeptide Golgi as described above.

One or more nucleic acids encoding a polypeptide having GnTIII activity can be expressed under the control of a constitutive promoter or, in another embodiment, the regulated expression system. Such systems are well known in this area, and these include the systems described above. If several different nucleic acids that encode a hybrid polypeptide having GnTIII activity and comprising the domain of localization of the Golgi heterological resident polypeptide Golgi included in the system of the host cell, some of them may be expressed under the control of a constitutive promoter, while others are expressed under the control of a regulated promoter. The levels of expression of hybrid polypeptides with GnTIII activity, determine ways, usually familiar to specialists in this field, for example, Western-blot, Northern-blot analysis of expression of the reporter gene or the activity measurement GnTIII. In another embodiment, can be used a lectin, which is good for�ivalsa with products of biosynthesis GnTIII, for example, a lectin (E4-RNA. In addition, can be carried out functional studies, allowing to measure the increased binding of Fc-receptor or increased effector function, which is mediated by antibodies produced by the cells engineered with a nucleic acid that encodes a polypeptide with GnTIII activity.

Identification of transfectants or transformants that Express the protein having a modified form of glycosylation

The host cell, which contain the sequence encoding the modified AFM of the present invention and Express the biologically active gene products may be identified using at least four main approaches: (a) hybridization of DNA-DNA, (b) determining the presence or absence of the functions of "marker" gene, (b) assessing the level of transcription as measured by expression of the respective mRNA transcripts in the host cell; and (d) identifying the gene product, the amount of which is determined by immunoassay or by its biological activity.

When you first approach the presence of the coding sequence of the modified AFM of the present invention and the coding sequence of the polypeptide having GnTIII activity can be detected by hybridization DNA-DNA or DNA-RNA using probes, incl�ing nucleotide sequence, homologous to the respective coding sequences, or portions thereof, or derivatives thereof.

In the second approach, the recombinant expression vector/host can be identified and selected by the presence or absence of certain functions of "marker" gene (e.g., activity timedancing, resistance to antibiotics, resistance to methotrexate is used, transformation phenotype, formation of occlusive cells by the baculovirus, etc.). For example, if the coding sequence of the modified AFM of the present invention or its fragment, and encodes the sequence of the polypeptide having GnTIII activity, insertion in the sequence of the marker gene of the vector, recombinant containing the appropriate coding sequence can be identified by the absence of marker gene function. In another embodiment, the marker gene can be placed in tandem with coding sequences under the control of the same or a different promoter used to control expression of the coding sequences. Expression of the marker in response to induction or selection indicates expression of the coding sequence of the modified AFM of the present invention and the coding sequence of the polypeptide having GnTIII activity.

P�and the third approach, transcriptional activity of the coding region of the modified AFM of the present invention or its fragment and encodes a polypeptide sequence, having GnTIII activity can be determined by hybridization. For example, RNA can be isolated and analyzed by Northern-blotting, using a probe homolog coding sequences of the modified AFM of the present invention or its fragment, and the encoding sequence of the polypeptide having GnTIII activity, or certain parts of it. In another embodiment, the amount of nucleic acids of the host cell can be Proektirovanie and analyzed for hybridization to such probes.

In the fourth approach, the expression of the protein product can be assessed immunologically, for example, Western-blotting, immunoassays, for example, radioimmunoprecipitation, parentclassname immunostimulant etc Effective analysis to determine the success of the expression system, however, involves the identification of biologically active gene products.

Preparation and use of modified antigen-binding molecules (AFM), having increased effector function including antibody-dependent cellular cytotoxicity

In preferred embodiments, the present invention provides glycoform chimeric modified by AFM, has almost the same binding specificity of the antibody B-Ly1 rodents and with enhanced effector �the function, including antibody-dependent cellular cytotoxicity. Designing glycosylation of antibodies was described previously. See, for example, patent US 6602684, the essence of which is included in the present invention by reference.

Clinical studies of the use of unconjugated monoclonal antibodies (MAB) for the treatment of certain types of cancer have given encouraging results. Dillman, Cancer Biother. &Radiopharm. 12, 1997, cc.223-225, Deo, etc., Immunology Today 18, 1997, p. 127. Confirmed that unconjugated chimeric IgGI can be applicable for the treatment of mild or follicular b-cell non-Hodgkin's lymphoma. Dillman, Cancer Biother. &Radiopharm. 12, 1997, cc.223-225, while another unconjugated Mat, humanized IgG1 against solid breast tumors, has also shown promising results in phase III clinical trials. Deo, etc., Immunology Today 18, 1997, p. 127. The antigens of these two Mat expressed at a high level in the respective tumor cells and antibodies mediate intensive destruction of the tumor effector cells in vitro and in vivo. On the contrary, many of unconjugated Mat with strict specificity against the tumor are not able to induce effector function with intensity sufficient for clinical use. Frost, etc., Cancer 80, 1997, cc.317-333, Surfus, etc., J. Immunother. 19, 1996, cc.184-191. Currently undergoing adjuvant therapy CIT�Keene for some of these weakened Mat. The addition of cytokines can stimulate antibody-dependent cellular cytotoxicity (ADCC) by increasing the activity and number of circulating lymphocytes. Frost, etc., Cancer 80, 1997, cc.317-333, Surfus, etc., J. Immunother. 19, 1996, cc.184-191. ADCC, a lytic attack on the target cell antibodies, induced by the binding of receptors of leukocytes with the constant region (Fc) of antibodies. Deo, etc., Immunology Today 18, 1997, p. 127.

Different, but complementary, approach to increase ADCC activity of unconjugated IgG1 lies in the design of the Fc region of the antibody. Research in the field of protein engineering has determined that FcγR interacts with the lower hinge region, CH2 domain in IgG. Lund and others, J. Immunol. 157, 1996, cc.4963-4969. However, FcγR binding also requires the presence of oligosaccharides covalently linked to a conservative amino acid residue Asn 297 in the CH2 region. Lund and others, J. Immunol. 157, 1996, cc.4963-4969, Wright and Morrison, Trends Biotech. 15, 1997, cc.26-31 confirmed that or oligosaccharide and polypeptide are both directly involved in the interaction with the site, or that the oligosaccharide is essential for maintaining the active conformation of CH2 polypeptide. Modification of the oligosaccharide structure can therefore be made as a means to increase the affinity of this interaction.

The IgG molecule has two N-linked oligosaccharide in the Fc region, one on each heavy chain. Like any �glycoprotein, the antibody is produced in the form of a population of glycoforms, which have a common frame polypeptide molecules, but with different oligosaccharides attached to the glycosylation sites. Usually oligosaccharides found in the Fc region of serum IgG are of complex of complex bi-antennary type (Wormald et, Biochemistry 36, 1997, cc.130-138) low terminal sialic acid and branched N-acetylglucosamine (GlcNAc), and variable degree of end galactosylceramide and measles fokusirovanie. A number of studies have confirmed that the minimal carbohydrate structure required for FcγR binding, lies at the core oligosaccharide chains. Lund and others, J. Immunol. 157, 1996, cc.4963-4969.

Cell lines derived from mice and hamsters are used in industry and science to obtain the unconjugated Mat therapeutic purposes, usually attached oligosaccharide chains required determinants for the Fc sites. Forms of IgG, expressed in these cell lines, however, lost a branched N-acetyl-D-glucosamine (GlcNAc) were detected in low amounts in serum immunoglobulins. Lifely, etc., Glycobiology 318, 1995, cc.813-8. On the contrary, it had been shown that cells of rat myeloma produce humanized IgG1 (CAMPATH-1H), having branched GlcNAc in some of its glycoforms. Lifely, etc., Glycobiology 318, 1995, cc.813-822.

The antibodies produced by the cells of the rat.�have similar maximum ADCC activity in vitro like antibodies SAMRAT-1H produced in standard cell lines, but at significantly lower antibody concentrations.

Antigen SAMRAT is normally present in large numbers on the lymphoma cells and chimeric Mat has high ADCC activity in the absence of GlcNAc branched. Lifely, etc., Glycobiology 318, 1995, cc.813-822. Metabolic pathways associated with N-linked glycosylation, branched GlcNAc added with GnTIII. Schachter, Biochem. Cell Biol. 64, 1986, cc.163-181.

Previous studies have used a line of Cho cells producing a single antibody that has previously been constructed for expression, while externally, different levels of enzyme cloned GnTIII gene (Umana, P., et al., Nature Biotechnol. 17, 1999, cc.176-180). This approach was first established strict correlation between the expression of GnTIII and the ADCC activity of the modified antibody. Thus, the present invention provides a recombinant chimeric antibody or a fragment thereof with binding specificity of the antibody B-Ly1 rodents with altered glycosylation, leading to increased GnTIII activity. Increased GnTIII activity leads to an increase in the percentage of branched oligosaccharides, as well as a decline in the percentage fokusnik residues in the Fc region of the modified AFM. Such an antibody or a fragment thereof has an increased Fc-re�eptor binding affinity and increased effector function. In addition, the present invention is connected with a fragment of the antibody and hybrid proteins containing a region equivalent to the Fc region of immunoglobulins.

Medical application of modified AFM according to the methods of the present invention

In the widest sense of the modified AFM of the present invention can be applied to target cells in vivo or in vitro, which Express antigen-targeted, in particular, where the specified antigen target is expressed on the cell surface. Cells expressing the antigen target, can be applied for diagnostic or therapeutic purposes. In one of the embodiments of the present invention modified AFM of the present invention can be used to modify cellular signaling activity in cells expressing the antigen target. In another embodiment of the present invention modified AFM of the present invention can be used for cross-linkage and/or oligomerization of one or more antigens-targets. Antigens-targets modified by AFM of the present invention may be cell surface receptors, including, but not limited to, receptors, CD20, CD21, CD22, CD19, CD47, CD99, CD2, CD45, Her1 (EGFR), a receptor Her2/neu, Her3, Her4, TRAIL (for example, TRAILR1, TRAILR2), TNFR, FGF (e.g., FGFR1), d�atory IGF, the PDGF receptor, VEGF receptor, and others associated with the cell surface receptors. In one specific embodiment of the present invention, the antigen target antigen is CD20. Modified AFM of the present invention also interrupt the cell cycle, induce apoptosis of target cells (e.g. tumor cells), inhibit angiogenesis and/or induce differentiation of target cells.

According to another object of the present invention it relates to a method of treating a disease that is treatable with altered cell signaling activity of the antigen target and/or altered ability to mediate cross-linking of one or more antigens-targets, comprising administering a therapeutically effective amount of a modified AFM of the present invention to a subject who is in need. In one embodiment of the present invention, a modified AFM is an antibody. In one specific embodiment of the present invention, the antibody is humanised. Examples of diseases for treatment of which can be entered AFM are, but their list is not limited to, diseases or disorders with cell proliferation, autoimmune diseases or disorders, and diseases or disorders associated with bacterial or viral infections.

In one embodiment of the present invention, the disease or disorder associated with cell proliferation. Examples of proliferative diseases that can be treated with the AFM of the present invention are, but not limited to, neoplasms, carcinoma, malignant diseases and/or tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid glands, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (Central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic part of the trachea and the urinary system. Separate neoplasms, cancer, malignant diseases and/or tumors that can be treated using AFM of the present invention include, but are not limited to, epidermal and squamous cell carcinoma, glioma, pancreatic cancer, ovarian cancer, prostate cancer, breast cancer, lung cancer, brain cancer, malignant melanoma, leukemia, lymphoma, T-cell lymphoma, multiple myeloma, stomach cancer, cervical cancer, endometrial carcinoma, esophageal cancer, liver cancer, skin cancer, carcinoma of the urinary tract, choriocarcinoma, pharyngeal cancer, larynx cancer, hyperplasia of the stroma of the ovary, and�troblesome, endometrial hyperplasia, embryoma, fibrosarcoma, hemangioma, cavernous hemangioma, angioblasts, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartomata, osteoblastoma, leiomyosarcoma, sarcoma of the thyroid gland, sarcoma of Wenge and Wilms tumor.

Similarly other proliferative diseases may also be treated with modified AFM of this disease. Examples of such proliferative diseases include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, macroglobulinemia purpura's macroglobulinemia, Gaucher's disease, histiocytosis, and any other proliferative disease, besides neoplasia, localized in the systems of the bodies listed above.

Examples of autoimmune diseases or disorders include, but is not limited to, immunopositive thrombocytopenia, for example acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham chorea, lupus nephritis, rheumatic fever, plyuriglandulyarnaya syndrome, purpura Schonlein purpura-Henoch, post-streptococcal nephritis, erythema nodosum, arteritis Takas�, Addison's disease, exudative mnogoformnaya erythema, periarteritis nodosa, ankylosing spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary cirrhosis, Hashimoto thyroiditis, graves ' disease, chronic active hepatitis, polymyositis/dermatomyositis, polyandria, pemphigus vulgaris (pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal tabes, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis, inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (e.g. atopic dermatitis), systemic scleroderma and sclerosis, responses, associated with inflammation of the bowel (such as Crohn's disease and ulcerative colitis), respiratory distress syndrome (including respiratory distress syndrome adult - SDNV), dermatitis, meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions such as eczema and asthma and other conditions, including infiltration of T cells and chronic inflammatory responses, atherosclerosis, failure, leukocyte adhesion, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus (e.g., diabetes mellitus of the first type or insulin-savisky sugar d�abet), multiple sclerosis, Raynaud's phenomenon, autoimmune thyroiditis, allergic encephalomyelitis, Sjogren's syndrome, juvenile diabetes, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, usually manifested in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis, pernicious anemia (Addison's disease), diseases involving leukocytosis diabetes, inflammatory disease of the Central nervous system (CNS), the syndrome of multiple organ failure, hemolytic anemia (including, but not limited to, cryoglobulinemia or positive anemia Coombs), myasthenia gravis plus celiac, disease, mediated complex antigen-antibody, the disease anti-glomerular basement membrane, antiphospholipid syndrome, allergic neuritis, graves ' disease, myasthenic syndrome of Lambert-Eaton, bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, disease, Reiter's syndrome, stiff person syndrome, Bechet, arteritis of gigantochloa, immune complex nephritis, IgA nephropathy, IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc.

Modified AFM of the present invention can be used alone or in combination with other treatment modalities Il� with other therapeutic targets for the treatment of disorders are treatable by increasing or decreasing cellular signaling activity and/or cross-stitching one or more antigens-targets. In one embodiment of the present invention, the AFM of the present invention are usually applied separately for targeting cancer cells and their destruction in vivo. Modified AFM can also be used in conjunction with an appropriate therapeutic agent for the treatment of carcinoma of man. For example, the modified AFM can be used in combination with standard or conventional methods of treatment, e.g., chemotherapy, radiotherapy, or can be anywhereman or linked to a therapeutic drug or toxin, as well as lymphokine or a tumor inhibitory growth factor-releasing agent in the field of cancer. In other embodiments of the present invention conjugates modified by AFM of the present invention (1) immunotoxins (conjugates of modified AFM and the cytotoxic portion of the molecule) and (2) labeled (e.g., with radiometal associated with the enzyme or associated with fluorochromes) modified AFM, in which the label is a means of identification of immune complexes in the composition of which contains labeled ASM. Modified AFM can also be used to ind�functions lysis through the natural complement pathway and interact with those present in normal antibody-dependent cytotoxic cells.

The cytotoxic portion of the molecule immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment ("A chain") of such a toxin. Enzymatically active toxins and fragments are a chain of diphtheria toxin, non-binding active fragments of diphtheria toxin, a chain of exotoxin (from the bacterium Pseudomonas aeruginosa), chain A of ricin, a chain And abrina, chain And modeccin, alpha sarcin, proteins Aleurites fordii, a protein diantin, proteins, Phytolacca American (Phytolacca americana) (PAPI, PAPII, and PAP-S), inhibitor of bitter melon (Momordica charantia), Curtin, krotin, inhibitor mylnjanki medicinal (Sapaonaria officinalis), gelonin, mitogillin, restrictocin, vanomycin and anomity. In another embodiment of the present invention modified AFM anywhereman with low molecular weight antitumor agents. Conjugates of modified AFM and such cytotoxic portion of the molecule get, using a variety of bifunctional protein-binding agents. Examples of such reagents are SPDP, IT, bifunctional derivatives of imidapril, for example, dimethylacetamide HCl, active esters, for example, disuccinimidyl suberate, aldehydes, e.g., glutaraldehyde, bis-azido compound, such as bis (p-azidobenzoyl)g�canadiain, derivatives of bis-diakonia, for example, bis-(p-disoriented)-Ethylenediamine, diisocyanates, for example, tailen-2,6-diisocyanate and compounds bis-active fluorine, for example, 1,5-debtor-2,4-dinitrobenzene. Lysed a portion of a toxin may be joined to the Fab fragment modified by AFM. Additional toxins that may also be applicable, known in this area that it was found, for example, in published patent application US 2002/0128448, the essence of which is provided in the present invention by reference.

