Fc-versions with changed binding with fcrn
SUBSTANCE: claimed are versions of polypeptides with Fc-segment from IgG, which possess increased binding FcRn due to introduction of mutation 308C, 308F, 308W, 308Y or modified binding with introduction of mutation 252Y/308F, 257L/308F, 257L/308Y, 257N/308Y, 279Q/308F, 279Y/308F, ^281S/308F, ^A281S/308Y, 284E/308F, 298A/308F/333A/334A, 308F/332E, 308F/311V, 308F/G385H, 308F/428L, and 308F/434Y. Described is antibody with said mutations in Fc region, as well as application of said Fc regions for obtaining fused protein. Application of the invention provides novel Fc-versions with modified FcRn binding.
EFFECT: possibility of application in medicine for obtaining various constructions, with prolonged time of preservation in blood serum in vivo or, vice versa, in case of therapy with application of radioactive medications, with reduced time of preservation in blood serum in vivo.
14 cl, 44 dwg, 4 ex
This application claims rights under 35 U.S.. §119(e) on all the advantages of bids US 60/627,763, registered 12 November 2004; US 60/642,886, registered on January 11, 2006; US 60/649,508 registered 2 February 2005; US 60/662,468, registered on March 15, 2005; US 60/669,311, registered on 6 April 2005; US 60/681,607, registered on 16 may 2005, US 60/690,200, registered on June 13, 2005; US 60/696,609, registered 5 July 2005; US 60/703,018, registered on July 27, 2005 and US 60/726,453, registered August 12, 2005, which are hereby incorporated by reference in its entirety.
The technical field to which the invention relates.
This invention relates to optimized variants of immunoglobulins IgG, to design methods for their preparation and to their application, particularly for therapeutic purposes.
The level of technology
Antibodies are immunologically proteins that bind a specific antigen. Most mammals, including humans and mice, antibodies constructed from paired heavy and light polypeptide chains. Each circuit is assembled from individual domains of immunoglobulin (Ig), and thus, these proteins use the General term immunoglobulin. Each circuit is assembled from two different areas related to variable and constant region is I. Variable region light and heavy chains show considerable differences in sequence among antibodies and are responsible for binding to the target antigen. Constant region show smaller differences in sequence and are responsible for binding with a large number of natural proteins to achieve significant biochemical effects. In humans there are five different classes of antibodies, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4 and IgM. A characteristic feature of these classes of antibodies are their constant region, although more subtle differences may exist in the V-region. Figure 1 shows the IgG1 antibody used here as an example to describe the basic structural features of immunoglobulins. IgG antibodies are protein tetramer, consisting of two heavy chains and two light chains. Heavy chain of IgG is composed of four immunoglobulin domains that are attached from N-Terminus to the C-end in the order VH-SN-CH2-CH3, related to the variable domain of the heavy chain constant domain 1 heavy chain constant domain 2 heavy chain and the constant domain 3 heavy chains, respectively (also referred to as VH-Cγ1-Cγ2-Cγ3 related to the variable domain of the heavy chain constant domain gamma-1, stuck-up to the ENU gamma-2 and a constant domain gamma-3, respectively). Light chain IgG consists of two immunoglobulin domains that are attached from N-Terminus to the C-end in the order VL-CL-related variable domain light chain and the constant domain of the light chain, respectively.
Variable region of the antibody includes antigen-binding determinants of the molecule and, thus, determines the specificity of the antibody against the target antigen. Variable area is called so because it most strongly differs in sequence from other antibodies within the same class. The greatest differences in sequence are found in the hypervariable sites (CDR). There are a total of 6 plots CDR, three for each heavy and light chain, denoted as VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3. Variable area outside the plots CDR refers to a frame (FR). Among different antibodies observed sequence differences in the FR plots, although not as significant as in the areas of CDR. In General, this is a typical structure of antibodies provides a stable basis (section FR), which was considerable variation in the binding of antigens (CDR) can be used by the immune system to achieve specificity for a wide variety of antigens. For many fragments of variable regions of different organisms there are several structures the ur, certain high resolution, some of them are free and some are complex with antigens. Sequence and structural features of the variable regions of antibodies are well characterized (Morea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267-279, work incorporated by reference in its entirety), the conservative properties of antibodies allowed us to develop many engineering technologies for the production of antibodies (Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376, work incorporated by reference in its entirety). For example, it is possible to transplant the CDR of one of the antibodies, such as mouse antibodies, wireframe plot of the other antibodies, such as human antibodies. This process, referred to in this technical field "humanization", allows you to get a less immunogenic therapeutic antibodies from antibodies that are unrelated to human. Fragments comprising the variable region can exist in the absence of other regions of antibodies, such fragments include, for example, the fragment antigen binding (Fab), including VH-Cγ1 and VH-CL; variable fragment (Fv)comprising VH and VL; single-chain variable fragment (scFv)comprising VH and VL connected in the same circuit; and many other fragments of variable regions (Little et al., 2000, Immunol Today 21:364-370, work included by reference and in its entirety).
Plot antibody Fc interacts with various Fc receptors and ligands, which give him a lot of important functionality, so-called effector functions. The immunoglobulins IgG Fc region as shown in figures 1 and 2, includes an immunoglobulin domain γ2 and γ3 and the N-terminal hinge leading into γ2. An important family of receptors for the Fc of IgG class includes gamma Fc receptors (FcγRs). These receptors communicate between antibodies and the cellular mechanisms of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, all works incorporated by reference in its entirety). In humans this family of proteins includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotype N and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotype V158 and F158) and FcγRIIIb (including allotype FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, work incorporated by reference in its entirety). These receptors usually have an extracellular domain that mediates binding to Fc, membrane-associated plot and intracellular domain, which may mediate some of the signaling events inside the cell. These receptors are expressed in various immune cells, including monocytes, macrophages, neutrophils, dendritic to EDI, eosinophils, mast cells, platelets, b cells, large granular lymphocytes, Langerhans cells, natural killer cells (NK) and γγ T cells. The formation of the Fc/FcγR directs these effector cells to bind antigens, which usually leads to a holding signal in cells and important subsequent immune responses such as release of inflammatory mediators, activation of b-cells, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and photoxicity effector function is a potential mechanism by which antibodies destroy target cells. Mediated by the cell reaction in which nonspecific cytotoxic cells that Express FcγRs, learn the antibody is bound on the surface of target cells, and then cause lysis of the target cells, called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Gheitie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, all works incorporated by reference in its entirety). Mediated by the cell reaction in which nonspecific cytotoxic cells, expressindia FcγRs, learn the antibody is bound on the surface of target cells, and then cause phagocytosis of the target cells, called antibody-dependent cell-mediated phagocytosis of cells is to target (ADCP). It was determined a number of structures of the extracellular domains of human FcγRs, including FcγRsIIa (access code in the database pdb 1H9V, incorporated by reference in its entirety) (Sonderman et al., 2001, J Mol Biol 309:737-749, work incorporated by reference in its entirety) (access code in the database pdb 1FCG, incorporated by reference in its entirety) (Maxwell et al., 1999, Nat Struc Biol 6:437-442, work incorporated by reference in its entirety), FcγRsIIb (access code in the database pdb 2FCB, incorporated by reference in its entirety) (Sondermann et al., 1999, Embo J 18:1095-1103, work incorporated by reference in its entirety); and FcγRsIIIb (access code to the database pdb 1E4J, incorporated by reference in its entirety) (Sondermann et al., 2000, Nature 406:267-273, work incorporated by reference in its entirety). All FcγRs bind the same Fc-plot in the N-terminal segment of the domain γ2 and the preceding hinge section, shown in figure 1. This interaction is well characterized structurally (Sondermann et al., 2001, J Mol Biol 309:737-749, work incorporated by reference in its entirety), and has identified a number of structures, Fc human, to bind to the extracellular domain FcγRsIIIb man (access code in the database pdb 1E4K, incorporated by reference in its entirety) (Sondermann et al., 2000, Nature 406:267-273, work incorporated by reference in its entirety) (access code in the database pdb 1IIS and 1IIX included PU is eaten reference in its entirety) (Radaev et al., 2001, J Biol Chem 276:16469-16477, work incorporated by reference in its entirety), as well as the structure of the complex Fc/FcєRIα human IgE (access code in the database pdb 1F6A, incorporated by reference in its entirety) (Garman et al., 2000, Nature 406:259-266, work incorporated by reference in its entirety).
The different IgG subclasses have different affinity for FcγRs, usually IgG1 and IgG3 bind to receptors significantly better than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett. 82:57-65, work incorporated by reference in its entirety). All FcγRs bind the same Fc-plot IgG, but with different affinity: FcγRI binding with high affinity, Kd for IgG1, equal to 10-8M, whereas receptors with low affinity, FcγRII and FcγRIII, bound at the 10-6M and 10-5M respectively. The extracellular domains FcγRIIIa and FcγRIIIb identical to 96%, but FcγRIIIb has no intracellular signaling domain. In addition, as FcγRI, FcγRIIa/C and FcγRIIIa are positive regulators of activation triggered by the immune complex, they are characterized by the presence of the intracellular domain, which includes immunoreceptor tyrosine-dependent activation motif (ITAM), FcγRIIb is composed of immunoreceptor tyrosine-dependent inhibitory motif (ITIM) and is therefore an inhibitor. Thus, the first receptors belong to the activating of prescriptions is oram, a FcγRIIb refers to inhibitory receptors. The receptors also differ in the nature and levels of expression of various immune cells. Another difficulty is the existence of FcγR polymorphism in the human proteome. Especially important polymorphism with clinical relevance are forms V158/F158 FcγRIIIa. IgG1 person binds with a higher affinity with the V158 allotype compared with F158 allotype. It is shown that the difference in affinity and presumably the impact of this difference on ADCC and/or ADCP are a significant indicator of the effectiveness of antibodies to CD20 rituximab (Rituxan®, BiogenIdec). Patients with V158 allotype respond favorably to treatment with rituximab, however, patients with F158 allotype possessing lower affinato, weakly respond to such treatment (Cartron et al., 2002, Blood 99:754-758, work incorporated by reference in its entirety). Approximately 10-20% of people are homozygotes V158/V158, 45% are heterozygotes V158/F158 and 35-45% of people are homozygotes F158/F158 (Lehmbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758, work incorporated by reference in its entirety). Thus, 80-90% of people are poorly reactive, as they have at least one allele F158 FcγRIIIa.
Overlapping, but individual plot on Fc, shown in figure 1, serves as a contact surface for protein complement C1q. The W is way wherein binding of the Fc/FcγR mediates ADCC, linking Fc/C1q mediates dependent complement cytotoxicity (CDC). To form the complex C1, C1q forms a complex with the serine protease SG and C1s. C1q is able to bind with six antibodies, although activation Complementos cascade linking with two IgG. Like the Fc interaction with FcγRs, different IgG subclasses have different affinity for C1q, usually IgG1 and IgG3 better contact with FcγRs than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett. 82:57-65, work incorporated by reference in its entirety.
In the IgG site on Fc between domains γ2 and γ3 (figure 1) mediates interaction with the neonatal receptor FcRn, the binding of which results in the endocytotic recycling antibodies from endosome back to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, both works are incorporated by reference in its entirety). This process, coupled with the elimination of renal filtration due to the large size of a full-sized molecules, leads to the fact that the half-life of appropriate antibodies in the serum is the range from one to three weeks. The binding of Fc to FcRn also plays a key role in the transport of antibodies. The binding site for FcRn at Fc is also the area in which contact of the bacterial proteins a and G. the Strong binding of these protein and is usually used as a method of purification of antibodies when using protein A - or protein G - affinity chromatography during protein purification. Thus, the accurate reproduction of this site on Fc is very essential for clinical properties of antibodies and their treatment. Available patterns of rat complex Fc/FcRn (Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, both works are incorporated by reference in its entirety) and complexes with proteins a and G (Deisenhofer et al., 1981, Biochemistry 20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiro et al., 1995, Curr Opin Struct Biol 5:471-481, all works incorporated by reference in its entirety) provide an understanding of how the interaction of Fc with these proteins. The FcRn receptor is also responsible for the transfer of IgG in the gastrointestinal tract of newborns and into the lumen of the intestinal epithelium in adults (Ghetie and Ward, Annu. Rev. Immunol., 2000, 18:739-766; Yoshida et al., Immunity, 2004, 20(6):769-783, both works are incorporated by reference in its entirety).
Research conducted on domains Fcγ rats and humans have demonstrated the importance of certain residues of the Fc binding to FcRn. Sequence in Fc-sites in rats and humans is identical approximately 65% (residues 237-443 numbering according to Kabat et al.). Cm. figures 3, 4 and 5, showing the results of the alignment of the Fc-FcRn heavy chain and light chain of FcRn (beta-2-microglobulin) rat/human. Based on the existing structure of the complex Fc/FcRn rat model was constructed complex Fc/FcRn man (Martin et al., 2001, Mol. Cell 7:867-877, the work included the way the reference in its entirety). Sequence from rat and human contain some common residues that are critical for binding of FcRn, for example N310 and N (Medesan et al., 1997, J. Immunol. 158(5):221-7; Shields et al., 2001, J. Biol. Chem. 276(9):6591-6604, both works are incorporated by reference in its entirety). In many provisions, however, proteins in rat and human have different amino acid, provided that residues in the sequence of a person other places, and possibly other features compared to the sequence in rats. This variability limits the ability to transfer characteristics of one homologue on the other homolog.
In murine Fcγ random mutation and selection using phage display areas T, T and T lead to triple mutant T252L/T254S/T256F, whose affinity to FcRn is increased 3.5 times and increased to 1.5 times the half-life (Ghetie et al., 1997, Nat. Biotech. 15(7):637-640, work incorporated by reference in its entirety).
The crystal structure of the complex Fc/FcRn rats identified the remains of Fc that are important for the binding of FcRn (Burmeister et al., Nature, 372:379-383 (1994); Martin et al. Molecular Cell. 7:867-877 (2001), both works are incorporated by reference in its entirety). The initial structure of the complex Fc/FcRn was obtained in 1994 with a resolution of 6 S (table 2A, Burmeister et al., Nature, 372:379-383 (1994), incorporated by reference in its entirety). The structure of the higher is m resolution, completed in 2001, Martin et al., has shown a more detailed view of the arrangement of the side chains (Martin et al. Molecular Cell. 7:867-877 (2001), incorporated by reference in its entirety). This crystal structure of Fc rats associated with rat FcRn, was resolved using the Fc dimer with one monomer containing mutations 252G/I253G/254G/NE/NE/NE, which disrupted binding to FcRn, and one monomer containing a monomer Fc wild-type.
Mutational studies of human Fcγ were performed on some of the residues that are important for binding to FcRn and which have been shown to have an increased half-life in serum. Hinton et al. in human Fcγ mutated three residue separately from the 19 common amino acids. Hinton et al. found that some point mutations double mutant increased the binding affinity FcRn (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, work incorporated by reference in its entirety). Two mutations increased the half-life in monkeys. Shields et al. mutated residues almost exclusively on Ala and studied their binding to FcRn and FcγR's (Shields et al., 2001, J Biol Chem 276:6591-6604, work incorporated by reference in its entirety).
In order to select the Fc mutants that bind FcRn with increased affinity, Dall'acqua et al. used phage display (Dall'acqua et al., 2002, J. Immunol. 169:5171-5180, work incorporated by reference in its entirety)Otobrannye DNA sequences were double and triple mutants. The link presents the proteins encoded by many of the selected sequences, and found some proteins that bind to FcRn more strongly than wild-type Fc.
For the introduction of antibodies and proteins, fused with the Fc, as a therapeutic means necessary injections prescribed by a frequency-dependent characteristics of breeding time and half-life of proteins. Longer half-life in vivo makes possible more rare injection or low dosages, which is undoubtedly beneficial. Although mutations made earlier in the Fc domain, has resulted in some proteins with high binding affinity to FcRn and increased at times half-life in vivo, these mutations have not figured out the optimal mutation and increased half-life in vivo.
A key feature of the Fc-region is conservative N-glycosylation, are shown in figure 1, which is found on the balance N297. This carbohydrate or oligosaccharide, as it is sometimes called, plays an important structural and functional role in the antibody, which is one of the principal reasons that antibodies must be produced with the use of gene-expression system mammalian (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J Biol Chem 276:45539-45547; Radaev et al., 2001, J Biol Chem 276:16478-16483; Shields et al., 2001, J Biol Chem 276:6591-6604; Shields et al., 2002, J Biol Chem 277:26773-26740; Simmons et al., 2002, J Immunol Metods 263:133-147). However, the influence of carbohydrate slightly, if you have any specific contact with FcγR (Radaev et al., 2001, J Biol Chem 276:16469-16477; Radaev et al., 2001, J Biol Chem 276:16469-16477 and Krapp et al., 2003, J. Mol. Biol. 325:979-989, work incorporated by reference in its entirety).
Antibodies have been developed for therapeutic applications. The most typical publications related to such treatment include Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; Cragg et al., 1999, Curr. Opin. Immunol. 11:541-547; Glennie et al., 2000, Immunol. Today 21:403-410; McLaughlin et al., 1998, J. Clin. Oncol. 16:2855-2833 and Cobleigh et. al., 1999, J. Clin. Oncol. 17:2639-2648, all works incorporated by reference in its entirety. Now for anticancer therapy any small improvement in the mortality rate is defined as success. Specific IgG variants disclosed here, at least in part, increase the effectiveness of the antibodies in limiting the further growth of or destroy cancer cells-targets.
Antitumor efficacy of antibodies is implemented through enhancing their ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC. Examples include work Clynes et al., 1998, Proc Natl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446 and Cartron et al., 2002, Blood 99:754-758, all works incorporated by reference in its entirety.
IgG1 human antibody, most frequently used for therapeutic purposes, and a large part of the con is traktorki research was performed in this context. Different isotypes of IgG class, including IgG1, IgG2, IgG3 and IgG4, however, have unique physical, biological and clinical properties. In this field of technology there is a need to develop ways to improve binding to FcRn and/or increase half-life in vivo in comparison with the natural polypeptides IgG. This application meets these and other requirements.
Disclosure of inventions
The present invention discloses the creation of new versions of the Fc domains, including those found in antibodies, fused Fc and immunoadhesin that have increased binding to FcRn receptor and increased retention in serum in vivo. An additional aspect of the invention is increased binding to FcRn as compared with the wild type, especially at low pH values, approximately 6.0 in order to ensure the binding of the Fc/FcRn in the endosomes. An additional aspect of the present invention is the preferred binding of the designed variants at pH of about 6 compared with their binding at pH of about 7.4 in order to facilitate the release of Fc in the blood after cell recycling.
