Fused proteins binding immunoglobulin domain
SUBSTANCE: offered is a fused protein containing from an amino-terminus to carboxyl terminus: binding domain polypeptide which is bound with a biological target molecule; human one-cysteine "link" region IgGl peptide; immunoglobulin heavy chain CH2 constant region polypeptide and immunoglobulin heavy chain CH3 constant region polypeptide. The fused protein represents mixed monomers and dimers, has ADCC, CDC or both cytotoxicities. Also, described are a pharmaceutical composition and a method of treating a B-cell disorder with using the fused protein.
EFFECT: use of the invention can find the further application in medicine in treating B-cell disorders.
26 cl, 26 dwg, 9 ex
The premise of the INVENTIONS
This invention in General relates to immunologically active recombinant binding proteins and, in particular, to the designed molecules fused protein binding domain-immunoglobulin, including fused protein of single-chain Fv immunoglobulin. This invention also relates to compositions and methods for treatment of malignant conditions, and b-cell disorders, including diseases, which are characterized by production of autoantibodies.
The immunoglobulin molecule consists of two identical light chains and two identical heavy chains, which are connected in a macromolecular complex megamachine disulfide bonds. Interchain disulfide bonds link the different areas of the same polypeptide chain, and this leads to the formation of loops, which together with the adjacent amino acid to form the immunoglobulin domains. Each light chain and each heavy chain has one variable region, which displays considerable variability in amino acid composition from antibody to antibody. Variable region light chain, VLassociated with the variable region of the heavy chain VHforming antigennegative site immunoglobulin Fv. Light chains have one domain of a constant region and a heavy chain have multiple domains constant the second area. Classes IgG, IgA and IgE have three domain constant region, which are labeled CH1, CH2 and CH3, and the classes IgM and IgE have four domain constant region.
The heavy chain of immunoglobulins can be divided into three functional areas: Fd, hinge and Fc. The area contains Fd domains VHand CH1 and with the light chain forms a Fab. Usually consider that the Fc fragment is responsible for the effector functions of an immunoglobulin, such as the fixation of complement and binding to Fc-receptors. The hinge area is identified in the classes IgG, IgA, and IgD, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions of the hinge domains are diverse in structure, and vary both in sequence and length among the classes and subclasses of immunoglobulins. For example, three subclasses of IgG human IgG1, IgG2 and IgG4 are the hinge region of 12-15 amino acids, whereas IgG3 contains about 62 amino acids, including 21 residue of Proline and 11 cysteine residues. According to crystallographic studies of the hinge, in addition, can functionally be divided into three areas: the upper hinge, the Central part and the lower hinge (Shin et al., Immunological Reviews 130: 87 (1992)). The upper hinge includes amino acids from the carboxyl end of the CH1 to the first residue in the hinge, which restricts movement, usually before the first octadecylamine, which forms miaocheng disulfide bonds between the two heavy chains. The length of the upper hinge region correlates with the flexibility plots antibodies. The Central hinge region contains disulfide bridges within the heavy chains, and the lower hinge region associated with aminocom.com CH2 domain and includes residues in the CH2 (ibid.). The Central hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys, which in the formation of disulfide bonds gives cyclic oktapeptid, presumably acting as a pivot point, thereby imparting flexibility. The hinge region may also contain sites of attachment of carbohydrates. For example, IgA1 contains five sites for carbohydrates within the area of the hinge region of 17 amino acids, giving the hinge exceptional resistance to proteases of the intestine, which is the property that gives the advantage Sekretareva immunoglobulin.
Conformational changes allowed by the structure and flexibility of the hinge region can affect the effector functions of the Fc portion of antibodies. Three main categories of effector functions associated with the Fc-region include (1) activation of the classical complement cascade, (2) interaction with effector cells, and (3) the compartmentalization of immunoglobulins. Different human IgG subclasses vary in their from oxytelinae efficiency in the activation and amplification stages of the complement cascade. In total, IgG1 and IgG3 is the most effective fix complement, IgG2 less effective and IgG4 does not activate complement. Activation of complement is initiated by the binding of C1q, subunit of the first component C1 in cascade, with the complex antigen-antibody. Although the C1q binding site localized in the CH2 domain of the antibody hinge region affects the ability of the antibody to activate the cascade. For example, recombinant immunoglobulins lacking the hinge region, is not able to activate the complement (ibid.). In the absence of flexibility, attached to the hinge region, the Fab part of the antibodies bound to the antigen, are not able to adopt a conformation that is required in order to enable C1q contact CH2 (see ibid.). Studies have shown that the length of the hinge and the flexible sections correlates with activation of complement; however, the correlation is not absolute. Molecules IgG3 human modified hinge regions, which are as inflexible as IgG4, still effectively activate the cascade.
No hinge region also affects the ability of IgG-immunoglobulin human contact with Fc receptors on immune effector cells. Binding of antibody to Fc receptor contributes dependent antibodies cellular cytotoxicity (ADCC), which presumably is an important way elimin the tion of tumor cells. The family of IgG Fc receptors person is divided into three groups, FcγRI (CD64), which is able with high affinity to bind IgG, FcγRII (CD32) and FcγRIII (CD16), both of which are low-affinity receptors. Molecular interaction between each of the three receptors and immunoglobulin is not precisely determined, but experiments indicate that residues in the proximal relative to the hinge region, CH2 domain are important for the specificity of the interaction between the antibody and the Fc receptor. In addition, proteins IgG1 myeloma and recombinant chimeric IgG3 antibodies lacking the hinge region, is not able to bind FcγRI, probably because of reduced accessibility to CH2 (Shin et al., Intern. Rev. Immunol. 10: 177, 178-79 (1993)).
The technology of monoclonal antibodies and genetic engineering methods has led to the rapid development of molecules of immunoglobulins for the diagnosis and treatment of human diseases. Designing proteins was used to increase the affinity of antibodies against them recognizable antigen to reduce the problems associated with immunogenicity, and to change the effector functions of antibodies. The domain structure of immunoglobulins is amenable to construction, which can be exchanged antihistamine domains and domains, giving effector functions between classes and subclasses of immunol Bulanov.
In addition, designed smaller molecules of immunoglobulins, in order to overcome the problems associated with the treatment on the basis of whole immunoglobulins. Single-chain Fv (scFv) contain the variable domain of the heavy chain, linked by a short linker peptide with the variable domain of the light chain (Huston et al. Proc. Natl. Acad. Sci. USA, 85: 5879-83, 1988). Because of the small size of scFv molecules is observed very rapid clearance from plasma and tissues and more effective penetration into tissue than the whole immunoglobulin. Antitumor scFv showed a more rapid penetration into the tumor and a more uniform distribution in the tumor weight than the corresponding chimeric antibody (Yokota et al., Cancer Res. 52, 3402-08 (1992)). Fusion of scFv with another molecule, such as a toxin, has the advantage of specific antigennegative activity and the small size of scFv to deliver the toxin to the target tissue (Chaudary et al., Nature 339: 394 (1989); Batra et al., Mol. Cell. Biol. 11: 2200 (1991)).