In one of the embodiments of the present invention the antigen-binding molecule of the present invention anywhereman with additional molecules, for example, radiometal or toxin. Such modified conjugated AFM can be obtained by various methods known to experts in this field.

Different radionuclides may be applicable in the present invention, and specialists in this field can determine which radionuclide is most appropriate, taking into account various specific circumstances. For example, radionuclide131I used for targeted immunotherapy. However, the clinical application of131I may be limited by several factors, including: the elimination half-life duration of 8 days, dehalogenation iodirovannoi antibodies and �Rowe, and in the tumor area, properties (e.g., large gamma component) which can be suboptimal for deposits localized in the tumor dose. When receiving the highest chelating agents, the opportunity for attaching metal chelating groups to proteins increases the opportunity to utilize other radionuclides such as111In and90Y. Radionuclide90Y provides some useful properties for application in radioimmunotherapy: the half-life of90Y is 64 hours, which is quite a lot for accumulation in the tumor, and, on the contrary, unlike, for example,131I, radionuclide90Y is the radiation source only beta particles are high energy with no accompanying gamma radiation during its decay, with the irradiation zone with a diameter of 100-1000 cells. In addition, a minimal amount of penetrating radiation can be entered outpatient treatment in the form of antibodies labeled with90Y. in addition, does not require the internalization of labeled antibodies for the destruction of cells, and the local emission of ionizing radiation can be lethal for adjacent tumor cells that lose their antigen target.

Effective dose for a single treatment (i.e., therapeutically effective amount) of radioisotope labeled90Y modified by the AFM of the present invention are in the range of approx�RNO from 5 to about 75 mcure, more preferably from about 10 to about 40 mcure. Effective dose for a single treatment is not rejected bone marrow labeled131I AFM of the present invention vary from about 5 to about 70 mcure, more preferably from about 5 to about 40 mcure. Effective single treatment and do not cause rejection (i.e. not requiring autologous bone marrow transplant) doses of the antibodies of the present invention, labeled131I vary from about 30 to about 600 mcure, more preferably from about 50 to about 500 mcure. In conjugation with the chimeric antibody of the present invention due to the longer half-life compared with rodent antibodies effective dose for a single treatment is not rejected bone marrow labeled131I chimeric antibodies of the present invention vary from about 5 to about 40 mcure, more preferably approximately less than 30 mcure. Criteria for visualization, for example, the tags111In, usually less than about 5 mcure.

In the present invention, therapy with the use of labeled antibodies with radionuclides may be in the form of a single treatment or multiple. Due to the radionuclide component, preferably before treatment were "collected" peripheral stem cells or cells casinogaming patients which can be subjected to fatal bone marrow toxicity caused by radiation. Cells in the bone marrow and/or peripheral stem cells collected by standard methods, then cleaned and frozen for possible reinfusion. In addition, most preferably, before the treatment the patient has produced its diagnostic dosimetry study using a diagnostic labeled antibody (e.g., using111In), which aims to establish that the labeled antibody therapeutic purposes (e.g., radionuclide90Y) does not "concentrate" unexpectedly in any healthy organs or tissues.

In one of the embodiments of the present invention is a chimeric modified method of glycoengineering AFM of the present invention anywhereman with a chain of ricin A. the Most profitable for the chain A of ricin deglycosylated and get methods of recombination. A valuable way of obtaining immunotoxin ricin is described in the publication Vitetta, etc., Science 238, 1987, p. 1098, included in the present invention by reference.

If you want to destroy human cancer cells in vitro for diagnostic purposes, the conjugates can usually be added to the medium for cell culture at a concentration equal to at least about 10 nm. The composition and method of administration when in vitro assays are not so important. About�commonly used aqueous formulations are compatible with the culture or perfusion medium. Cytotoxicity can be determined by conventional methods for the detection of cancer or the extent of its development.

Above it was discussed that the cytotoxic radiopharmaceutical agent for the treatment of cancer can be obtained by conjugation of a radioactive isotope (e.g., I, Y, Pr) Jiminy subjected to glycoengineering and/or modified AFM of the present invention. The term "cytotoxic portion of the molecule" in the context of the present invention include isotopes.

In another embodiment of the present invention the liposomes are filled with a cytotoxic drug and coated AFM of the present invention. Since many target molecules identified for the AFM of the present invention expressed on the cell surface (for example, a large number of molecules of the antigen CD20 on the surface of malignant b-cells), the present method makes it possible to release large quantities of drugs to correct cell type.

Methods of conjugating such therapeutic agents to antibodies are well known (see, e.g., Arnon and others in kN.: "Monoclonal Antibodies and Cancer Therapy", ed. by Reisfeld and others, 1985, publ Alan R. Liss, Inc., cc.243-256, Hellstrom and others in kN.: "Controlled Drug Delivery", 1987, 2nd ed., ed Robinson and others, publ Marcel Dekker, Inc., cc. 623-653, Thorpe in the book: "Monoclonal ntibodies '84: Biological And Clinical Applications", 1985, ed. by Pinchera, etc., cc.475-506, Thorpe, etc., Immunol. Rev., 62, 1982, cc.119-158 (each of the publications included in the present invention by reference).

There are also other therapeutic applications of the AFM of the present invention, including conjugation or linkage, e.g., techniques of recombination DNA with the enzyme, capable of converting a prodrug into a cytotoxic drug and the use of this conjugate antibody with the enzyme in combination with prodrug for the Convention of the prodrug to the cytotoxic agent at the tumor site (see, e.g., Senter et, Proc. Natl. Acad. Sci. USA 85, 1988, cc.4842-4846 and Cancer Research 49, 1989, cc.5789-5792, Senter, FASEB J. 4, 1990, cc.188-193).

Another use of the AFM of the present invention is to use or unconjugated antibodies (in the presence of complement), or part of the conjugate antibodies with drug or antibody with a toxin, to remove tumor cells from the bone marrow of patients with cancer. With the help of this approach, autologous bone marrow may be purged ex vivo by antibody treatment, after which the bone marrow is returned to the patient by infusion (see, e.g., Ramsay, etc., J. Clin. Immunol., 8, 1988, cc.81-88).

In addition, the present invention provides a single-stranded immunotoxin comprising antigen binding domains, which provides practically the same specificity tie�tion, the original antibody (for example, polypeptides comprising the CDRs of the source antibody), and also includes a polypeptide toxin. The single-stranded immunotoxin of the present invention can be used to treat carcinoma in vivo.

Similarly, a fusion protein comprising at least one antigen-binding region of the AFM of the present invention, connected least a functionally active portion of a second protein having antitumor activity, for example, lymphokine or oncostatin, and can be used to treat human carcinoma in vivo.

The present invention provides a method of selective destruction of tumor cells expressing cell surface receptors, including, but not limited to, the CD20 receptor, Her1 (EGFR), Her2/neu, Her3, Her4, TRAIL (for example, TRAILR1, TRAILR2), TNFR, FGF (e.g., FGFR1), IGF, PDGF, VEGF and other receptors associated with the surface of the cell. This method represents the response of the modified AFM of the present invention (conjugated, for example, immunotoxin, or unconjugated) with the indicated tumor cells. These tumor cells may be derived from a human carcinoma.

In addition, the present invention provides a method of treating cancer (e.g., carcinoma human) in vivo. This method presents an introduction to the subject Phar�cally effective amount of the composition, containing at least one of the modified AFM of the present invention (conjugated, for example, immunotoxin, or unconjugated).

In yet another embodiment of the present invention involves an improved method for the treatment of b-cell proliferative diseases, including b-cell lymphomas and autoimmune diseases caused wholly or partly by pathogenic antibody-based depletion of b-cells and comprising administering a therapeutically effective amount of ACM present invention to a person in need of such treatment. In a preferred embodiment of the present invention obtained by the AFM method glycoengineering anti-CD20 antibody with a binding specificity that is almost the same as that of the B-Ly1 antibody rodent. In another preferred embodiment of the present invention, the antibody is humanised. In this embodiment of the present invention, the AFM of the present invention is used to reduce normal b-cells in the blood for a long period.

In accordance with the practice of application of the present invention the subject may be a person, horse, pig, cow, representatives of families of rodents, canine, feline, and birds. Other warm-blooded animals can also be included in the present�tense invention.

The object of the present invention also provides methods of inhibiting the growth of cancer cells, treating cancer in the subject and treatment of proliferative disease type in a subject. The methods include the introduction to the subject an effective amount of a composition of the present invention.

In one of the embodiments of the present invention relates to the AFM of the present invention for use for the treatment or prevention of cancer, precancerous conditions, or damage or an autoimmune disorder. In yet another embodiment of the present invention relates to the AFM of the present invention for use as pharmaceuticals for the treatment or prevention of cancer, precancerous conditions or injuries, or autoimmune diseases. Cancer can be, for example, b-cell lymphoma, lung cancer, non-small cell lung cancer, broncho-alveolar lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system, cancer mitovich�, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepato-cellular cancer, gallbladder cancer, chronic or acute leukemia, lymphocytic lymphomas, neoplasms of the Central nervous system (CNS), spinal tumors, glioma of the brain stem glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Precancerous conditions or injuries include, for example, leukoplakia of the oral cavity, actinic keratosis (senile warts), precancerous polyps of the colon or rectum, gastric epithelial dysplasia, adenomatous dysplasia syndrome hereditary nonpolyposis colon cancer, ulcers Barrett's esophagus, bladder dysplasia, and precancerous cervical conditions.

Preferably the cancer is selected from the group including b-cell lymphoma, breast cancer, bladder cancer, head and neck cancer, skin cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer and racemosa. Examples of autoimmune diseases listed above.

In yet another embodiment of the present invention describes the use of AFM according to the present invention for obtaining a medicinal product for the treatment or prophylaxis of a precancerous condition or damage. Cancers and precancerous conditions described above.

Thus, the present invention provides pharmaceutical compositions, combinations, applications and methods of treatment of human carcinoma. For example, the present invention provides pharmaceutical compositions for use for the treatment of human carcinoma, including pharmaceutically effective amount of the antibody of the present invention and a pharmaceutically acceptable carrier.

The composition of the AFM of the present invention may be administered using conventional methods of administration, including, but not limited to: intravenous, intraperitoneal, oral, WinUtilities or directly into the tumor. Intravenous administration is preferred.

In one embodiment of the present invention, therapeutic compositions comprising the AFM of the present invention, are prepared for storage by mixing antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipient�AMI or stabilizers (see kN.: "Remington's Pharmaceutical Sciences, 1980, 16th ed., the EDS A. Osol) in the form of freeze-dried formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers, such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyltrimethylammonium ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzene chloride, phenol, butyl or benzyl alcohol, alkylarene, for example, methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol), low molecular weight polypeptides (less than 10 bases), proteins, such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, e.g., glycine, glutamine, asparagine, histidin, arginine or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins, chelating agents, e.g. EDTA, sugars, such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions, for example, sodium, metal complexes (e.g., complexes of Zn-protein) and/or non-ionic surface active compounds, for example, TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

<> Typical formulations of anti-CD20 AFM is described in WO98/56418, included in the present invention by reference. In this publication describe the multi-dose liquid formulations comprising 40 mg/ml rituximab, 25 mm acetate, 150 mm trehalose, 0.9% of benzyl alcohol, 0.02% Polysorbate 20, pH value of 5.0, the expiry date that is at least two years at 2-8°C. Another anti-CD20 studied composition comprises 10 mg/ml retuximab at 9.0 mg/ml sodium chloride, of 7.35 mg/ml sodium citrate, 0.7 mg/ml Polysorbate 80 and sterile water for injection, pH 6.5. In the present invention, the rituximab may be substituted with a modified AFM of the present invention.

Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801. For the application of such lyophilized formulations may be recovered by appropriate breeding, which produces a solution with a high concentration of protein, and the recovered solution may be injected subcutaneously to a mammal exposed to treatment.

In the present invention the composition may also contain a number of active connections for a particular indication, and it is preferable that these compounds have additional activities that do not exert on one another negative effect. For example, it may be appropriate PR�replace additionally chemotherapeutically agent, cytokine or an immunosuppressive agent (e.g., agent, acting on T cells, e.g., cyclosporine or antibody that binds T cells, for example, linking LFA-1). An effective amount of such additional agents depends on the amount of antagonist in the composition, the type of disease or the type of disorder or treatment, and other factors discussed above. Typically, they are used in the same dosages and same techniques described above, or in the amount of about 1-99% from the previous dosages.

The active ingredients can also be enclosed in microcapsules prepared, for example, methods using coacervates or polymerization on the border of media, e.g. hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal systems drug release means (for example, lysosomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in the book: "Remington's Pharmaceutical Sciences", 16th ed., 1980, edited by Osol A.

Can be prepared the drugs sustained release. Examples of these drugs are semi-permeable matrices of solid hydrophobic polymers containing the antagonist, and the matrix have a specific shape, for example,films or microcapsules. Examples of matrices for sustained release are polyesters, hydrogels (e.g., poly(2-hydroxyethylmethacrylate), polyvinyl alcohol, polylactic acid called PLA (US 3773919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, indestructible vinyl acetate, destructible copolymer of lactic and glycolic acids, for example, the product LUPRON DEPOT™ (microspheres for injection, consisting of a copolymer of lactic and glycolic acids and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

The compositions for administration in vivo, must be sterile. This is easy to achieve sterilization by filtration through a membrane.

The compositions of the present invention can be in various dosage forms, including, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microbubbles, liposomes, and injectable solutions or solutions for infusion. Preferred forms depend on the method of administration and therapeutic application.

The compositions of the present invention also preferably include conventional pharmaceutically acceptable carriers and adjuvants known in this field, for example, human serum albumin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, glycine, sorbic acid, potassium Kal�I and salts or electrolytes, for example, Protamine sulfate.

The most effective method of administration and dose regimen for the pharmaceutical compositions of the present invention depend on the severity and course of the disease, health condition of the patient, response to treatment and the attending physician's opinion. In this regard, the dose and composition should be selected for patients individually. Nevertheless, an effective dose of the compositions of the present invention may generally be in the range of from about 0.01 to about 2000 mg/kg.

The molecules described in this invention may be in various dosage forms, including in the form of solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, solutions for injection or infusion, but their list is not limited. The preferred form depends on the method of administration and therapeutic application.

The dosage of the present invention in some cases may be determined depending on the biomarkers. Biomarkers or molecular markers, which are used to determine (i.e. qualitative and/or quantitative) type of the expression and/or activation are associated with tumors of genes or proteins, or cellular components associated with tumors metabolic signaling pathways. Assessment of biological effects of targeted therapies on tumor� tissue and the correlation of these effects with clinical response allows you to identify the preferential growth and metabolic pathways of survival, active in tumors, thereby, setting the profile of likely responders, and Vice versa, providing a rational approach for the development of strategies to overcome resistance to treatment. For example, if a modified AFM is an antibody specific against the EGFR receptor, biomarkers for anti-EGFR therapy may include one or more molecules that are located below the signal of the metabolic pathways of EGFR, leading to cellular proliferative disease, including, but not limited to, Akt, RAS, RAF, MARK, ERK1, ERK2, PKC, STAT3, STAT5 (Mitchell, Nature Biotech. 22, 2004, cc.363-364, Becker, Nature Biotech 22, 2004, cc.15-18, Tsao, Herbst, Signal 4, 2003, cc.4-9). Biomarkers for anti-EGFR therapy may also include receptors of the growth factor, for example, EGFR, ErbB-2 (HER2/neu and ErbB-3 (HER3), and can be positive or negative signs of the patient response to anti-EGFR therapy. For example, it was found that the receptor for the growth factor (ErbB-3 (HER3) is a negative biomarker for anti-EGFR antibodies amplitude-time characteristic-EGF (US patent application 2004/0132097 A1).

Biomarkers for prediction of treatment effectiveness can be measured by the methods of analysis cells are well known in the art, including, but not limited to, immunohistochemistry, liquid cytometry, immunofluorescence, methods of capture and detection methods and the antiphase, and/or through the research, specified�CSOs previously in the patent application US 2004/0132097 A1, the contents of which are included in the present invention by reference. Biomarkers to predict the efficacy of anti-EGFR therapy can be identified using the methods described previously in the patent application US 2003/0190689 A1, the contents of which are included in the present invention by reference.