Another aspect of the present invention relates to the construction of Fc variants with reduced binding to FcRn and a reduced half-life in vivo. Such proteins, including mutations to reduce sredstv is to FcRn and/or time half-life in vivo, useful for many treatment options and diagnosis, including the delivery and monitoring of radioactive medicines, where in the ideal case, the half-life of a radioactive label is approximately equal to the time of half-life in vivo its protein conjugate.
Another aspect of the present invention is related to changes in the binding of the Fc domain with FCR's, for example, FcgRI, FcgRIIa, FcgRIIb, FcgRIIIa people. These receptors are responsible for the induction of the different effector functions of antibodies. Therefore, another aspect of the invention relates to changes in the effector functions of the Fc-domain, such as dependent antibody-dependent cell-mediated cytotoxicity (ADCC) - dependent complement cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP).
Another aspect of the present invention relates to Fc variants that have altered binding to FcRn, and altered binding to Fcg to affect the half-life in vivo, and effector functions of the protein, including Fc. For example, these options may have an increased half-life in vivo, and enhanced ADCC. Options, for example, may have an increased half-life in vivo and reduced CDC.
In another aspect, the invention provides recombinant nucleic acid encoding a variant Fc proteins, expression vectors and cell the owner.
In an additional aspect, the invention provides methods of producing a protein comprising an Fc, comprising culturing host cells of the invention under conditions suitable for expression of the protein.
In another aspect the invention provides pharmaceutical compositions comprising a variant Fc protein of the invention and a pharmaceutical carrier.
In another aspect, the invention provides methods of treating disorders, comprising the administration to a patient of a protein comprising a variant Fc invention.
In an additional aspect, the invention provides Fc-plot option in the source Fc-polypeptide that includes at least one modification in the Fc-specified area of the original polypeptide, where the specified variant protein demonstrates an altered binding to FcRn compared with the original polypeptide, and where the specified Fc variant includes at least one modification selected from the group consisting of:
where the numbering corresponds to the EU index, and the symbol "^" means the insertion after the specified position, and the symbol "#" indicates a deletion in the specified position.
In another aspect the invention provides Fc variants comprising at least one modification selected from the group consisting of:
In an additional aspect, the invention provides Fc variants comprising at least one modification selected from the group consisting of:
Brief description of drawings
Figure 1: Structure and function of antibodies. Shows the full-size model of a human antibody IgG1 modeled using patterns gumanitarnogo Fab fragment with access code SE in the pdb (James et al., 1999, J Mol Biol 289:293-301, work incorporated by reference in its entirety) and the structure of the Fc fragment of human IgG1 with access code 1DN2 in the pdb (DeLano et al., 2000, Science 287:1279-1283, work incorporated by reference in its entirety). A moveable hinge, which connects the sections of the Fab and Fc, not shown. IgG1 is a glycosilated composed of heterodimers composed of two light chains and two heavy chains. The Ig domains, which constitute the antibody marked and include VLand CLin the light chain and VHC-gamma-1 (Cγ1), gamma 2 (γ2) and gamma-3 (γ3) for the heavy chain. Fc-plot marked. Binding sites important proteins are marked and include the binding site of the antigen in the variable regions and binding sites FcγRs, FcRn, C1q, and proteins a and G in the plot Fc.
Figure 2: Sequence human IgG used in the present invention, with numbering according to EU index database d is the R Kabat et al.
Figure 3: Example sequences of IgG human and rodents used in the present invention, with numbering according to EU index database Kabat et al.
Figure 4: Example of sequences of the heavy chains of human FcRn and rodents used in the present invention.
Figure 5: Example sequences of beta-2-microglobulin human and rodents used in the present invention.
Figure 6: Model of the complex Fc/FcRn man, created on the basis of the structures of the rat (Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, both works are incorporated by reference in its entirety). Some histidine residues are shown as filling the space of the atoms in the chains FcRn (light grey) and Fc-polypeptide (dark gray).
Figure 7: Illustration of some concepts used in the design options, including insertions and deletions.
Figure 8: Variations of the present invention.
Figure 9: Variations of the present invention.
Figure 10: Variations of the present invention.
Figure 11: Scheme vector pcDNA3.1 Zeo+, which can be used in the construction of Fc-options.
Figure 12: Data on competition of binding of wild-type Fc and Fc variants of the present invention with FcRn. Ha each panel Fc variants of the present invention shown as the left curve (red or dark grey)and wild-type trastuzumab for the azan in the right curve (blue or gray).
Figure 13: results from FcRn-binding properties of Fc variants. The column on the right to the left show a modification of the binding of FcRn used immunoglobulin, other modifications, the relative affinity for FcRn measured using competitive analysis AlphaScreen™, compared with wild type (median value), the number of analyses and the identification number of the protein. The relative binding affinity for FcRn, which is higher than 1.0, shows improved binding compared to wild type.
Figure 14: Data binding FcRn-Fc variants of the present invention. Fc variants of the present invention are alemtuzumab and trastuzumab. Shows fold increased binding compared to wild type.
Figure 15: Summary of the experiments carried out by means of surface plasma resonance with Fc variants with improved binding to FcRn. The bar graph shows the fold increase of binding affinity to FcRn each option relative to the Fc domain of the wild type.
Figure 16: the Experiments carried out by means of surface plasma resonance with antibodies to wild type and variants of the present invention. Curves represent the Association and dissociation of Fc variant antibodies to FcRn at pH 6.0.
Figure 17: Analysis of binding of Fc variants of the present invention with FcRn. Pok is explained the results of the analysis of the direct binding method AlphaScreen™ at pH 6.0 (a and b) and at pH 7.0 (C).
Figure 18: Analysis of binding of Fc variants of the present invention with FcRn. Given unit of surface plasmon resonance, obtained by binding of Fc variants with attached on the surface of FcRn.
The implementation of the invention
The present invention discloses the creation of new versions of the Fc domains, including those found in antibodies, fused Fc and immunoadhesin, which have enhanced binding to FcRn receptor. As stated here, binding to FcRn leads to longer retention in serum in vivo.
To increase the retention Fc-proteins in vivo, increased binding affinity should be approximately at pH 6.0 without an accompanying increase in affinity at approximately pH 7.4. It turns out, I believe that Fc-sites have a longer half-life in vivo, as binding to FcRn at pH 6 in endosome leads to the capture of Fc (Ghetie and Ward, 1997, Immunol. Today, 18(12):592-598, work incorporated by reference in its entirety). Endosomal compartment then again returns Fc on the cell surface. After the compartment is opened in the extracellular space, higher values of pH about 7.4 induce the release of Fc back into the bloodstream. Dall' Acqua et al. showed that Fc mutants with increased binding to FcRn at pH 6 and 7.4 in fact have reduced the end of the ation in the serum and the same half-life, as Fc wild-type (Dall' Acqua et al., 2002, J. Immunol., 169:5171-5180, work incorporated by reference in its entirety). Consider that the high affinity Fc to FcRn at pH 7.4 does not allow Fc to be released back into the blood. Thus, mutations Fc, which will increase the half-life of Fc in vivo, will ideally increase the FcRn binding at low pH values, and at the same time still provide release Fc at higher pH values. The amino acid histidine changes its charge in the pH range from 6.0 to 7.4. Therefore, it is not surprising to find traces of His in important positions in the complex Fc/FcRn (figure 6).
In Figures 8-10 and 17-19 presents additional data on the variants disclosed in the originally filed description. In Figures 20, 27 and 30 shows the data obtained in vivo.
Figure 20 presents a summary of the results of measurement of the affinity of binding the Fc variants of the present invention with FcRn human, macaque and mouse obtained by the method of surface plasma resonance (SPR). Values greater than one indicate an increase in the binding of Fc variants of the present invention with FcRn. The measurements were carried out using the approximation curve obtained by the SPR method, in accordance with the model binding on Langmore when the stoichiometry of binding is 1:1.
Figure 27 presents the results obtained in ivo. Shows the values of the concentration of the antibodies wild-type (WT) and variants in the serum of mice with Nokin human FcRn. We used the following antibodies against VEGF: antibody wild-type (WT) - open squares, V308F closed squares, P257L closed triangles, and P257N - TIC.
Figure 30 presents data on the binding of the antibody wild-type and variants to FcRn on the cell surface T.
An additional aspect of the invention is increased binding to FcRn as compared with the wild type, especially at low pH values of about 6.0 to facilitate the binding of the Fc/FcRn in endosome. Also disclosed in the application Fc variants with altered binding to FcRn and changed the link to another class Fc-receptors, FcγR's because it has been shown that various binding to FcγRs, particularly the increased binding FcγRIIIb and reduced binding to FcγRIIb, leads to increased efficiency.
So the application could understand more fully, below are some definitions. Of course, that such definitions include grammatical equivalents.
The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity", which is used here, means-mediated cell reaction in which nonspecific cytotoxic cells that Express FcγRs, recognize antibodies related to CL is TKE target and subsequently cause lysis of the target cells.
The term "ADCP" or "antibody-dependent cell-mediated phagocytosis", which is used here, means-mediated cell reaction in which nonspecific cytotoxic cells that Express FcγRs, recognize antibodies related to the target cell and subsequently cause phagocytosis of the target cell.
The term "modified amino acid" means here the substitution, insertion and/or deletions in the polypeptide sequence.
The terms "replacement of amino acids" or "replacement" means here the substitution of amino acid at a certain position in the original polypeptide sequence with another amino acid. For example, replacing E272Y refers to the variant polypeptide, in this case, the Fc variant, in which the glutamic acid at position 272 replaced by tyrosine.
The term "insert amino acids" or "insert" here means the addition of amino acids at a particular position in the original polypeptide chain. For example, E or ^E means the insertion of glutamic acid after position 233 and to position 234. In addition, 233ADE or ^233ADE means the insertion AlaAspGlu after position 233 and before position 234.
The term "deletion of amino acids" or "deletion", which is used here, means the destruction of amino acids, in particular, in a situation the outcome is Oh polypeptide sequence. For example, He or A# denote a deletion of glutamic acid at position 233. In addition, EDA233 or EDA233# a deletion deletion sequence GluAspAla, which starts at position 233.
The term "variant protein"or "variant protein"or "variant", which is used here, means a protein which differs from the original protein at least one amino acid modification. Variant protein may refer to the protein itself, to a composition that includes a protein or amino acid sequence that it encodes. Preferably, when the variant protein has at least one amino acid modification compared to the original protein, for example from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the original protein. Sequence variant protein here preferably will have at least 80%homology with the sequence of the original protein, most preferably at least about 90%homology, more preferably at least about 95%homology. Variant protein can refer to variant protein itself, to the composition, which includes a variant protein, or amino acid sequence, to the which it encodes. Accordingly, the term "variant antibody or variant antibody", which is used here, refers to an antibody that differs from the original antibody, at least one amino acid modification, the term "IgG-variant" or variant "IgG", as used here, refers to an antibody that differs from the original IgG, at least one amino acid modification, and the term "immunoglobulin" or "variant immunoglobulin", which is used here, refers to immunoglobulin sequence that differs from the original immunoglobulin sequence at least one amino acid modification. Options may include unnatural amino acids. Examples include US 6586207; WO 98/48032; WO 03/073238; US 2004-021988 A1; WO 05/35727A2; WO 05/74524A2; J.W.Chin et al., (2002) Journal of the AmericanChemical Society 124:9026-9027; J.W.Chin & P.G.Schultz, (2002), ChemBioChem 11:1135-1137; J.W.Chin et al., (2002), PICAS United States of America 99:11020-11024; and L.Wang & P.G.Schultz, (2002), Chem. 1-10, all works incorporated by reference in its entirety).
The term "protein" refers to at least two covalently linked amino acids, the term includes proteins, polypeptides, oligopeptides and peptides. Piptadenia group may include naturally occurring amino acids and peptide bonds, or synthetic peptideprophet patterns, i.e., "analogs", such as peptide (see imon et al., PNAS USA 89(20):9367 (1992), the work incorporated by reference in its entirety). Amino acids can be either natural, or not found in nature, as is well known in this technical field. For example, homophenylalanine, citrulline and norleucine treated as amino acids, used for the purposes of the present invention, and can be used both D - and L- (R or S) - configuration amino acids. Variants of the present invention may include modifications that include the use of unsaturated amino acids, including the use of, for example, technologies developed Schultz et al., including, but not limited to the methods described Cropp&Schultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci. USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7, all works incorporated by reference in its entirety). In addition, the polypeptides can include synthetic derivatives of one or more side or main chains, glycosylation, Pegylation, cyclic rearrangement, cyclization, the linkers to other molecules, fusion proteins or protein domains and protein accession tags or labels.
The term "residue", which is used here, means the position in the protein and associated with him amino acid identity. For example, the asparagine-297 (also called Asn297, also called N297) is Statcommittee human IgG1.
The term "Fab" or "Fab-site", as used here, means polypeptides that include VH, CH1-, VL and CL domains of immunoglobulins. Fab may be relevant to this site in isolation, or this area in the context of full-length antibody or antibody fragment.
The term "modification of IgG subclass"used here means an amino acid modification that converts one amino acid isotype of IgG in the corresponding amino acid in the other, aligned with the sequence of the IgG isotype. For example, as IgG1 in EU-position 296 includes tyrosine, a IgG2 - phenylalanine, replacement F296Y in IgG2 means the modification of the IgG subclass.
The term "non-naturally occurring modification", as used here, means an amino acid modification which is not izotopicheskoj. For example, because none of the IgG does not include glutamic acid at position 332, replacement I332E in IgG1, IgG2, IgG3 or IgG4 considered as not occurring modification.
The terms "amino acid" or "amino acid identity" is used here to denote one of the 20 naturally occurring amino acids or any of their non-natural counterparts, which may be present in a specific position.
The term "effector function" refers to a biochemical phenomenon that occurs in the interaction is the major Fc-region of an antibody to an Fc receptor or ligand. Effector functions include, but are not limited to ADCC, ADCP, and CDC.
"Effector cell" means a cell of the immune system, which expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, b cells, large granular lymphocytes, Langerhans cells, natural killer cells (NK) and γδ-cells, which may be from any organism, including, but not limited to, humans, mice, rats, rabbits and monkeys.
The term "Fc-ligand IgG, which is used here, means a molecule, preferably a polypeptide, from any organism that binds to the Fc-part of IgG antibodies with the formation of the complex Fc/Fc ligand. Fc ligands include but are not limited to, FcγRI, FcγRII, FcγRIII, FcγRn, C1q, C3, mannan binding lectin, mannosyl receptor, staphylococcal protein a, streptococcal protein G, and viral FcγR. Fc ligands also include homologues of Fc-receptor (FcRH), which belong to the family of Fc receptors that are homologous to FcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, work incorporated by reference in its entirety). Fc ligands may include still not open molecules that bind to Fc. Special Fc-what ihandle IgG are FcRn and Fc-gamma receptors. The term "Fc-ligand", which is used here, means a molecule, preferably a polypeptide, from any organism that binds to the Fc-part of the antibody with the formation of the complex Fc/Fc ligand.
The terms "Fc-gamma receptor" or "FcγR", which is used here, means any member of a family of proteins that bind the Fc fragment of IgG antibodies and which are encoded by the genome of FcγR. In humans this family includes but is not limited to, FcγRI (CD64), including isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotype N and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotype V158 and F158)and FcγRIIIb (including allotype FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, work incorporated by reference in its entirety), and any still not open to human FcγRs or isoforms or allotype FcγR. FcγR can be from any organism, including humans, mice, rats, rabbits and monkeys, but not limited by them. Murine FcγRs include, but are not limited to the mentioned above, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD 16-2), as well as any still not open murine FcγRs or isoforms or allotype FcγR.
The term "FcRn" or "neonatal Fc receptor", as used here, means a protein which binds to the Fc-part of IgG antibodies and is encoded at least partially genome Fcn. FcRn may be from any organism, including humans, mice, rats, rabbits and monkeys, but not limited to them. As is well known in this technical field, a functional FcRn protein comprises two polypeptide, often called heavy chain and light chain. Light chain - beta-2-microglobulin, and the heavy chain is encoded by gene FcRn. If the text is not specifically limited, or FcRn FcRn protein are complex FcRn heavy chain with beta-2-microglobulin. Sequence FcRn, special interest, in particular the sequence of the person shown on the drawings.
The term "original polypeptide", which is used in the text, means polypeptide that significantly modified to generate a variant. The original polypeptide can be a polypeptide that is found in nature, or a variant or engineered variant of a naturally occurring polypeptide. The original polypeptide can refer to the polypeptide itself, to the composition, which includes the original polypeptide or amino acid sequence that it encodes. Accordingly, the term "source immunoglobulin, which is used here, means unmodified immunoglobulin polypeptide, which is then modified to generate a variant, and the term "original antibody, which are used here, oznachaet the unmodified antibody, which is then modified to generate a variant antibody. It should be noted that "the original antibody" includes well-known commercially available antibodies, recombinante-produced antibodies, as noted below.
The term "position", which is used here, means a certain place in the sequence of the protein. Provisions may be numbered sequentially, or in accordance with the established format, for example in accordance with the EU index in Kabat database. For example, the position 297 is the position in the human antibody IgG1.
The term "antigen-target", which is used here, means a molecule that specifically binds with the variable region of a specific antibody. Antigen-target can be a protein, carbohydrate, lipid, or other chemical substance.
The term "cell-target", which is used here, means a cell which expresses the antigen target.
The term "variable region", which is used here, means a portion of an immunoglobulin that comprises one or more Ig domains, mainly encoded by any of the Vκ, Vλ - and/or VHgenes that contain genetic loci Kappa, lambda, and heavy chain locus immunoglobulin, respectively.
The term "wild type or WT", which is used here, refers to the amino acid sequence or Amu is amidou sequence, which can be found in nature, including allelic variations. WT-protein has the amino acid sequence or nucleotide sequence, that are not intentionally modified.
The present invention is directed to antibodies that exhibit modulated binding to FcRn (modulation, including high or low binding). For example, in some cases, increased binding leads to cell recycling antibodies and thus to increased time-life, for example, in the case of therapeutic antibodies. Alternatively, the required reduced binding to FcRn, for example in the case of diagnostic antibodies or therapeutic antibodies that contain radioactive label. In addition, in the present invention find their application antibodies, demonstrating an increased binding to FcRn and altered binding to other Fc receptors, such as with FcγRs.
The present invention is directed to antibodies that include amino acid modifications that modulate the binding FcRw. Of particular interest are antibodies that minimally include Fc-plot, or a functional variant that show increased means of binding to FcRn at low pH and do not show significantly altered binding for more than you is okeh pH.