Despite the advantages that bring scFv molecules to serotherapy, there are several disadvantages of this therapeutic approach. Although rapid clearance of scFv may reduce toxic effects in normal cells, this rapid clearance may prevent the delivery of the minimum effective dose in the target tissue. The production of adequate quantities of scFv for introducing patie there was a difficult task due to the difficulty, associated with the expression and secretion of scFv that adversely affect the output. During the expression of the scFv molecule is unstable and often aggregate in the mating variable regions from different molecules. In addition, the levels of production of scFv molecules in expressing systems of mammals is low, which limits the possibility of effective production of scFv molecules for therapy (Davis et al., J. Biol. Chem. 265: 10410-18 (1990); Traunecker et al., EMBO J. 10: 3655-59 (1991)). Identified ways to improve products, including the addition of glycosylation sites to the variable regions (Jost, C. R. U.S. Patent No. 5888773, Jost et al., J. Biol. Chem. 269: 26267-73 (1994)).
Conjugation or fusion toxins with scFv provides a very effective molecule, but dosing is limited by the toxicity caused by the toxin molecule. Toxic effects include increased levels of liver enzymes and syndrome semeneya vascular permeability. In addition, immunotoxins are highly immunogenic and antibodies owner made against toxin, limit the possibility of re-treatment.
An additional disadvantage of using scFv for therapy is the lack of effector functions. scFv in the absence of cytolytic functions, ADCC and dependent complement cytotoxicity (CDC), associated with the constant region of the immunoglobulin may be effective in treatment the disease. Although the development of methods of scFv started 12 years ago, there is currently no scFv-products approved for therapy.
The advantage of the effector functions associated with the constant region of the antibody, for the treatment of the disease has prompted the development of a fused protein in which nimmanahaeminda sequence replace the variable region of the antibody. For example, CD4 surface protein T-cell recognized HIV, recombinant method was merged with the effector domain, immunoglobulin Fc (See Sensel et al., Chem. Immunol. 65: 129-158 (1997)). The biological activity of this molecule in part will depend on the class and subclass of the selected constant region. Protein IL-2-IgG1 influenced mediated complement lysis of cells bearing the receptor of IL-2 (See ibid.). The application of the constant regions of immunoglobulins to construct these and other fused proteins can also give improved pharmacokinetic properties.
Diseases and disorders that are deemed amenable to certain types of immunoglobulin therapy include cancer and disorders of the immune system. Cancer encompasses a broad range of diseases that affect approximately one in four people worldwide. Rapid and uncontrolled proliferation of malignant cells is the hallmark of many types of malignant tumors, including hematologic the ski malignancy. The greatest benefit of advances in the treatment of malignant tumors in the last two decades have brought patients with hematological malignant state (Multani et al., J. Clin. Oncology 16: 3691-3710, 1998). Although the increased proportion of remission, most patients still relapse and they die because of the disease. Barriers to treatment of cytotoxic drugs include the resistance of tumor cells and high toxicity of chemotherapy that interfere with optimal dosing for many patients. New treatments based on the fact that to send to the target molecules that are specific associated with malignant cell, including monoclonal antibodies (MAB)that increase efficiency without increasing toxicity.
Since in 1975 were first described Mat (Kohler et al., Nature 256: 495-97 (1975)), many patients were treated Mat to antigens expressed on tumor cells. These studies have yielded important experience concerning the selection of antigenic targets that are suitable for therapy. First, and most important antigenic target should not be expressed critical normal tissues. Fortunately malignant blood cells Express many antigens that are not expressed in stem cells or other vital cells. Treatment heme is tological malignant condition, which depletes populations of both normal and malignant cells of hematological origin, acceptable, because after the end of therapy is the regeneration of normal cells from precursors. Secondly, antigenic target should be expressed at all count of clonogenic populations of tumor cells, and expression should continue, despite the selective pressure immunoglobulin therapy. Thus, the choice of surface idiotype for therapy of b-cell malignancy is limited by the growth options of tumor cells with altered expression of surface idiotype, even if the antigen shows high degree of selectivity towards tumor (Meeker et al., N. Engl. J. Med. 312: 1658-65 (1985)). Thirdly, the selected antigen should properly be moved after binding of immunoglobulin. Reset or the internalization of antigenic targets after the immunoglobulin binds to the antigen, can give the ability of tumor cells to escape destruction, thereby limiting the effectiveness of serotherapy. Fourthly, the binding of antibody with antigen targets that transmit signals activation can be enhanced functional responses in tumor cells, which lead to a stop of growth and apoptosis. Although all these important t the VA, the apoptosis after binding of the immunoglobulin to the antigen can be a critical factor in achieving a successful serotherapy.
The antigens that were tested as targets for serotherapy B - and T-cell malignancies include idiotype Ig (Brown et al., Blood 73: 651-61 (1989)), CD19 (Hekman et al., Cancer Immunol. Immunother. 32: 364-72 (1991); Vlasveld et al., Cancer Immunol. Immunother. 40: 37-47 (1995)), CD20 (Press et al., Blood 69: 584-91 (1987); Maloney et al., J. Clin. Oncol. 15: 3266-74, (1997)), CD21 (Scheinberg et. al., J. Clin. Oncol. 8: 792-803, (1990)), CD5 (Dillman et al., J. Biol. Respn. Mod. 5: 394-410 (1986)) and CD52 (CAMPATH) (Pawson et al., J. Clin. Oncol. 15: 2667-72, (1997)). Of them the greatest success was achieved with the use of CD20 as a target for therapy of B-cell lymphoma. Each of the other targets was limited biological properties of the antigen. For example, surface idiotype can be modified somatic mutation, providing salvation tumor cells. CD5, CD21 and CD19 quickly internalized after bonding the Mat, allowing tumor cells to escape destruction if the Mat is not conjugated to a toxic molecules. CD22 is expressed only on a subset of b-cell lymphomas, whereas CD52 is expressed on T-cells and B-cells and causes immunosuppression due to the depletion of populations of T cells.
CD20 meets the above criteria for selecting a suitable antigenic target for therapy of B-cell malignant condition. L is the treatment of patients with b-cell lymphoma, low-grade or follicular b-cell lymphoma using chimeric CD20-Mat induces partial or complete response in many patients (McLaughlin et al., Blood 88: 90a (abstract suppl. 1) (1996); Maloney et al., Blood 90: 2188-95 (1997)). However, usually the tumor recurrence during the period of time from six months to one year. Thus, further improvements are required of serotherapy in order to induce a more prolonged response in B-cell lymphoma low-grade malignancy and to ensure the possibility of effective treatment of lymphoma high grade and other b-cell diseases.
One approach to improve serotherapy CD20 was targeted delivery of radioisotopes to B-cell lymphomas using the Mat, it is specific to CD20. Although increasing the effectiveness of therapy also increases related toxicity because of the long half-life radioactive antibodies in vivo, sometimes requiring in order to expose the patient to the procedure of salvation stem cells (Press et al., N. Eng. J. Med. 329: 1219-1224, 1993; Kaminski et al., N. Eng. J. Med. 329: 459-65 (1993)). Mat CD20 were digested with proteases, receiving the fragments F(ab')2or Fab to bind to the radioisotope. This increased penetration of radioisotope conjugate in the tumor and shortened the half-life in vivo, thereby reducing toxicity to normal tissues. However, losing the advantages of effector functions including complement fixation and ADCC, which provides Fc-about the region CD20-mattack way for improved delivery of radioisotopes required method of deriving CD20-Mat, which keeps Fc-dependent effector functions, but smaller in size, thereby enhancing penetration into the tumor and shortening the half-life of the Mat.