Thus, the present invention provides a method of treating disorders associated with altered or unregulated transmission of the cellular signal is the antigen target and/or with an altered ability to mediate cross-linking and/or oligomerization of one or more antigens of the target, comprising: obtaining a prediction on the treatment by using a modified AFM the person in need of such treatment, by studying the sample obtained from the individual before treatment, with the use of one or a large number of reagents that detect expression and/or activation of predictive biomarkers for the disease, associated with altered or unregulated transmission of the cellular signal is the antigen target and/or with an altered ability to mediate cross-linking and/or oligomerization of one or more antigens-targets (e.g., cancer); the type definition of the expression and/or activation of one or more predictive biomarkers on the basis of which predict the CTE� person for treatment of the modified AFM; and introduction to the person who predicts positively respond to treatment with a modified AFM, a therapeutically effective amount of a composition comprising a modified AFM of the present invention. In the context of the present invention, the term "person, which predicts a positive response to the treatment of the modified AFM" refers to the subject, which is a modified AFM causes a measurable effect on a disease or a disorder associated with altered or unregulated transmission of the cellular signal is the antigen-targeted, and/or with an altered ability to mediate cross-linking and/or oligomerization of one or more antigens-targets (e.g., cross-stitching/shrinkage of the tumor), and which benefit from therapy modified AFM does not overlap the side effects (e.g., toxicity). In the context of the present invention, the term "sample" means any biological sample withdrawn from the body, in particular from the human body, comprising one or more cells, including single cells of any origin, tissue or biopsy samples, which had been removed from organs, such as the Breasts, lungs, gastrointestinal tract, skin, cervical, ovarian, kidney, brain, head and neck or from any other bodies and�and tissues of the body, and other samples obtained from an organism, including, but not limited to, smears, sputum, secrets, cerebrospinal fluid, bile, blood, lymph, urine and feces.

Composition comprising a modified AFM of the present invention, can be recycled, measured and entered by a way that is consistent with conventional medical practice. In this context, among the considered factors include: the specific disease or disorder being treated, the type of mammal being treated, the clinical condition of the individual patient, the cause of the disease or disorder, the location of the release agent, the method of administration, the scheme of administration and other factors known to practitioners. A therapeutically effective amount of the antagonist for administration may be determined taking into consideration such factors.

Typically, a therapeutically effective amount administered parenterally antibodies in a dose within the range of about 0.1 to 20 mg/kg of body weight of the patient per day, with the usual initial range of antagonist used in the range of about 2-10 mg/kg.

Also preferably modified AFM is used in a therapeutically effective amount of about 1.0 mg/kg to about 15 mg/kg.

Also more preferably, the modified AFM used� in therapeutically effective amounts, lag of about 1.5 mg/kg to about 12 mg/kg.

Also more preferably, the modified AFM is used in a therapeutically effective quantity of approximately 1.5 mg/kg to about 4.5 mg/kg.

Also more preferably, the modified AFM is used in a therapeutically effective quantity of approximately 4.5 mg/kg to about 12 mg/kg.

More preferably, the modified AFM is used in a therapeutically effective quantity of approximately 1.5 mg/kg.

In addition, most preferably a modified AFM is used in a therapeutically effective quantity of approximately 4.5 mg/kg.

In addition, most preferably a modified AFM is used in a therapeutically effective quantity of approximately 12 mg/kg.

In a preferred embodiment of the present invention, a modified AFM is an antibody, preferably a humanized antibody. Suitable dosages of an unconjugated antibody are, for example, from about 20 mg/m2to about 1000 mg/m2. In one of the embodiments of the present invention, the dosage of the antibody is different from the dosage of rituximab, currently appointed. For example, when administered to the patient one or more doses, significantly smaller than the dose of the antibody, 375 mg/m 2for example , in the range of from about 20 mg/m2to about 250 mg/m2for example , from about 50 mg/m2to about 200 mg/m2.

In addition, there may be one or more initial doses of the antibody followed by introduction of one or more sequential doses, and the dose in mg/m2the antibody in the subsequent dose (dose) is greater than the dose in mg/m2the antibody in the initial dose (doses). For example, the initial dose may be from about 20 mg/m2to about 250 mg/m2(for example, from about 50 mg/m2to about 200 mg/m2), and the subsequent dose may be in the range of from about 250 mg/m2to about 1000 mg/m2.

However, as indicated above, such proposed amount modified AFM is subjected to a large number of therapeutic limitations. A key factor in the selection of the appropriate dose and treatment regimen is the result obtained above. For example, relatively higher doses may be needed initially for the treatment of diseases, including flowing in the acute form. For more effective results, depending on the disease or disorder, antagonist is administered as soon as possible after the first symptoms, diagnosis, manifestations of the disease, or when possible appearance�Oia disease or disorder, or during remissions of the disease or disorder.

Modified AFM of the present invention introduces one of the ways, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if required by local immunosuppressive treatment, the introduction into the wound. For parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antagonist can introduce pulse infusion, e.g., at lower doses of the antagonist. Preferably, the dosing is carried out by injections, most preferably intravenous or subcutaneous injection, partly depending on whether the introduction is brief or chronic.

In the present invention it is also possible to introduce other compounds, such as cytotoxic agents, chemotherapeutic agents, immunosuppressive agents and/or cytokines, which are also the antagonists. The combined introduction includes co-administration, using separate formulations or a single pharmaceutical composition, and, in another embodiment, consistently, preferably, both (or all) active agents simultaneously showed their inherent biological activity during the same time period.

It is obvious that the dose of the composition of the present invention, req�supported to achieve a therapeutic effect, can be further reduced through optimization of the treatment regimen.

In accordance with the practice of the present invention, the pharmaceutical carrier may be a lipid carrier. The lipid carrier may be a phospholipid. In addition, the lipid carrier may be a fatty acid. In addition, the lipid carrier may be a detergent. In the context of the present invention, the term "detergent" means any substance that modifies the surface tension of the liquid, usually lowering it.

In one example of the present invention, the detergent may be a nonionic detergent. Examples of nonionic detergents can be, but their list is not limited to, Polysorbate 80 (also known as Tween 80 or polyoxyethylenesorbitan monooleate), Brij, Triton (e.g., Triton WR-1339 and Triton A-20).

In another embodiment, the surfactant may be ionic detergent. Example ionic detergent is alkyltrimethylammonium, but the list is not limited.

In addition, in accordance with the present invention the lipid carrier may be a liposome. In the context of the present invention, the term "liposome" refers to a membrane surrounded vesicles that contain any molecules of the present invention or a combination thereof.

The examples below explain in more detail the present invention. Given the lower� preparations and examples are provided for a clearer understanding of specialists in the field of the present invention and its application. However, the present invention is not limited to the examples thereof, which are given as illustrations of individual objects of the present invention, and functionally equivalent methods are also applicable in the present invention. There is no doubt that various modifications of the present invention described in addition to clear to experts in this field thanks to the above description and figures. Such modifications correspond to reproduced the claims.

Examples

Unless otherwise specified, references to provisions of amino acid residues in the examples below correspond to the numbering system of Kabat.

Example 1

Materials and methods

Cloning and expression of recombinant antibody B-Ly1

Cells of hybridomas expressing the antibody B-Ly1 (see, for example, Poppema S., and others in kN.: "Proceedings of the 9thBiotest Symposium", 1987, ed. by H. H. Sonneborn and Tills D, publishing house of the Institute of Education, London; N. R. Ling and others in kN.: "Leucocyte Typing III Conference: White cell differentiation antigens", 1987, cc.302-355, ed. by A. J. McMichael, publishing house of the Oxford University Press, Oxford; W. Knapp in the book: "Leukocyte Typing IV Conference Proceedings", 1990, publishing house of the Oxford University Press, Oxford), grown in RPMI medium containing 10% FSB and 4 mm L-glutamine. 6×106cells with a survival rate of >90% harvested and isolated total RNA using a medium size kit Qiagen RNAeasy. The cDNA molecule encoding the variable heavy and light chain �-Ly1, amplificateur using the RV-PCR. The reaction RV-PCR performed under the following conditions: 30 min at 50°C for the synthesis of the first chain cDNA, 15 min at 95°C initial denaturation, 30 cycles with a duration of 1 min at 94°C, 1 min at 45°C, 1.5 min at 72°C and final stage of elongation for 10 min at 72°C. the Expected size of the PCR products confirmed by gel electrophoresis. PCR products are cloned in suitable vectors E. coli, the DNA sequence confirms that the selected genes encoding variable light and heavy chain genes.

For construction of chimeric B-Ly1 vectors expressing a synthetic signal sequence and appropriate restriction sites hybridizing with variable chains with additional PCR reactions. After final confirmation of conformity of the DNA sequence of the variable chains, combine them with the corresponding constant regions of human IgG1. After constructing their genes cloned under control of the MPSV promoter and located above the chain synthetic website cobordism using two independent vectors, one for each circuit, to form plasmid pETR1808 (expression vector of the heavy chain) and pETR1813 (expression vector light chain). Each vector carries a sequence of EBV OriP.

Chimeric antibody B-Ly1 get joint cell transfection HEK293-EBNA century�the ora pETR1808 and pETR1813, using the technique of transfection using calcium phosphate. Exponentially growing cells HEK293-EBNA subjected to transfection method using calcium phosphate. Cells grown as attached monolayers in T-flasks in culture medium DMEM with addition of 10% FTS and transferout when you merge cells 50-80%. For transfection into the vial Kzt75 seeded 8 million cells 24 hours before transfection in 14 ml culture medium DMEM with the addition of the FTS (10% of final volume), 250 µg/ml neomycin, after which the cells were placed in an incubator with temperature of 37°C in atmosphere of 5% CO2for the night. For each vial Kzt75 when conducting transfection, prepare the DNA solution, CaCl2and water by mixing 47 μg total plasmid vector DNA divided into two equal vector expression of light and heavy chains, 235 μl of 1M solution of CaCl2and adding water to a final volume of 469 μl. To this solution was added 469 μl of a solution of 50 mm HEPES, 280 mm NaCl, 1.5 mm Na2HPO4pH 7,05, immediately stirred for 10 h and allowed to stand at room temperature for 20 h. The suspension is diluted in 12 ml of DMEM with the addition of 2% FTS and bring in Kzt75 instead of the existing environment. Cells were incubated at 37°C in atmosphere of 5% CO2for about 17-20 hours, then the medium is replaced! 2 ml of DMEM 10% FTS. To generate unmodified antibody, referred to as "chB-Ly1", cell� transferout only vectors expressing antibodies, pETR.1808 and pETR.1813 in 1:1 ratio. To generate antibodies, referred to as "chB-Ly1-ge" subjected to glycoengineering, the cells transform together four plasmid: two plasmid for the expression of antibodies (pETR.1808 and pETR.1813), one for expression of the hybrid polypeptide GnTIII (pETR1519) and one for expression of mannosidase II (pCLF9) in the ratio 4:4:1:1, respectively. 5 days after transfection, collect the supernatant, centrifuged 5 min at 1200 rpm, then centrifuged again for 10 min at 4000 rpm and stored at 4°C.

Antibody chB-Ly1 and chB-Ly1-ge purified from culture supernatant using three sequential steps of chromatography: chromatography protein A, cation exchange chromatography and exclusion size chromatography PA column Superdex 200 (firm Amersham Pharmacia), replacing the buffer in phosphate saline buffer (FSB) and collecting the monomer peak antibody during this final stage. The concentration of the antibiotic evaluated using a spectrophotometer, since the absorption at 280 nm.

Analysis of oligosaccharides

Oligosaccharide release from the antibody using enzymatic cleavage enzyme PNGaseF, the antibodies are in solution or immobilized on a membrane of polyvinylidene fluoride (PVDF).

Obtained after cleavage solution which contains Visvaldis�yosia oligosaccharides, or used directly for enzyme-linked immunosorbent assay MALDI/TOF-MS, or again subjected to cleavage by an enzyme EndoH glycosidase before sample preparation for analysis by MALDI/TOF-MS.

Means of releasing the oligosaccharides from the antibody immobilized on the membrane of polyvinylidene fluoride (PVDF)

The wells of 96-hole tablet, equipped with a PVDF membrane (product Immobilon P, the firm Millipore, Bedford, mA), wetted with 100 µl of methanol, and the liquid is passed through the PVDF membrane using multi-charged vacuum device Multiscreen vacuum manifold (firm Millipore, Bedford, mA). The PVDF membrane was washed three times with 300 μl of water. Then the wells are washed in 50 μl of RCM buffer (8m urea, 360mm Tris, 3.2 mm EDTA, pH 8,6). The antibody in the amount of 30-40 µg loaded into the hole containing 10 μl of RCM buffer. The liquid from the wells extend through the membrane by applying a vacuum, and the membrane is then washed twice with 50 μl of RCM buffer. Reduction of disulfide bridges is carried out by application of 50 μl of 0.1 M dithiothreitol in RCM buffer and incubated at 37°C for 1 h.

During subsequent recovery use the vacuum to remove the solution of dithiothreitol from the wells. The wells are washed three times with 300 ál of water before karboksimetilirovaniya of cysteine residues by application of 50 μl 0.1 M Vodokanal acid in RCM buffer and incubation at room temperature in the dark in t�within 30 min.

After karboksimetilirovaniya the wells dried by vacuum and then washed three times with 300 μl of water. Then the membrane polyvinylidene fluoride (PVDF) block to prevent the absorption of endoglycosidase by incubation of 100 μl of 1% aqueous solution of polyvinylpyrrolidone 360 at room temperature for 1 h. the Blocking reagent is then removed with a weak vacuum with subsequent three-time washing with 300 μl of water.

N-linked oligosaccharides release the introduction of 2.5 IU peptide-N-glycosidase F (recombinant N-G, firm GLYKO, Novato, CA) and 0.1 IU of sialidase (firm GLYKO, Novato, CA) to remove any potentially charged monosaccharide residues, in a final volume of 25 µl 20 mm NaHCO3, pH 7.0). Cleavage was carried out for 3 h at 37°C.

Method of releasing oligosaccharides for antibodies in solution

The antibody in the amount of 40-50 µg mixed with 2.5 IU of enzyme PNGaseF (firm Glyko, USA) in 2 mm Tris, pH 7.0 to a final volume of 25 µl, and the resulting mixture was incubated for 3 h at 37°C.

Cleavage enzyme endoglycosidase H oligosaccharides released as a result of processing by the enzyme PNGaseF, for the distribution of neutral oligosaccharide peaks MALDI/TOF-MS

The oligosaccharides released by treatment with the enzyme PNGaseF, subsequently cleaved by the enzyme endoglycosidase� H (EndoH, EC 3.2.1.96). For splitting 15 IU of enzyme EndoH (Roche, Switzerland) was added to the mixture after cleavage by the enzyme PNGaseF (the above-described antibody solution) to obtain a final volume of 30 μl, and the mixture was incubated for 3 h at 37°C. EndoH cleaves between residues of N-acetylglucosamine chitobiose core N-linked oligosaccharides. The enzyme can cleave only oligomannose and most glycans, hybrid type, but does not hydrolyze the oligosaccharides of the complex type.

Preparation of sample for MALDI/TOF-MS

After enzymatic digestion, the released oligosaccharides were incubated 3 h at room temperature after the addition of acetic acid to a final concentration of 150 mm, and then passed through 0.6 ml of cation-exchange resin (resin AG50W-X8, hydrogen form, 100-200 cells, firm BioRad, Switzerland), which is Packed chromatographic column (micro bio-spin (firm BioRad, Switzerland) to remove cations and proteins. One microliter of the resulting sample is applied to the stainless steel Cup and mix in a Cup with 1 µl of sDHB matrix. The sDHB matrix is prepared by dissolving 2 mg 2,5-dihydrobenzoic acid plus 0.1 mg of 5-methoxystilbene acid in 1 ml of ethanol/10 mm aqueous sodium chloride solution (the volume ratio of 1:1). Samples dried in the air, add 0.2 ál of ethanol and finally samples were subjected to paracrystalline�and in the air.

MALDI/TOF-MS

The mass spectrometer Voyager Elite (firm Perspective Biosystems) used for MALDI-TOF to obtain the mass spectrum. A device is used in a linear configuration with an acceleration of 20 kB and a delay of 80 NS. Calibrated using standards of oligosaccharides for correlating the mass of the ions. Spectra 200 laser pulses summed to obtain the final spectrum.

Depletion of b-cells whole blood

495 µl of heparinized blood from a healthy donor were placed in test tubes of polystyrene 5 ml, 5 μl of 100-fold concentrated samples of antibodies (to final concentration of 1-1000 ng/ml) or PBS were placed in test tubes and incubated at 37°. After 24 h, 50 µl of blood is transferred to a new tube and stained with anti-CD3-FITC, anti-CD19-PE and anti-CD45-CyChrome (firm Becton-Dickinson) for 15 min at room temperature in the dark. Before analysis 500 µl FACS buffer (PBS containing 2% PBS and 5 mm EDTA) is introduced into the tube. The fluorescence of CD3-FITC and CD19-PE samples of liquid blood is determined by cytometry for setting the threshold CD45-CyChrome. Depletion of b-cells is determined by the ratio of CD19+B-cells to CD3+The t-cells.

Binding of anti-CD20 antibodies cell one

200000 cells in 180 µl of FACS buffer (PBS with 2% FTS and 5 mm EDTA) was transferred into test tubes made of polystyrene with a volume of 5 ml. Then bring in 20 µl of 10-fold concentrated samples anti-CD20 (to final concentration of 1-5000 ng/ml) or PBS and the tubes were incubated at 4°C for 30 min. Then the samples are washed twice with FACS buffer and precipitated with 300 g for 3 min. the Supernatant is aspirated and discarded, and the cells were placed in 100 µl of FACS buffer. Then add 1 ál anti-Fc-specific F(ab')2-FITC fragments (firm Jackson Immuno Research Laboratories, USA) and the tubes were incubated at 4°C for 30 min, the Samples washed twice with FACS buffer and placed in 500 µl of FACS buffer, 0.5 μg/ml PI for analysis by the method of the flow cytometry analysis. The binding is determined by the application of points of geometric mean values of fluorescence relative to the antibody concentration.