Traditional structural units antibodies typically comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair has one "light" (usually with a molecular mass of about 25 kDa) and one "heavy" chain (typically with a molecular weight of about 50-70 kDa). Human light chains are classified as light chains Kappa and lambda. Heavy chains are classified as chain mu, Delta, gamma, alpha, or Epsilon, and define these isotypes of antibodies, as IgM, IgD, IgG, IgA, and IgE, respectively. Immunoglobulin IgG has several subclasses, including, but unlimited IgG1, IgG2, IgG3 and IgG4. Immunoglobulin IgM has subclasses, including, but not limited to IgM1 and IgM2. Thus, the term "isotype", which is used here, means any of the subclasses of immunoglobulins with defined chemical and antigenic characteristics of their constant regions. Known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE.
N-terminal portion of each chain includes a variable region, consisting of approximately 100-110 or more amino acids, which source is responsible for antigen recognition. In variable regions in each of the V-domain of the heavy chain and light chain were collected at three loops for the formation of the antigen-binding site. Each loop refers to the hypervariable segment (is hereafter referred to as "CDR"), in which the difference of amino acid sequence of the most significant.
Carboxy-terminal portion of each chain defines a constant region, the source responsible for effector function. Kabat et al. gathered a large number of primary sequences of the variable regions of the heavy chains and light chains. On the basis of the degree of conservatism of the sequences they classified the individual primary sequence for CDR and frame part and created a list of them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5™ edition, NIH publication. No. 91-3242, Kabat et al., the work incorporated by reference in its entirety).
In IgG-subclass of immunoglobulin heavy chain there are several immunoglobulin domains. The term "immunoglobulin (Ig) domain here refers to the area of immunoglobulin having separate tertiary structure. In the present invention are of interest in the domains of the heavy chains, including constant heavy (CH) domains, and the domains of the hinged section. In the context of IgG antibodies each of the IgG-isotypes has three CH-plot. Accordingly SN-domains in the context of IgG are: "SN"relevant to the provisions of 118-220 in accordance with the EU index in Kabat database. "CH2" refers to the provisions 237-340 in accordance with the EU index in Kabat database and "CH3" refers to the provisions 341-447 in accordance with EU-index in the database given the Kabat.
Another type Ig-domain of the heavy chain belongs to the hinge region. The terms "joint" or "hinge area", or articulated plot antibody", as used here, refers to rolling polypeptide comprising amino acids between the first and second constant domains of the antibody. Structural SN domain of IgG ends in EU-position 220, and the CH2 domain of IgG begins at residue at EU position 237. Thus, IgG hinge antibodies are defined here as the domain that incorporates provisions from 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering corresponds to the EU index in Kabat database. In some embodiments, for example in the context of Fc-area included the lower hinge, usually related provisions from 226 or 230.
Of particular interest in this invention are Fc-plots. The terms "Fc" or "Fc-plot" means here the polypeptide comprising the constant region of an antibody excluding the first constant area immunoglobulin domain and in some classes the portion of the hinge. Thus, Fc refers to the last two constant sections immunoglobulin domains of IgA, IgD and IgG, and the last three constant sections immunoglobulin domains of IgE and IgM and rolling stock N-terminal hinge of these domains. Fc IgA and IgM may include a j chain As shown in figure 1 for IgG, Fc comprises immunoglobulin domains-gamma-2 and gamma-3 (Cg2 and Cg3) and the Astok lower hinge between C-gamma-1 (Cg1) and C-gamma-2 (Cg2). Despite the fact that the interface Fc-phase may vary, Fc-part of the heavy chain of human IgG usually includes balances S or R-end, where the numbering corresponds to the index EU Kabat database. Fc may be relevant to this site in isolated form or to the site in the context of an Fc polypeptide, as described below. The term "Fc polypeptide"as used here, means a polypeptide which includes the whole or part of the Fc section. Fc-polypeptides include antibodies, fused Fc-proteins isolated Fc and Fc-fragments.
In some embodiments the antibodies are full-sized. Under the "full-size antibody" here mean a structure that creates a natural biological form of an antibody comprising the variable and constant region comprising one or more modifications, as it was mentioned here.
Alternatively, antibodies can be represented as a set of structures, including but not limited to them alone, fragments of antibodies, monoclonal antibodies, bespecifically antibodies, Manantial, domain antibodies, synthetic antibodies (sometimes referred to here as mimetics of antibodies), chimeric antibodies, humanized antibodies, fused antibodies (sometimes referred to as "conjugates of antibodies and their fragments, respectively.
In one embodiment the antibody of t is possessing a fragment of antibodies. Of particular interest are antibodies that include Fc-sections, merged Fc and the constant region of the heavy chain (SN-hinge-CH2-CH3), again including fused constant heavy sections.
Specific antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CN domains, (ii) the Fd fragment consisting of the VH - SM domains, (iii) an Fv fragment consisting of the VL and VH domains single antibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546, work incorporated by reference in its entirety), which consists of a single variable segment, (v) isolated areas CDR, (vi) F(AB')2 fragments, a bivalent fragment comprising two linked Fab-fragment (vii) monoepoxide Fv molecules (scFv), where VH and VL domains are linked via a peptide linker which allows the two domains to associate to form an antigen-binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, work incorporated by reference in its entirety), (viii) bispecific single-chain Fv (WO 03/11161 included here by reference) and (ix) the dimers and trimers of small fragments of antibodies, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, work incorporated by reference in its entirety). Antibody fragments which may be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature. Biotech. 14:1239-1245, work incorporated by reference in its entirety).
Chimeric and humanized antibodies
In some embodiments the components of the skeleton can be a mixture of different species. Actually, if the antibody is any antibody, the antibody may be a chimeric antibody and/or humanized antibody. Basically, as a "chimeric antibody"or "humanized antibody" refers to antibodies which bind the region from more than one species. For example, "chimeric antibodies" usually include variable area (s) from mouse (or, in some cases, from rats) and constant region (area) from the person. "Humanized antibodies" are usually antibodies, non-human, which include wireframe plots variable domain of replacing them with sequences found in human antibodies. Usually humanitariannet the antibody is a whole antibody, with the exception of CD-R's, is coded polynucleotides human processes or identical to this antibody, except for its CD-R's. CD-R's, some or all of which are encoded by nucleic acids originating from organisms that are not human, pereshagivayut beta-folded frame plot of the variable segment of human antibodies to create antibodies, the specificity of which is determined by the transplanted CD-R's. The creation of such antibodies are described, for example, in WO 92/11018, Jones et al., 1986, Nature 321:522-525; Verhoeyen et al., 1988, Science, 239:1534-1536, work incorporated by reference in its entirety. "Reverse mutation" selected amino acid residues of the acceptor frame portion at the corresponding donor amino acid residues are often necessary to restore the affinity that is lost in the original design for the transplant (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337; US 6054297; US 6407213, all works incorporated by reference in its entirety). Optimally humanitariannet antibody will also include at least part of a constant region of immunoglobulin, usually belongs to human immunoglobulin, and, therefore, will include human Fc-plot. Humanized antibodies can also be obtained using mice with genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654, work incorporated by reference in its entirety. In this technology is well known to many methods of humanization and restore the original form of antibodies that do not belong to the person (see Tsurushita &Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of Cells, 533-545, Elsevier Science (USA), and references cited there, all works incorporated by reference in its entirety). Methods GU is anizatio include, but not limited to the methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, science, 239:1534-1536; Queen et al., 1989, Proc. Natl. Acad. Sci., USA 86:10029-33; He et al., 1998, J. Immunol. 160:1029-1035; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'connor et al., 1998, Protein Eng 11:321-8, all works incorporated by reference in its entirety. Humanization or other methods to reduce the immunogenicity of the variable regions of antibodies, non-human, may include surface restoration, as described, for example, Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, incorporated by reference in its entirety. In one of the incarnations of the original antibody is afinno ripe, as it is known in this technical field. For humanization and affinity maturation can be applied structural methods, such as described in US 11/004,590. For humanization and affinity maturation of the variable regions of the antibodies can be applied selection methods, which include, but are not limited to the methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, incorporated by reference in its entirety. Other methods of humanization may include transplanting only parts CDR, these methods is clucalc, but are not limited to methods described in US 091810,502; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084 included here by reference in its entirety.
In one of the embodiments of the antibodies of the invention are multispecific antibody and exclusively bispecific antibody, also sometimes called "diately". These are antibodies that bind two (or more) different antigens. Diately can be manufactured in a variety of ways known in this technical field (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, work incorporated by reference in its entirety), for example, by chemical means or from hybrid hybridomas.
In one embodiments the antibody is Minatitlan. Manantial are reduced to an absolute minimum antitelephone proteins, including scFv attached to the CH1 domain. Hu et al., 1996, Cancer Res. 56:30553061, work incorporated by reference in its entirety. In some cases, the scFv can be attached to the Fc-section and may include some or all of the hinge section.
In one embodiments the antibody is a fully human antibody, at least, with one modification, as already mentioned. "Fully human antibody" or "completely human antibody" regarding the Xia to the human antibody, having the sequence of antibody derived from a human chromosome with modifications specified in the proposal.
In one of the embodiments of the antibodies of the invention are fused proteins antibodies (sometimes referred to here as the "conjugate antibodies"). One type of fused antibody includes fused Fc, where Fc is the area associated with the merger partner. The term "fused Fc". which is used in the text, means a protein in which one or more polypeptides effectively attached to the Fc-part. Fused Fc here is synonymous with immunoadhesin", "merged Ig", "Ig Chimera", and "receptor globulin" (sometimes written by a dash)used previously (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997 Curr Opin Immunol 9:195-200). Fused Fc combines the Fc-part of an immunoglobulin with a partner on the merger, which, basically, can be any protein or small molecule. Virtually any protein or small molecule may be attached to Fc to create a merged Fc. Protein partners in the merger are not limited to these examples, but may include variable region of the antibody binding site of the receptor-target adhesion molecule, ligand, enzyme, cytokine, chemokine, or any other protein or protein domain. Small molecule as partners merger may include any the e therapeutic agent, which sends the fused Fc to therapeutic targets. Such targets can be any molecule, preferably the extracellular receptor, which is involved in the disease. Thus, the IgG variants may be attached to one or more partners in the merger. In one alternative embodiment of the IgG-option anywhereman or effectively connected to another therapeutic compound. therapeutic compound may be a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, cytokine or other therapeutically active agent. IgG can be attached to one of the many non-protein polymers, such as polyethylene glycol, propylene glycol, polyalkylene or copolymers of polyethylene glycol and propylene glycol.
In addition to Fc merged, merged antibodies include the merger of the constant region of the heavy chain with one or more partners in the merger (also including variable region of any of the antibodies), while others merged antibodies are largely full of full-length antibodies or antibody with partners to merge. In one of the embodiments of the role of the partner in the merger consists in mediating binding to the target and, thus, it is the functional analogue of the variable regions of the antibodies (and actually can b the th). Virtually any protein or small molecule may be attached to Fc to create a merged Fc (or merged antibodies). Protein partners in the merger are not limited to these examples, but may include binding site of the receptor-target adhesion molecule, ligand, enzyme, cytokine, chemokine, or any other protein or protein domain. Small molecule as partners merger may include any therapeutic agent that directs fused Fc to therapeutic targets. Such targets can be any molecule, preferably the extracellular receptor, which is involved in the disease.
Partner in the merger may be protein and non-protein; the latter is usually created with the use of functional groups located on the antibody and the merger partner. For example, in this area linkers known in the art; for example, the well-known Homo - or hetero-bifunctional linkers (see catalog 1994 Pierce Chemical Company technical section related to cross-linking agents, page 155-200, included here by reference).
Suitable conjugates include, but are not limited to, a label, as described below, medicines and cytotoxic means, including but not limited to cytotoxic drugs (e.g., chemotherapeutics or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria a chain, a chain of exotoxin a chain of ricin, a chain abrina, Curtin, krotin, vanomycin, inomycin etc. Cytotoxic tools also include radioactive chemical substances made by the merger of radioisotopes to antibodies or by binding of radionuclides with chelating agents that have been covalently attached to the antibody. Additional embodiments use calicheamicin, auristatin, geldanamycin, maytansine and duocarmycin and analogues (last described in US 2003/0050331 A1, incorporated by reference in its entirety).
Covalent modifications of the antibody
Covalent modifications of the antibody included in the scope of this invention and, mostly, but not always, made excision. For example, several types of covalent modifications of the antibody is introduced into the molecule by reaction of specific amino acid residues of the antibody with the organic derivatizing agent, which is capable of reacting with selected side chains or the N - or C-terminal residues.
Residues of cysteine most often react with α-halogenation (and corresponding amines), such as Chloroacetic acid or chloroacetamide, education carboxymethyl or carboxylatomethyl derivatives. Residues of cysteine can also pens the STI in derivatives through reaction with BROMOTRIFLUOROMETHANE, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetylation, N-alkylamide, 3-nitro-2-pyridyldithio, methyl-2-pyridyldithio, para-chloromercuribenzoate, 2-chloromercuri-4-NITROPHENOL, or chloro-7-nitrobenzo-2-oxa-1,3-diazoles etc.
Residues of histidine in turn derived through reaction with diethylpyrocarbonate at pH 5,5-7,0, as this compound is relatively specific in relation to the side chain of histidine. Also useful para-brompheniramine; the reaction is preferably carried out in 0.1 M cacodylate sodium at pH 6.0.
Residues lysine and terminal amino group is introduced into reaction with succinic anhydrides and other carboxylic acids. Deriving from the use of these agents has the effect of changing the charge of lysine residues. Other suitable reagents for obtaining derivatives residues containing alpha-amino group include imidiately, such as methyl ether of picolinamides; pyridoxal phosphate; pyridoxal; harborhead; trinitrobenzenesulfonic acid; O-methylisoleucine; 2,4-pentandiol and reaction with glyoxylate, being catalyzed by transaminases.
Residues of arginine modified through reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butandiol, 1,2-cyclohexandione and ninhydrin. For the conversion of arginine residues in their derivatives necessary is IMO, to the reaction was carried out in alkaline conditions because of the high pKa value guanidino functional groups. Furthermore, these reagents may react with the lysine groups, as well as with the Epsilon-amino group of arginine.
Can be made specific modification of tyrosine residues, and of particular interest is the introduction of tyrosine residues spectral labels reactions with aromatic compounds, page or tetranitromethane. Often for the formation of O-acetyltyrosine derivatives and 3-nitro-derivatives used N-acetylimidazole, tetranitromethane respectively. Residues of tyrosine idiot using125I or131I get labeled proteins for use in radioimmunoassay analyses, the appropriate method is using chloramine T as described above.
Side carboxyl groups (aspartamine or glutamina) selectively modified through reaction with carbodiimides (R'-N=C=N-R', where R and R' are not necessarily different alkyl groups such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4 - dimethylpentyl)carbodiimide. In addition, residues of aspartic and glutamic acids turn in residues asparagine and glutamine through reaction with ammonium ions.
The derivatization with bifunctional agents is good for stitching antibodies with a water-insoluble support matrix or surface for use in many methods, in addition to the methods described below. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaric aldehyde, N-hydroxysuccinimide esters, for example esters with 4-azidoaniline acid, homophonically imidiately, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylester) and bifunctional maleimide, such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(para-azidophenyl)dithio]propionamide allow you to get photoactivated intermediates, which are capable of forming cross-links in the presence of light. Alternatively, reactive water-insoluble matrices such as activated cyanogenmod carbohydrates and the reactive substrates described in US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440 included here by reference in its entirety, is used for immobilization of proteins.
The remains of glutamine and asparagine often diamidino to obtain the corresponding residues of glutamic and aspartic acids. Alternatively, these residues can be deliberate in mild acidic conditions. Both ways of formation of these residues are included in the scope of the present invention.
Other modifications include hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups of residues with the Rina and threonine, methylation of the α-amino group lysine, arginine and his-tag side chains (I.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman&Co., San Francisco, pp.79-86 , work incorporated by reference in its entirety), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification is glycosylation. In another embodiment of the IgG variants disclosed herein may be modified to incorporate one or more constructed glycoform. The term "constructed glycoform", which is used here, means a mixture of carbohydrates, which is covalently attached to IgG, where these carbohydrates are chemically different from the carbohydrate source of IgG. Designed glycoform can be useful for many purposes, including but not limited to increase or decrease effector function. Designed glycoform can be created using a variety of methods known in this technical field (Umaňa et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; US 6,602,684; US 10/277,370; US 10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1, all sources incorporated by reference in its entirety (PotelligentTMtechnology [Biowa, Inc., Princeton, NJ]; GlycoMAb™ glycosylation engineering technology [GLYCART biotechnology AG, Zűrich, Switzerland]), Many of these techniques are based on the regulation fokusirovannyi and/or branching of the oligosaccharide, which are covalently attached to the Fc-section, for example, using the expression of IgG in various organisms or cell lines, engineered or otherwise (for example, in cells Lec-13 Cho or cells of rat hybridoma YB2/0), by regulating enzymes involved in the path of glycosylation (e.g., FUT8 [α1,6-fucosyltransferase] and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrate(s) after the IgG expression. Designed glycoform usually relates to the different carbohydrate or oligosaccharide; therefore, IgG-option, for example, the antibody or Fc merged, can include constructed glycoform. Alternative, designed glycoform may be relevant to the variant IgG, which includes the different carbohydrate or oligosaccharide. As is well known in this technical field, methods of glycosylation may depend on the protein sequence (for example, from the presence or absence of particular amino acid residues suitable for glycosylation), or from the host cell or organism, which produce the protein. Individual expression systems are discussed below.
Glycosylation of polypeptides usually occurs either through N-bond or through O-link. N-glycosylation is related to the attachment of a molecule of carbohydrate to the side chain of residue is asparagine. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are the recognition sequences for enzymatic attachment molecule of carbohydrate to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-glycosylation is related to the accession of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, often serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.
The inclusion of sites of glycosylation of the antibody is efficiently performed by changing the amino acid sequence such that it contains one or more Tripeptide sequences described above (related to the N atom of glycosylation sites). The change can be done with the addition or substitution of one or more residues of serine or threonine for the beginning of the sequence (associated with the atom Of glycosylation sites). For convenience, the amino acid sequence of the antibody is preferably altered through changes at the DNA level, particularly by mutation engine DNA that encodes a polypeptide target, in a pre-selected basis, so that brazavilis codons, which will be broadcast in the desired amino acids.
Other ways of increasing the number of carbohydrate molecules in the antibody is a chemical or enzymatic joining of glycosides to the protein. These procedures are useful in those methods that do not require the production of protein in the cell host, which has the ability to glycosylation by N - and O-linkages. Depending on the applied method of attaching sugar(sugar) can join (a) arginine and histidine, (b) free carboxyl groups, (C) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine, or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 and work in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306, included here by reference in its entirety.