CD20 was the first surface molecule person-specific B-cell lines, identified by monoclonal antibodies, but the function of CD20 in the biology of B-cells is not yet fully understood. CD20 is deglycosylation hydrophobic phosphoprotein with Mm 35 KD, amino and carboxyl ends of which are located in the cytoplasm (Einfeld et al., EMBO J. 7: 711-17 (1988)). Natural ligands CD20 were not identified. The CD20 antigen is expressed in all normal Mature B-cells, but not expressed by precursor B-cells.
CD20-Mat delivers the signals to normal B-cells, which affect the viability and growth (Clark et al., Proc. Natl. Acad. Sci. USA 83: 4494-98 (1986)). Recent data showed that extensive crosslinking of CD20 can induce apoptosis of cell lines b-cell lymphoma (Shan et al., Blood 91: 1644-52 (1998)). The crosslinking of CD20 on the cell surface increases the value and the kinetics of signal transduction, which was recorded by measuring the phosphorylation of cellular substrates on tyrosine residues (Deans et al., J. Immunol. 146: 846-53 (1993)). It is important that the apoptosis of B-cell lymphoma Ramos was also induced Shiva the receiving CD20-Mat adding cells, positive in relation to the Fc-receptor (Shan et al., Blood 91: 1644-52 (1998)). Thus, in addition to depleting populations of cells through mechanisms based on complement fixation and ADCC binding of Fc-receptor CD20-Mat in vivo can stimulate apoptosis of malignant B-cells by crosslinking of CD20. This theory is consistent with experiments showing that the effectiveness of therapy using human CD20 lymphoma model in SCID mice was dependent on the binding of Fc-receptor Mat to CD20 (Funakoshi et al., J. Immunotherapy 19: 93-101 (1996)).
The CD20 polypeptide contains four transmembrane domain (Einfeld et al., EMBO J. 7: 711-17, (1988); Stamenkovic et al., J. Exp.Med. 167: 1975-80 (1988); Tedder et al., J. Immunol. 141: 4388-4394 (1988)). Multiple domains, passing in the membrane, prevents the internalization of CD20 after binding with the antibody. Specified as CD20 recognized as an important property for the effective therapy of b-cell malignancies, when murine CD20-Mat, 1F5, were injected with patients with B-cell lymphoma, which led to a significant depletion of the population of malignant cells and partial clinical responses (Press et al., Blood 69: 584-91 (1987)).
Because normal Mature B-cells also Express CD20, the population of normal B-cells are depleted during therapy CD20-antibody (Reff, M. E. et al., Blood 83: 435-445, 1994). However, after treatment of normal B-cells recovered from CD20-negative PressTV nikov B-cells; thus, patients who were treated with anti-CD20 therapy, do not have significant immunosuppression. The depletion of populations of normal B-cells may be useful in cases involving unusual production of autoantibodies, or other diseases, which can play the role of B-cells. Chimeric Mat, it is specific to CD20, consisting of the variable regions of the heavy and light chains of murine origin, merged with the constant regions of the heavy chain of human IgG1 and Kappa light-chain person, retain the ability to bind to CD20 and to mediate ADCC and fix complement (Liu et al., J. Immunol. 139: 3521-26 (1987); Robinson et al., U.S. patent No. 5500362). This work led to the development of chimeric CD20-Mat, rituximab (RituximabTM),recently approved by the food and drug administration (FDA) for the treatment of B-cell lymphoma. Although after treatment with rituximab clinical responses have been observed with high frequency in patients often relapse after about 6-12 months.
For intravenous infusion requires high doses of rituximab, because the molecule is large, approximately 150 KD, and diffusion in lymphoid tissue, where many tumor cells, is limited. Consider that the mechanism of antitumor activity of rituximab is combined, not the how many activities including ADCC, complement fixation, and the launch of signals in malignant B-cells, which stimulate apoptosis. The large size of rituximab reduces the diffusion of molecules in lymphoid tissues that contain malignant B-cells, thereby limiting these antitumor activity. As discussed above, the splitting of the CD20-mate proteases to Fab or F(ab')2fragments makes them smaller and can better penetrate into lymphoid tissue, but effector functions that are important for antitumor activity, is lost. Although fragments of CD20-Mat can be more effective for delivery of radioisotopes than intact antibody, it would be desirable design derived CD20-Mat which retains the effector functions of the Fc-portion, but smaller in size, facilitating penetration into tumors and characterized by the shorter half-life.
CD20 is expressed by malignant cells of B-cell nature, including B-cell lymphoma and chronic lymphocytic leukemia (CLL). CD20 is not expressed in malignant tumors of the pre-B-cells, such as acute lymphoblastic leukemia. Thus, CD20 is a good target for therapy of B-cell lymphoma, CLL and other diseases in which B cells are involved in the disease activity. Other B-cell disorders in luchot autoimmune diseases, in which autoantibodies are produced during the differentiation of B-cells into plasma cells. Examples of B-cell disorders include autoimmune thyroid disease, including graves ' disease and Hashimoto's thyroiditis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogren syndrome, immune thrombocytopenic purple (ITP), multiple sclerosis (MS), myasthenia gravis (MG), psoriasis, scleroderma and inflammatory bowel disease, including Crohn's disease and ulcerative colitis.
From the above clear need for improved compositions and methods of treatment of malignant conditions, and B-cell disorders. Compositions and methods according to this invention overcomes limitations of the prior art by providing a protein binding domain-immunoglobulin containing polypeptide binding domain fused with a polypeptide of the hinge region of the immunoglobulin, which merged with the polypeptide constant region CH2 heavy chain immunoglobulin fused with polypeptide constant region CH3 heavy chain immunoglobulin, while the protein binding domain-immunoglobulin able to mediate ADCC or complement fixation. In addition, the compositions and methods provide other related advantages.
Aspect on the frame of the invention to provide a fused protein binding domain-immunoglobulin, containing (a) a polypeptide binding domain fused with a polypeptide of the hinge region of the immunoglobulin, where the specified polypeptide hinge region selected from the group consisting of (i) a mutant polypeptide of the hinge region that contains no cysteine residues and which is derived from a polypeptide of the hinge region of the immunoglobulin wild-type, having one or more cysteine residues, (ii) a mutant polypeptide of the hinge region, which contains one cysteine residue and which is derived from a polypeptide of the hinge region of the immunoglobulin wild-type, having two or more cysteine residues, (iii) the polypeptide of the hinge region of IgA wild-type human (iv) a mutant polypeptide of the hinge region of IgA person that contains no cysteine residues and which is derived from a polypeptide region IgA wild-type person, and (v) a mutant polypeptide of the hinge region of human IgA, which contains one cysteine residue and which is derived from a polypeptide region IgA wild-type person; (b) a polypeptide constant region CH2 heavy chain of immunoglobulin, which merged with the polypeptide hinge region; and (c) a polypeptide constant region CH3 heavy chain of immunoglobulin, which merged with the polypeptide constant region CH2, where: (1) protein binding domain immunoglobulin able to show that at m is re, one immunological activity selected from the group consisting of mediated antibody-dependent cell cytotoxicity and complement fixation, and (2) a polypeptide binding domain capable of specific contact with the antigen. In one embodiment, the polypeptide of the hinge region of the immunoglobulin is a mutant polypeptide of the hinge region and exhibits a reduced ability to timeresults compared to the polypeptide of the hinge region of human immunoglobulin G wild type. In another embodiment, the polypeptide binding domain contains at least one polypeptide variable region of the immunoglobulin, which is a polypeptide variable region of the light chain of the immunoglobulin or polypeptide variable region of the heavy chain of immunoglobulin. In the following variant polypeptide variable region of an antibody derived from a human immunoglobulin.