Example 2

The approach of using highly homologous acceptor

The study of the acceptor skeleton of the plot is highly homologous antibodies is carried out by comparing the protein sequence of the murine antibody B-ly1 with a collection of sequences of the germline of the person and selecting the sequence of a person that shows the highest sequence identity. In this case, the sequence VH1_10 (locus 1, the accepted number DP-88) from the database VBase chosen as acceptor sequences frame the heavy chain sequence IGKV2-40 (accepted room H) from the database IMGT choose as frame acceptor for the light chain. These two acceptor skeleton of the plot transplanted three complementary d�terminated region (CDR) of the heavy and light variable domains of the mouse. Since frame section 4 is not part of the variable region gene V germ line, Bank reconciliations for this position is carried out separately. The JH4 region chosen for the heavy chain, and the area JK4 opt for light chain. Molecular modeling design domain of immunoglobulin identifies a single point, potentially requiring amino acid residues of the rodent instead of the amino acid residue sequence of a person outside the CDR. Re-introduction of amino acid residues of rodent in the frame sequence of a person can generate so-called reverse mutation. For example, acceptor amino acid residue in the sequence of a person in position 27 according to the Kabat numbering is reverse mutation and returns to the tyrosine residue. Variants of the humanized antibody thus obtained, or what they include or do not include any reverse mutation. Light chain is a humanized antibody does not require any reverse mutations. After designing protein sequences, DNA sequences encoding these proteins, prepared in a manner described in detail below. Approach using mixed frame section in order to avoid the introduction of backward mutations on the provisions of the fundamentally important residues of amino acids (essential to the preservation of the choir�higher antigen-binding affinity or function of antibodies) acceptor framework of human rights, investigate whether, or the whole frame area 1 (FR1), or frame sections 1 (FR1) and 2 (FR2) together, replaced by sequences of antibodies, already bearing donor residues, or functionally equivalent sequences, these important provisions in the natural sequences of the germline of the person. For this frame sections VH 1 and 2 sequences of the antibody B-ly1 align individually with sequences of the germline of the person. In the present invention the highest sequence identity is not important and not used to select the frame acceptor sites, and on the contrary, a comparison of a few crucial residues presumably more important. Such a fundamentally important amino acid residues at positions 24, 71 and 94 (Kabat numbering), and the amino acid residues at positions 27, 28 and 30 (Kabat numbering), which are localized outside the CDR1 Kabat numbering, often involved in binding to the antigen. The IMGT sequence IGHV3-15 (adopted room H) is chosen as a sequence. After designing protein sequences synthesized DNA sequences that encode these proteins, the method, described in detail below. With the help of this approach does not require reverse mutation neither for light nor for the heavy chain, in order �to protect high levels of binding to the antigen.

Synthesis of antibody genes

After designing the amino acid sequence region V receive a humanized antibody DNA sequence. Information about the DNA sequences of individual frame sections are drawn in databases of sequences of germ line person. The DNA sequence of the CDR regions derived from the corresponding cDNA databases rodents. Using these sequences actually collect the full DNA sequence. Using data about the sequence of such DNA diagnostic sites of restriction implemented in the actual sequence of the introduction of silent mutations, creating recognition sites for restriction endonucleases. To obtain chains of material conduct DNA synthesis of the gene (for example, the method of Wheeler and others 1995). According to this method design the oligonucleotides with the target gene so that the encoding circuit receives a series of oligonucleotides, and other series - with non-coding circuit. The ends 3' and 5' of each oligonucleotide (excluding the first and last in the series) always show the complementary sequences of the two primers derived from the opposite chain. When these oligonucleotides in the reaction buffer for heat-resistant polymerase with the addition of Mg2+dinucleotides and DNA polymerase each of�gonucleotide increased from 3'-end. The newly formed 3'-end of one primer is then annealed with the following primer opposite the chain and increasing the sequence under conditions suitable for dependent matrix chain elongation of DNA. The final product is cloned in the usual vector for propagation in E. coli.

Antibodies

Leader sequence (for secretion) heavy and light chain human presence adds higher in the chain of upstream sequences of the variable regions and their link above for Kappa chain sequences of the heavy and light chain of a human IgG1, respectively, using standard methods of molecular biology. The resulting DNA sequences of the heavy and light chains of all antibodies saute in expressing vectors mammals (one vector for light chain and one heavy chain) under the control of the MPSV promoter above synthetic website cobordism of each vector carrying the sequence of the EBV OriP as described above in example 1. Antibodies get according to the description in example 1 above, namely, by co-transfection of HEK293-EBNA expression vectors of the heavy and light chains of an antibody of a mammal, getting some culture medium for 5-7 days after transfection, and purifying the synthesized antibody affinity chromatography protein A, then cation exchange chromatography and was�to the amount of displacement (size) chromatography to obtain pure Monomeric IgG1 antibodies. According to the recipe antibodies develop in a solution of 25 mm potassium phosphate, 125 mm sodium chloride, 100 mm glycine at pH of 6.7. Variants of the humanized antibodies, exposed glycoengineering receive joint transfection of vectors expressing the antibody, together with expression vectors GnT-III glycosyltransferases or together with the vector of expression of GnT-III plus expression vector of mannosidase II Golgi, as described for the chimeric antibody in example 1 above. Created by glycoengineered antibodies cleaned and processed as described above for antibodies, not exposed to glycoengineering. The oligosaccharides attached to the Fc region of antibodies analyzed by enzyme immunoassay MALDI/TOF-MS as described above.

Analysis of oligosahara

Method of releasing oligosaccharides for antibodies, present in the solution

The antibody in the amount of 40-50 μg was mixed with 2.5 IU of enzyme PNGaseF (firm Glyko, USA) in 2 mm Tris, pH 7.0 in a final volume of 25 μl and the mixture incubated for 3 h at 37°C.

Sample preparation for MALDI/TOF-MS

After enzymatic digestion, the released oligosaccharides were incubated 3 h at room temperature after the addition of acetic acid to a final concentration of 150 mm, and then passed through 0.6 ml of cation-exchange resin (resin AG50W-X8, hydrogen form, 100-200 cells, Fi�mA BioRad, Switzerland), which is Packed chromatographic column (micro bio-spin (firm BioRad, Switzerland) to remove cations and proteins. One microliter of the resulting sample is applied to the stainless steel Cup and mix in a Cup with 1 µl of sDHB matrix. The sDHB matrix is prepared by dissolving 2 mg 2,5-dihydrobenzoic acid plus 0.1 mg of 5-methoxystilbene acid in 1 ml of ethanol/10 mm aqueous sodium chloride solution (the volume ratio of 1:1). Samples dried in the air, add 0.2 ál of ethanol and finally samples were subjected to recrystallization in the air.

MALDI/TOF-MS

The mass spectrometer Voyager Elite (firm Perspective Biosystems) used for MALDI-TOF to obtain the mass spectrum. A device is used in a linear configuration with an acceleration of 20 kV and a delay of 80 NS. Calibrated using standards of oligosaccharides for correlating the mass of the ions. Spectra 200 laser pulses summed to obtain the final spectrum.

The study of binding antigens

Purified Monomeric variants of humanized antibodies are tested to determine the binding to human CD20 on target cells lymphoma b-cells one, using a method based on the flow cytometry analysis as described above in example 1 for the chimeric antibody B-ly1.

Binding of Monomeric glucobalance IgG1 with natural killer cells (GAC) and the cells Lin�and SSS expressing FcγRIIIA

PAC man is isolated from freshly isolated mononuclear cells from peripheral blood (PBMC) using negative selection for enrichment of CD16 - and CD56-positive cells (MACS system, the company Miltenyi Biotec GmbH, Bergisch Gladbach/Germany). Purity is determined by the expression of CD56, is 88-95%. Freshly isolated GAC were incubated in PBS without calcium and magnesium (3×105cells/ml) for 20 min at 37°C to remove the PAC-associated IgG. Cells were incubated in the amount of 106cells/ml with different concentrations of anti-CD20 antibodies(0, 0,1, 0,3, 1,3, 10 μg/ml) in PBS with 0.1% BSA. After several washes, the antibody binding is determined by incubation with 1:200 FITC-conjugated F(ab')2goat antibody to human F(ab')2 specific IgG (firm Jackson ImmunoReasearch, West Grove, PA/USA) and the antibody to human CD56-PE (company BD Biosciences, Allschwil/Switzerland). Anti-FcγRIIIA 3G8 F(ab')2 fragments (firm Ancell, Bayport, mn/USA) was added at a concentration of 10 μg/ml to ensure complete binding glucobalance antibodies (3 µg/ml). The intensity of fluorescence corresponding to the bound antibody variants, to determine CD56-positive cells on a FACSCalibur instrument (firm BD Biosciences, Allschwil/Switzerland). Cell lines SNO carry electroportatil (280 V, 950 μf, 0.4 cm) with an expression vector, encoding FcγRIIIA-Val158 α-chain and γ-chain. Transfectant otber�t make 6 µg/ml puromycin, and examined resistant clones by FACS, using 10 µl of FITC-conjugated-anti-FcγRIII 3G8 monoclonal antibody (firm BD Biosciences, Allschwil/Switzerland) for 106cells. Binding of IgG1 to FcγRIIIA-Val158-expressing cell lines of the SSS is carried out analogously to the above linking the GAC.

Investigation of antibody-dependent cellular cytotoxicity (ADCC)

Mononuclear cells of peripheral blood (PBMC) of a person used as effector cells and get by using Histopaque-1077 (Sigma Diagnostics Inc., St. Louis, MO63178 USA), following the manufacturer's instructions. Briefly, venous blood is taken from the volunteers using heparinized syringes. The blood was diluted FSB (without CA++or Mg++) in the ratio 1:0.75 to 1.3 and put on Histopaque-1077. The gradient was centrifuged at 400 g for 30 min at room temperature (CT) without interruption. The interphase containing the PBMC, collected, washed in PBS (50 ml for cells of the two gradients) and collected by centrifugation at 300 g for 10 min at room temperature. After resuspension of the precipitate in the FSB count ciav and washed a second time by centrifugation at 200 g for 10 min at room temperature. Then the cells was resuspended in the appropriate medium for further treatments.

The ratio of effector to target used to study ADCC, is 25:1 and 10:1 for PBMC cells and the GAC, COO�respectively. Effector cells are prepared in the medium AIM-V in the appropriate concentration in order to make 50 μl in round-bottomed wells of 96-hole tablet. The target cells are b-cell lymphomas (for example, cell one), grown in DMEM containing 10% FTS. Target cells were washed in PBS, counted and was resuspended in AIM-V in the amount of 300000 in ml to make 30,000 cells in 100 μl in a well of. Antibodies are diluted with medium, add 50 ál of pre-cultivated target cells and to bind with targets for 10 min at room temperature. Then adding Effector cells and the Ouija Board were incubated for 4 h at 37°C in a humid atmosphere with 5% CO2. The destruction of target cells is assessed by release of lactate dehydrogenase (LDH) from damaged cells, using a set of reagents for the determination of cytotoxicity Cytotoxicity Detection kit (Roche Diagnostics, Rotkreuz, Switzerland). After 4-hour incubation the plates centrifuged in a mode 800 g. Supernatant (100 μl from each well is transferred to a transparent flat-bottomed wells of 96-hole tablet. 100 µl of buffer for staining of the substrate from the kit to make the hole. The magnitude of Vmax color reaction was determined by ELISA reader at 490 nm for at least 10 min with the use of the software SOFTmax PRO (company Molecular Devices, Sunnyvale, CA94089, �SHA). Spontaneous LDH release determined in the wells containing only target cells and Effector cells, but not containing antibodies. Maximum release is determined in the wells containing only target cells and 1% Triton X-100. The percentage of dead cells, mediated by specific antibodies, is calculated as follows: (x-SR)/(MR-SR)*100, where x denotes the average value of Vmax at a specific antibody concentration, SR denotes the average value Vmax of the spontaneous release and MR denotes the average value Vmax of the maximum release.

A study of complement-dependent cytotoxicity

Target cells are counted, washed with PBS, was resuspended in the medium AIM-V (firm Invitrogen) at a concentration of 1 million cells per ml 50 ál of cells plated into flat-bottomed wells of 96-hole tablet. Dilutions of the antibodies prepared in the medium AIM-V and in a volume of 50 µl contribute to the cells. Antibodies bind to the cells for 10 min at room temperature. Svezheraspilennaya serum complement (firm Quidel) diluted with three times medium AIM-V and in a volume of 50 µl contribute to the wells. The rabbit complement (company Cedarlane Laboratories) is prepared based on the recommendations of the manufacturer, diluted threefold medium AIM-V and in a volume of 50 μl added to the wells. As a control, before examining the sources of complement was heated for 30 min �ri 56°C before making. The plates were incubated for 2 h at 37°C. the Destruction of cells is determined by measuring the release of LDH. Briefly, the plates centrifuged in mode 300 g for 3 min In 50 µl of supernatant from the wells is transferred to a new 96-well plate and add 50 μl of the reagent from the kit Cytotoxicity Kit (Roche). Kinetic measurement using ELISA reader determines the value of Vmax, the corresponding concentration in the supernatant. Maximum release was determined by incubating cells in the presence of 1% Trition X-100.

Study of the depletion of b-cells in whole blood

The depletion of normal b cells In whole blood anti-CD20 antibodies is performed as described above in example 1.

The study of apoptosis

The ability of antibodies to apoptosis examined by incubation of the antibody at a concentration of 10 μg/ml (under saturated conditions for binding of the antigen) with target cells (load target cells is 5×105cells/ml) overnight (16-24 h). Samples stained with AnnV-FITC and analyzed by FACS. The analysis is carried out in three replicates.

Detection of apoptosis is carried out by the method of the flow cytometry analysis, following the emergence of markers of apoptosis, such as annexin V and phosphatidylserine. In the negative control (without induction of apoptosis), there is no antibody is present only phosphate buffered saline. In positive control the maximum expression of apoptosis) contains 5 mol of camptothecin, intensively inducing apoptosis.

Results and discussion

Comparison of binding the human antigen CD20 antibodies with options IN-N, IN-N, IN-N, combined with a light chain of a humanized antibody B-ly1 (BKV1), and the original chimeric antibody chB-ly1 (described above in example 1) shows that all antibodies have similar values of the EU50, but the design IN-N binds with a lower intensity/stoichiometry than options IN-N and IN-N (Fig.11). It is possible to distinguish IN-NN from IN-N and IN-N by the presence of part regions CDR1 and CDR2 (Kabat numbering), and Ala/Thr polymorphism at position 28 (Kabat numbering). This suggests that either position 28, or complete the sequence of CDR1, and/or complete the sequence of CDR2 is important for the interaction of an antibody/antigen.

Comparison of B-HL1, IN-N and chimeric chB-ly1 source of the antibody indicates the absence of any binding activity in the design in HL1 and about half the intensity/stoichiometry of binding IN-N compared with the antibody B-ly1 (Fig.12). And B-HL1, and IN-N designed on the basis of the framework acceptor obtained from the class of VH1. Among other differences is important difference at position 71 (according to Kabat numbering) design B-HL1, presumably important for binding antigen. Amino acid residue in position 71 is one of aminoxy�acidic residues, which define the canonical loop structure of the CDR2 of the heavy chain. Alanine or its functional equivalents, e.g., leucine, valine or threonine (see, for example, Morea, etc., Methods 20, 2000, cc.267-279), presumably important for antigen binding and arginine for binding to the antigen adversely affected.

When comparing the data for the binding of antigen presented on Fig.2, 9-13, options BHH2-BKV1, BHL8-BKV1 and BHL11-BKV1 among different variants of a humanized antibody showed good binding affinity with human antigen CD20 on the surface of human cells. Despite similar values of the magnitude / EC50 for binding to the antigen, these variants differ in their ability to induce apoptosis in CD20-positive target cells (see Fig.4-6, 14, 15). Because the original antibody B-ly1 induces apoptosis at a low level, the present invention highlights the difference between the design of the original antibody B-ly1 and B-HL8 and b-NN. Revealed seven amino acid residues in the heavy chain IN-N, which are absent in the B-HL8 source or heavy chains of the antibody B-ly1: Gln1, Ala9, Val11, Lys12, Ser16, Val20 and Met48. All of these seven amino acid residues localized in frame sections VH domain. The likelihood of direct contact with the antigen is impossible, with the exception of Gln1. In order to determine whether one of these amino acid of ostatka� be responsible for newly developed property to induce apoptosis, get seven variants of the heavy chain of the antibody B-HL8, which can potentially recover appticable action design IN-N: B-HL11 (carries the mutation E1Q), B-HL12(G9A, V48M), B-HL13(L11V, V48M), B-HL14(V12K, V48M), B-HL15(G16S, V48M), B-HL16(L20V, V48M) and b-HL17(V48M). Cm. the following sequence (SEQ ID NO):32, 34, 36, 38, 40, 42 and 44 (which in the sequence listing not numbered according to the Kabat numbering, however, for the specialist is not difficult to transfer them to the Kabat numbering system). Antigen-binding properties of these variants do not differ significantly by size EU50 and stoichiometry (see Fig.2). However, a marked difference can be determined by their inherent ability to induce apoptosis (Fig.4-6, 14, 15 and 24). Design with L11V, V48M modifications, B-HL13 significantly increase the ability to induce apoptosis (see Fig.24). However, one modification V48M has no visible effect (see Fig.5). Therefore, the amino acid residues at positions 11 and 12 according to the Kabat numbering is most strongly affect apoptosis. These residues do not directly affect binding to the antigen, but significantly affect the boundary between the domains VH and CH1 and thus act through the modification of the "elbow angle".