Removal of the carbohydrate part present in the original antibody can be carried out chemically or enzymatically. For chemical deglycosylation must impact on protein compounds triftormetilfullerenov acid or equivalent connection. This treatment results in the cleavage of most or all sugars except the linked sugars (N-acetylglucosamine or N-atsetilgalaktozamin), lying is as outgoing protein remains intact. Chemical deglycosylation described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131, both works are incorporated by reference in its entirety. Enzymatic cleavage of carbohydrate part of the polypeptide may be achieved using a variety of endo - and ectoparasites, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350, incorporated by reference in its entirety. Glycosylation at potential sites of glycosylation can be prevented when using connection tunicamycin, as described in the work Duskin et al., 1982, J. Biol. Chem. 257:3105, included here by reference in its entirety. Tunicamycin blocks the formation of protein-N-glycosidic linkages.
Another type of covalent modification of the antibody comprises attaching antibodies to various non-protein polymers, including, but not limited to various polyols, such as polyethylene glycol, polypropyleneglycol or polyoxyalkylene, as described, for example, in 2005-2006 PEG Catalog from Nektar Therapeutics (available on the web site Nektar), US Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, they are all incorporated by reference in its entirety. In addition, as is well known in this technical field, in different positions inside the antibodies can be done amino acid substitutions, to facilitate the joining of polymers such as PEG. See, for example, the publication US Publicatin No. 2005/A, incorporated by reference in its entirety.
In some embodiments covalent modification of the antibody of the invention includes the introduction of one or more labels. In some cases they are considered as a fusion antibody. The term "aiming group" means any detektiruya tag. In some embodiments of the labeled group attached to the antibody through spacer elements legs of various lengths to reduce potential steric hindrance. Various methods of obtaining labeled proteins known in this technical field and can be used in implementing the present invention.
Basically, the labels are of three classes, depending on the analysis, in which these labels find: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); (C) a redox-active agent; (d) optical colors; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); (e) biotinylated group and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., a pair of sequences "latinboy lightning"connecting places for secondary antibodies, metal binding domains, epitope tags and so on). In some embodiments of the labeled group process is Diana to the antibody via spacer elements legs of various lengths to reduce potential steric inconsistencies. Various methods of obtaining labeled proteins known in this technical field and can be used in implementing the present invention.
Specific labels include optical dyes, including but not limited to them alone, chromophores, fluorescent and fluorophores, the latter in many cases are specific. The fluorophores can be either fluorescent small molecules", or fluorescent-labeled protein nature.
The term "fluorescent label" refers to any molecule that can be detected using its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarins, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, CY 5, CY 5.5, LC Red 705, Oregon green dye Alexa-Fluor (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, rhodamine, and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores described in the book Molecular Probes Handbook by Richard P. Haugland, included here by reference in its entirety.
Suitable protein fluorescent labels include, but are not limited to, green flu is rescently protein, including GFP type Renilla, Ptilosarcus, or Aequorea (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., access code in Genbank U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Helm et al., 1996, Cur. Biol. 6:178-182), yellow, protein-enhanced fluorescence (EYFP, Clontech Laboratories, Inc.), the luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 98/26277, WO 99/49019, US Patent Nos. 5292658, 5418155, 5683888, 5741668, 5777079, 5804387, 5874304, 5876995, 5925558). All of the above sources in this paragraph directly incorporated by reference.
In one of the embodiments of the invention provides a variant IgG proteins. At least IgG-options include a fragment of antibodies, including CH2-CH3-part of the heavy chain. In addition, appropriate IgG-options include Fc domains (e.g., including the lower hinge section), and IgG variants, including the constant region of the heavy chain (SN-hinge-CH2-CH3), are also useful in the present invention, they can be attached to the partners in the merger.
IgG-variant comprises one or more amino acid modifications related to the original IgG-polypeptide, in some cases related to the wild type IgG. IgG variant may have one or more optimized properties. IgG-variant differs in amino acids of the second sequence from its original IgG in fact, at least one amino acid modification. Thus, the IgG variants have at least one amino acid modification compared to the original protein. Alternatively, IgG variants may have more than one, for example, from about one to fifty amino acid modifications, preferably from about one to ten amino acid modifications, and most preferably from about one to about five amino acid modifications compared to the original protein.
Thus, the sequence of IgG variants and the original Fc-polypeptide substantially homologous. For example, the variant sequence IgG-options here will have approximately 80%homology with the sequence of the original IgG solution, preferably at least about 90%homology and more preferably at least about 95%homology. Modifications can be made genetically using molecular biology, or can be made enzymatically or chemically.
Specialists in this area of technology will recognize that the above list of targets refers not only to pacificism proteins and biomolecules but to biochemical pathway or pathways that include them. For example, the reference CTLA-4 as a target antigen implies that ligands and receptors that comprise T-cell co-stimulating way, including CTLA-4, B7-1, B7-2, CD28, and any other not open currently ligands and receptors that bind these proteins are also targets. Thus, the term "target", which is used here, refers not only to specific biomolecules, but also to a number of proteins that interact with the specified target and participants in the biochemical pathway that belongs to the specified target. Specialists in this field of technology in the future will understand that any of the above antigens are targets, ligands or receptors to which they bind, or other participants in the relevant biochemical pathway can be effectively attached to the Fc variants of the present invention for the formation of fused Fc. So, for example, fused Fc, which targets EGFR, can be constructed effectively attach Fc-option to EGF, TGF-b, or any other ligand, investigated or not open at the present time, which binds to EGFR. Accordingly, an Fc variant of the present invention can be effectively attached to the EGFR for the formation of fused Fc that binds EGFR, TGF-b, or any other ligand, the research is or is not currently open, which binds to EGFR. Thus, virtually any polypeptide or ligand, or receptor, or some other protein or protein domain that includes, but is not limited to the above targets and proteins that make up the relevant biochemical pathway can be effectively attached to the Fc variants of the present invention to develop fused Fc.
The selection of the appropriate antigen depends on the desired practical application. For cancer treatment it is desirable to have the target, the expression of which is restricted in cancer cells. Some of the targets for which it is proved that they are treated particularly well by antibodies, are targets with alarm functions. Other therapeutic antibodies exert their effects by blocking the signaling receptor by inhibiting the binding of receptor and ligand recognized by this receptor. Another mechanism of action of therapeutic antibodies is that they cause suppressive regulation of the receptor. Other antibodies do not function by passing the signal through its antigen target. In some cases, using antibodies directed against agents that cause infectious diseases.
In one of the embodiments of the Fc variants of the present invention is introduced into the antibody together with a cytokine. Alternatively, Fc-var is the ants poured or kongugiruut with the cytokine. The term "cytokine"as used here means a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and traditional peptide hormones. For example, as described in the work Penichet et al., 2001, J Immunol Methods 248:91-101, is expressly incorporated here by reference, cytokines can be fused with the antibody to provide a set of desired properties. Examples of such cytokines are lymphokines, Monokini and normal polypeptide hormones. The cytokines are growth hormone such as human growth hormone, N-nationally the human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follikulostimuliruyuschy hormone (FSH), thyroidstimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogenic; tumor necrosis factor-alpha and-beta; antimullerian hormone; mouse gonadotropin-associated peptide; inhibin; activin; endothelial growth factor vascular; integrin; thrombopoietin (SRW), nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGFs)such as TGF-alpha and TGF-beta; insulin-like growth factor I and II; retrop etin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and-gamma; colony stimulating factors (CSFs)such as macrophage-colony stimulating factor (N-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (ILs)such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; Sa and other polypeptide factors including LIF and Kit ligand (KL). Used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of cytokines with natural sequences.
Cytokines and soluble targets, like the members of the TNF superfamily, are preferred targets in the application options of the present invention. For example, anti-VEGF antibody and antibodies to CTLA-4 and antibodies to TNF or fragments thereof, are particularly good antibodies for use of Fc variants with increased binding to FcRn. Therapies aimed at these targets, often used in the treatment of autoimmune diseases and require multiple injections over a long period of time. It is therefore particularly preferable increased half-life in serum and less frequent exposure, provide variants of the present invention.
For some antibodies and Fc merged, which are approved for use, are currently in clinical trials or development, can be useful Fc variants of the present invention. These antibodies and Fc merged called "clinical products and candidates." Thus, in the preferred embodiment of the Fc polypeptides of the present invention can find application in a range of clinical products and candidates. For example, some antibodies targeting CD20 is, can be useful Fc polypeptides of the present invention. For example, the Fc polypeptides of the present invention may find use in an antibody that is substantially similar to rituximab (Rituxan®, IDEC/Genentech/Roche) (see for example, US 5,736,137), chimeric antibody to CD20, approved for the treatment of non-Hodgkin lymphoma; HuMax-CD20, an antibody to CD20, currently being developed by the company Genmab, antibody to CD20 that is described in the patent US 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedic, Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled "Immunoglobulin Variants and Uses Thereof"). For some antibodies, the target of which are representatives of the family of receptors for epidermal growth factor, including EGFR (rb-1), Her2/neu (ErbB-2), Neg (ErbB-3), Her4 (Erb-4), can be useful Fc polypeptides of the present invention. For example, the Fc polypeptides of the present invention can find use in antibodies is, largely similar to trastuzumab (Herceptin®, Genentech) (see for example, US 5,677,171), humanized antibody to Her2/neu, approved for the treatment of breast cancer; pertuzumab (rhuMab-2C4, Omnitarg™), currently developed by the company "Genentech"; antibody to Neg described in US 4,753,894; cetuximab (Erbitux®, Imclone) (US 4,943,533; PCT WO 96/40210), chimeric antibody to EGFR undergoing clinical trials for use in many types of cancers; ABX-EFG (US 6,235,883), currently developed by the company "Abgenix/Immunex/Amgen"; HuMax-EGFr (US 10/172,317), currently being developed by the company Genmab"; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (US 5558864; Murthy et al., 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al., 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al., 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (US 5,891,996; US 6,506,883; Mateo et al., 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al., 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2) and SC100 (Scancell) (PCT WO 01/88138). In another preferred embodiment of the Fc polypeptides of the present invention can find application in alemtuzumab (Campath®, Millenium), humanitariannet monoclonal antibody currently approved for treatment of chronic In-KL is accurate lymphocytic leukemia. Fc polypeptides of the present invention can find application in a multitude of antibodies or Fc merged, which is largely similar to other clinical products and candidates, including, but not limited by them, muromonab-CD3 (Orthoclone OKT3®), an antibody to CD3, developed by Ortho Biotech/Johnson & Johnson", ibritumomab tiuxetan (Zevalin®), an antibody to CD20, developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), antibody to CD33 (protein P67), developed by Celltech/Wyeth", alefacept (Amevive®), antibody to LFA-3 fused with Fc, developed by Biogen", abciximab (ReoPro®), developed by Centocor/Lilly", basiliximab (Simultec®), developed by Novartis, palivizumab (Sinagis®), developed by MedImmune, infliximab (Remicade®), an antibody to TNF-alpha, developed by Centocor", adalimumab (Humira®), an antibody to TNF-alpha, developed by Abbott, Humicade™"antibody to TNF-alpha, developed by Celltech, etanercept (Enbrel®), an antibody to TNF-alpha, merged with Fc, developed by Immunex/Amgen, AVH-CBL, antibody to CD 147, developed by the company "Abgenix", ABX-IL8, an antibody to IL8, developed by the company "Abgenix", ABX-MA1, an antibody to MUC18, developed by the company "Abgenix", pemtumomab (R1549,90Y-muHMFG1), antibody to MUC1 in development by the company Antisoma", Therex (R1550), an antibody to MUC1, developed by the company Antisoma", AngioMab (AS1405), once emotively company Antisoma", HuBC-1, being developed by the company Antisoma", Thioplatin (AS1407), developed by the company Antisoma", Antegren® (natalizumab), an antibody to alpha-4-beta-1(VLA-4) and alpha-4-beta-7, developed by the company "Biogen", VLA-1 mAb, an antibody to the integrin VLA-1, developed by the company "Biogen", LTBR mAb, an antibody to the receptor lymphotoxin-beta (LTBR), developed by the company "Biogen", CAT-152, an antibody to TGFβ2 that is being developed by Cambridge Antibody Technology, J695, an antibody to IL-12, being developed by companies "Cambridge Antibody Technology and Abbott, CAT-192, an antibody to TGFβ1, developed by companies "Cambridge Antibody Technology and Genzyme, CAT-213, an antibody to eotaxin-1 that is being developed by Cambridge Antibody Technology, LymphoStat-B™ anti-Blys antibody being developed by companies "Cambridge Antibody Technology and Human Genome Sciences, Inc.", TRAIL-R1mAb, antibody to TRAIL-R1, developed by companies "Cambridge Antibody Technology and Human Genome Sciences, Inc.", Avastin™ (bevacizumab, rhuMAb-VEGF), an antibody to VEGF, developed by the company "Genentech", antibody to the family of HER receptors, developed by the company "Genentech", Anti-Tissue Factor (ATF), antibody to tissue factor, developed by the company "Genentech", Xolair™ (omalizumab), an antibody to the immunoglobulin IgE, developed by the company "Genentech", Raptiva™ (efalizumab), antibody to CD-11, developed by the company "Genentech and Xoma, antibody MLN-02 (formerly LDP-02), developed by companies Genentech and Millenium Pharmaceuticals, HuMax CD4, antibodies to CD4, the bit is nativemode company Genmab", HuMax-IL15, an antibody to IL15 developed by companies "Genmab and Amgen, HuMax-Inflam, being developed by companies Genmab and Medarex", HuMax-Cancer, an antibody to heparinase I, developed by the company Genmab and Medarex and Oxford GcoSciences, drug HuMax-Lymphoma, being developed by companies Genmab and Amgen, HuMax-TAC, being developed by the company Genmab", IDEC-131, and antibody to CD40L, developed by the company "IDEC Pharmaceuticals, IDEC-151 (clenoliximab)the antibody to CD4, developed by the company "IDEC Pharmaceuticals, IDEC-114, an antibody to CD80, developed by the company "IDEC Pharmaceuticals, IDEC-152, an antibody to CD23 being developed by the company "IDEC Pharmaceuticals, antibodies to factor migration inhibitory macrophages (MIF), developed by the company "IDEC Pharmaceuticals, BEC2, antiidiotypic antibody, developed by the company "Imclone", IMC-1C11, an antibody to KDR, developed by the company "Imclone", DC101, an antibody to flk-1, developed by the company "Imclone", antibodies to VE-cadherin, developed by the company "Imclone", CEA-Cide™ (labetuzumab), antibody to carcinoembryonic antigen (CEA), developed by the company "Immunomedics", LymphoCide™ (epratuzumab), antibody to CD22, developed by the company "Immunomedics", AFP-Cide, being developed by the company Immunomedics", MyelomaCide developed by the company Immunomedics", LkoCide developed by the company Immunomedics", ProstaCide, being developed by the company Immunomedics", MDX-010, an antibody to CTLA4, developed by the company "Medarex", MDX-060 anti the ate to CD30, developed by the company "Medarex", MDX-070 being developed by the company "Medarex", MDX-018 being developed by the company "Medarex", Osidem™ (IDM-1), and antibody to Her2, developed by companies "Medarex" and "Measurement Designed Moleculas", HuMax™-CD4 antibody to CD-4, developed by the company Medarex and Genmab", HuMax-IL15, an antibody to IL15, developed by the company Medarex and Genmab", CNTO 148, an anti-TNFα antibody being developed by companies "Medarex and Centocor/J&J, CNTO 1275, an antibody to the cytokine, developed by the company "Centocor/J&J, MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54), developed by the company "MorphoSys", MOR201, the antibody to the receptor 3 fibroblast growth factor (FGFR-3), developed by the company "MorphoSys", Nuvion® (visilizumab), antibody to CD-3, developed by the company "Protein Design Labs, HuZaF™antibody to interferon-gamma, developed by the company "Protein Design Labs, antibody to integrin α5β1, developed by the company "Protein Design Labs, antibody to IL-12, developed by the company "Protein Design Labs, ING-1, an antibody to Er-ITSELF, developed by the company "Xoma" and MLN01, antibody to beta2-integrin, being developed by the company "Xoma", all of the above sources in this paragraph is expressly incorporated by reference.
Fc polypeptides of the present invention can be included in the above clinical candidates and products, or antibodies and Fc merged, which are largely similar to it is. Fc polypeptides of the present invention can be included in a variation on the above clinical candidates and products, which are humanized, affinity-matured, constructed or modified in any way varieties.
In one of the embodiments of the Fc polypeptides of the present invention is used for the treatment of autoimmune, inflammatory diseases or indications in transplantation. The antigens of the target and clinical products and candidates that are important in such diseases include, but are not limited to, antibodies against α4β7-integrin, such as LDP-02, antibodies to beta 2-integrin, such as LDP-01, antibodies to the complement (C5), such as 5G1.1, antibodies to CD2, such as BTI-322, MEDI-507, antibodies to CD3, such as OCT, SMART anti-CD3, antibodies to CD4, such as IDEC-151, MDX-CD4, OCTA, antibodies to CD11, antibodies to CD14, such as IC14, antibodies to CD18 antibodies to CD23, such as IDEC 152, antibodies to CD25, such as zenapax, antibodies to CD40L, such as s, Antova, IDEC-131, CD64 antibodies such as MDX-33, anti-CD80, such as IDEC-114, CD147 antibodies such as ABX-CBL antibodies to E-selectin, such as CDP850, antibodies to gpIIb/IIIA, such as ReoPro/Abcixima antibodies to ICAM-3, such as ICM3, antibodies to ICE, such as VX-740, antibodies to FcR1, such as MDX-33, anti-IgE, such as rhuMab-E25, antibodies to IL-4, such as SB-240683, antibodies to IL-5, such as SB-240563, SCH55700, antibodies to IL-8, is such as ABX-IL8, antibodies to interferon-gamma, antibodies to TNF (TNF, TNFa, TNFa, TNF-alpha), such as CDP571, CDP870, D2E7, infliximab, MAK-195F and antibodies to VLA-4, such as antegren.
Fc variants of the present invention with increased binding to FcRn can be used in TNF-inhibitory molecules to provide improved properties. Useful TNF-inhibitory molecules include any molecule that inhibits the action of TNF-alpha in mammals. Suitable examples include fused Fc-protein Enbrel® (etanercept) and antibody Humira® (adalimumab) and Remicade® (infliximab). Monoclonal antibodies (such as Remicade and Humira), constructed using the Fc variants of the present invention, for improved binding to FcRn can be converted to antibodies with the best effectiveto by increasing the time half-life.