In another embodiment, the polypeptide binding domain fused protein Fv binding domain-immunoglobulin contains (a) at least one polypeptide variable region light chain immunoglobulin; (b) at least one polypeptide variable region of the heavy chain of immunoglobulin and (c) at least one linker peptide, which merged with the polypeptide (a) and the polypeptide (b). In the following embodiment, the polypeptides of variab the school region light chain immunoglobulin and variable regions of the heavy chain of immunoglobulin derived from human immunoglobulins.
In another embodiment, at least one of the polypeptides is a polypeptide constant region CH2 heavy chain immunoglobulin and a polypeptide constant region CH3 heavy chain immunoglobulin derived from the heavy chain of human immunoglobulin. In another embodiment, polypeptides constant regions CH2 and CH3 heavy chain of immunoglobulin belong to the isotype selected from human IgG and IgA person. In another embodiment, the antigen is selected from the group consisting of CD19, CD20, CD37, CD40, and L6. In some of the following embodiments described above fused protein linker polypeptide contains at least one polypeptide having the amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 21], and in some other embodiments, the linker polypeptide contains at least three repeats of the polypeptide having the amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 21]. In some embodiments, the polypeptide of the hinge region of the immunoglobulin polypeptide contains the hinge region of human IgA. In some embodiments, the polypeptide binding domain contains the extracellular domain of CD154. In some embodiments, the polypeptide binding domain contains the extracellular domain of CD154 and at least one polypeptide variable region of the immunoglobulin.
In other embodiments, the invention relates to selected polynucleotide, codereuse any of the above with itih protein binding domain-immunoglobulin, in related embodiments, the invention relates to recombinant expressing constructs containing such polynucleotide, and in some of the following embodiments, the invention relates to the cell host transformed or transtitional such recombinant expressing the design. In another embodiment, the invention relates to a method for producing a fused protein binding domain-immunoglobulin, including the stage of (a) culturing disclosed here, the host cell under conditions that provide for expression of the fused protein binding domain-immunoglobulin; and (b) the allocation of fused protein binding domain-immunoglobulin from the culture of host cells.
This invention also relates to certain variants of a pharmaceutical composition containing a protein binding domain-immunoglobulin, which is described above, in combination with a physiologically acceptable carrier. In another embodiment presents a method of treatment of a subject who has or is alleged to have malignant condition or a b-cell disorder, comprising the administration to a patient a therapeutically effective amount of the above-described fused protein binding domain-immunoglobulin. In some of the following options malignant condition or a b-cell disorder is a b-cell lymphoma and the and disease, which is characterized by production of autoantibodies, and in some other embodiments, a malignant condition or a b-cell disorder is rheumatoid arthritis, myasthenia gravis, graves disease, diabetes type I, multiple sclerosis or autoimmune disease.
These and other aspects of the present invention will become more clear upon reference to the following detailed description and the accompanying drawings. All these sources of information included in this description by reference in full, as would have been included separately.
Brief description of drawings
The figure 1 shows the DNA sequence and the decoded amino acid sequence [SEQ ID NO: 15] 2H7scFv-Ig, fused protein binding domain immunoglobulin capable of specific bind CD20.
The figure 2 shows the levels of production IN scFv-Ig transfitsirovannykh stable lines of the SNO and the generation of a standard curve using bind purified N scFv-Ig cells SNO expressing CD20.
The figure 3 shows the analysis in SDS-page of various drugs selected protein 2H7scFv-Ig.
The figure 4 shows the fixation of complement (figa) and mediating antibody-dependent cellular cytotoxicity (ADCC, pigv)) by means of 2H7scFv-Ig.
The figure 5 shows the effect of simultaneous ligation of CD20 and CD40 on the growth of normal the cells.
The figure 6 shows the effect of simultaneous ligation of CD20 and CD40 on the expression of CD95 and the induction of apoptosis in lymphoblastoid b-cell line.
The figure 7 shows the DNA sequence and the decoded amino acid sequence of the fused protein binding domain-immunoglobulin 2H7scFv-CD154 L2 (figa, SEQ ID NO:21 and 33) and 2H7scFv-CD154 34 (7, SEQ ID NO:22 and 34)capable of specific bind CD20 and CD40.
The figure 8 shows the binding of slit proteins binding domain-immunoglobulin 2H7scFv-CD154 with SNO-cells CD20+using flow immunocytometry.
The figure 9 shows the binding of annexin V with lines b-cells Ramos, JAB and Kzt51 after binding to cells fused protein binding domain-immunoglobulin 2H7scFv-CD154.
The figure 10 shows the effect on the proliferation of b-cell line Kzt51 after binding fused protein binding domain-immunoglobulin 2H7scFv-CD154.
Figure 11 depicts a schematic representation of the structures of fused proteins, 2H7ScFv-Ig [SEQ ID NO:16, 17 and 18], called derivatives CytoxB or CytoxB: CytoxB-MHWTG1C (2H7-ScFv, mutant hinge, an Fc domain of human IgG1 wild type), CytoxB-MHMG1C (2H7-ScFv, mutant hinge, the mutant Fc domain of human IgG1) and CytoxB-IgAHWTHG1C (2H7-ScFv, hinge, derived from human IgA, respectively, Fc domain of IgG1 wild-type person). Arrows indicate the numbers of the provisions of the amino acid residues that assume is Ino participate in the FcR binding and ADCC activity (dark arrow), and fixation of complement (light arrows). Megamachine disulfide bond is not specified.
The figure 12 shows the analysis in SDS-page dedicated fused protein binding domain-immunoglobulin CytoxB and 2H7scFv-CD154.
The figure 13 shows mediated antibody-dependent cell cytotoxicity (ADCC) activity of derivatives CytoxB.
The figure 14 shows the complement-dependent cytotoxicity (CDC) derivatives CytoxB.
The figure 15 shows the definition of time half-life in serum CytoxB-MHWTG1C in blood samples macaques.
The figure 16 shows the effect CytoxB-MHWTG1C on the levels of circulating b cells CD40+in blood samples macaques.
The figure 17 shows the levels of production HD37 (CD19-specific)-ScFv-Ig transfitsirovannykh lines of mammalian cells and the generation of a standard curve using bind purified HD37-ScFv-Ig with cells expressing CD19.
The figure 18 shows the levels L6 (antigen carcinoma)-ScFv-Ig transfitsirovannykh stable lines CHO and the generation of a standard curve using bind purified L6-ScFv-Ig with cells expressing the antigen L6.
The figure 19 shows ADCC activity fused protein binding domain-immunoglobulin 2H7-ScFv-Ig, HD37-ScFv-Ig and G28-1 (CD37-specific)-ScFv-Ig.
The figure 20 shows ADCC activity fused protein L6-ScFv-Ig.
The figure 21 shows the analysis in SDS-PAG fused protein L6-ScFv-Ig and 2H7-ScFv-Ig.
On the figures is 22 shows the analysis in SDS-PAG fused proteins G28-1-ScFv-Ig and HD37-ScFv-Ig.