Design B-HL4 is prepared from antibodies-NN replacement frame section FR1 of antibody IN-NN on a wireframe plot of the sequence IGHV1-45 germ line person (ustanovlennymi H). The antigen-binding ability of this design has been significantly weakened, although differ in only four amino acids on four provisions in the framework region FR1. These residues are localized in positions 2, 14, 28 and 30 (Kabat numbering). Of clauses 28 and 30 can be important, as they form part of the area of the CDR1 Chothia definition. In embodiments, IN-N and 9 (both in BKV1 light chain) regulations 28 and 30 are modified compared with the sequence of the original antibody B-ly1. Fig.22 shows that the binding capacity does not change significantly, if at position 28 is present and threonine at position 30 is also present threonine (Fig.22). In humanitarian light chain, which has CDR1, CDR2 and CDR3 Kabat numbering, has not been introduced reverse mutation. Most strongly induces apoptosis variant BHH2-BKV1 humanized antibody B-ly1 (even stronger than the original antibody chB-ly1 and much stronger than the antibody with sequence identical to rituximab, SW; see Fig.14, 15 and 21). Other humanized variants, which can cause increased apoptosis following: B-HL13 and B-HL14 (derivatives BHL8), VNN ("mixed frame"), VNN ("mixed frame" with one reverse mutation to evaluate the effect S30T) and IN-N (M34I derived IN-NN). Option VNN is another variant humanized antibody B-ly1, for�which is not introduced additional sequences, not belong to man. Options IN-N, IN-N and IN-N derive design IN-NN with partially humanized region CDR1 Kabat numbering.

Important properties of a humanized antibody B-ly1 are that it is an antibody type II anti-CD20 antibody according to the description of M. S. Cragg and Glennie M. J. Blood 103, 2004, cc.2738-2743. In this regard, it induces the binding with CD20 any substantial resistance to the non-ionic detergent extraction of CD20 antigen from the surface of CD20+ cells using the method described for this purpose M. J. Polyak and Deans, J. P., Blood 99, 2002, cc.3256-3262. It induces significantly less tolerance to the non-ionic detergent extraction CD20 than the antibody C2 B8 (another anti-CD20 antibody with sequence identical to rituximab (see US 2003 0003097)). According to the expected anti-CD20 antibodies type II, a humanized antibody B-ly1 does not have any significant complement-mediated lytic activity and exhibits a much smaller complement-mediated lytic activity compared to anti-CD20 antibody SV (chimeric IgG1 sequence with sequence identical to rituximab). Another important property of the humanized antibody B-ly1 (option-N B-KV1) is that it is very actively for research gomotopicheskoi aggregation. During this �the study of CD20-positive human cells, the Daudi cells incubated in culture medium for the cells for 24 h at 37°C in an atmosphere of 5% CO2in the incubator of mammalian cells according to the described method, Deans, etc., with the antibody at a concentration of 1 μg/ml and in parallel at a concentration of 5 μg/ml For comparison is carried out parallel control incubation of cells in identical conditions, using anti-CD20 antibody, SW. At various times, including after 8 and 24 h of incubation, the cells viewed with a microscope. It is established that a humanized antibody B-ly1 leads to hard gomotopicheskoi aggregation with formation of aggregates, which are significantly greater than those that induce the introduction of a control antibody SV. In addition, compatibility with an antibody specific to anti-CD20 type II, causes elevated levels of apoptosis, if CD20-positive human cells were incubated with humanized antibody B-ly1, relative to the control under identical conditions using SV chimeric IgG1 antibody with a sequence identical to the sequence of rituximab.

Glycoengineered options humanized antibodies get a joint expression of GnTIII-glycosyltransferases with antibody genes in mammalian cells. This results in a higher fraction nefokusirana oligosaccharides attached to the Fc region of antibodies, including extensive nave�kazimirovsky oligosaccharides, described in WO 2004/065540 (Fig.17-19). Glycoengineered antibodies have significantly higher levels of binding to the receptor FcγRIII (Fig.20), as well as enhanced ADCC activity (Fig.16), compared to non-exposed glycoengineered antibody and the antibody SV. A humanized antibody B-ly1 also largely induces depletion of b-cells in whole blood (Fig.16) compared with the control antibody SV. This includes not exposed to glycoengineering the antibody B-ly1, and his glycoengineering version. Subjected to glycoengineered antibody is about 1000 times more intense depletes b cells in whole blood of comparison with SW control anti-CD20 antibody. This comparison is important and not exposed to glycoengineering, and for glycoengineered humanized form of the antibody B-ly1, as it was found that such combined Fc-receptorpositive activity, e.g., ADCC, plus complement-mediated lysis, plus the induction of apoptosis, In both forms-ly1 significantly more active compared to SW, although both forms In-ly1 exhibit a significantly reduced complement-mediated lytic activity. ADCC, Fc receptor-cell killing and induction of apoptosis are indicated the activity of the humanized antibody variants B-ly1. In addition, in the study of apoptosis Akti�ness showed and exposed, and have not been glycoengineering form of anti-CD20 antibodies type II with Fc-engineered variants with increased binding affinity to Fcγ-receptors, and induction of apoptosis more effectively than non-Fc-engineered variants, and all variants that are significantly more active compared with the control antibody SV. A clear mechanism to increase gomotopicheskoi aggregation and induction of apoptosis mediated by anti-CD20 antibodies type II, unknown, and concominant binding with other molecules on the surface of CD20-positive cells, for example, with Fcγ-receptors, can affect this important property. It is therefore important to show that anti-CD20 antibodies type II, which have been modified in the Fc region for enhanced binding affinity to Fc receptors gamma, including FcγRIII, and with an associated increase in the activity of ADCC, retain the ability to induce strong apoptosis, even in a greater degree than when not exposed to the design of the Fc region, and gomotopicheskoi aggregation. Induction of apoptosis is important because in vivo in the body there are places that can be detected CD20-positive target cells, but in which access to FcγRIII-positive cells is more difficult than access to blood (e.g., lymph nodes). At such locations the induction of apoptosis by anti-CD20 antibody, in itself, can be decisive d�I good the effectiveness of therapy of anti-CD20 antibody in humans and for the treatment of malignant hematological diseases, for example, non-Hodgkin's lymphoma and b-cell chronic lymphocytic leukocytosis, as well as for the treatment of autoimmune diseases such as rheumatoid arthritis and lupus, by depletion of b-cells. Increased binding affinity to FcγRIII and increased antibody-dependent cellular cytotoxicity (ADCC) is a humanized, Fc-engineered anti-CD20 antibodies type II can also be a very important attribute for such therapy. Thus, the lowered or minor complement-mediated lytic activity of anti-CD20 antibodies type II, including humanized and Fc-engineered variants, can also be important, because the increased complement activation anti-CD20 antibodies correlates with increased unwanted side effects.

Example 3

For further study of amino acid residues that affect ipoptions activity, receive six options heavy chain VNN: VN-A (V11L) (SEQ ID NO:124), VNN-IN (K12V) (SEQ ID NO:125), BHH2-C (A9G) (SEQ ID NO:126), BHH2-D (E10G) (SEQ ID NO:127), BHH2-E (T110I) (SEQ ID NO:128), BHH2-F (S112I) (SEQ ID NO:129). These design variants were examined for the ability to bind to the antigen target (CD20) methods described above. All six designs have retained binding activity.

The design options for the heavy chain also examined for adoptionthe above ways.Five of these structures, VNN-IN, BHH2-C, BHH2-D, UNN 2E and BHH-2F, have apoptosis effect to the same extent as the original design VNN. However, the ability of the design VN-A (V11L) to induce apoptosis significantly lower than that of VN (see Fig.23).

Also explore appticable the effect of single substitutions of amino acids in the light chain of a humanized antibody B-ly1 (BKV1) by obtaining five design options: BKV-10 (P40A) (SEQ ID NO:130), BKV-11 (A80P) (SEQ ID NO:131), BKV-12 (V83F) (SEQ ID NO:132), BKV-13 (E105A) (SEQ ID NO:133) and BKV-14 (I106A) (SEQ ID NO:134). Linking structures BKV-11, BKV-12, BKV-13 and BKV-14 antigen target (CD20) investigated the methods described above, and it was found that all four constructs retain binding activity. These four kinds of the light chain also tested for adoptionthe above ways. The ability to apoptosis designs BKV-11, BKV-12 and BKV-13 has not changed as a result of replacements. However, the design BKV-14 exhibits a reduced ability to induce apoptosis compared to BKV1 (see, e.g., Fig.25).

All publications (manuals, journal articles, databases, patent applications) cited in the present description, are provided in the present invention is in the form of links in the extent to which each of them specifically had to mention.

SEQUENCE LISTING

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SIGNAL ACTIVITY

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Thr Cys Ala Gly Gly Thr Ala Ala Gly Gly Thr Cys Thr Cys Cys Thr

50 55 60

Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Cys Gly Gly Ala Thr Ala

65 70 75 80

Cys Gly Cys Cys Thr Thr Cys Ala Gly Cys Thr Ala Thr Thr Cys Thr

85 90 95

Thr Gly Gly Ala Thr Cys Thr Cys Gly Thr Gly Gly Gly Thr Gly Cys

100 105 110

Gly Gly Cys Ala Gly Gly Cys Gly Cys Cys Thr Gly Gly Ala Cys Ala

115 120 125

Ala Gly Gly Gly Cys Thr Cys Gly Ala Gly Thr Gly Gly Ala Thr Gly

130 135 140

Gly Gly Ala Cys Gly Gly Ala Thr Cys Thr Thr Thr Cys Cys Cys Gly

145 150 155 160;

Gly Cys Gly Ala Thr Gly Gly Gly Gly Ala Thr Ala Cys Thr Gly Ala

165 170 175

Cys Thr Ala Cys Ala Ala Thr Gly Gly Gly Ala Ala Ala Thr Thr Cys

180 185 190

Ala Ala Gly Gly Gly Cys Ala Gly Ala Gly Thr Cys Ala Cys Ala Ala

195 200 205

Thr Thr Ala Cys Cys Gly Cys Cys Gly Ala Cys Ala Ala Ala Thr Cys

210 215 220

Cys Ala Cys Thr Ala Gly Cys Ala Cys Ala Gly Cys Cys Thr Ala Thr

225 230 235 240

Ala Thr Gly Gly Ala Gly Cys Gly Thr Ala Gly Cys Ala Gly Cys Cys

245 250 255

Thr Gly Ala Gly Ala Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys

260 265 270

Gly Gly Cys Cys Gly Gly Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr

275 280 285

Gly Cys Ala Ala Gly Ala Ala Ala Thr Gly Thr Cys Thr Thr Thr Gly

290 295 300

Ala Thr Gly Gly Thr Thr Ala Cys Thr Gly Gly Cys Thr Gly Thr Thr

305 310 315 320

Thr Thr Ala Thr Cys Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Ala

325 330 335

Ala Cys Cys Cys Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr

340 345 350

Cys Cys Thr Cys Ala

355

<210> 15

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Design B-HH8 modified anti�svyazivalsa molecules of mouse/human

<400> 15

caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctggcgcctc agtgaaggtc 60

tcctgcaagg cttccggata caccttcaca tacagctgga tgaactgggt gcggcaggcc 120

cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 16

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH8 modified antigen-binding molecules of the mouse/human

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 17

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Design B-HH9 modified antigen-binding molecules of the mouse/human

<400> 17

caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctggcgcctc agtgaaggtc 60

tcctgcaagg cttccggata caccttcagc tattcttgga tgaactgggt gcggcaggcc 120

cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 18

<211> 119

<212> PROTEIN

<13> Artificial

<220>

<223> Design B-HH9 modified antigen-binding molecules of the mouse/human

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 19

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Design B-HL1 modified antigen-binding molecules of the mouse/human

<400> 19

caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttccggata caccttcacc tattcttgga tgcactgggt gcggcaggcc 120

cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

gcacagaaat tccaaggaag agtcacaatg acacgggaca cgtccacttc caccgtctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 20

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL1 modified antigen-binding molecules of the mouse/human

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Lys Gl Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 21

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Design B-HL2 modified antigen-binding molecules of the mouse/human

<400> 21

gaggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctggggccac cgtgaagatc 60

tcctgcaagg tgtccggata caccttcacc tattcttgga tgcactgggt gcagcaggcc 120

cctggaaagg ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

gcagagaaat tccaaggaag agtcacaatc acagccgaca cgtccactga caccgcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aaccaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 22

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL2 modified antigen-binding molecules of the mouse/human

<400> 22

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Ser

20 25 30

Trp Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Tyr Ala

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 23

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Design B-HL3 molecules of mouse/human

<400> 23

gaggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctggggccac cgtgaagatc 60

tcctgcaagg tgtccggata caccttcacc tattcttgga tgaactgggt gcagcaggcc 120

cctggaaagg ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

aatgggaaat tcaagggaag agtcacaatc acagccgaca cgtccactga caccgcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aaccaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 24

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL3 modified antigen-binding molecules of the mouse/human

<400> 24

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Ser

20 25 30

Trp Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Tyr Ala

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 25

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Design B-HL4 modified antigen-binding molecules of the mouse/human

<400> 25

cagatgcaat tggtgcagtc tggcgctgaa gttaagaaga ccgggagttc agtgaaggtc 60

tcctgcaagg cttccggata caccttcacc tattcttgga tgagctgggt gcggcaggcc 120

cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180

gcacagaaat tccaaggaag agtcacaatt accgccgaca aatccactag cacagcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctcagct 360

agcacc 366

<210> 26

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL4 modified antigen-binding molecules of the mouse/human

<400> 26

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 27

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Design B-HL8 modified antigen-binding molecules of the mouse/human

<400> 27

gaagtgcagc tggtggagtc tggaggaggc ttggtcaagc ctggcgggtc cctgcggctc 60

tcctgtgcag cctctggatt cacatttagc tattcttgga tgaactgggt gcggcaggct 120

cctggaaagg gcctcgagtg ggtgggacgg atctttcccg gcgatgggga tactgactac 180

aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 28

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL8 modified antigen-binding molecules of the mouse/human

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Th Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 29

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design B-HL10 modified antigen-binding molecules of the mouse/human

<400> 29

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca 120

agcctggcgg gtccctgcgg ctctcctgtg cagcctctgg attcgcattc agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgac tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcc tatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 30

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL10 modified antigen-binding molecules of the mouse/human

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 31

<211> 357

<212> DNA

<13> Artificial

<220>

<223> Design B-HL11 modified antigen-binding molecules of the mouse/human

<400> 31

caggtgcagc tggtggagtc tggaggaggc ttggtcaagc ctggcgggtc cctgcggctc 60

tcctgtgcag cctctggatt cacatttagc tattcttgga tgaactgggt gcggcaggct 120

cctggaaagg gcctcgagtg ggtgggacgg atctttcccg gcgatgggga tactgactac 180

aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300

tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 32

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL11 modified antigen-binding molecules of the mouse/human

<400> 32

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 33

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design B-HL12 modified antigen-binding molecules of the mouse/human

<400> 33

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagctcac tccgaagtgc agctcgtgga gtctggagca ggcttggtca 120

agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgc tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcc tatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 34

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL12 modified antigen-binding molecules of the mouse/human

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Ala Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 35

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design B-HL13 of the modified antigen-binding molecules of the mouse/human

<400> 35

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagctcac tccgaagtgc agctcgtcga gtctggagga ggcgtggtca 120

agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgac tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcc tatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 36

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL13 of the modified EN�eigenvazue molecules of mouse/human

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 37

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design B-HL14 modified antigen-binding molecules of the mouse/human

<400> 37

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagctcac tccgaagtgc agctggtcga gtccggagga ggcttgaaga 120

agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgac tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcc tatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 38

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL14 modified antigen-binding molecules of the mouse/human

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Lys Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Ly Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 39

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design B-HL15 modified antigen-binding molecules of the mouse/human

<400> 39

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca 120

agcctggctc ttccctgcgg ctctcctgcg cagcctctgg attcacattt agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgac tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcc tatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 40

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL15 modified antigen-binding molecules of the mouse/human

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 41

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design�I B-HL16-modified antigen-binding molecules of the mouse/human