In some embodiments of the used antibodies against infectious diseases. Antibodies against eukaryotic cells include antibodies directed to yeast cells, including but not limited to Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces fragilis and K.lactis, Pichia guillerimondii and .pastoris, Schizosaccharomyces pombe, Plasmodium falciparium, and Yarrowia lipolytica.
Also useful antibodies to other cells of fungi, including antigens of the target associated with Candida species, including Candida glabrata, Candida albicans, .krusei, .lusitaniae and .maltosa, as well as representatives of the genus Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces, and Pnicillium and other
Antibodies directed against antigens of the target associated with protozoa include, but are not limited to antibodies, associated with Trypanosoma, representatives of the genus Leishmania, including Leishmania donovanii, Plasmodium spp., Pneumocystis carinii, Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, and Cyclospora cayetanensis.
Also useful antibodies against antigens of prokaryotes, including antibodies against the appropriate bacteria, such as pathogenic and non-pathogenic prokaryotes, including but not limited to them alone, Bacillus, including Bacillus anthracis; Vibrio, e.g. V. cholerae; Escherichia, e.g. enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium, such as M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C. perfringens; Corynebacterium, such as C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, for example P. aureus; Haemophilus, such as H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g Y. lamblia, Ypestis, Pseudomonas such as P. aeruginosa, P. putida; Chlamydia, e.g .trachomatis; Bordetella, e.g Century pertussis; Treponema, e.g. T. palladium; B. anthracis, Y pestis, Brucella spp., F. tularensis, B. mallei, B. pseudomallei, C. botulinum, Salmonella spp., SEB V. cholerae toxin B, E. coli O157:H7, Listeria spp., Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Enterobacter sp., Klebsiella sp., Listeria sp., Mycoplasma sp. etc.
In some aspects, the antibodies directed against viral infections; these viruses include, but are not limited to, orthomyxoviruses (e.g., influenza virus), paramyxoviruses (e.g. the R, respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g., rubella virus), parvoviruses, poxviruses (e.g. variola virus, vaccinia virus), enteroviruses (e.g. poliovirus, Coxsackievirus), hepatitis viruses (including a, b and C), herpesviruses (e.g., herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr), rotaviruses, viruses, Norfolk, Hantavirus causing hemorragic fever, arenavirus, rhabdovirus (for example, rabies virus), retroviruses (including HIV, HTLV-I and II), papovaviruses (for example, the human papilloma virus), polyomaviruses and picornaviruses, and the like,
Properties optimized IgG-options
The present invention also provides IgG variants that are optimized for a wide range of therapeutically important properties. IgG-variant, designed or predicted to demonstrate one or more optimized properties, called here "optimized IgG-option". The most preferable properties that can be optimized include, but are not limited to increasing or decreasing the affinity to FcRn and increase or decrease the half-life in vivo. Suitable embodiments include antibodies that exhibit enhanced binding affinity to FcRn at lower pH values, such the AK pH, associated with endosomes, for example at pH 6.0, and at the same time do not show a corresponding improved binding affinity at higher pH values, for example at pH 7.4, which improves the absorption of endosome at normal speeds the release. Similarly, these antibodies with modulated binding to FcRn may not have other desirable properties, such as modulated binding to FcγR, such as described in US 11/174,287, 11/124,640, 10/822,231, 10/672,280, 10/379,392 and patent application entitled "IgG Immunoglobulin variants with optimized effector function" and filed on October 21, 2005 under number ________. Thus, the optimized properties also include, but are not limited to, the increase or decrease in the affinity to FcγR. In one optional embodiment of the IgG variants are optimized in order to increase their affinity for the activator human FcγR, preferably to FcγRIIIa in addition to the FcRn-binding profile. In yet another optional alternative preferred embodiment of the IgG variants are optimized to reduce their affinity for the inhibitory receptor FcγRIIb person. Therefore, particular embodiments include the use of antibodies that exhibit increased binding to FcRn and increased binding to FcγRIIIa. In other embodiments employ antibodies that exhibit high St. the statements with FcRn and increased binding to FcγRIIIa. These embodiments provide IgG-polypeptides with enhanced therapeutic properties for people, for example, with enhanced effector function and higher anticancer efficacy. In an alternate embodiment of the IgG variants are optimized so that they have increased or reduced affinity for FcRn and increased or reduced affinity for human FcγR, including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb, including their allelic variations. These embodiments provide IgG-polypeptides with enhanced therapeutic properties for people, for example, with increased half-life in serum and reduced effector function. In other embodiments IgG-options provide increased affinity for FcRn and increased affinity for one or more FcγRs, and, in addition, reduced affinity to one or more FcγRs. For example, IgG-option may have enhanced binding to FcRn and FcγRIIIa and, in addition, reduced binding to FcγRIIb. Alternatively, IgG-option may have reduced binding to FcRn and FcγR's. In another embodiment of the IgG variant may have a reduced affinity for FcRn and increased affinity to FcγRIIb, and, in addition, a reduced affinity to one or more activator FcγRs. In yet another embodiment of the IgG variant may have an increased half-life in SIV is the pigs blood and impaired effector function.
Preferred embodiments include optimizing the binding to human FcRn and FcγR, however, in alternative embodiments of the IgG variants have increased or reduced affinity for FcRn and FcγRs from organisms that are not people, including but not limited to rodents and primates, are not people. IgG variants that have optimized binding to FcRn, non-human, can find application in research. For example, the model in mice suitable for studies of a variety of diseases and allow you to test properties such as efficacy, toxicity and pharmacokinetics of a particular candidate in medicine. As is known to experts in this field of technology, the cancer cells can be grafted or introduced into a mouse to simulate human cancer, this process is known as xenotransplantation. Test IgG variants that include IgG variants that are optimized for FcRn can provide useful information about characteristics of the clearance of the protein, the mechanism of its clearance, etc. IgG variants can also be optimized to enhance the functionality and/or enhance the solubility in glycosylamines form. Fc ligands include but are not limited to the mentioned above, FcRn, FcγRs, C1q and proteins a and G, and can be from any source, including but not limited the ne, mouse, rat, rabbit or monkey, but the preferred source is the man. In an alternative preferred embodiment of the IgG-optimize options so that they are more stable and/or more soluble than glikozilirovana form of the original IgG-option.
IgG variants may include modifications that modulate the interaction with Fc-ligands that do not belong to FcRn and FcγRs, which include, but are not limited to the above, complement proteins and homologues Fc-receptors (FcRHs). FcRHs include, but are not limited to the mentioned above, FcRH1, FcRH2, FcRH3, FcRH4, FcRH5 and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, work incorporated by reference in its entirety).
Preferably, the specificity of IgG-option to Fc-ligand will determine its therapeutic application. The applicability of specific IgG options for therapeutic purposes will depend on the epitope or shape of the target antigen and the disease or symptom, which is subjected to treatment. For most targets and indications, it may be preferable increased binding to FcRn as increased binding to FcRn may lead to longer half-life in serum. Increased half-life in serum allows you to use a lower frequency of doses or low dose drugs. This can be in particular a preference for the equipment, if a therapeutic tool used in response to indications that require repeated administration tools. For some targets and indications, it may be preferable reduced affinity for FcRn. This can be especially preferred if it is desirable to have an Fc variant with increased clearance or decreased half-life in serum, such as Fc-polypeptide used as physiographically funds or in the form of a radioactive therapeutic agents.
You can apply IgG variants that include IgG variants, providing increased affinity for FcRn with reinforced activator FcγR and/or reduced affinity for the inhibitory FcγR. For some targets and indications, in addition, it may be useful to use IgG variants that provide different selectivity for different activator FcγR; for example, in some cases it may be desirable enhanced binding to FcγRIIa and FcγRIIIa but not FcγRI, whereas in other cases it may be preferable increased binding only with FcγRIIa. For some targets and indications, it may be preferable application of the IgG variants that alter the binding of FcRn and enhance effector function, as mediated by FcγR and effector functions mediated by complement, whereas in other cases it may be useful to use the best of IgG variants, to enhance the binding of FcRn or increase the half-life in serum and enhance effector function, as mediated by FcγR and effector functions mediated by complement. For some targets and cancer indications may be useful to reduce or completely eliminate one or more effector functions, for example using knockout binding C1q, one or more FcγR, FcRn, one or more other Fc ligands. For other targets and indications, it may be preferable to apply IgG variants that provide enhanced binding to the inhibitory FcγRIIb and, in addition, provide the binding of an activator FcγR-level WT, at a lower level or not contacted with an activator FcγR at all. This can be especially useful, for example, if the purpose of IgG-variant is the inhibition of inflammation or autoimmune disease or modulate in some way the immune system. As an autoimmune disease primarily continue for a long period of time and are used for treatment repeated dose, particularly preferably for the treatment of these diseases with the use of Fc variants with increased half-life due to increased FcRn.
To improve the stability, solubility, function, and clinical application of IgG, you can spend a mod is the ion. In the preferred embodiment of the IgG variants may include modifications to reduce immunogenicity in humans. In the preferred embodiment, the immunogenicity of IgG-variant reduced using the methods described in patent US 11/004,590, incorporated by reference in its entirety. In alternative embodiments of the IgG variants humanitarian (Clark, 2000, Immunol Today 21:397-402, work incorporated by reference in its entirety).
IgG variants may include modifications that reduce immunogenicity. Modifications to reduce immunogenicity may include modifications that reduce the binding protestirovannyx peptides derived from the sequence of the precursor, protein major histocompatibility complex (MHC). For example, the amino acid modifications can be designed so that they would not be at all or would be the minimum number of immune epitopes, presumably bind with high affinity with any prevailing MHC alleles. In this technical field, there are several methods to identify protein sequences of epitopes that bind to MHC, and they can be used to determine the total score for epitopes in the variant IgG. See, for example, WO 98/52976; WO 02/079232; WO 00/13317; US 09/903,378; US 10/039,170; US 60/222,697; US 10/754,296; PCT WO 01/21823 and PCT WO 02/00165; Mallios, 1999, Bioinformatis 15: 432-439; Mallios, 2001, Bioinformatics 17: 942-948 (in Russian); Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO 98/59244; WO 02/069232; WO 02/77187; Marshall et al., 1995, J. Immunol. 154: 5927-5933 and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358, all works incorporated by reference in its entirety. To determine the amount of points to associate with the specific interaction of peptide and MHC, you can use the information based on sequence (see, for example, Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: p942-948; Sturniolo et. al., 1999, Nature Biotech. 17: 555-561, all works incorporated by reference in its entirety).
Variants of the present invention can be constructed in various ways. Options, as described here, can be insertions, deletions, substitutions, and other modifications or combinations and other changes. In particular, the latest embodiment of the present invention is the construction of insertions and deletions, which both improve and worsen the binding of the Fc-polypeptide with Fc-ligand. As disclosed in this application can be made insertions and deletions that increase or decrease the affinity of the Fc-polypeptide for FcRn. Insertions and deletions can be constructed by using rationally designed approaches or approaches that include the use of random components, such as the creation of libraries of random or polyleucine options or screening. In Alta is native to the embodiment disclosed replacement which increase or decrease the affinity of the Fc-polypeptide for FcRn.
Insertions and deletions
Variant Fc polypeptides can be generated by replacing the original amino acid at any position of the Fc polypeptide variant amino acid. By replacing one or more amino acids in the Fc-polypeptide variant amino acids at these positions are modified side chains. The most useful replacement modify the properties of the Fc by changing the side chains of the Fc. Substituted side chains can interact directly or indirectly with a partner to bind to the Fc associated with the function or property of Fc. At least one replacement changes the covalent structure of one or more side chains of the original Fc polypeptide.
Alternatively, it may be created variant Fc polypeptides that alter the covalent structure of the main chain of the original Fc polypeptide. The atoms of the protein included in the main chain of the molecule include peptide nitrogen atom, the alpha carbon atom, or peptide carbonyl carbon atom and the carbonyl oxygen. The change in the covalent structure of the main chain provides additional ways to modify the properties of Fc-polypeptides. Covalent structure of the main chain of the Fc can be changed by adding atoms in the main chain of the molecule, for example by inserting one who does several amino acids, or removing atoms from the main chain, for example by deletion of one or more amino acids. The covalent structure of the main chain can also be modified by changing the individual atoms of the main chain to other atoms (Deechongkit et al., J. Am. Che. Soc. 2004, 126(51):16762-71, work incorporated by reference in its entirety). As is well known in this technical field and illustrated here, insertions and deletions of amino acids in the Fc-polypeptides can be made using inserts or deletions of the corresponding nucleotides in the DNA encoding the Fc-polypeptide. Alternatively, as is well known in this technical field, insertions and deletions can be made during the synthesis of Fc-polypeptides.
The design of insertions and deletions of amino acids that alter the interaction of the Fc-polypeptide with one or more binding partners (for example, Fc-R's, FcRn, C1q) can be made on the structure of the Fc-polypeptide and its binding partner. In a less preferred embodiment of the design you can spend based on the structure of the Fc-polypeptide and information about Fc-region involved in binding to the binding partner. This information can be obtained through experiments on the mutagenesis experiments using phage display, by comparison homology, using computer modeling and friends and ways.
The preferred position in the amino acid sequence of insertions and deletions that affect the interaction with the binding of the Fc, but do not affect the overall structure, stability, expression or application of the Fc-polypeptide, are the sites of interaction of the Fc/Fc partner binding. To modify the binding of FcRn with Fc-polypeptide that are preferred locations loops for insertions and deletions are provisions 244-257, 279-284, 307-317, 383-390 and 428-435 (numbering according to EU index in the database Kabat et al., Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, Mol Cell 7:867-877, all works incorporated by reference in its entirety). To modify the binding of Fc-gamma receptor with Fc-polypeptide that are preferred locations loops for insertions and deletions are provisions 229-239, 266-273, 294-299 and 324-331 (numbering according to EU index in the database Kabat et al., code pdb 1E4K.pdb Sondermann et al., 2000, Nature 406:267, all works incorporated by reference in its entirety). Loops are parts of the polypeptide are not involved in the structure of alpha-helices and beta-pleated (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, Chapter 1 pp2-67, work incorporated by reference in its entirety). The position of the hinges are preferred, since the main chain atoms of a molecule are usually more mobile and less likely including both the HN in the formation of hydrogen bonds compared to the atoms of the main chain alpha-helices and beta-pleated. So it is less likely that the lengthening or shortening of the loops due to insertions or deletions of one or several amino acids will lead to large, destructive changes in the Fc-polypeptide, including its stability, expression and other problems.
Insertions and deletions can be used to change the length of the polypeptide. For example, in the area of the loops change the loop length will result in a modified mobility and conformational entropy of the loop. Insert in the loop will increase the conformational entropy of the loop, which can be defined as the Boltzmann constant multiplied by the natural logarithm of the number of possible conformations (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, Chapter 1 pp78, work incorporated by reference in its entirety). When inserted into the polypeptide, at least one amino acid the total number of conformations that are possible in the polypeptide, is increased. These additional conformations can be useful for the formation of favorable interactions Fc/Fc partner binding, as the Fc-polypeptide may use one additional conformations when binding protein, to bind to the Fc. In this case, the insert may lead to stronger interactions of the Fc/Fc partner binding. If additional conformations are not used on the surface of the binding, insertion meters which can lead to weaker interactions Fc/Fc partner binding, as an additional conformation must compete with a conformation that is competent in relation to linking. Similarly, the deletion polypeptide segment also can lead to more robust and less strong interactions Fc/partner Fc binding. If the deletion of a segment, which reduces the possible number of main chain conformations of the molecule, eliminate conformation that is competent in relation to the binding, the deletion can lead to weaker interactions Fc/partner Fc binding. If the deletion of a segment does not treat the conformation that is competent in relation to the binding, the deletion can lead to stronger interactions of the Fc/Fc partner binding, because the deletion can eliminate conformation competing with conformation that is competent in relation to the binding.
Insertions and deletions can be used to change the position and orientation of the amino acids in the Fc-polypeptide. Since insertions and deletions can cause changes in the covalent structure of the main chain, they will cause a change in the positions of the atoms of the main chain. Figure 7 compares the position of the main chain in certain segments of the loop, labeled L1-L4, in three different basic circuits. The original main structure contains four segments of the loop, at the same time deletion in the main chain leads to p is Tere segment L1, and inserting segment includes an additional segment preceding the segment L1, for example, before the N-end. Deletions and insertions causing large-scale changes in the structure of the main chain near insertions or deletions. Due to the removal of a segment located near the N-terminal part of the loop, such as a segment of the L1 loop is shortened, and the remaining segments are shifting their position closer to the N-end of loop. This has an effect on the movement of the segment L2 in the direction to the former location of the segment L1 and towards the N end of the loop. This change of position of the segment L2 relative to the segment L1 can enhance the binding of the complex Fc/partner Fc binding and is preferred if there is prior information suggesting that the amino acid or amino acids located in the L2, make favorable interactions with a partner on the binding of Fc in L1. For example, if L2 contains alanine and tyrosine, and the replacement of two amino acids in L1 by alanine and tyrosine previously led to Fc-variant with improved binding, deletion of L1 can create Fc-variant with increased affinity to the binding partner with Fc.
Similarly, insertion of the polypeptide segment in the Fc-polypeptide at the N-terminal part of the loop will cause the position of the segments of the loop will be displaced towards the C-terminal part of the PE the Lee. In figure 7 inserting one or more amino acids before the segment L1, for example, N-end, changes the conformation of the main chain, including moving segment L1 in the direction From the end of the loop. This type of insert is preferred, if it is known that amino acids located in the segment L1, favor the interaction, when they are in position L2, since the insert can lead to stronger interactions Fc/partner Fc binding. If you want a weaker interaction of the Fc/Fc partner binding, you can use insert to offset adverse amino acids in the new position. Inserted, deleted and the original segments (L1-L4 in figure 7) can be one or several amino acids in the Fc-polypeptide.
Alternatively, similar to insertions and deletions at the N-terminal segment of the loops can be used for insertions and deletions at the C-terminal segment of the loops. Insert-the end loops can cause movement of the N-terminal segment of the insert towards the N end of the loop. Deletions at the C-end loops can cause movement of the N-terminal segment deletions in the direction From the end of the loop. The choice of using paste or deletions at the N-terminal or C-terminal section of the loop based on the amino acids located in the loop, it is necessary to increase or mind is isenia affinity complex Fc/Fc partner binding and the desired positional shift.