DETAILED description of the INVENTION
This invention is directed to fused proteins binding domain-immunoglobulin and related compositions and methods that will be used in immunotherapy and immunodiagnostics applications and which provide certain advantages compared to the antigen-specific polypeptides of the prior art. Slit proteins according to the invention preferably represent a single polypeptide chain that contain substantial portions of the following fused domains: polypeptide binding domain polypeptide of the hinge region of the immunoglobulin polypeptide constant region CH2 heavy chain immunoglobulin and a polypeptide constant region CH3 heavy chain immunoglobulin. In particularly preferred embodiments, the polypeptide domain that consists of a protein binding domain-immunoglobulin, or are derived from polypeptides that are products of gene sequences person, but the invention should not be limited thereby, and in fact it may be fused proteins binding domain-immunoglobulin, which are presented in this description, which is obtained from any natural or artificial source, including genetically engineered and/or Muta is fair polypeptides.
This invention belongs partly to the unexpected observation that described in this description of fused proteins binding domain immunoglobulin capable to demonstrate immunological activity. More specifically, these proteins retain the ability to participate in the well-known immunological effector activities, including mediated antibody-dependent cell cytotoxicity (ADCC, for example, occur after binding of the antigen on the cell surface contacts and the induction of cytotoxic effector cells bearing the appropriate Fc receptors, such as natural killer cells (NK), bearing FcRγIII, in appropriate circumstances) and/or fixation of complement in the case of the complement-dependent cytotoxicity (CDC, for example, occur after binding of the antigen on the cell surface, the recruitment and activation of cytolytic proteins that are components of the complement cascade in the blood), despite the availability of structures for which you did not expect that they are able to stimulate effector activity. As described in more detail below, ADCC and CDC are unexpected functions for Monomeric proteins containing region of the heavy chain of immunoglobulin, which are preferred due to the structures selected for this fused proteins, and, in particular, due to the Bor polypeptides hinge region, with impaired ability to form megamachine, disulfide bonds of homodimers.
Another advantage provided by this invention is a fused polypeptide binding domain-immunoglobulin, which can be obtained in large quantities, as a rule, larger than typically achieved number in the case of structures of single-chain antibodies of the prior art. In preferred embodiments, the slit polypeptides binding domain-immunoglobulin according to the invention recombinante Express in expressing systems of mammals, which give the advantage of providing polypeptides that are stable in vivo (e.g., at physiological conditions). According to non-limiting theory, the stability can partly be derived from post-translational modifications, in particular glycosylation fused proteins. Product data fused protein binding domain-immunoglobulin by recombinant expression in mammalian cells was achieved in a stationary cell cultures at levels in excess of 50 mg protein per liter nadeshiko culture, and is usually observed in such cultures at the level of 10-50 mg/l, preferably in a stationary culture, you can get at least 10-50 mg/l; it is also assumed uselen the products I fused proteins using generally accepted in the field of large-scale methods of production, such as the method of obtaining from "intermittent feeding" (i.e. non-stationary), which receive the outputs comprising at least 5-500 mg/l, and in some cases at least 0.5-1 g/l depending on the specific protein product.
The polypeptide binding domain according to this invention may be any polypeptide that has the ability to recognize specific and stable or temporarily to contact recognizable biological molecule or a complex of several molecules or with a group or Assembly of such molecules that include a protein, polypeptide, peptide, amino acid or derivative; lipid, fatty acid and the like; carbohydrate, saccharide or the like, or their derivative, nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative, or the like; or any combination of, for example, glycoprotein, glikopeptid, glycolipid, lipoprotein, proteolipid; or any other biological molecule that can be present in the biological sample. The biological sample can be prepared, receiving a blood sample, a biopsy sample, the Explant tissue culture of organs, biological fluids, or any other drug tissue or cells from a subject or biological source. The subject or biological source may be man or animal, different from human primary cell culture or cell line adapted in culture, including, but without limiting the above, the genetically engineered cell lines that may contain integrated into the chromosome or epilimnia recombinant nucleic acid sequences, immortalized or naimportovane cell line, cell line hybrids somatic cells, differentiated or undifferentiated cell lines, transformed cell lines and the like. In some preferred embodiments of the invention the subject or biological source may be suspected to be at risk or may be at risk of having a malignant condition, or B-cell disorders, which are presented in this description and which in some additional preferred embodiments, can be an autoimmune disease, and in some other preferred embodiments of the invention may be known that the subject or biological source is not exposed, or has such a disease.
Thus, the polypeptide binding domain can be any of natural origin or obtained recombinante partner linking relevant biological molecules, which is represented in the data description and which is of interest structure-target called herein "antigen", but the meaning described in this structure, covering any biological molecule-target, which requires specific binding of the fused protein according to this invention. Slit proteins binding domain-immunoglobulin defined as "immunospecific" or capable of specific contact, if they are associated with desired molecule-target, such as presented in this description of the antigen, with Kagreater than or equal to about 104M-1preferably greater than or equal to about 105M-1more preferably greater than or equal to about 106M-1and even more preferably greater than or equal to about 107M-1. The affinity of binding the fused protein binding domain-immunoglobulin according to the invention can be easily determined using conventional methods, for example, methods described by Scatchard et al., Ann. N. Y. Acad. Sci. 51: 660 (1949). This binding definition fused protein of interest antigenic targets also can be performed using any of a number of known methods of identifying and obtaining proteins that have been specifically interact with other proteins or polypeptides, for example, yeast two-hybrid screening system such as the system described in U.S. Pat is the USA No. 5283173 and U.S. patent No. 5468614, or equivalent.
Preferred variants fused protein binding domain-immunoglobulin according to the invention contain binding domains, which include at least one polypeptide variable region of immunoglobulin, such as all or a portion or fragment of the V-region heavy chain or light chain, provided that it is capable of specific bind the antigen or other desired interest in the structure of the target given in this description. In other preferred embodiments, the binding domain contains a single-stranded derived from immunoglobulin Fv-product, which may include all or part of at least one V-region light chain immunoglobulin and all or part of at least one V-region heavy chain immunoglobulin and which further comprises a linker, merged with V-regions; obtaining and testing of such structures described in this description in more detail and is known in this field. Other polypeptide binding domain can contain any protein or part thereof that retains the ability of specific contact with the antigen, which is described in this description, including proteins other than immunoglobulins. Thus, the invention encompasses fused proteins containing polypeptides binding domain, is that derived from a polypeptide ligands, such as hormones, cytokines, chemokines and the like; receptors, cell surface or soluble receptors for such polypeptide ligands, lectins, receptors, intercellular adhesion, such as specific leukocyte integrins, selectins, members of the superfamily of immunoglobulin genes, molecules intercellular adhesion (ICAM-1,-2,-3) and the like; antigens of the major histocompatibility etc.