<400> 41

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca 120

agcctggcgg gtccctgcgg gtcagctgcg cagcctctgg attcacattt agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgac tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcc tatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 42

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL16-modified antigen-binding molecules of the mouse/human

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 43

<211> 456

<212> DNA

<213> Artificial

<220>

<223> Design B-HL17 modified antigen-binding molecules of the mouse/human

<400> 43

cggaattcgg cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60

cagcagccac aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca 120

agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt agctattctt 180

ggatgaactg ggtgcggcag gctcctggaa agggcctcga gtgggtggga cggatctttc 240

ccggcgatgg ggatactgac tacaatggga aattcaaggg cagagtcaca attaccgccg 300

acaaatccac tagcacagcctatatggagc tgagcagcct gagatctgag gacacggccg 360

tgtattactg tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa 420

ccctggtcac cgtctcctca gctagcgaat tctcga 456

<210> 44

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HL17 modified antigen-binding molecules of the mouse/human

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 45

<211> 57

<212> DNA

<213> Homo sapiens

<400> 45

atggactgga cctggaggat cctcttcttg gtggcagcag ccacaggagc ccactcc 57

<210> 46

<211> 19

<212> PROTEIN

<213> Homo sapiens

<400> 46

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 47

<211> 345

<212> DNA

<213> Artificial

<220>

<223> B-KV1 of the modified antigen-binding molecules of the mouse/human

<400> 47

gatatcgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc 60

attagctgca ggtctagcaa gagcctcttg cacagcaatg gcatcactta tttgtattgg 120

tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc caaccttgtc 180

tctggcgtcc ctgaccggtt ctccggatcc gggtcaggca ctgatttcac actgaaaatc 240

agcagggtgg aggctgagga tgttggagtt tattactgcg ctcagaatct agaacttcct 300

tacaccttcg gcggagggac caaggtggag atcaaacgta cggtg 345

<210> 48

p> <211> 114

<212> PROTEIN

<213> Artificial

<220>

<223> B-KV1 of the modified antigen-binding molecules of the mouse/

<400> 48

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr

<210> 49

<211> 66

<212> DNA

<213> Homo sapiens

<400> 49

atggacatga gggtccccgc tcagctcctg ggcctcctgc tgctctggtt cccaggtgcc 60

aggtgt 66

<210> 50

<211> 22

<212> PROTEIN

<213> Homo sapiens

<400> 50

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys

20

<210> 51

<211> 360

<212> DNA

<213> Artificial

<220>

<223> Design I-HHD-modified antigen-binding molecules of the mouse/human

<400> 51

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cctctggttt cacattcact gactacaaga tacactgggt gcgacaggcc 120

cctggacaag ggctcgagtg gatgggatat ttcaacccta acagcggtta tagtacctac 180

gcacagaagt tccagggcag ggtcaccatt accgcggaca aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagactatcc 300

ccaggcggtt actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 360

<210> 52

<211> 120

<212> PROTEIN

<213> Artificial

<220>

<223> Designed�modernization I-HHD-modified antigen-binding molecules of the mouse/human

<400> 52

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 53

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Design M-HHA modified antigen-binding molecules of the mouse/human

<400> 53

gaagtgcagc tggtggagtc tggaggaggc ttggtcaagc ctggcgggtc cctgcggctc 60

tcctgtgcag cctccggatt cacatttagc aactattgga tgaactgggt gcggcaggct 120

cctggaaagg gcctcgagtg ggtgggagag atcagattga aatccaataa cttcggaaga 180

tattacgctg caagcgtgaa gggccggttc accatcagca gagatgattc caagaacacg 240

ctgtacctgc agatgaacag cctgaagacc gaggatacgg ccgtgtatta ctgtaccaca 300

tacggcaact acgttgggca ctacttcgac cactggggcc aagggaccac cgtcaccgtc 360

tccagt 366

<210> 54

<211> 122

<212> PROTEIN

<213> Artificial

<220>

<223> Design M-HHA modified antigen-binding molecules of the mouse/human

<400> 54

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Glu Ile Arg Leu Lys Ser Asn Asn Phe Gly Arg Tyr Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu As Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Tyr Gly Asn Tyr Val Gly His Tyr Phe Asp His Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 55

<211> 122

<212> PROTEIN

<213> Artificial

<220>

<223> IF5-VH - chimeric polypeptide of the mouse/human

<400> 55

Gln Val Gln Leu Arg Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser His Tyr Gly Ser Asn Tyr Val Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Thr

115 120

<210> 56

<211> 120

<212> PROTEIN

<213> Artificial

<220>

<223> B9E9-VH - chimeric polypeptide of the mouse/human

<400> 56

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Gln Leu Arg Pro Asn Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser

115 120

<210> 57

<211> 121

<212> PROTEIN

<213> Artificial

<220>

<223> C2B8-VH - chimeric polypeptide of the mouse/human

<400> 7

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 58

<211> 121

<212> PROTEIN

<213> Artificial

<220>

<223> 2H7-VH - chimeric polypeptide of the mouse/human

<400> 58

Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Thr Gly Thr Thr Val Thr Val Ser

115 120

<210> 59

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> B-lyl-VH - chimeric polypeptide of the mouse/human

<400> 59

Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Lys Leu Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tr

65 70 75 80

Met Gln Leu Thr Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Leu Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 60

<211> 122

<212> PROTEIN

<213> Artificial

<220>

<223> 2F2-VH - chimeric polypeptide of the mouse/human

<400> 60

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Tyr Leu

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 61

<211> 122

<212> PROTEIN

<213> Artificial

<220>

<223> 7D8-VH - chimeric polypeptide of the mouse/human

<400> 61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Asp Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Thr Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 62

<211> 125

<212> PROTEIN

<213> Artificial

<220>

<223> 11B8-VH chimeric polypeptide of the mouse/human

<400> 62

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Gly Thr Gly Gly Val Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Tyr Gly Ala Gly Ser Phe Tyr Asp Gly Leu Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 63

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 63

Gly Ala Glu Val Lys Lys

1 5

<210> 64

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 64

Gly Pro Thr Leu Val Lys

1 5

<210> 65

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence according to the Kabat numbering modified

<400> 65

Gly Pro Val Leu Val Lys

1 5

<210> 66

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 66

Gly Pro Ala Leu Val Lys

1 5

<210> 67

<211> 6

lt; 212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 67

Gly Gly Gly Leu Val Gln

1 5

<210> 68

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 68

Gly Gly Gly Leu Val Lys

1 5

<210> 69

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 69

Gly Gly Gly Leu Val Glu

1 5

<210> 70

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 70

Gly Gly Gly Val Val Arg

1 5

<210> 71

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 71

Gly Gly Gly Val Val Gln

1 5

<210> 72

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 72

Gly Gly Val Val Val Gln

1 5

<210> 73

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 73

Gly Gly Gly Leu Ile Gln

1 5

<210> 74

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 74

Arg Gly Val Leu Val Gln

1 5

<210> 75

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 75

Gly Pro Gly Leu Val Lys

1 5

<210> 76

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 76

Gly Ser Gly Leu Val Lys

1 5

<210> 77

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 77

Gly Ala Gly Leu Leu Lys

1 5

<210> 78

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modificar�bathroom antigen-binding molecules of the mouse/human

<400> 78

Gly Ser Glu Leu Lys Lys

1 5

<210> 79

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 8-13 modified antigen-binding molecules of the mouse/human

<400> 79

Gly His Glu Val Lys Gln

1 5

<210> 80

<211> 987

<212> DNA

<213> Homo sapiens

<400> 80

accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 60

gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 120

tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 180

tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 240

tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagcaga gcccaaatct 300

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 360

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 420

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 480

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 540

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 600

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 660

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 720

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 780

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 840

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 900

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 960

agcctctccc tgtctccggg taaatga 987

<210> 81

<211> 328

<212> PROTEIN

<213> Homo sapiens

<400> 81

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

1 5 10 15

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

20 25 30

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

35 40 45

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

5 55 60

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

65 70 75 80

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala

85 90 95

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

100 105 110

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

115 120 125

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

130 135 140

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

145 150 155 160;

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

165 170 175

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

180 185 190

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

195 200 205

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

210 215 220

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

225 230 235 240

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

245 250 255

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

260 265 270

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

275 280 285

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

290 295 300

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

305 310 315 320

Ser Leu Ser Leu Ser Pro Gly Lys

325

<210> 82

<211> 618

<212> DNA

<213> Homo sapiens

<400> 82

ctcatgaata tgcaaataac ctgagattta ctgaagtaaa tacagatctg tcctgtgccc 60

tgagagcatc acccagcaac cacatctgtc ctctagagaa tcccctgaga gctccgttcc 120

tcaccatgga ctggacctgg aggatcctct tcttggtggc agcagccaca ggtaagaggc 180

tccctagtcc cagtgatgag aaagaggatt gagtccagtc cagggagatc tcatccactt 240

ctgtgttctc tccacaggag cccactccca ggtgcagctg gtgcagtctg gggctgaggt 300

gaagaagcct ggggcctcag tgaaggtctc ctgcaaggct tctggataca ccttcaccgg 360

ctactatatg cactgggtgc gacaggcccc tggacaaggg cttgagtgga tgggacggat 420

caaccctaac agtggtggca caaactatgc acagaagttt cagggcaggg tcaccagtac 480

cagggacacg tccatcagca cagcctacat ggagctgagc aggtgagat ctgacgacac 540

ggtcgtgtat tactgtgcga gagacacagt gtgaaaaccc acatcctgag ggtgtcagaa 600

accccaggga ggaggcag 618

<210> 83

<211> 613

<212> DNA

<213> Homo sapiens

<400> 83

ccagctccac cctcctctgg gttgaaaaag ccgagcacag gtaccagctc agtgactcct 60

gtgcaccacc atggacacac tttgctccac gctcctgctg ctgaccatcc cttcatgtga 120

gtgctgtggt cagggactcc ttcacgggtg aaacatcagt tttcttgttt gtgggcttca 180

tcttcttatg ctttctccac aggggtcttg tcccagatca ccttgaagga gtctggtcct 240

acgctggtga aacccacaca gaccctcacg ctgacctgca ccttctctgg gttctcactc 300

agcactagtg gagtgggtgt gggctggatc cgtcagcccc caggaaaggc cctggagtgg 360

cttgcactca tttattggaa tgatgataag cgctacagcc catctctgaa gagcaggctc 420

accatcacca aggacacctc caaaaaccag gtggtcctta caatgaccaa catggaccct 480

gtggacacag ccacatatta ctgtgcacac agaccacaaa gacacagccc agggcacctc 540

ctgtacaaaa acccaggctg cttctcattg gtgctccctc cccacctctg cagaacagga 600

aagtctgtct gct 613

<210> 84

<211> 546

<212> DNA

<213> Homo sapiens

<400> 84

agtgactcct gtgccccacc atggacacac tttgctacac actcctgctg ctgaccaccc 60

cttcctgtga gtgctgtggt cagggacttc ctcagaagtg aaacatcagt tgtctccttt 120

gtgggcttca tcttcttatg tcttctccac aggggtcttg tcccaggtca ccttgaagga 180

gtctggtcct gtgctggtga aacccacaga gaccctcacg ctgacctgca ccgtctctgg 240

gttctcactc agcaatgcta gaatgggtgt gagctggatc cgtcagcccc cagggaaggc 300

cctggagtgg cttgcacaca ttttttcgaa tgacgaaaaa tcctacagca catctctgaa 360

gagcaggctc accatctcca aggacacctc caaaagccag gtggtcctta ccatgaccaa 420

catggaccct gtggacacag ccacatatta ctgtgcacgg ataccacaga gacacagccc 480

aggatgcctc ctgtacaaga acctagctgc atctcagtgg tgctccctcc ctacctctgc 540

agaaca 546

<210> 85

<211> 460

<212> DNA

<213> Homo sapiens

<400> 85

atggacatac tttgttccac gctcctgcta ctgactgtcc cgtcctgtga gtgctgtggt 60

caggtagtac ttcagaagca aaaaatctat tctctccttt gtgggcttca tcttcttatg 120

tcttctccac aggggtctta tcccaggtca ccttgaggga gtctggtcct gcgctggtga 180

aacccacaca gaccctcaca ctgacctgca ccttctctgg gttctcactc agcactagtg 240

gaatgtgtgt gagctggatc cgtcagcccc cagggaaggc cctgggtgg cttgcactca 300

ttgattggga tgatgataaa tactacagca catctctgaa gaccaggctc accatctcca 360

aggacacctc caaaaaccag gtggtcctta caatgaccaa catggaccct gtggacacag 420

ccacgtatta ctgtgcacgg ataccacaga gacacaccca 460

<210> 86

<211> 877

<212> DNA

<213> Homo sapiens

<400> 86

acagcctatt cctccagcat cccactagag cttcttatat agtaggagac atgcaaatag 60

ggccctccct ctactgatga aaaccaaccc aaccctgacc ctgcaggtct cagagaggag 120

ccttagccct ggactccaag gcctttccac ttggtgatca gcactgagca cagaggactc 180

accatggaat tggggctgag ctgggttttc cttgttgcta ttttagaagg tgattcatgg 240

aaaactagga agattgagtg tgtgtggata tgagtgtgag aaacagtgga tttgtgtggc 300

agtttctgac cttggtgtct ctttgtttgc aggtgtccag tgtgaggtgc agctggtgga 360

gtctggggga ggcttggtcc agcctggggg gtccctgaga ctctcctgtg cagcctctgg 420

attcaccttt agtagctatt ggatgagctg ggtccgccag gctccaggga aggggctgga 480

gtgggtggcc aacataaagc aagatggaag tgagaaatac tatgtggact ctgtgaaggg 540

ccgattcacc atctccagag acaacgccaa gaactcactg tatctgcaaa tgaacagcct 600

gagagccgag gacacggctg tgtattactg tgcgagagac acagtgaggg gaagtcagtg 660

tgagcccaga cacaaacctc cctgcagggg tcccttggga ccaccagggg gcgacagggc 720

attgagcact gggctgtctc cagggcaggt gcaggtgctg ctgagggctg gcttcctgtc 780

gcggtctggg gctgcctcgt cgtcaaattt ccccaggaac ttctccagat ttacaattct 840

gtactgacat ttcatgtctc taaatgcaat acttttt 877

<210> 87

<211> 557

<212> DNA

<213> Homo sapiens

<400> 87

ccaggagttt ccattcggtg atcagcactg aacacagagg actcaccatg gagtttgggc 60

tgagctgggt tttccttgtt gctataataa aaggtgattt atggagaact agagacattg 120

agtggacgtg agtgagataa gcagtgaata tatgtggcag tttctgacta ggttgtctct 180

gtgtttgcag gtgtccagtg tcaggtgcag ctggtggagt ctgggggagg cttggtcaag 240

cctggagggt ccctgagact ctcctgtgca gcctctggat tcaccttcag tgactactac 300

atgagctgga tccgccaggc tccagggaag gggctggagt gggtttcata cattagtagt 360

agtggtagta ccatatacta cgcagactct gtgaagggcc gattcaccat ctccagggac 420

aacgccaaga actcactgta tctgcaaatg aacagcctga gagccgagga cacggccgtg 480

tattactgtg cgagagacac agtgagggga agtcagtgtg agcccagaca caaacctccc 540

tgcagggggt cccttgg 557

<210> 88

<211> 727

<212> DNA

<213> Homo sapiens

<400> 88

agatttaaga accttgcacc tggtacccgt tgctcttctt gtaaccattt gtcttttaag 60

ttgtttatca ctctgtaact attttgatta ttttgattct tgcatgtttt tacttctgta 120

aaattattac atttgagtcc ctctcccctt cctaaaccta ggtataaaat ttactcgagc 180

cccttcctcg tggccgagag aattttgagc atgagctgtc tctttggcag ccggcttaat 240

aaaggactct taattcgtct caaagtgtgg cgttttctta actcacctgg gtacaacagt 300

gcagctggtg gagtctgggg gaggcttggt agagcctggg gggtccctga gactctcctg 360

tgcagcctct ggattcacct tcagtaacag tgacatgaac tgggtccgcc aggctccagg 420

aaaggggctg gagtgggtat cgggtgttag ttggaatggc agtaggacgc actatgcaga 480

ctctgtgaag ggccgattca tcatctccag agacaattcc aggaacttcc tgtatcagca 540

aatgaacagc ctgaggcccg aggacatggc tgtgtattac tgtgtgagaa acactgtgag 600

aggacggaag tgtgagccca gacacaaacc tcctgcagga acgttggggg aaatcagctg 660

cagggggcgc tcaagaccca ctcatcagag tcaaccccag agcaggtgca catggaggct 720

gggtttt 727

<210> 89

<211> 514

<212> DNA

<213> Homo sapiens

<400> 89

ggactcgcca tggagtttgg gctgagctgg gttttccttg ttgctatttt aaaaggtgat 60

tcatggatca atagagatgt tgagtgtgag tgaacacgag tgagagaaac agtggatttg 120

tgtggcagtt tctgaccagg gtgtctctgt gtttgcaggt gtccagtgtg aggtgcagct 180

ggtggagtct gggggaggtg tggtacggcc tggggggtcc ctgagactct cctgtgcagc 240

ctctggattc acctttgatg attatggcat gagctgggtc cgccaagctc cagggaaggg 300

gctggagtgg gtctctggta ttaattggaa tggtggtagc acaggttatg cagactctgt 360

gaagggccga ttcaccatct ccagagacaa cgccaagaac tccctgtatc tgcaaatgaa 420

cagtctgaga gccgaggaca cggccttgta tcactgtgcg agagacacag tgaggggaag 480

ccagtgagag cccagacaca aacgtccctg cagg 514

<210> 90

<211> 412

<212> DNA

<213> Homo sapiens

<400> 90

tgattcatgg agaaatagag agactgagtg tgagtgaaca tgagtgagaa aaactggatt 60

tgtgtggcat tttctgataa cggtgtcctt ctgtttgcag gtgtccagtg tcaggtgcag 120

ctggtggagt ctgggggagg cgtggtccag cctgggaggt ccctgagact ctcctgtgca 180

gcgtctggat tcaccttcag tagctatggc atgcactggg tccgccaggc tccaggcaag 240

gggctggagt gggtggcagt tatatggtat gatggaagta ataaatacta tgcagactcc 300

gtgaagggcc gattcaccat ctccagagac aatccaaga acacgctgta tctgcaaatg 360

aacagcctga gagccgagga cacggctgtg tattactgtg cgagagacac ag 412

<210> 91

<211> 870

<212> DNA

<213> Homo sapiens

<220>

<221> mixed properties

<222> (43)..(43)