Insertions and deletions can be used in any part of the Fc-polypeptide, including parts of loops, alpha-helices and beta-pleated. The preferred location of insertions and deletions include lots of loops, which are not parts of alpha-helices and beta-pleated. Loops are preferred because they generally perceive changes in the main chain is better than alpha-helix and beta-folds. Especially preferred locations of insertions or deletions that lead to a stronger protein/protein interactions, which are N-terminal and C-terminal region of the loop. If the side chain loops involved in the interaction of the Fc/Fc partner binding, then it is less likely that the insertion or deletion at the edges will lead to very unhealthy changes in binding interactions. It is more likely that deletion of the exact center of the loop eliminate important residues on the surface interaction of the Fc/Fc partner binding and paste the exact center of the loop is more likely to create adverse interaction on the surface of the binding of the Fc/Fc partner binding. The number of deleted or inserted residues can be determined by the amount of desired changes in the main chain, preferably about 15 insertions or deletions or less, more preferably about 10 insertions or deletions or less and most preferably about 5 inserts and deletions or less.
Once the location and size of the deletion Fc-variants were designed to definitively determine the complete sequence of the polypeptide, and the polypeptide can be created using methods known in this technical field.
Insertion Fc-variants, however, have additional stages of the design sequence, at least one inserted amino acids. Insert polar residues, including Ser, Thr, Asn, Gln, Ala, Gly, His, preferable in the provisions that are expected to be in the Fc-polypeptide exposed. Particularly preferred smaller amino acids, including Ser, Thr and Ala, as it is less likely that the small size will sterically hinder the interaction of the Fc/Fc partner binding. Ser and Thr also have the ability to form hydrogen bonds with atoms partner Fc binding.
The insert may also have additional flexibility, which can be constructed in the insertion of the polypeptide to create a positive interaction with partner Fc binding, as would be necessary if it is desirable stronger binding of the Fc/Fc partner binding. The length of the insert in the main chain can be determined by modeling the variance of the main chain with a simple base sequence that is used for getting the I. For example, you can design and simulate insertion polyserena, polyglycine or polyalanine different lengths. Modeling can be performed using a variety of methods, including homology modeling based on the known three-dimensional structures of homologues, including insert, and with the help of computer simulation, including MODELLER (M.A. Marti-Renom et al., Annu. Rev. Biophys. Biomol. Struct. 29, 291-325, 2000) and ROSETTA (Kuhlman et al. (2003). Science 302, 1364-8), both works are incorporated by reference in its entirety. Typically, first create a different conformation of the main chain, and the final structure of the main chain can be defined after you have installed the identity of the side chain. Side chains can be designed using algorithms PDA® (US 6,188,965; 6,269,312; 6,403,312; 6,801,861; 6,804,611; 6,792,356, 6,950,754 and US 09/782,004; 09/927,790; 10/101,499; 10/666,307; 10/666311; 10/218,102, all incorporated by reference in its entirety).
Insertions and deletions can be made in any polypeptides in addition to Fc-polypeptides using the methods described here. For example, insertions or deletions in the representative of the superfamily, TNF, APRIL, you can design using its three-dimensional structure (PDB code IXU1.pdb, Hymowitz, et al. (2005) J.Biol.Chem. 280:7218, incorporated by reference in its entirety). Insertions or deletions can be designed to enhance the APRIL binding to its receptor, TACI. Preferred OST the kami loop as sites for insertion or deletions are the remains of Ser118-Val124, Asp164-Phe167, Pro192-Ala198, Pro221-Lys226. These loops interact with TACI in complex APRIL/TACI and mediates the binding.
Polypeptides, including variants
IgG variants may be based on the IgG sequences, and thus, the sequence of human IgG is used as the "main" sequence with which to compare the other sequences, including but not limited to sequences from other organisms, for example sequences of rodents and primates. IgG variants may also include sequences from other classes of immunoglobulins, such as IgA, IgE, IgD, IgM, etc. Suggest that although IgG-design options in the context of a single source of IgG variants may be constructed and transferred in the context of another, the second source of IgG. Do this by defining "equivalent" or "relevant" residues and substitutions between the first and second IgG, which is typically based on sequence or structural homology between the sequences of IgGs. To establish the homology of amino acid sequence of the first IgG specified here, directly compared with the sequence of the second IgG. After alignment of the sequences using one or more programs establish homology, known to specialists in this field (for example, by using OST Dow, conserved in different species), allowing, if necessary, the existence of insertions and deletions in order to maintain alignment (i.e. to avoid exceptions conservative residues due to random insertions and deletions), set of amino acid residues equivalent to certain amino acids in the primary sequence of IgG-option. Alignment conservative residues preferably should retain 100% of such residues. However, the alignment of more than 75% or at least 50% conservative residues are also sufficient to determine equivalent residues. Equivalent residues can also be set using the definition of homology between the first and second IgG at the level of tertiary structure for IgGs, for which the tertiary structure is already installed. In this case, the equivalent is called residues, for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue in the original polypeptide or its precursor (N on N, CA on CA, C on C, O, O) in the alignment are within 0.13 nm and preferably in the range of 0.1 nm. Alignment is achieved after the best model compliance oriented and disposed in such a way as to ensure maximum overlap of atomic coordinates, related to the protein atoms that are not atoms is hydrogen. Regardless of how many define equivalent or corresponding residues, and regardless of the features of the original IgG, on the basis of which create IgGs, we are talking about what is known IgG variants can be designed in any second source of IgG, which has significant homology in sequence and structure with IgG-option. Thus, for example, if you get a variant antibody in which the antibody is human IgG1, using the methods described above or other methods for determining equivalent residues, the variant body can be designed in different source IgG1 antibody that binds a different antigen in the original antibody IgG2 man, the original antibody IgA man, the original murine antibody IgG2a or IgG2b, etc. in Addition, as described above, the context of the original IgG-option does not affect the possibility of transfer of IgG variants in other source IgGs.
Provides methods for creating, producing and screening of IgG variants. This does not mean that the described methods are limited to any practical application or mechanism of action. Rather, the provided methods are intended to illustrate in General terms the fact that one or more IgG-options you can design, produce and select experimentally, in order to obtain the IgG variant is optimized effector function. Many methods for the design, production and testing of antibodies and source variations are described in US 10/754,296 and US 10/672,280, both incorporated by reference in its entirety.
For the design of IgG variants with optimized effector function, you can apply many methods of constructing proteins. In one embodiment can be applied based on the known structure construction method, in which control substitutions apply available information about the structure, where replacement project, based on their energy according to computer calculations. See, for example, US 10/754,296 and US 10/672,280 and the references cited in them, they are all incorporated by reference in its entirety.
To manage changes in established positions may be used in the sequence alignment. Professionals in this area of technology will be clear that the use of sequence information can restrain the introduction of substitutions that are potentially harmful to the protein structure. Sources of sequences can vary greatly and include one or more well-known databases, including but not limited to the above sources, Kabat database (Northwestern University); Johnson & Wu, 2001, Nucleic Acids Res 29:205-206; Johnson & Wu, 2000, Nucleic Acids Res 28:214-218), database IMGT (IMGT, between Narodnoy information system ImMunoGeneTics information system®; Lefranc et al., 1999, Nucleic Acids Res 27:209-212; Ruiz et al., 2000 Nucleic Acids Res 28:219-221; Lefranc et al., 2001, Nucleic Acids Res 29:207-209; Lefranc et al., 2003, Nucleic Acids Res 31:307-310), and VBASE, all incorporated by reference in its entirety. Information on the sequences of the antibodies can be obtained, collect and/or create from alignments of sequences germ line cells, or existing in nature antibodies from any organism, including, but not limited only mammals. Professionals in this area of technology will be clear that the use of sequences that are human or mostly human, may also benefit due to the reduced immunogenicity when administered to man. Other databases, which are usually a database of nucleic acids or proteins, that is not relevant only to the antibodies include, but are not limited to SwissProt, GenBank, Entrez and EMBL Nucleotide Sequence Database. Align sequences can include sequences of VH, VL, CH and/or CL. There are a large number of alignment programs based on the sequences, and methods known in this technical field, and all of them are used to create a sequence alignment.
Alternatively, you can apply the methods of random or polyleucine mutagenesis to obtain amino acid modifications of the desired positions. In these cases, the position is chosen randomly or amino acid substitutions produce using simplified methods. For example, all the remnants can be motivovany in alanine, this process is called alanine scanning. Such methods can be paired with more complex engineering approaches, in which for the selection of higher levels of species sequences used selection methods. As is well known in this technical field, there is a diverse selection of technologies that can be used in such approaches and which include, for example, display technologies, for example, phage display, ribosomal display, the display on the surfaces of cells, etc. as described below.
Methods of obtaining and selection IgG variants are well known in this technical field. The basic methods of molecular biology, antibodies, expression, purification, and screening are described in the book of Antibody Engineering, edited by Duebel&Kontermann, Springer-Verlag, Heidelberg, 2001; and in the work Hayhurst&Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard&Georgiou, 2000, Annu Rev Biomed Eng 2:339-76. See also the methods described in US 10/754,296; US 10/672,280 and US 10/822,231 and 11/124,620, all incorporated by reference in its entirety.
Preferred variants of the present invention include those that are presented on figure 8. Alternative preferred variants of the present invention include those that depict what aulani in figure 9. In addition, an alternative preferred variants of the present invention include those that are presented on figure 10. Particularly preferred variants of the present invention include G358H and N434Y. The most preferred variants of the present invention include S, M, 257L, 257N, 257Y, 279Q, 279Y, 308F and 308Y.
Production of IgG-options
IgG variants can be produced by any means known in this technical field. In one of the embodiments to generate nucleic acids that encode the sequence of any representative and which can then be cloned into host cells, expressed and, if necessary, analyzed, applied sequence of IgG variants. These methods execute, using well-known procedures, and a variety of methods which can be used, as described in the book Molecular Cloning - A Laboratory Manual, 3ndEd. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley&Sons), both incorporated by reference in its entirety. For the expression of protein nucleic acids that encode the IgG variants may be included in the expression vector. Expression vectors typically include protein, effectively connected, that is in functional relationship with management or regulatory sequences, the selective marker is mi and partners to merge and/or additional elements. IgG variants can be generated by culturing the host cell, transformed with nucleic acid, preferably the expression vector including nucleic acid encoding the IgG variants, in the appropriate conditions to induce or cause expression of the protein. Can be used a huge variety of suitable host cells, including but not limited to the above, mammalian cells, bacteria, insect cells and yeast. For example, many cell lines, which may find use in the present invention, described in the catalogue of cell lines of ADS available in the American type culture collection (American Type Culture Collection) and incorporated here by reference in its entirety. Methods of introducing exogenous nucleic acid into a host cell are well known in this technical field and will change depending on what the host cell used.
In the preferred embodiment of the IgG variants cleanse or release after expression. Proteins can be selected or cleared by using numerous methods known to experts in this field of technology. Standard purification methods include chromatographic techniques, electrophoresis, immunological methods, precipitation, dialysis, isoelectrofocusing. As is well known in this technical field, the variety of the knowledge of natural proteins bind antibodies, for example, the bacterial proteins A, G and L, and these proteins can be used when cleaning. Cleaning frequently can be done with a specific partner to merge. For example, proteins can be purified using glutathione resin if used fused GST; N+2-affinity chromatography, if you are using the His-tag, or using immobilized antibodies against flag when using flag-tag. To familiarize yourself with suitable purification techniques, see the book Antibody Purification: Principles and Practice, 3rdEd., Scopes, Springer-Verlag, NY, 1994, incorporated here by reference in its entirety.
IgG variants may be subjected to screening using a variety of methods, including but not limited to the above, the ways in which applied in vitro, in vivo analyses, cellular analysis and selection methodology. In the screening procedures can be automated and high-technology screening. The screening can be applied partner in the merger, or label, for example the immune label, isotope label, or a label, which is a small molecule, such as fluorescent or colorimetric dye.
In the preferred embodiment in the analysis of in vitro selected functional and/or biophysical properties of IgG variants. In the preferred embodiment the protein is selected on the basis of his record and the national opportunities for example, the ability to catalyze a reaction or binding affinity to its target. Analyses linking can be carried out using a number of methods known in this field of technology, including but not limited to the above, assays based on FRET (resonance energy transfer fluorescence) and BRET (resonance energy transfer bioluminescence), AlphaScreen™ (homogeneous proximity analysis using enhanced luminescence), scintillation proximity analysis, ELISA (enzyme-linked immunosorbent assay), SPR (surface plasma resonance, also known as BIACORE®), calorimetry isothermal titration and differential scanning calorimetry, gel electrophoresis and chromatography, including gelfiltration. These and other methods can take advantage of some of the partners in the merger or labels. In the analyses can be used a variety of methods of detection, including, but not limited to the above, chromogenic, fluorescent, fluorescent, isotopic labels. Biophysical properties of proteins, such as stability or solubility, can be selected using a variety of methods known in this technical field. The stability of the protein can be determined by measuring thermodynamic equilibrium between collapsed and expanded States. For example, IgG-VA is ianti can be deployed using chemical denaturing compounds, heat or pH, and this transition may be monitored using methods including, but not limited to the above, spectroscopy circular dichroism, fluorescence spectroscopy, absorption spectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As recognized experts in the field of technology, kinetic parameters of transitions collapsed and expanded state can also be traced using these and other technologies. The solubility and overall structural integrity of the IgG variants may be characterized qualitatively and quantitatively by using a wide range of ways that are known in this technical field. Methods that can be used to characterize the biophysical properties of the IgG variants include gel electrophoresis, chromatography, such as exclusion chromatography and inverse-phase high-performance liquid chromatography, mass spectrometry, ultraviolet absorption spectroscopy, fluorescence spectroscopy, spectroscopy circular dichroism, isothermal calorimetry titration, differential scanning calorimetry, analytical ultracentrifugation, dynamic light scattering, proteolysis and intermolecular crosslinking, the turbidity measurement, analysis delay on the filter, immunological tests, analysis of binding flu is rescently dyes, analysis of the staining of proteins, microscopy, and detection of aggregates using ELISA or other analysis of binding. Possible structural analysis using crystallographic methods using x-rays and NMR spectroscopy.
As is well known in this technical field, the group of methods of screening allows you to select useful members of the library. These methods refer to the methods of selection, and these methods are used when screening for IgG variants. If the library proteins are subjected to screening using the method of breeding, propagation, isolated and/or mark only those library members that are useful, that is, which meet certain criteria of selection. Consider that in connection with obtaining only the most suitable options such methods allow for the screening of libraries that are larger than libraries that are selected using methods that analyze individual suitability of members of the library. Selection can be performed by any method, technique or by using a partner to merge, which covalently or ecovalence connects the phenotype of the protein with its genotype, i.e. the function of a protein with nucleic acid that encodes. For example, the use of the method of selection of the method of phage l is splay becomes possible at the confluence of the library members with protein gene III. In this case, selection or obtaining IgG variants that meet certain criteria, such as the binding affinity with protein targets, also used for selection or obtaining a nucleic acid that encodes them. After selecting a gene or genes encoding the IgG variants that can then be amplified. This process of selection and amplification, called penninga, can be repeated and enrich library of useful options IgG. Finally, sequencing of the attached nucleic acid allows the identification of a gene.
In this field of technology there are many methods of selection, which can be used for screening of protein libraries. They include, but are not limited to the above methods, the method of phage display (Phage display of peptides and proteins: a laboratory manual, Kay et al., 1996 Academic Press, San Diego, CA, 1996; Lowman et al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science 228:1315-1317) and its modifications, such as a method of selective phage infection (Malmborg et al., 1997, J Mol Biol 273:544-551), the method selectively infective phage (Krebber et al., 1997, J Mol Biol 268:619-630) and panning delayed infectivity (Benhar et al., 200, J Mol Biol 301:893-904), the display on the cell surface (Wittrup, 2001, Curr Opin Biotechnol, 12:395-399), for example, the display on the bacteria (Georgiou et al., 1997, Nat Biotechnol 15:29-34; Geourgiou et al., 1993, Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648; Jim et al., 1998, Nat Biotechnol 16:576-80), nadarajah (Boder & Wittrup, 2000, Methods Enzymol 328:430-44; Boder &Wittrup, 1997, Nat Biotechnol 15:553-557), and in mammalian cells (Whitehom et al., 1995, Biotechnology 13:1215-1219), as well as technology display in vitro (Amstutz et al., 2001, Curr Opin Biotechnol 12:400-405nm), such as the display on polysome (Matheakis et al., 1994, Proc Natl Acad Sci USA 91:9022-9026), the display on the ribosomes (Hanes et al., 1997 Proc Natl Acad Sci USA 94:4937-4942), mRNA display (Roberts &Szostak, 1997, Proc Natl Acad Sci USA 94:12297-12302; Nemoto et al., 1997, FEBS Lett 414:405-408) and the system display the inactivation of ribosomes (Zhow et al., 2002, J Am Chem Soc 124:538-543). All sources included in this paragraph by reference in its entirety.
Other methods of selection, which can be used include techniques that are not related to the display, for example, that methods of selection in vivo, including, but not limited to the above, the expression in periplasm and cytometrics screening (Chen et al., 2001, Nat Biotechnol 19:537-542, work incorporated by reference in its entirety), complementation analysis of protein fragments (Johnsson &Varshavsky, 1994, Proc Natl Acad Sci USA 91:10340-10344; Pelletier et al., Proc Natl Acad Sci USA 95:12141-12146, all works incorporated by reference in its entirety) and yeast screening system double hybrids (Fields &Song, 1989, Nature 340:245-246, work incorporated by reference in its entirety), used in selective mode (Visintin et al., 1999, Proc Natl Acad Sci USA 96:11723-11728, work incorporated by reference in its entirety). In an alternate embodiment, the selection can wire the TB with the help of a partner to merge, which binds to a specific sequence of the expression vector, thereby covalently or ecovalence binding partner fusion and the associated party libraries Fc variant with nucleic acid that encodes them. For example, US patents 09/642,574; US 10/080,376; US 09/792,630; US 10/023,208; US 09/792,626; US 10/082,671; US 09/953,351; US 10/097,100; US 60/366,658; PCT WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO 01/28702; and PCT WO 02/07466, all incorporated here by reference in its entirety, describe such a partner in the mergers and technique, which can find application. In an alternate embodiment, the selection in vivo can occur if the expression of the protein confers cells are some of the benefits of growth, reproduction or survival.