Examples of receptors on the cell surface, which can be a polypeptide binding domain and which can also be selected as a target molecule or antigen, which preferably binds to a protein binding domain-Ig according to this invention, includes the following or similar receptors: HER1 (for example, with the Deposit number in GenBank No. U48722, SEG_HEGFREXS, K03193), HER2 (Yoshino et al., 1994 J. Immunol. 152: 2393; Disis et al., 1994 Canc. Res. 54: 16; see, for example, GenBank Depository No. X03363, M17730, SEG_HUMHER20), HER3, (for example, GenBank Depository No. U29339, M34309), HER4 (Plowman et al., 1993 Nature 366: 473; see also, e.g., GenBank Depository No. L07868, T64105), the receptor for epidermal growth factor (EGFR) (e.g., GenBank Depository No. U48722, SEG_HEGFREXS, KO3193), a growth factor, endothelial cells of blood vessels (e.g., GenBank No. M32977), growth factor receptor endothelial cells of blood vessels (e.g., GenBank Depository No. AF022375, 1680143, U48801, X62568), insulin-like growth factor I(for example, GenBank Depository No. X00173, X56774, X56773, X06043, see also European patent No. GB 2241703), insulin-like growth factor II (e.g., GenBank Depository No. X03562, X00910, SEG_HUMGFIA, SEG_HUMGFI2, M17863, M17862), transferrin receptor (Trowbridge and Omary, 1981 Proc. Nat. Acad. Sci. USA 78: 3039; see also, e.g., GenBank Depository No. X01060, M11507), estrogen receptor (for example, GenBank Depository No. M38651, X03635, X99101, U47678, M12674), progesterone receptor (e.g., GenBank Depository No. X51730, X69068, M15716), follicle-stimulating hormone receptor (FSH-R) (e.g., GenBank Depository No. Z34260, M65085), retinoic acid receptor (e.g., GenBank Depository No. L12060, M60909, X77664, X57280, X07282, X06538), MUC-1 (Barnes et al., 1989 Proc. Nat. Acad. Sci. USA 86: 7159; see also, e.g., GenBank Depository No. SEG_MUSMUCIO, M65132, M64928) NY-ESO-1 (e.g., GenBank Depository No. AJ003149, U87459), NA 17-A (for example, in European patent No. WO 96/40039), mesland-A/MART-1 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91: 3515; see also, e.g., GenBank Depository No. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat. Acad. Sci. USA 91: 9461; see also, e.g., GenBank Depository No. M26729, SEG_HUMTYR0, see also Weber et al., J. Clin. Invest. (1998) 102: 1258), Gp-100 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91: 3515; see also, e.g., GenBank Depository No. S73003, see also European patent No. EP 668350; Adema et al., 1994 J. Biol. Chem. 269: 20126), MAGE (van den Bruggen et al., 1991 Science 254: 1643; see also, e.g., GenBank Depository No. U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U1065, L18877, U10340, U10339, L18920, U03735, M77481), GAGE (for example, GenBank Depository No. U19180, see also U.S. patent No. 5683886 and 5571711), GAGE (for example, GenBank Depository No. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143, U19142), any of the CTA-class receptors, including, in particular, the antigen HOM-MEL-40, encoded SSX2 gene (e.g., GenBank Depository No. X86175, U90842, U90841, X86174), oncofetal antigen (CEA, Gold and Freedman, 1985 J. Exp.Med 121: 439; see also, e.g., GenBank Depository No. SEG_HUMCEA, M59710, M59255, M29540) and PyLT (for example, GenBank Depository No. J02289, J02038).
Additional cell surface receptors, which can be sources of polypeptides binding domain, or which may be relevant antigens include the following or similar examples: CD2 (e.g., GenBank Depository No. Y00023, SEG_HUMCD2, M16336, M16445, SEG_MUSCD2, M14362), 4-1BB (CDw137, Kwon et al., 1989 Proc. Nat. Acad. Sci. USA 86: 1963), the ligand of 4-1BB (Goodwin et al., 1993 Eur. J. Immunol. 23: 2361; Melero et al., 1998 Eur. J. Immunol. 3: 116), CD5 (for example, GenBank Depository No. X78985, X89405), CD10 (for example, GenBank Depository No. M81591, X76732), CD27 (for example, GenBank Depository No. M63928, L24495, L08096), CD28 (June et al., 1990 Immunol. Today 11: 211; see also, e.g., GenBank Depository No. J02988, SEG_HUMCD28, M34563), CTLA-4 (e.g., GenBank Depository No. L15006, X05719, SEG_HUMIGCTL), CD40 (e.g., GenBank Depository No. M83312, SEG_MUSC040A0, Y10507, X67878, X96710, U15637, L07414), interferon-γ (IFN-γ; see, e.g., Farrar et al. 1993 Ann. Rev. Immunol. 11: 571 and cited in this work references, Gray et al. 1982, Nature 295: 503, Rindernecht et al. 1984 J. Biol. Chem. 259: 6790, DeGrado et al. 1982 Nature 300: 379), interleukin-4 (IL-4; see, for example, 53rd Forum in Immunology, 1993 Research in Immunol. 144: 553-643; Banchereau et al., 1994 in The Cytokine Handbook, 2nd ed., A. Thomson, ed., Academic Press, NY, p.99; Keegan et al., 1994 J. Leukocyt. Biol. 55: 272, and cited in these works reference), interleukin-17 (IL-17) (for example, GenBank Depository No. U32659, U43088) and the receptor for interleukin-17 (IL-17R) (e.g., GenBank Depository No. U31993, U58917). Notwithstanding the foregoing, this invention is definitely not covered, in particular, immunoglobulin fused proteins, which are claimed in U.S. patent 5807734, 5795572 or 5807734.
Additional cell surface receptors, which can be sources of polypeptides binding domain, or which may be relevant antigens include the following or similar examples: CD59 (for example, GenBank Depository No. SEG_HUMCD590, M95708, M34671), CD48 (for example, GenBank Depository No. M59904), CD58/LFA-3 (e.g., GenBank Depository No. A25933, Y00636, E12817; see also JP 1997075090-A), CD72 (for example, GenBank Depository No. AA311036, S40777, L35772), CD70 (for example, GenBank Depository No. Y13636, S69339), CD80/B7.1 (Freeman et al., 1989 J. Immunol. 43: 2714; Freeman et al., 1991 J. Exp.Med. 174: 625; see also, e.g., GenBank Depository No. U33208, I683379), CD86/B7.2 (Freeman et al., 1993 J. Exp.Med. 178: 2185, Boriello et al., 1995 J. Immunol. 155: 5490; see also, e.g., GenBank Depository No. AF099105, SEG_MMB72G, U39466, U04343, SEG_HSB725, L25606, L25259), CD40 ligand (for example, GenBank Depository No. SEG_HUMCD40L, X67878, X65453, L07414), IL-17, (e.g. the, GenBank Depository No. U32659, U43088), CD43 (for example, GenBank Depository No. X52075, J04536) and VLA-4 (α4β7) (for example, GenBank Depository No. L12002, X16983, L20788, U97031, L24913, M68892, M95632). These receptors to the cell surface usually associated with B cells: CD19 (e.g., GenBank Depository No. SEG_HUMCD19W0, M84371, SEG_MUSCD19W, M62542), CD20 (e.g., GenBank Depository No. SEG_HUMCD20, M62541), CD22 (e.g., GenBank Depository No. I680629, Y10210, X59350, U62631, X52782, L16928), CD30 ligand (e.g., GenBank Depository No. L09753, M83554), CD37 (for example, GenBank Depository No. SEG_MMCD37X, X14046, X53517), CD106 (VCAM-1) (e.g., GenBank Depository No. X53051, X67783, SEG_MMVCAM1C, see also U.S. patent No. 5596090), CD54 (ICAM-1) (e.g., GenBank Depository No. X84737, S82847, X06990, J03132, SEG_MUSICAM0), interleukin-12 (see, e.g., Reiter et al., 1993 Crit. Rev. Immunol. 13: 1, and cited in this work links). Extra cellular agents can also include any of the following receptors on the cell surface, usually associated with dendritic cells (CD83 (for example, GenBank Depository No. AF001036, AL021918), DEC-205 (e.g., GenBank Depository No. AF011333, U19271).