<223> n denotes a, c, g, or t

<220>

<221> mixed properties

<222> (824)..(824)

<223> n denotes a, c, g, or t

<400> 91

catctgttac agaactcatt atatagtagg agacatccaa atngggtccc tccctctgct 60

gatgaaaacc agcccagccc tgaccctgca gctctgggag aggagcccca gccctgagat 120

tcccaggtgt ttccattcgg tgatcagcac tgaacacaga gaacgcacca tggagtttgg 180

actgagctgg gttttccttg ttgctatttt aaaaggtgat tcatggataa atagagatgt 240

tgagtgtgag tgaacatgag tgagagaaac agtggatatg tgtggcagtg tctgaccagg 300

gtgtctctgt gtttgcaggt gtccagtgtg aagtgcagct ggtggagtct gggggagtcg 360

tggtacagcc tggggggtcc ctgagactct cctgtgcagc ctctggattc acctttgatg 420

attataccat gcactgggtc cgtcaagctc cggggaaggg tctggagtgg gtctctctta 480

ttagttggga tggtggtagc acatactatg cagactctgt gaagggccga ttcaccatct 540

ccagagacaa cagcaaaaac tccctgtatc tgcaaatgaa cagtctgaga actgaggaca 600

ccgccttgta ttactgtgca aaagatacac agtgagggga agtcagcgag agcccagaca 660

aaaacctcgc tgcaggaaga caggaggggc ctgggctgca gaggccactc aagacacact 720

gagcataggg ttaactctgg gacaagttgc tcaggaaggt taagagctgg tttcctttca 780

gagtcttcac aaatttctcc atctaacagt ttccccagga accngtctag atctgtgatc 840

ttggatctgc tgaaactgcc tgtgtcacct 870

<210> 92

<211> 724

<212> DNA

<213> Homo sapiens

<220>

<221> mixed properties

<222> (560)..(560)

<223> n denotes a, c, g, or t

<400> 92

ccattcggtg atcagcactg aacacagagg actcaccatg gagttttggc tgagctgggt 60

tttccttgtt gctattttaa aaggtgattc atggagaact agagatattg agtgtgagtg 120

aacacgagtg agagaaacag tggatatgtg tggcagtttc taaccaatgt ctctgtgttt 180

gcaggtgtcc agtgtgaggt gcagctggtg gagtctggag gaggcttgat ccagcctggg 240

gggtccctga gactctcctg tgcagcctct gggttcaccg tcagtagcaa ctacatgagc 300

tgggtccgcc aggctccagg gaaggggctg agtgggtct cagttattta tagcggtggt 360

agcacatact acgcagactc cgtgaagggc cgattcacca tctccagaga caattccaag 420

aacacgctgt atcttcaaat gaacagcctg agagccgagg acacggccgt gtattactgt 480

gcgagagaca cagtgagggg aagtcattgt gcgcccagac acaaacctcc ctgcaggaac 540

gctgggggga aatcagcggn agggggcgct caggagccac tgatcagagt cagccccgga 600

ggcaggtgca gatggaggct gatttccttg tcaggatgtg gggacttttg tcttcttctg 660

acgggttccc caggggaacc tctctaagtt tagcattctg tgcctatgaa cgtcttctct 720

aagt 724

<210> 93

<211> 288

<212> DNA

<213> Homo sapiens

<400> 93

gaggtgcagc tggtggagtc tcggggagtc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccgtcagt agcaatgaga tgagctgggt ccgccaggct 120

ccagggaagg gtctggagtg ggtctcatcc attagtggtg gtagcacata ctacgcagac 180

tccaggaagg gcagattcac catctccaga gacaattcca agaacacgct gcatcttcaa 240

atgaacagcc tgagagctga ggacacggct gtgtattact gtaagaaa 288

<210> 94

<211> 626

<212> DNA

<213> Homo sapiens

<400> 94

catccctttt cacctcttta tacaaaggca ccacctacat gcaaatcctc acttaggcac 60

ccacaggaaa ccaccacaca tttccttaaa ttcagggtcc agctcacatg ggaaatactt 120

tctgagagct catgggcctc ctgcacaaga acatgaaaca cctgtggttc ttcctcctcc 180

tggtggcagc tcccagatgt gagtgtctca aggctgcaga catgggggta tgggaggtgc 240

ctctgatccc agggctcact gtgggtctct ctgttcacag gggtcctgtc tcaggtgcag 300

ctgcaggagt cgggcccagg actggtgaag cctccgggga ccctgtccct cacctgcgct 360

gtctctggtg gctccatcag cagtagtaac tggtggagtt gggtccgcca gcccccaggg 420

aaggggctgg agtggattgg ggaaatctat catagtggga gcaccaacta caacccgtcc 480

ctcaagagtc gagtcaccat atcagtagac aagtccaaga accagttctc cctgaagctg 540

agctctgtga ccgccgcgga cacggccgtg tattgctgtg cgagagacac agtgagggga 600

ggtgagtgtg agcccagaca caaacc 626

<210> 95

<211> 299

<212> DNA

<213> Homo sapiens

<400> 95

cagctgcagc tgcaggagtc cggctcagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcgctg tctctggtgg ctccatcagc agtggtggtt actcctggag ctggatccgg 120

cagccaccag ggaagggcct ggagtggatt gggtacatct atcatagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atatcagtag acaggtccaa gaaccagttc 240

tccctgagc tgagctctgt gaccgccgcg gacacggccg tgtattactg tgccagaga 299

<210> 96

<211> 410

<212> DNA

<213> Homo sapiens

<400> 96

gggtcctgtc ccaggtgcag ctacagcagt ggggcgcagg actgttgaag ccttcggaga 60

ccctgtccct cacctgcgct gtctatggtg ggtccttcag tggttactac tggagctgga 120

tccgccagcc cccagggaag gggctggagt ggattgggga aatcaatcat agtggaagca 180

ccaactacaa cccgtccctc aagagtcgag tcaccatatc agtagacacg tccaagaacc 240

agttctccct gaagctgagc tctgtgaccg ccgcggacac ggctgtgtat tactgtgcga 300

gaggcacagt gaggggaggt gagtgtgagc ccagacaaaa acctccctgc aggtaggcag 360

agggggcggg cgcaggtact gctcaagacc agcaggtggc gcgcggcgcc 410

<210> 97

<211> 700

<212> DNA

<213> Homo sapiens

<220>

<221> mixed properties

<222> (19)..(19)

<223> n denotes a, c, g, or t

<220>

<221> mixed properties

<222> (46)..(46)

<223> n denotes a, c, g, or t

<400> 97

aggttctggg ttataaacnc tgtagactcc tcccttcagg gcaggntgac caactatgca 60

aatgcaagtg ggggcctccc cacttaaacc cagggctccc ctccacagtg agtctccctc 120

actgcccagc tgggatctca gggcttcatt ttctgtcctc caccatcatg gggtcaaccg 180

ccatcctcgc cctcctcctg gctgttctcc aaggtcagtc ctgccgaggg cttgaggtca 240

cagaggagaa cgggtggaaa ggagcccctg attcaaattt tgtgtctccc ccacaggagt 300

ctgttccgag gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct 360

gaagatctcc tgtaagggtt ctggatacag ctttaccagc tactggatcg gctgggtgcg 420

ccagatgccc gggaaaggcc tggagtggat ggggatcatc tatcctggtg actctgatac 480

cagatacagc ccgtccttcc aaggccaggt caccatctca gccgacaagt ccatcagcac 540

cgcctacctg cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag 600

acacacagtg agagaaacca gccccgagcc cgtctaaaac cctccacacc gcaggtgcag 660

aatgagctgc tagagactca ctccccaggg gcctctctat 700

<210> 98

<211> 650

<212> DNA

<213> Homo sapiens

<220>

<221> mixed properties

<222> (621)..(621)

<223> n denotes a, c, g, or t

<400> 98

agggcagtca ccagagctcc agacaatgtc tgtctccttc ctcatcttcctgcccgtgct 60

gggcctccca tggggtcagt gtcagggaga tgccgtattc acagcagcat tcacagactg 120

aggggtgttt cactttgctg tttccttttg tctccaggtg tcctgtcaca ggtacagctg 180

cagcagtcag gtccaggact ggtgaagccc tcgcagaccc tctcactcac ctgtgccatc 240

tccggggaca gtgtctctag caacagtgct gcttggaact ggatcaggca gtccccatcg 300

agaggccttg agtggctggg aaggacatac tacaggtcca agtggtataa tgattatgca 360

gtatctgtga aaagtcgaat aaccatcaac ccagacacat ccaagaacca gttctccctg 420

cagctgaact ctgtgactcc cgaggacacg gctgtgtatt actgtgcaag agacacagtg 480

aggggaagtc agtgtgagcc cagacacaaa cctccctgca gggatgctca ggaccccaga 540

aggcacccag cactaccagc gcagggccca gaccaggagc aggtgtggag ttaagcaaaa 600

atggaacttc ttgctgtgtc ntaaactgtt gttgtttttt tttttttttt 650

<210> 99

<211> 388

<212> DNA

<213> Homo sapiens

<400> 99

taaggggctc cccagtcact gggctgaggg agaaaccagc acagtcaagt gagacttcat 60

gcactcccat ctcctctcca caggtgccca ctcccaggtg cagctggtgc aatctgggtc 120

tgagttgaag aagcctgggg cctcagtgaa ggtttcctgc aaggcttctg gatacacctt 180

cactagctat gctatgaatt gggtgcgaca ggcccctgga caagggcttg agtggatggg 240

atggatcaac accaacactg ggaacccaac gtatgcccag ggcttcacag gacggtttgt 300

cttctccttg gacacctctg tcagcacggc atatctgcag atctgcagcc taaaggctga 360

ggacactgcc gtgtattact gtgcgaga 388

<210> 100

<211> 294

<212> DNA

<213> Homo sapiens

<400> 100

caggtgcagc tggtgcagtc tggccatgag gtgaagcagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cagtttcacc acctatggta tgaattgggt gccacaggcc 120

cctggacaag ggcttgagtg gatgggatgg ttcaacacct acactgggaa cccaacatat 180

gcccagggct tcacaggacg gtttgtcttc tccatggaca cctctgccag cacagcatac 240

ctgcagatca gcagcctaaa ggctgaggac atggccatgt attactgtgc gaga 294

<210> 101

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 101

Gly Ala Glu Leu Lys Lys

1 5

<210> 102

<211> 6

< 212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 102

Gly Ala Glu Val Val Lys

1 5

<210> 103

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 103

Gly Gly Glu Val Lys Lys

1 5

<210> 104

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Vh Sequence at Kabat positions 108 to 113 of mouse/human modified

antigen binding molecule

<400> 104

Gly Ala Gly Val Lys Lys

1 5

<210> 105

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 105

Gly Gly Gly Val Val Lys

1 5

<210> 106

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 106

Leu Val Thr Val Ser Ser

1 5

<210> 107

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 107

Leu Val Ile Val Ser Ser

1 5

lt; 210> 108

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 108

Leu Val Thr Val Ile Ser

1 5

<210> 109

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 109

Leu Val Ile Val Ile Ser

1 5

<210> 110

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 110

Leu Val Gly Val Ser Ser

1 5

<210> 111

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 111

Leu Val Thr Val Gly Ser

1 5

<210> 112

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 112

Leu Val Gly Val Gly Ser

1 5

<210> 113

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigens�asiausa molecules of mouse/human

<400> 113

Leu Val Ala Val Ser Ser

1 5

<210> 114

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 114

Leu Val Thr Val Ala Ser

1 5

<210> 115

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 115

Leu Val Ala Val Ala Ser

1 5

<210> 116

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 116

Leu Val Val Val Ser Ser

1 5

<210> 117

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 117

Leu Val Thr Val Val Ser

1 5

<210> 118

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 118

Leu Val Val Val Val Ser

1 5

<210> 119

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh on well�erachi Kabat 108-113 modified antigen-binding molecules of the mouse/human

<400> 119

Leu Val Leu Val Ser Ser

1 5

<210> 120

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 120

Leu Val Thr Val Leu Ser

1 5

<210> 121

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 121

Leu Val Leu Val Leu Ser

1 5

<210> 122

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 122

Leu Val Ser Val Ser Ser

1 5

<210> 123

<211> 6

<212> PROTEIN

<213> Artificial

<220>

<223> Sequence Vh Kabat numbering 108-113 modified antigen-binding molecules of the mouse/human

<400> 123

Leu Val Thr Val Thr Ser

1 5

<210> 124

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH2A modified antigen-binding molecules of the mouse/human

<400> 124

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pr Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 125

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH2B modified antigen-binding molecules of the mouse/human

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 126

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH2C modified antigen-binding molecules of the mouse/human

<400> 126

Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val hr Val Ser Ser

115

<210> 127

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH2D modified antigen-binding molecules of the mouse/human

<400> 127

Gln Val Gln Leu Val Gln Ser Gly Ala Gly Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 128

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH2E modified antigen-binding molecules of the mouse/human

<400> 128

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Ile Val Ser Ser

115

<210> 129

<211> 119

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-HH2F modified antigen-binding molecules of the mouse/human

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ile Ser

115

<210> 130

<211> 115

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-KV10 modified antigen-binding molecules of the mouse/human

<400> 130

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val

115

<210> 131

<211> 115

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-KV11 modified antigen-binding molecules of the mouse/human

<400> 131

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met SerAsn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val

115

<210> 132

<211> 115

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-KV12 modified antigen-binding molecules of the mouse/human

<400> 132

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Phe Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val

115

<210> 133

<211> 115

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-KV13 modified antigen-binding molecules of the mouse/human

<400> 133

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Ala Ile Lys

100 105 110

Arg Thr Val

115

<20> 134

<211> 115

<212> PROTEIN

<213> Artificial

<220>

<223> Design B-KV14 modified antigen-binding molecules of the mouse/human

<400> 134

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ala Lys

100 105 110

Arg Thr Val

115

1. Modified anti-CD20 antibody or antigen-binding fragment, and the indicated antibody or antigen-binding fragment cause higher levels of apoptosis in the formation of complex with CD20, compared with the chimeric B-Ly1 antibody in the formation of the complex with a CD20, and where the specified antibody or antigen-binding fragment comprises: a first polypeptide containing the variable region of the heavy chain with the sequence SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127; and a second polypeptide containing the variable region light chain with the sequence SEQ ID NO:48.

2. The antibody or antigen-binding fragment according to claim 1, wherein the said antibody or antigen-binding fragment include a first polypeptide comprising a variable region heavy �ETUI with sequence SEQ ID NO: 125, and a second polypeptide comprising a variable region light chain with the sequence SEQ ID NO: 48.

3. The antibody or antigen-binding fragment according to claim 1, wherein the said antibody or antigen-binding fragment include a first polypeptide comprising the variable region of the heavy chain with the sequence SEQ ID NO: 126, and a second polypeptide comprising a variable region light chain with the sequence SEQ ID NO: 48.

4. The antibody or antigen-binding fragment according to claim 1, wherein the said antibody or antigen-binding fragment include a first polypeptide comprising the variable region of the heavy chain with the sequence SEQ ID NO: 127, and a second polypeptide comprising a variable region light chain with the sequence SEQ ID NO: 48.

5. The antibody or antigen-binding fragment according to any one of claims.1-4, wherein the said antibody or antigen-binding fragment comprises an Fc region, which was held Glyco-engineering modification to increase the number of branched nefokusirana oligosaccharides.