Subgroup of methods of selection related to the "directed evolution", is a means, which include the connection or reproduction of useful sequences during breeding, sometimes with the inclusion of new mutations. As recognized experts in the field of technology, the methods of directed evolution can help identify the most useful sequences of the library and can increase the diversity of sampled sequences. In this field of technology there are many methods of directed evolution, which can knight is used for screening libraries of IgG variants and which include, but not limited to the mentioned above, the permutation DNA (PCT WO 00/42561 A3; PCT WO 01/70347 A3), the permutation of the exon (US 6,365,377; Kolkman & Stemmer, 2001, Nat Biotechnol 19:423-428), the permutation family (Crameri et al., 1998, Nature 391:288-291; US 6,376,246), RACHITT™ (Coco et al., 2001, Nat Biotechnol 19:354-359; PCT WO 02/06469), STEP and random priming recombination in vitro (Zhao et al., 1998, Nat Biotechnol 16:258-261; Shao et al., 1998, Nucleic Acids Res 26:681-683), the Assembly of genes dependent ectonucleoside (US 6,352,842; US 6,361,974), the method of Gene Site Saturation Mutagenesis™ (US 6,358,709), the method of Gene ' s reassembly™ (US 6,358,709), SCRATCHY (Lutz et al., 2001, Proc Natl Acad Sci USA 98:11248-11253), methods of DNA fragmentation (Kikuchi et al., Gene 236:159-167), the permutation of single-stranded DNA (Kikuchi et al., 2000, Gene 243:133-137) and technology design directed evolution of proteins AMEsystem™ (applied molecular evolution) (US 5,824,514; US 5,817,483; US 5,814,476; US 5,763,192; US 5,723,323). All sources cited in the previous paragraph are hereby incorporated by reference in its entirety.
In the preferred embodiment of the IgG variants is subjected to screening using one or more cellular assays or assays in vivo. For such analyses usually add exogenous purified or crude proteins, so that the cells were exposed to certain options or pool of variants belonging to the library. These tests are usually, but not always, based on functions of IgG, i.e. on the ability of IgG to bind its antigen target and mediate some biochemical event, for example effector function, inhibition of binding of a ligand/receptor, apoptosis, etc. Such assays often involve monitoring the response of cells to IgG, such as the survival of cells, cell death, changes in cell morphology or activation of transcription, such as cellular expression of a natural gene or reporter gene. For example, such assays may measure the ability of IgG variants to induce ADCC, ADCP or CDC. For some analyses in addition to the target cells may be necessary to add more cells or components, such as serum complement or effector cells, such as monocytes peripheral blood (RVMS), NK-cells, macrophages, etc. Such additional cells may be from any organism, but it is preferable that they were human cells, mice, rats, rabbits and monkeys. Antibodies can induce apoptosis in some cell lines expressing the target, or they can mediate the attack on target cells with immune cells that were added for analysis. In this area of technology known methods of monitoring cell death or viability, and they include the use of dyes, immunochemical, cytochemical and radioactive reagents. For example, analysis by staining caspase gives the possibility to measure the level of apoptosis, and the absorption and release of RA is ioactive substrates or fluorescent dyes, such as Alumaloy blue, it helps to observe cell growth or cell activation. In the preferred embodiment applied to the analysis of cytotoxicity kits are used®EuTDA-based cytotoxicity assay (Perkin Elmer, MA). Alternatively, for dead or damaged target cells can be monitored using the release of one or more natural intracellular proteins such as lactate dehydrogenase. Activation of transcription can also serve as a method of analysis functions in the cell dimensions. In this case, the response can be monitored using natural genes or proteins that can be regulated through the activation, for example, it is possible to measure the release of certain interleukins, or, alternatively, the response may be mediated reporter construct. Cellular analyses may also include the measurement of morphological changes of cells in response to the presence of protein. The types of cells in these assays may be prokaryotic or eukaryotic, and you can apply a variety of cell lines, which are known in this technical field. Alternatively, the selection using cells performed using cells that have been transformed or transliterowany using nucleic acid coding options. That is, the IgG variants are not added exogenously to cells. For example, in one of the embodiments in the selection, the basis of the data on the use of cells, use the view method on the cell surface. You can apply for partner to merge, which makes possible the representation of the IgG variants on the cell surface (Witrrup, 2001, Curr Opin Biotechnol, 12:395-399, work incorporated by reference in its entirety).
In the preferred embodiment, the immunogenicity of IgG variants determined experimentally, using one or more cellular analyses. For experimental confirmation epitopes you can apply several methods. In the preferred embodiment for experimental quantitative evaluation of the immunogenicity apply the activation of T cells ex vivo. In this method, antigen-presenting cells and native T-cells from a matching donor one or more times provoke peptide or whole protein of interest. Activation of T cells detected using a number of methods, for example by monitoring the production of cytokines or measurements of absorption thymidine labeled with tritium. In the most preferred embodiment of the monitor gamma-interferon products using Elispot analysis (Schmittel et al., 2000, J. Immunol. Meth. 24:17-24, work incorporated by reference in its entirety).
Biological properties of IgG variants can be characterized in experiments on cells, tissues, or whole organism. As is well known in this technical field, karstenia drugs are often tested on animals, including but not limited to the above, mice, rats, rabbits, dogs, cats, pigs, and monkeys, in order to determine the effectiveness of drugs for treatment of illness or disease models; or to measure the pharmacokinetics, toxicity, and other properties of drugs. Animals can be classified as disease models. A therapeutic agent is often experienced in mice, including but not limited to the above, hairless mice, SCID mice, xenotransplantation mice and transgenic mice (including nocini and knockouts). Such an experiment can provide very important information to identify potential protein used as a therapeutic agent. Any organism, preferably a mammal, can be used in the tests. For example, due to the fact that genetically monkeys is similar to a person, they can be suitable therapeutic models, and thus can be used to test the efficacy, toxicity, pharmacokinetics, or other properties of IgG. Ultimately for testing them as medicines required trials in humans, and, of course, therefore, such experiments are provided. Thus, IgG can be tested on humans to determine therapeutic efficacy, toxicity, immunogenicity is of farmacocinetica and/or other clinical properties.
IgG variants may find application in a wide range of products. In one of the embodiments of the IgG-option is therapeutic, diagnostic, or a research reagent, preferably a therapeutic tool. IgG-option can be used in the compositions of the antibodies can be monoclonal or polyclonal. In the preferred embodiment of the IgG variants are used for the destruction of target cells, which are antigen-target, for example cancer cells. In an alternate embodiment of the IgG variants are used to block the antigen is a target to be antagonists or agonists of the target antigen, for example, to be antagonists of cytokine or cytokine receptor. In an alternative preferred embodiment of the IgG variants are used to block the antigen is a target to be antagonists or agonists of the target antigen and to kill target cells bearing the antigen target.
IgG variants can be used for different therapeutic purposes. In the preferred embodiment of the antibodies, including IgG-option, enter the patient for the treatment of antibody-dependent disorders. The term "patient" for purposes of the present invention includes both humans and other animals, preferably mammals and most preferably people who. The terms "antibody-dependent disorder"or "antibody-responsive disorder"or "condition"or "disease" here refers to the disorder, which can be enhanced by the introduction of a pharmaceutical composition comprising IgG-option. Antibody-dependent disorders include, but are not limited to, autoimmune diseases, immunological diseases, infectious diseases, inflammatory diseases, neurological diseases and cancer and neoplastic diseases, including cancer. The terms "cancer" and "cancerous" relate or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, Sanoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
In one embodiment of the IgG-option is the only therapeutically active agent, administered to the patient. Alternatively, IgG-option administered together with one or more therapeutic means, including, but not limited by them, cytotoxic tools, chemotherapeutic agents, cytokines, means inhibiting growth, antihormone medium spans the VA, kinase inhibitors, antiangiogenic tools, cardioprotector or other therapeutic agent. IgG variants may be introduced in addition to one or more other therapeutic regimens. For example, IgG-variant can be administered to the patient along with chemotherapy, radiotherapy or concurrent with chemotherapy and radiotherapy. In one of the embodiments of the IgG variant can be introduced in combination with one or more antibodies, which may or may not be IgG-option. In accordance with another embodiment for effects on cancer cells ex vivo use of IgG-option and one or more kinds of anti-cancer therapy. I think, what is the impact of ex vivo may be useful in bone marrow transplantation and, in particular, when autologous bone marrow transplantation. Of course, I believe that IgG variants can be applied also in combination with other therapeutic techniques such as surgery.
For the introduction together IgG variants may find applications in many other therapeutic agents. In one of the embodiments of IgG administered together with an antiangiogenic agent. The term "angiogenic agent", which is used here, means a compound that blocks or to some extent hinders the development of blood vessels. Antiangiogenic agent, for example, may be a small the molecule or protein, for example antibody fused Fc or cytokines that are associated with growth factor or growth factor receptor involved in the stimulation of angiogenesis. Preferred angiogenic factor is an antibody that binds to endothelial growth factor vascular (VEGF). In an alternate embodiment of IgG administered together with a therapeutic tool that enables or enhances the adaptive immune response, for example, an antibody, a target which is CTLA-4. In an alternate embodiment of IgG administered together with a tyrosine kinase inhibitor. The term "tyrosine kinase inhibitor". who here used, means a molecule, which to some extent inhibits tyrosinekinase tyrosine kinase activity. In an alternate embodiment of the IgG variants are administered together with a cytokine.
Provides pharmaceutical compositions, which include IgG-option and one or more therapeutically active agents. Composition IgG-options receive for storage by mixing the IgG having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed., 1980, work incorporated by reference in its entirety) in the form of lyophilised compositions or aqueous solutions. Compositions used for suggesting the in vivo preferably are sterile. This can be easily done by filtration through sterile filtration membranes or in other ways. IgG variants and other therapeutically active products disclosed here can be used in compositions in the form of immunoliposome and/or put in microcapsules.
The concentration of therapeutically active IgG-variant in the composition can vary from about 0.1 to 100% by weight. In the preferred embodiment, the IgG concentration is in the range of from 0.003 to 1.0 M For the treatment of the patient can enter a therapeutically effective dose of IgG-option. The term "therapeutically effective dose" means the dose that causes the effects for which it is injected. The exact dose will depend on the goals of treatment and is installed by the technician using known techniques. The dosage may range from 0.01 to 100 mg/kg body weight or more, such as 0.01, of 0.1, 1.0, 10, or 50 mg/kg body weight, preferably from 1 to 10 mg/kg As is well known in this technical field may need to be adjusted, taking into account the degradation of the protein, the ratio of system delivery versus localized delivery, and rate of synthesis of new proteases, as well as the age, body weight, General health, sex, diet, time of administration, drug interaction and the severity of the condition, and this will be confirmed by experts in PR is the process routine experimental work.
Introduction pharmaceutical composition comprising IgG-variant, preferably in the form of a sterile aqueous solution, may be done in many ways, including, but not limited only to them, oral, subcutaneous, intravenous, parenteral, intranasal, custom instruments, intraocular, rectal, vaginal, dermal, surface (for example, when using gels, ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intra-lungs (for example, when applying the method of inhalation, AERx®, commercially available from Aradigm, or in the application delivery system in light Inhance™, commercially available from Nektar Terapeutics") routes of administration, etc. of a Therapeutic agent described herein, can be added together with other therapies, that is, a therapeutic agent described herein, can be introduced in conjunction with other therapies or in combination with therapies that include, for example, small molecules, and other biological compounds, radiation therapy, surgery, etc.
Below are examples illustrating the present invention. This does not mean that these examples limit the scope of the present invention any private ways or theories DEISTVIYa all positions discussion in the present invention, the numbering corresponds to the EU index in Kabat database (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Intitules of Health, Bethesda, work incorporated by reference in its entirety). Specialists in this area of technology will recognize that this arrangement includes inconsistent numbering in certain parts of the sequence of immunoglobulins, which allows normalizename to refer to a conservative position in the collections of immunoglobulins. Thus, the position of any given immunoglobulin determined using the index EU, not necessarily coincide with its sequential numbering.
Example 1: Construction of DNA, expression and purification of Fc-options
Fc variants were constructed using the Fc-domain of human IgG1 and the variable regions of trastuzumab (Herceptin®, Genentech). Fc-polypeptides were part of alemtuzumab, trastuzumab or AS. Alemtuzumab (Campath®, brand of the company "Millenium") is humanitariannet monoclonal antibody approved currently for the treatment of chronic b-cell lymphocytic leukemia (Hale et al., 1990. Tissue Antigens 35:118-127, work incorporated by reference in its entirety). Trastuzumab (Herceptin®, brand of the company "Genentech") is an antibody to Her2/neu to ensure metastatic the ski breast cancer. AS is a monoclonal antibody to CD30. Variable region of Herceptin was using the method of recursive PCR. This variable region was then cloned together with IgG1 in the vector pcDNA3.1/Zeo(+) (Invitrogen), shown in figure 11. Plasmids were propagated in cells One Shot TOP10 E. coli (Invitrogen) and purified using the set Hi-Speed Plasmid Maxi Kit (Qiagen). Plasmids sequenced to verify the presence of the cloned inserts.
Site-directed mutagenesis was performed using the method Quikchange™ (Stratagene). Plasmids containing the desired substitutions, insertions and deletions, were propagated in cells One Shot TOP10 E. coli (Invitrogen) and purified using the set Hi-Speed Plasmid Maxi Kit (Qiagen). DNA sequenced to confirm the accuracy of the sequence.
Plasmids containing heavy chain gene (VH-Cγ1-Cγ2-γ3) (wild type or variants), Ko was transfusional with plasmid containing light chain gene (VL-Cκ), in cells C. The medium was harvested 5 days after transfection and antibodies from the supernatant was purified using affinity chromatography on a column of protein A (Pierce). The antibody concentration was determined by analysis with bicinchoninic acid (ICA) (Pierce).
Example 2: Measurement of the affinity binding
The binding of the Fc-polypeptide with Fc-ligands was analyzed by measuring the surface plasma resonance. Measurement of surface plasma resonance (SPR) is bodily device BIAcore 3000 (BIAcore AB). Antibody wild-type or variant antibody recorded using immobilized protein L (Pierce Biotechnology, Rockford, IL) and measured the binding of the receptor with the analyte. Protein L is covalently linked with the touch chip CM at a concentration of 1 μm in 10 mm sodium acetate, pH 4.5 on the sensor chip CM using N-hydroxysuccinimide/N-ethyl-N'-(-3-dimethylamino-propyl)carbodiimide (NHS/EDC) with a flow rate of 5 ál/min Flow cell 1 touch each chip was treated with NHS/EDC as a negative control binding. Assay buffer was 0.01 M HEPES pH 7.4, 0,15M NaCl, 3 mm EDTA, 0.005% mass/mass surfactant P20 (HBS-EP, Biacore, Upsala, Sweden), and the buffer for the regeneration of the chip was 10 mm glycine model HC1 pH of 1.5. 125 nm antibody trastuzumab, wild-type or variant was associated with protein L-SM chip in HBS-EP at a speed of 1 μl/min for 5 minutes. Analyte FcRn-His-GST, FcRn, fused with His-tag and glutathione-S-transferase, in serial dilution ranging from 1 to 250 nm, was administered by injection to the Association within 20 minutes for dissociation within 10 min in HBS-EP at a speed of 10 ál/min, the Response measured in resonance units (RU) and reflecting close to the stationary link, found over 1200 seconds after the injection of the receptor. The cycle is carried out only with the antibody, and a buffer provided base level of the signal. Build a dependency graph EN 1/log of concentration and was approximatively in a sigmoidal curve dose-response using a non-linear regression, using the program GraphPad Prism.
The binding of the Fc-polypeptide with Fc ligands also was performed using analysis of AlphaScreen™ (homogeneous proximity analysis using enhanced luminescence). AlphaScreen™ - non-radioactive luminescent analysis measuring the vicinity. The excitation laser donor beads stimulates the oxygen, which in the case of closely spaced acceptor beads will cause a cascade of chemiluminescent phenomena, leading eventually to the fluorescence emission at 520-620 nm. The fundamental advantage of the analysis AlphaScreen™ is its sensitivity. As one donor bead allocates up to 60,000 of the excited oxygen molecules per second, the amplification of the signal becomes extremely high, which enables to detect the magnitude attomolar level (10-18). Antibody wild type was biotinilated using standard methods for joining streptavidine the donor beads and labeled Fc-ligand, such as FcRn, was attached to the acceptor beads, chelating glutathione. AlphaScreen™ was used as a analysis direct linking, in which the interaction of the Fc/Fc ligand led to the unification of the donor and acceptor beads. In addition, AlphaScreen™ was used in the competitive analysis for the screening of engineered Fc-polypeptides. In the absence of contention Fc-polypeptide antibody wildly what about the type and FcRn cooperated and gave the signal at 520-620 nm. Unlabeled Fc domains competed with the interaction of the wild-type Fc/FcRn, quantitatively reducing the fluorescence, which allows to determine the relative affinity of binding.
Example 3: FcRn-binding properties of the Fc-options.
The affinity of binding of the IgG1 Fc to FcRn was measured with variant antibodies using AlphaScreen™. Fc-polypeptides were part of alemtuzumab or trastuzumab. Alemtuzumab (Campath®, Ilex) is humanitariannet monoclonal antibody approved currently for the treatment of chronic b-cell lymphocytic leukemia (Hale et al., 1990. Tissue Antigens 35:118-127, work incorporated by reference in its entirety). Trastuzumab (Herceptin®, "Genentech") is an antibody to Her2/neu for the treatment of metastatic breast cancer.
Collected data competitive analysis AlphaScreen™ to measure the relative binding of Fc variants compared with the antibody wild type at pH 6.0. Examples of signal AlphaScreen™ as a function of competitive antibodies presented on figure 12. Curves for 12 options: P257L, P257N, V279E, V279Q, V279Y, ^281S, E283F, V284E, L306Y, T307V, V308F and Q311V, show an increased affinity, as in the graph at the appropriate curve for each case is shifted to the left from the curve for the wild type. Data competitive analysis AlphaScreen™ Fc variants of the present invention are summarized in figures 13 and 14. With the given table relative binding FcRn variants compared to wild type. Values greater than 1 show enhanced binding of Fc variants to FcRn compared to a wild type. For example, option E283L and V284E demonstrate binding to 9.5 and 26 times stronger than that of wild type, respectively. Measurement of surface plasma resonance many variants also show an increased binding to FcRn as shown in figures 15 and 16.
At position 257 all variants, which removed iminocyclitol Proline and replaced amino acid, which in the main circuit no N atom, the covalent bond of the side chain, provide greater mobility of the main chain, which provides greater freedom Fc-domain to the best of FcRn binding. In particular, variants with L and N at position 257 have a strong binding to FcRn at pH 6, which demonstrates that the 4-atom side chain and gamma-branched element side chain helps Fc-domain to do productive, that is, the stronger the interaction with FcRn. Position 308 interacts with the position 257. Both of these provisions, in turn, interact with the N310, which is directly involved in the interaction of the Fc/FcRn (table 2, Burmeister et al., 1994, Nature, 372:379-383, work incorporated by reference in its entirety). The affinity of Fc variants V308F and V308Y to FcRn 2.9 times and 4.3 times higher in comparison with the wild type (figure 13). Position 279 and 385 interact with FcRn, as all options V279E, V279Q and V279Y, G385, and G385N stronger contact with FcRn. All of these options are related to amino acids, which are capable of forming hydrogen bonds.