The polypeptide of the hinge region of the immunoglobulin, which is discussed above, includes any peptide or polypeptide of the hinge, which has a natural origin, obtained in the form of artificial peptide or as a result of genetic engineering and which is located in the polypeptide heavy chain immunoglobulin between Amin is acid residues, responsible for the formation of interchain disulfide bonds domains of immunoglobulin in the areas CH1 and CH2; polypeptides hinge region for use in this invention can also include mutant polypeptide of the hinge region. Thus, the polypeptide of the hinge region of the immunoglobulin can be obtained from part or fragment, or it can be part or fragment (i.e. one or more amino acids linked by peptide bond, usually 5-65 amino acids, preferably 10-50, more preferably 15-35, even more preferably 18-32, even more preferably 20-30, even more preferably 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acids) region of polypeptide chain of the immunoglobulin, which according to the classical view has the function of a hinge, which is described above, but the polypeptide of the hinge region for use in the present invention should not be limited thereby, and it may include amino acids located (in accordance with the structural criteria for the placement of a particular residue in a specific domain, which may vary, as is known in this field) in the surrounding immunoglobulin domain, such as domain CH1 or CH2 domain, or in the case of some artificially constructed structures immunoglobulin domain of the variable region of the immunoglobulin.
Poly is atidy the hinge region of the immunoglobulin wild type include any area of the hinge of natural origin, which is located between the domains of the constant region CH1 and CH2 immunoglobulin. The polypeptide of the hinge region of the immunoglobulin wild-type and preferably is a polypeptide hinge region of human immunoglobulin, preferably containing the hinge region of the immunoglobulin IgG human, and more preferably the polypeptide of the hinge region of IgG1 isotype person. As is well known in this area, despite the huge total diversity of amino acid sequences of immunoglobulin, in the primary structure of the immunoglobulin has a high degree of conservatism sequences in specific parts of the polypeptide chains of immunoglobulins, especially in relation to the location of the cysteine residues, which because of its sulfhydryl groups provide the opportunity for the formation of disulfide bonds with other available sulfhydryl groups. Thus, in the context of this invention, the polypeptides of the hinge region of the immunoglobulin wild type can be considered as polypeptides, characterized by one or more highly conservative (for example, prevailing in the population in a statistically significant way) cysteine residues, and in some preferred embodiments, it is possible to choose a mutant polypeptide of the hinge region, which is not with the contains or contains one cysteine residue and which is obtained from such hinge region of the wild type.
Mutant polypeptide of the hinge region of the immunoglobulin may contain a hinge region, which is the origin of the immunoglobulin species, isotype or class of immunoglobulin or subclass of immunoglobulin, which is different from that for domains CH2 and CH3. For example, in some embodiments of the invention protein binding domain-immunoglobulin may contain a polypeptide binding domain fused with a polypeptide of the hinge region of immunoglobulin-containing polypeptide of the hinge region of human IgA wild-type or mutant polypeptide of the hinge region of human IgA, which does not contain or contains only one cysteine residue, as specified in this description. Such polypeptide hinge region may be fused with polypeptide region CH2 heavy chain immunoglobulin of another Ig isotype or class, such as IgG subclass, which in some preferred embodiments, will be the IgG1 subclass.
For example, and as described in more detail hereinafter, in some embodiments of the present invention is selected polypeptide hinge region of the immunoglobulin, which is derived from the hinge region of IgA wild-type person, which in nature contains three cysteine, however, the selected polypeptide hinge region is shortened compared to the full hinge region so that remains then is are one cysteine residue (for example, SEQ ID NO: 35-36). Similarly, in some other embodiments of the invention protein binding domain-immunoglobulin polypeptide contains a binding domain that is fused with a polypeptide of the hinge region of the immunoglobulin containing mutant polypeptide of the hinge region, in which the number of cysteine residues reduced by amino acid substitutions or deletions. Thus, the mutant polypeptide of the hinge region may be obtained from the hinge region of the immunoglobulin wild type, which contains one or more cysteine residues. In some embodiments, the mutant polypeptide of the hinge region may not contain or contain only one cysteine residue, while the mutant polypeptide of the hinge region derived from the hinge region of the immunoglobulin wild type, which contain, respectively, one or more or two or more cysteine residues. In the mutant polypeptide of the hinge region cysteine residues of the hinge region of the immunoglobulin wild-type preferred substitute amino acids that are unable to form disulfide bonds. In one embodiment of the invention, a mutant polypeptide of the hinge region derived from the polypeptide of the hinge region of IgG wild-type person, which may include any of the four subclasses of IgG isotype human IgG1, IgG2, IgG3 or IgG4. In some pre is respectful embodiments, the mutant polypeptide of the hinge region derived from the polypeptide of the hinge region of IgG1 wild-type person. As an example, the mutant polypeptide of the hinge region derived from a polypeptide of the hinge region of IgG1 wild-type person, may contain mutations in two of the three cysteine residues in the hinge region of the immunoglobulin wild type or mutation in all three cysteine residues.
Residues of cysteine, which are present in the hinge region of the immunoglobulin wild-type and which is removed as a result of mutagenesis according to a particularly preferred variants of the present invention include cysteine residues, which form or are capable of forming megamachine disulfide bonds. Not wanting to associate with theory, in this invention suggest that mutation of these cysteine residues of the hinge region, which presumably involved in the formation of miaocheng disulfide bonds, reduces the ability of the fused protein binding domain-immunoglobulin according to the invention to timeresults (or to form oligomers of higher order) through the formation of disulfide bonds, thus unexpectedly without compromising the ability of the fused protein to stimulate mediated antibody-dependent cell cytotoxicity (ADCC) or fix complement. In particular, Fc-receptors (FcR), which mediate ADCC (for example, FcRIII, CD16), have a low affinity in relation to the Fc-domain of IME is of noglobulin, that suggests that the functional binding with Fc FcR requires stabilization of the avidity of the complex Fc-FcR due to the dimeric structure of the heavy chains in a conventional antibody, and/or aggregation and crosslinking of FcR with conventional Fc-patterns At. (Sonderman et al., 2000 Nature 406: 267; Radaev et al., 2001 J. Biol. Chem. 276: 16469; Radaev et al., 2001 J. Biol. Chem. 276: 16478; Koolwijk et al., 1989 J. Immunol. 143: 1656; Kato et al., 2000 Immunol. Today, 21: 310). Therefore, the slit proteins binding domain-immunoglobulin according to this invention provide the advantages associated with single-fused proteins of the immunoglobulin, while also unexpectedly maintaining immunological activity. Similarly, the ability to fix complement is usually associated with immunoglobulins, which are dimeric with respect to the constant regions of the heavy chain, such as areas that contain Fc, despite this fused protein binding domain-immunoglobulin according to the invention show unexpected ability to fix complement.
As noted above, suggest that slit proteins binding domain-immunoglobulin according to non-limiting theory violated their ability to timeresults and, in addition, according to theory of the specified property is a consequence of reducing the number of cysteine residues present in the polypeptide of the hinge region immunoglo Olina, selected for inclusion in the design of the fused protein. The determination of the relative ability of the polypeptide to timeresults well known in the relevant field, you can use any one of a number of the developed techniques to detect dimerization of the protein (see, e.g., Scopes, Protein Purification: Principles and Practice, 1987, Springer-Verlag, New York). For example, methods of biochemical separation to separate proteins based on size of molecules (for example, gel electrophoresis, gel filtration chromatography, analytical ultracentrifugation, and so on) and/or comparison of physico-chemical properties of proteins before and after the introduction of active towards sulfhydryl groups (for example, todatetime, N-ethylmaleimide) or reducing the formation of disulfide bonds (e.g., 2-mercaptoethanol, dithiothreitol) agents or other equivalent method, all these methods can be used to determine the degree of dimerization or oligomerization of polypeptides and to determine the possible contribution of disulfide bonds in such possible Quaternary structure. In some embodiments, the invention relates to fused protein binding domain-immunoglobulin, which shows reduced (i.e. statistically significant compared with the corresponding control derived from IgG) ability to timeresults compared to the polypeptide ø miRNAs human immunoglobulin G wild-type, as specified in this description. Thus, the experts in this field will easily be able to determine, is there a specific protein such reduced ability to dimerization.