6. The antibody or antigen-binding fragment according to any one of claims.1-4, wherein the said antibody or antigen-binding fragment comprises an Fc region N-linked oligosaccharides, which are modified to increase at least one effector function, zoom�of the binding affinity Fc receptor, or increasing at least one effector function and increase the affinity of binding of the Fc receptor.

7. A mixture of antibodies or antigen-binding fragments to enhance effector function and/or increased affinity to Fc-receptor containing the antibody or antigen-binding fragment according to claim 6, wherein at least 20% of the oligosaccharides in the Fc region of the indicated antibodies or antigen-binding fragment have a branched chain and not fuckalicious.

8. Mixture according to claim 7, wherein at least 50% of the oligosaccharides in the Fc region is not fuckalicious.

9. A host for expression of the antibody or antigen-binding fragment according to any one of claims.5-6 or mixture according to claims.7-8, with the specified host includes one or more of the polynucleotide encoding these heavy and light chains, and at least one nucleic acid encoding a polypeptide having the activity of β(1,4)-N-acetylglucosaminyltransferase III, which are expressed in quantities sufficient for Glyco-engineering and modification of the Fc region.

10. A host according to claim 9, wherein the specified host additionally expresses the nucleic acid encoding the polypeptide with the activity of mannosidase II.

11. A host according to claim 9, wherein the said antibody or antigen-binding fragment produced by a given cell-hosaina�, demonstrates increased binding affinity Fc receptor as a result of the Glyco-engineering modifications.

12. A host according to claim 11, wherein the specified Fc receptor is a receptor FcγRIIIA.
13 a host according to claim 9, wherein the said antibody or antigen-binding fragment produced by the specified host cell, demonstrates an enhanced effector function as a result of the Glyco-engineering modifications.

14. A host according to claim 12, wherein usilennaya effector function is increased Fc cell-mediated cytotoxicity.

15. A host according to claim 12, in which a specified enhanced effector function is an enhanced antibody-dependent cell-mediated cytotoxicity.

16. A host according to claim 9, comprising at least one transfected with the polynucleotide that encodes first and second polypeptides, which are: a first polypeptide comprising a variable region heavy chain sequences with SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127; and a second polypeptide comprising a variable region light chain with the sequence SEQ ID NO: 48; wherein the said polynucleotide comprises a sequence encoding a region equivalent to the Fc region of human immunoglobulin.

17. Pharmaceutical composition, on�consisting of a first antibody or antigen-binding fragment according to any one of claims.1-6, or a mixture thereof according to claim.7-8 in effective amounts, as well as pharmaceutically acceptable carrier for the manufacture of a medicine for the treatment of malignant haematological diseases or autoimmune diseases.

18. Pharmaceutical composition according to claim 17, wherein the specified malignant hematological disease is a b-cell lymphoma, nahodkinskuju lymphoma or b-cell chronic lymphocytic leukemia.

19. Pharmaceutical composition according to claim 17, wherein the specified autoimmune disease is a rheumatoid arthritis or lupus.

20. Pharmaceutical composition according to claim 17, wherein the specified malignant hematological disease is a B-cell lymphoma.

21. The selected polynucleotide encoding the variable region of the heavy chain of the antibody or antigen-binding fragment according to claim 1, comprising the sequence of SEQ ID NO: 125.

22. The selected polynucleotide encoding variable regions of heavy and light chain according to claim 1, comprising the sequence of SEQ ID NO: 125 and SEQ ID NO: 48.

23. The selected polynucleotide encoding the variable region of the heavy chain of the antibody or antigen-binding fragment according to claim 1, comprising the sequence of SEQ ID NO: 126.

24. The selected polynucleotide encoding variable regions of heavy and light chain according to claim 1, comprising posledovatelno�ü SEQ ID NO: 126 and SEQ ID NO: 48.

25. The selected polynucleotide encoding the variable region of the heavy chain of the antibody or antigen-binding fragment according to claim 1, comprising the sequence of SEQ ID NO: 127.

26. The selected polynucleotide encoding variable regions of heavy and light chain of the antibody or antigen-binding fragment according to claim 1, comprising the sequence of SEQ ID NO: 127 and SEQ ID NO: 48.

27. Expression vector comprising the polynucleotide according to any one of claims.21-26.

28. A host comprising the expression vector according to claim 27.

29. Method of production in the host cell anti-CD20 antibody or antigen-binding fragment to the Fc region with modified oligosaccharides and subjected to genetic engineering modifications to enhance effector function, wherein said method comprises: culturing the host cell is subjected to genetic engineering modifications for expression of one or more nucleic acid that encodes the said antibody or antigen-binding fragment and at least one nucleic acid that encodes a polypeptide having the activity of β(1,4)-N-acetylglucosaminyltransferase III; and the allocation of the specified antibodies or antigen-binding fragment; moreover, the specified antibody or antigen-binding fragment represent the antibody or antigen-binding fragment according to any one of claims.5-6 Il� mixture according to claim.7-8.

30. A method according to claim 29, in which the specified host is subjected to additional genetic engineering modifications for expression of the nucleic acid that encodes a polypeptide having the activity of mannosidase II.

31. A method according to claim 29, where the specified modified oligosaccharides have a lower degree of fokusirovanie compared to unmodified oligosaccharides.

32. A method according to claim 29, wherein said antibody or antigen-binding fragment produced by the specified host cell, include a higher percentage of branched nefokusirana oligosaccharides in the Fc region.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to method of obtaining bivalent bispecific antibody, which includes transformation of host cell by vectors, containing molecules of nucleic acids, coding first light chain and first heavy chain of bivalent bispecific antibody, and vectors, containing molecules of nucleic acids, coding second light chain and second heavy chain of bivalent bispecific antibody, cultivation of host cell under conditions, providing synthesis of molecule of bivalent bispecific antibody from said culture. Said antibody contains first light chain and first heavy chain of antibody, specifically binding with first antigen, and second light chain and second heavy chain of antibody, specifically binding with second antigen, in which variable domains VL and VH of second light chain and second heavy chain are replaced by each other and constant domains CL and CH1 of second light chain and second heavy chain are replaced by each other.

EFFECT: invention makes it possible to increase output of correct bispecific antibody by increasing the level of correct heterodimerisation of heavy chains of wild type and modification of heavy chains resulting from crossing over.

2 cl, 31 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.

EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.

25 cl, 24 dwg, 8 tbl, 17 ex

Csf-1r antibody // 2547586

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented an antibody and its antigen-binding fragment specifically binding human colony-stimulating factor-1 receptor (CSF-1R) characterised by sequences of complementary-determining regions (CDR). There are also disclosed a nucleic acid coding the antibody according to the invention or its antigen-binding fragment, a vector providing the expression of the antibody and its antigen-binding fragment, and a pharmaceutical composition applicable in treating the diseases associated with an inflammation or an autoimmunity, or cancer.

EFFECT: invention can find further application in diagnosing and therapy of the CSF-1 associated diseases.

23 cl, 18 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, in particular to peptydoglycane hydrolase biosynthesis, and represents a protein with the peptydoglycane hydrolase activity, a plasmid, containing a peptydoglycane hydrolase-coding fragment, a bacterium-producer, a method of microbiological peptydoglycane hydrolase synthesis, as well as a pharmaceutical composition, containing the obtained peptydoglycane hydrolase, for the therapy of diseases, caused by Gram-negative microflora.

EFFECT: elaborated method of microbiological synthesis makes it possible to obtain bacteriophage S-394 peptydoglycane hydrolase in an effective way.

22 cl, 2 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: inventions relate to chimeric proteins, nucleic acid, coding such a protein, an expression cassette, providing the expression of nucleic acid, a vector, including the expression cassette, a method of diagnostics and a set for diagnostics. The characterised chimeric Borrelia protein includes at least one sequence of an extracellular domain of the VlsE Borrelia protein of the first type, corresponding to a certain strain, and at least one sequence of IR6 area of the VlsE Borrelia protein of the second type or Borrelia of the first type, but corresponding to a strain, different from the strain of the first type, with Borrelia being selected from Borrelia stricto-sensu, Borrelia afzelii and Borrelia garinii.

EFFECT: claimed inventions make it possible to carry out diagnostics of Lyme-borreliosis with an increased specificity and sensitivity.

15 cl, 8 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Presented are antibodies targeting integrin α2β1 containing humanised anti-integrin alpha-2 (α2) antibodies, as well as a method of treating by the integrin α2 antibodies. The humanised integrin α2 antibodies comprise a variable region of a light chain domain, a constant human light chain domain and a variable constant heavy chain domain of human IgG1, which exhibit the altered effector function. The variable constant heavy chain domain of human IgG1 comprises an S324N substitution. The invention can be used in medicine.

EFFECT: antibodies exhibit complement-dependent cytotoxicity, improved antibody-dependent cell-mediated cytotoxicity and improved CDC and ADCC.

33 cl, 3 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is a host cell of Bordetella pertussis, Bordetella bronchiseptica or Bordetella parapertussis, which is used as an adjuvant or for preventing or treating whooping cough, having the low activity of endogenous glycosyltransferase at least 98% identical to the amino acid sequence SEQ ID NO: 2 as compared to the activity of glycosyltransferase of a relative parent strain, wherein the low activity is ensured by using an inactivating vector, which causes the inactivation of expression of a sequence of endogenous nucleic acid coding glycosyltransferase, or reduces to a low level of expression of the sequence of endogenous nucleic acid coding glycosyltransferase by the fusion of nucleic acid coding glycosyltransferase with a low-level or inducible promotor. What is disclosed is a preparation consisting of LPS of the above host cell with an increased replacement of hexosamine 1' or 4' phosphate groups of LPS referred to a lipid A, as compared to a LPS preparation from the related parent strain; thereby LPS is characterised by producing at least 8 ions in the ESI-MS spectrum, wherein the preparation is used as an adjuvant or for preventing or treating whooping cough. What is presented is using the above host cells or the LPS preparation for producing the preparation for preventing and/or treating whooping cough, or producing the drug preparation for immunising a mammal, wherein the host cells or LPS is used as an adjuvant. What is described is a pharmaceutical composition used as the adjuvant or for preventing or treating Bordetella infection containing the above host cell or above LPS preparation in an effective amount and a pharmaceutically acceptable carrier.

EFFECT: invention enables producing the pharmaceutical preparation of Bordetella cells or LPS, possessing the high immunogenicity as compared to the preparation of the related parent strain Bordetella.

12 cl, 7 dwg, 3 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. Presented are versions of an antigen-binding polypeptide specific to LIGHT polypeptide characterised by a variable domain of heavy and light chains, as well as versions of based conjugates for treating a disease or a condition. What is described is a pharmaceutical composition for inhibiting apoptosis induced by human LIGHT based on a therapeutically effective amount of the polypeptide. There are disclosed: a method of treating or a diagnosing the disease or condition on the basis of the composition. There are described versions of the recovered polynucleotide or a cell transformed by the polynucleotide for preparing the antigen-binding polypeptide, as well as a method for producing the antigen-binding polypeptide on the basis of the cell.

EFFECT: using the invention provides the antibodies, which block the human or macaque LIGHT interaction to LIGHT receptors that can find application in treating various diseases related to high T-cell activity.

23 cl, 11 dwg, 8 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to biospecific antibodies, and can be used in medicine. Constructed is an antibody containing one of the following groups of six hypervariable region (HVR) sequences: (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2, containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NIKDTY; (e) HVR-H2 containing the sequence RIYPTNGYTR; and (f) HVR-H3 containing the sequence WGGDGFYAMD; or (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2 containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NISGTY; (e) HVR-H2 containing the sequence RIYPSEGYTR; and (f) HVR-H3 containing the sequence WVGVGFYAMD. The produced antibody specifically binds human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) The invention also refers to a recovered Fab fragment of the above antibody, a polynucleotide coding it, to an expression vector, a host cell, a method for producing it, as well as to using it for treating HER2-mediated diseases.

EFFECT: present invention enables producing the bispecific high-affinity antibody able to bind VEGF and HER2 simultaneously.

14 cl, 65 dwg, 16 tbl, 8 ex

Csf-1r antibody // 2547586

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented an antibody and its antigen-binding fragment specifically binding human colony-stimulating factor-1 receptor (CSF-1R) characterised by sequences of complementary-determining regions (CDR). There are also disclosed a nucleic acid coding the antibody according to the invention or its antigen-binding fragment, a vector providing the expression of the antibody and its antigen-binding fragment, and a pharmaceutical composition applicable in treating the diseases associated with an inflammation or an autoimmunity, or cancer.

EFFECT: invention can find further application in diagnosing and therapy of the CSF-1 associated diseases.

23 cl, 18 dwg, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. Presented are versions of an antigen-binding polypeptide specific to LIGHT polypeptide characterised by a variable domain of heavy and light chains, as well as versions of based conjugates for treating a disease or a condition. What is described is a pharmaceutical composition for inhibiting apoptosis induced by human LIGHT based on a therapeutically effective amount of the polypeptide. There are disclosed: a method of treating or a diagnosing the disease or condition on the basis of the composition. There are described versions of the recovered polynucleotide or a cell transformed by the polynucleotide for preparing the antigen-binding polypeptide, as well as a method for producing the antigen-binding polypeptide on the basis of the cell.

EFFECT: using the invention provides the antibodies, which block the human or macaque LIGHT interaction to LIGHT receptors that can find application in treating various diseases related to high T-cell activity.

23 cl, 11 dwg, 8 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology. Disclosed is a method of improving functional properties of an antibody or antigen-binding fragment thereof, for which the heavy chain belongs to the human VH1b family, which comprises performing mutagenesis in frameworks with replacement of the amino acid in position 78 of the heavy chain in the AHo numbering system (position 67 in the Kabat numbering system) into leucine or isoleucine.

EFFECT: method improves the stability and solubility of antibodies and antigen-binding fragments thereof.

2 cl, 17 dwg, 33 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are described antibodies and their functional equivalents specifically binding to RSV. Also, the invention refers to nucleotide sequences coding the above antibody, as well as to the cells producing antibodies, and to methods for producing the above antibodies.

EFFECT: invention can be used in medicine.

23 cl, 4 dwg, 3 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology and biotechnology. Described are: humanised antibody and its antigen-binding fragment (Fab), which selectively bind human IFN-γ and contain variable site of heavy chain (VH) and variable site of light chain (VL), where VH and VL have sequences of amino acids respectively SEQ ID NO:1 and 2, presented in description. Also disclosed are fragments of DNA, which code said antibody and its Fab-fragment; plasmid DNA for expression of said specific proteins; and modified cells of bacteria and eukaryotes, which contain said plasmid DAN and intended for expression of said antibody and its Fab-fragment. Claimed are methods of obtaining said antibody and its Fab-fragment, which include cultivation of said modified cells in nutritional medium and isolation of antibody and its Fab-fragment by claimed invention from cultural liquid.

EFFECT: invention makes it possible to obtain humanised antibody or its Fab-fragment, binding with IFN-γ with kD 4,6 and IC50 not less than 1,5 nM in test on cells U937, with preservation of affinity of initial mouse monoclonal antibody.

13 cl, 3 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology, more specifically to granulocyte-macrophage colony-stimulating factor (GM-CSF) antagonists, and can be used in medicine. The invention consisting in using the GM-CSF specific antibody in treating or preventing multiple sclerosis in the patients with multiple sclerosis.

EFFECT: invention enables delaying the onset of multiple sclerosis recurrences.

9 cl, 5 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented: an antibody binding to interleukin-17 (IL-17) characterised by 6 CDR of a light and heavy chain, as well as a coding nucleic acid and a vector for expression of the above antibody. What is described is a pharmaceutical composition for treating a patient with multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn's disease, chronic obstructive pulmonary disease, asthma, graft rejection on the basis of the above antibody. What is disclosed is a method for preparing the antibody by means of expressing the respective nucleic acid and recovering the antibody from a cell culture or a cell culture supernatant.

EFFECT: using this invention provides the antibody with IC50 twice as much as shown by in vitro IL-6 and IL-8 neutralisation as compared to the known NVP-AIN-497 antibody, which binds human IL-17A and IL-17F that can find application in medicine in therapy of various inflammatory diseases.

9 cl, 6 tbl, 11 ex

Humanised antibody // 2538709

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to ophthalmology, and can be used for treating ocular diseases associated with amyloid-beta related pathological abnormalities/changes in the visual system tissues. That is ensured by administering a pharmaceutical composition, which contains a therapeutically effective amount of a humanised antibody or antigen-binding fragment, wherein the humanised antibody or its fragment is able to bind amyloid-beta. Presented are preventing, treating or relieving symptoms of an ocular disease, reducing the plaque load of retinal ganglion cells, diagnosing the ocular disease and diagnosing a predisposition to the ocular disease, prolonging the patient's sensitivity when treating with the pharmaceutical composition for treating the ocular disease.

EFFECT: group of inventions provides the effective treating of the above ocular pathology by using the composition containing the high-specific antibodies, which specifically recognise and bind to specific epitopes of various β-amyloid proteins.

20 cl, 18 dwg, 9 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

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