Fc-variant N434Y has a particularly strong binding to FcRn at pH 6.0, as shown in figure 13. A single option N434Y has a 16-fold increased binding. Combinations of these with other modifications lead to stronger binding. For example, P257L/N434Y, ^281S/N434Y and V308F/N434Y demonstrate increased binding of FcRn in 830,180 and 350 times.
Example 4: Alternatives, including insertions and deletions
Were designed insertions and deletions that alter the interaction force Fc/FcRn and were measured their binding properties towards different Fc ligands. To enhance FcRn-binding properties of the Fc-domain was engineered Fc-variant inserted with a Ser residue between 281 and 282 residues, in accordance with the EU index in Kabat et al. This option navamsa ^281S, where the symbol "^" means the insertion immediately after the specified position. The inserted sequence, which may include more than one residue assign the following position number. This Fc variant was constructed in Kappa-IgG1-the antibody trastuzumab (Herceptin®, "Genentech"), using the methods disclosed in this application. The inset on the site between residues 281 and 282 moves the remains of the Fc loop to the C-terminal side of residue 281 toward the C-terminal cha the t loop and changes the location of the side chain. Assume that the shift in the C-terminal part of the loop at Fc variants comprising substitutions at positions 282, 283 and 284, was best (see figure 14). To move the position of the FcRn-binding loop was also constructed another variant with a deletion of the N286, sometimes called N286#. Both of these options demonstrate increased affinity for the FcRn at pH 6.0.
The results of the analysis AlphaScreen™ show link option ^281S and other variants to FcRn. These results AlphaScreen™ was collected as a direct analysis of binding. Higher levels of the chemiluminescent signal show a more durable binding. With increasing concentrations of cases in the analysis get more powerful signals. The data at pH 6.0, shown in figures 17A and 17b show an increased affinity variants ^281S, P257L, P257L/^281S (combination of replacement and insertion) and other variants compared to wild-type Fc. Also shows a double substitution, T250Q/M428L, which, as was shown earlier, has an increased half-life in serum of monkeys (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, work incorporated by reference in its entirety). Insert ^281S itself enhances the binding of the Fc/FcRn. In addition, ^281S enhances the binding P257L, if option P257L/^281S combine two modifications, as shown in the results for the points ~40 nm. The results, shown in figure 17c, the dem will Instituut, what these options do not have an increased binding to FcRn at pH 7.0. Low affinity at pH 7.0, it is desirable to increase the half-life in vivo, as it allows the release of Fc polypeptides of the FcRn in the extracellular space, which is an important stage in the recycling of Fc.
Experiments with surface plasma resonance also exhibit improved binding ^281S with FcRn. Figure 18 shows the units of the signal arising from the binding of different Fc variants to FcRn on the surface of the chips. Once the option is fully associated with the chip, write unit signal, which is shown on the ordinate. Insert ^281S shows binding properties comparable to the other options shown here, which have an increased affinity for FcRn compared to a wild-type (see, for example, figures 13, 14 and 15).
Deletion variant N286#, including a deletion N286, also shows increased affinity for FcRn compared to a wild type. This option has a 2-fold increased affinity for FcRn, as shown in figure 13, there are also data analysis AlphaScreen™ research competition at pH 6.0. For inhibition of binding to Fc wild-type, attached to the donor beads, FcRn, is attached to the acceptor beads, used different ways. For inhibition of binding the Oia donor and acceptor beads through the complex Fc/FcRn was required in two times less free N286# compared with the free wild-type Fc. This shows 2-fold increased binding N286# compared to wild type.
Other Fc variants with insertions and division, had a reduced affinity for FcRn. Insertional option ^254N has significantly reduced FcRn binding, as would be expected from the nature of the option and its location. In this embodiment, the insert Asn placed in the middle of the FcRn-binding loop. This insert has only 1.1% of the binding affinity compared with the wild type (figure 13).
Despite the fact that the particular embodiment of the present invention have been described above for purposes of illustration, the experts in this field of technology it will be obvious that it is possible to make many modifications of parts without departure from the invention as described in the attached at the end of the text of the claims. All publications cited herein are hereby incorporated by reference in its entirety.
1. Polypeptide demonstrating an altered binding to FcRn, including Fc-variant of the original Fc-IgG polypeptide containing a modification in the Fc-section selected from the group consisting of S, 308F, 308W, 308Y, where the specified Fc-variant demonstrates increased binding to FcRn as compared with the initial Fc polypeptide, where the numbering corresponds U-index.
2. The polypeptide according to claim 1, where the specified Fc-variant contains at least one substitution selected from the group consisting the th from: 308F, 308W and 308Y.
3. The polypeptide according to claim 1 or 2, where the specified Fc-variant contains 308F.
4. The polypeptide according to any one of claims 1 to 3, where the specified Fc-variant demonstrates an altered binding to FcγR compared with the specified source Fc polypeptide.
5. The polypeptide according to any one of claims 1 to 4, where the specified Fc-variant demonstrates increased binding to FcγR.
6. The polypeptide according to any one of claims 1 to 4, where the specified Fc-variant shows reduced binding to FcγR.
7. The polypeptide according to any one of claims 1 to 4, where the specified Fc-variant demonstrates an altered binding to FcRn as compared with the initial Fc polypeptide, and where the specified polypeptide is additionally characterized by the fact that the polypeptide has specificity to the molecule target which is selected from the group consisting of a cytokine, a soluble protein factor and protein expressed on cancer cells.
8. Polypeptide demonstrating an altered binding to FcRn, including Fc-variant of the original Fc-IgG polypeptide, where the specified Fc-variant demonstrates increased binding to FcRn as compared with the initial Fc polypeptide, where the specified Fc-variant comprises a modification in the Fc-specified area of the original polypeptide selected from the group consisting of 252Y/308F, 257L/308F, 257L/308Y, 257N/308Y, 279Q/308F, 279Y/308F, ^281S/308F, ^281S/308Y, 284E/308F, 298A/308F/333A/334A, 308F/332E, 308F/311V, 308F/G385H, 308F/428L, and 308F/434Y, where ^ means the insertion, where num is the radio complies with EU-index.
9. The polypeptide of claim 8, where the specified Fc-variant demonstrates an altered binding to FcγR compared with the specified source Fc polypeptide.
10. The polypeptide according to any one of p and 9, where the specified Fc-variant demonstrates increased binding to FcγR.
11. The polypeptide according to any one of p and 9, where the specified Fc-variant shows reduced binding to FcγR.
12. The polypeptide according to any one of p and 9, where the specified Fc-variant demonstrates an altered binding to FcRn as compared with the initial Fc polypeptide, and where the specified polypeptide is additionally characterized by the fact that the polypeptide has specificity to the molecule target which is selected from the group consisting of a cytokine, a soluble protein factor and protein expressed on cancer cells.
13. Antibody, demonstrating an altered binding to FcRn, containing Fc variant according to any one of claims 1 to 12.
14. The use of Fc variants described in any one of claims 1 to 13, to obtain merged with Fc protein, demonstrating increased binding to FcRn.
SUBSTANCE: binding molecule represents a CD45RO and CD45RB chimeric antibody. The molecule contains two domains with consistent hypervariable sites CDR1, CDR2 and CDR3, and CDR1', CDR2' and CDR3', CDR1 has amino acid sequence NYIIH, CDR2 has amino acid sequence YFNPYNHGTKYNEKFKG, and CDR3 has amino acid sequence SGPYAWFDT. CDR1' has amino acid sequence RASQNIGTSIQ, CDR2' has amino acid sequence SSSESIS, and CDR3' has amino acid sequence QQSNTWPFT. Related coding polynucleotide is described.
EFFECT: use of the invention allows to induce immunosuppression, to inhibit T-cell response and primary lymphocyte reaction in the mixed culture, to prolong survival time in mice with severe combined immunodeficiency SCID.
6 cl, 5 dwg, 2 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention relates to immunology and biotechnology. The invention discloses versions of a cytotoxically active CD3-specific binding structure. The structure comprises a first domain specifically binding to human CD3 and an Ig-derived second binding domain which is specific to molecules on the cell surface. The invention describes a coding nucleic acid, a vector for expressing the structure and an eukaryotic cell transformed by the vector. The invention discloses versions of compositions based on the structure for treating, preventing or alleviating various diseases and corresponding methods of treating the diseases. A method of obtaining the structure is disclosed.
EFFECT: use of the invention provides a structure with low immunological potency, which has cytotoxicity comparable to the initial structure, which may find further use in medicine.
60 cl, 18 dwg, 15 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and is an antibody which bonds with OX40L and variants of this antibody which contain certain Fc-fragments obtained from the human body and do not bond with the complement factor Clq. The monoclonal antibody is produced by a cell line selected from a group which includes cell lines deposited in the German collection of microorganisms and cell cultures (DSMZ) under inventory numbers No. DSM ACC 2685, DSM ACC 2686, DSM ACC 2688, DSM ACC 2689. The invention also relates to a method of obtaining such antibody, to nucleic acid molecules which code the disclosed antibody. The disclosed antibody has an advantage for patients suffering from inflammatory diseases.
EFFECT: antibody is used in diagnostic composition for detecting OX40L in vitro, in a pharmaceutical composition for preventing and treating inflammatory diseases, as well as in preparing a medicinal agent for preventing and treating inflammatory diseases.
9 cl, 20 dwg, 7 tbl, 23 ex
SUBSTANCE: proposed is a chimeric or humanised monoclonal antibody against hepatocyte growth factor, produced from L2G7 antibody. Invented is a mouse antibody L2G7, produced by hybridoma ATCC PTA-5162, and the said hydbridoma. Described is a cell line, producing a chimeric or humanised monoclonal antibody against hepatocyte growth factor. Proposed is a pharmaceutical composition and a method of treating tumours based on the said antibody.
EFFECT: use of the invention provides for a neutralising antibody against hepatocyte growth factor, which can be used in treating human cancer.
7 cl, 12 dwg, 1 tbl, 3 ex
SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.
EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.
10 cl, 21 dwg, 11 ex
SUBSTANCE: present invention relates to biotechnology and immunology. Proposed here is a polynucleotide, encoding a cyclic single-stranded tri-specific antibody. The antibody is directed against human ovarian carcinoma in vitro, has mass of approximately 84 kD and consists of three components: an antibody against human ovarian carcinoma cells, anti-CD3 antibody and anti-CD28 antibody, which are joined together by peptide interlinks such that, they form a cyclic antibody. Invented is an expression vector, containing a coding polynucleotide and versions of E.coli host cell based on the polynucleotide and expression vector.
EFFECT: use of the invention provides for a stable antibody molecule, optimum for activation of T-cells, which can be used in curing human ovarian carcinoma.
8 cl, 12 dwg
SUBSTANCE: versions of the bond intended for linkage with the external domain B (ED-B) of a fibronectin are offered. The bond includes an antigen-binding fragment of one-chained antibody L19 and a cysteinum-containing linker for hanging of a radioactive label. Versions of a pharmaceutical composition for diagnostics and treatment of angiogenic diseases on the basis of the specified bond are opened. Application of bond for linkage with radioactive bond is described. The method of reception of bond in eucariotic cells is opened, including in Pichia pastoris and a set for reception is radioactive labelled agents on the basis of bond.
EFFECT: high-avid bond accumulation in solid tumours.
23 cl, 4 dwg, 5 tbl, 15 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.
EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.
22 cl, 13 dwg, 8 tbl
FIELD: medicine, microbiology.
SUBSTANCE: invention concerns biotechnology. It is described bispecific antibody which binds also the factor of blood coagulation IX or the activated factor of blood coagulation IX, and the factor of blood coagulation X, and functionally replaces the factor of blood coagulation VIII or the activated factor of blood coagulation VIII which strengthens enzymatic reaction. The pharmaceutical composition containing the described antibody is revealed. The present invention can be used as an alternative agent for functional replacement of cofactor which strengthens enzymatic reaction.
EFFECT: creation of bispecific antibody which can replace functional proteins, strengthens enzymatic reaction.
14 cl, 18 dwg, 37 ex
SUBSTANCE: invention relates to biotechnology and immunology. Claimed is therapeutically active fused protein with reduced immunogenicity. Protein consists of two proteins derived from human proteins connected through the fusion region. Connective region, which covers or surrounds fusion region within the limits from 1 to 25 amino acid residues, contains modification, which removes T-cell epitope, in norm absent in humans. Claimed is application of fused protein for obtaining pharmaceutical composition for tumour treatment. Claimed is nucleic acid coding fused protein. Method of reduction of fused protein immunogenicity by introduction of substitutes of corresponding amino acids is described. Application of the invention allows reducing ability of connective epitope of therapeutically active fused protein to bind with molecules of the main complex of hystocompatibility (MHC) of class II, which finally reduces interaction of epitope with receptors of T-cells and can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.
EFFECT: reduction of interaction of epitope with receptors of T-cells, which can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.
23 cl, 12 ex
SUBSTANCE: compound contains two or more V-areas of H-chain and two or more V-areas of L-chain of monoclonal antibody, capable of recognizing receptors of thrombopoietin and their transverse suturing in intercellular mode, and representing integral antibidy or F(ab')2. Compound may specifically recogniser and transversely suture receptor of thrombopoietin, manifesting, meanwhile, same or higher agonistic action (ED50) compared to thrombopoietin.
EFFECT: modification of antibody molecule results in reduction of side effects caused by formation of intercellular transverse ties, and compound of invention, having properties of thrombopoietin agonist of signal transduction, may be used as preventive or medicinal agent in case of various diseases, such as blood diseases, illnesses related to reduction of platelets number, thrombopenia, which accompanies chemotherapy in case of cancer or leukaemia.
7 cl, 60 dwg, 3 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to biotechnology. The invention describes an antibody which specifically identifies a membrane or secretory protein selected from several proteins with sequences given in the application materials, or functional fragment thereof. The invention discloses a hybridoma which produces the said antibody and is deposited under inventory No. FERM BP-10376. A medicinal agent used in an autoimmune disease and which contains the described antibody or its functional fragment as an active ingredient is proposed. Described also, is a T cell adhesion inhibiting agent which contains the described antibody or its functional fragment as an active ingredient. The invention discloses a method of screening a substance which inhibits dendritic cell activation, or its salts or solvate involving the following steps: bringing the said antibody or its functional fragment into contact with a dendritic cell in the presence or absence of the substance under test and measuring the activation level of the said dendritic cell.
EFFECT: invention widens the range of agents for treating rheumatoid arthritis.
18 cl, 24 dwg, 12 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discovers an antibody containing a Fc-version component with at least one amino acid substitution in a Fc-region of initial polypeptide. Said substitution is in the amino acid position matched with that of human Fc-polypeptide amino acid sequence (the positions are presented in the patent claim). The antibody exhibits a modulated binding with FcγR. There are described versions of said antibody with increased affinity to FcγR wherein said substitution is in the amino acid position 239, 332 or 267 matched with that in human Fc-polypeptide amino acid sequence (numeration according to the EU index). The invention also describes a method of treating a mammal with an antibody-dependent disorder by using a pharmaceutical composition with the modulated linkage with FcγR and containing the antibody or its version.
EFFECT: according to the invention antibodies provide the affinity in 5 and more times higher in comparison with initial Fc-polypeptide.
43 cl, 44 dwg, 66 tbl, 12 ex
SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.
EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.
57 cl, 45 dwg, 4 tbl, 8 ex
SUBSTANCE: there is offered application of group of survival-improving polypeptide cone cells originated from rod cells and designated as RDCF, and also coding molecules of nucleic acid to prepare medicines, particularly pharmaceutical compositions used to treat retinal dystrophy. Methods for preparing RDCF by recombinant DNA technologies, and required aids, as well as preparation of antibodies distinguishing said polypeptides are described.
EFFECT: improved clinical effectivenesses.
12 cl, 19 dwg, 1 ex
SUBSTANCE: invention is related to biotechnology and represents monoclonal antibody or its antigen-binding fragment, containing antigen-binding site, which specifically binds with protein of prostatic stem cells antigen (PSCA). At the same time monoclonal antibody is produced with postfusional cell line, selected from group of postfusional cell lines deposited in American typical cultures collection (A.T.C.C.) under inventory No.PTA-6698, PTA-6699, PTA-6700, PTA-6701, PTA-6702 and PTA-6703. Besides invention is also related to expression vector, which contains polynucleotide coding this antibody, and also to method for analysis for detection of PSCA protein presence in biological sample with application of this antibody. Moreover, invention is related to method for delivery of cytotoxic agent or diagnostic agent to cell, which expresses PSCA protein, with application of this antibody, and also to method for detection of PSCA protein in biological sample with application of above-mentioned antibody.
EFFECT: invention may efficiently be used in active and passive immunisation against cancer diseases.
21 cl, 27 dwg, 11 tbl, 33 ex
SUBSTANCE: invention can be used for production of monoclonal antibodies (MCAs) to heat shock protein 70 (HSP 70). A hybridoma strain is made by immunisation of BALB/c mice with bovine HSP 70 within 78 days. For the third days, splenocytes of immune mice (108 cells) are hybridised with murine myeloma cells P3-X63 Ag/8-653 (107 cells). A fusion agent is polyethylene glycol of molecular weight 4000 (Merk, Germany). The hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma. Hybridoma 6G2 is deposited in the microorganism collections of "ГНТТ ПМБ" under No. H-2. MCA.
EFFECT: produced hybridoma under the invention is more evident to be detected as HSP 70 on the cell surfaces, and change of endocellular HSP 70 level when exposed to the stress factors.
4 dwg, 1 tbl, 6 ex
SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.
EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.
16 cl, 4 dwg, 1 tbl, 13 ex
SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.
EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.
16 cl, 34 dwg, 7 tbl, 20 ex
SUBSTANCE: invention relates to Bcl-2 proteins, fragments thereof, and to application thereof in patients with a malignant tumour. The declared proteins and peptide fragments particularly are applicable in vaccine compositions for treatment of malignant tumour. Besides, the invention concerns the methods of treatment with application of specified compositions. Also, an aspect of the invention is production of T-cells and receptors thereof which are specifically recognise declared proteins and peptide fragments.
EFFECT: higher clinical effectiveness with respect to tumours.
61 cl, 5 ex, 2 tbl, 12 dwg
FIELD: biotechnology, medicine, proteins.
SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.
EFFECT: valuable medicinal properties of polypeptide.
19 cl, 33 dwg, 1 ex