Compositions and methods for producing fused protein of the immunoglobulin is well known in this field, as described, for example, in U.S. patent No. 5892019 in which the claimed recombinant antibodies, which are products of one coding polynucleotide, but are not fused protein binding domain-immunoglobulin according to the invention.
For the fused protein of the immunoglobulin according to the invention, which is intended for use on the human constant region, typically will have the origin of the sequences of human, in order to minimize the potential immune response against human proteins and provide appropriate effector functions. Processing sequences encoding the constant region of the antibodies described in the application for the grant of a patent PCT Morrison and Oi, WO 89/07142. In particularly preferred embodiments, the domain CH1 deleterows, and the carboxyl end of the binding domain, or when binding domain contains two polypeptide variable region of the immunoglobulin, the second (i.e., more proximal with respect to the C-end) variable region is linked to aminocom.com CH2 through W is miRNAs area. Schematic diagram which shows the structure of two typical fused protein binding domain-immunoglobulin shown figure 11, it should be noted that in especially preferred embodiments megamachine disulfide bonds are absent, and in other embodiments may be a limited number miaocheng disulfide bonds relative to the number of such relationships that might be present, if available instead were the polypeptides of the hinge region of the wild type, and that in other variants of the protein contains a mutant polypeptide of the hinge region, which exhibits a reduced ability to timeresults compared to the polypeptide of the hinge region of IgG wild-type person. Thus, the selected molecule polynucleotide encodes a single-chain protein of the immunoglobulin having binding domain, which provides a specific binding affinity of the selected antigen.
As indicated above, in some embodiments, protein binding domain-immunoglobulin contains at least one polypeptide variable region of the immunoglobulin, which may be a polypeptide variable region of the light chain or the heavy chain, and in some embodiments, a protein contains at least one such V-region light chain and so is Yu V-region heavy chain and at least one of the linker peptide, which merged with each V-region. The design of such binding domains, such as single-chain Fv domains, it is well known in this field and are described in more detail in the examples below and are described, for example, in U.S. patent No. 5892019 and cited in the mentioned patent references; selection and Assembly of single-chain variable regions and linker peptides that may be fused with each V region derived from the heavy chain and derived from the light chain (for example, to create a binding domain that contains a single-chain Fv-polypeptide), also known in this field and described in this description and, for example, in U.S. patent No. 5869620, 4704692 and 4946778. In some embodiments, all or part of the sequence of immunoglobulin, which is obtained from a source other than human, may be "humanitarian" according to known methods of generating humanized antibodies, or sequences of immunoglobulins, in which the input sequence Ig a person to reduce the extent to which the human immune system can recognize such proteins as alien (see, for example, U.S. patent No. 5693762; 5585089; 4816567; 5225539; 5530101 and cited in these patent references).
After constructing fused protein binding domain-immunoglobulin, which is described in this about what Isani, it is possible to synthesize DNA encoding the polypeptide, by synthesis of oligonucleotides, as described, for example, Sinha et al., Nucleic Acids Res., 12, 4539-4557 (1984); to collect by PCR, as described, for example, in Innis, Ed., PCR Protocols, Academic Press (1990), as well as in Better et al. J. Biol. Chem. 267, 16712-16118 (1992); to clone and Express using standard methods, as described, for example, in Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley and Sons, New York (1989)and Robinson et al., Hum. Antibod. Hybridomas, 2, 84-93 (1991); and to test against specific antigennegative activity, as described, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988) and Munson et al., Anal. Biochem., 107, 220-239 (1980).
Obtain binding molecules Fv-area with a single polypeptide chain, single-chain Fv molecules described in U.S. patent No. 4946778, which is included in this description by reference. In this invention synthesize single-stranded Fv-like molecules by first encoding the variable region of the heavy or light chain, followed by one or more linkers to the variable regions of the respective light or heavy chain, respectively. The choice of an appropriate linker(ditch) between the two variable regions is described in U.S. patent No. 4946778. Typical examples in this description of the linker is Gly-Gly-Gly-Gly-Ser)3. The linker is used to convert the aggregated EU is the natural enemy conditions, but chemically separated heavy and light chains in aminobenzene antigennegative part of a single polypeptide chain, with the specified antigennegative part will be installed in a structure similar to the original structure, formed by two polypeptide chains, and thus retain the ability to bind with interest antigen. The nucleotide sequence encoding the variable regions of the heavy and light chains, connected by a sequence that encodes a linker, linked to a nucleotide sequence that encodes a constant region of the antibody. Constant regions are areas that allow the result to the polypeptide to form megamachine disulfide bonds to form a dimer, and which possess the desired effector functions, such as the ability to mediate antibody-dependent cellular cytotoxicity (ADCC). For such immunoglobulin molecules according to the invention, which is designed for use on the human constant region usually are largely human in order to minimize the potential immune response against human proteins and permitted to provide effector functions. Processing sequences encoding the constant region of the antibodies described in the application PCT Morrison and Oi, WO 89/07142, the which are included in this description by reference. In preferred embodiments, the domain CH1 deleterule and carboxyl end of the second variable region is linked to aminocom.com CH2 through the hinge region. The Cys residue of the hinge, which forms a disulfide bond with the corresponding Cys light chain, holding the heavy and light chain of the native molecule antibodies, can be deleterows or preferably substituted, for example, balance Pro, or the like.
As described above, the present invention relates to recombinant expressing structures that can control the expression of the fused protein binding domain-immunoglobulin, which are presented in this description. Amino acids, which are found in various specified in this description of the amino acid sequences identified in accordance with the well-known three-letter or one-letter abbreviations. Nucleotides that are found in various mentioned in this description of DNA sequences, or their fragments, specified standard single-letter designations commonly used in this field. Given amino acid sequence can also include similar amino acid sequences that have only small changes, for example, for the purpose of illustration and without limitation, covalent chemical modification, insertion, deletion and C the exchange, which additionally may include conservative substitutions. Amino acid sequences that are similar to each other, can have significant homology sequences. Similarly nucleotide sequence can include substantially similar nucleotide sequence, with only minor changes, for example, in the illustration, and without limitation, covalent chemical modifications, insertions, deletions and substitutions, in addition, may include silent mutations due to the degeneracy of the genetic code. Nucleotide sequences that are similar to each other, can have significant homology sequences.
The presence of a malignant condition in a subject refers to the presence of dysplastic, carcinogenic and/or transformed cells from the subject, including, for example, neoplastic, tumor, not contact-inhibited or oncogene transformed cells or the like. For example, in the preferred embodiments described in this invention, such cancer cells are malignant hematopoietic cells, such as transformed cells of the lymphoid lineage and, in particular, B-cell lymphoma, and the like; cancer cells in some preferred options, which the ants can also be epithelial cells, such as carcinoma cells. The invention also treated B-cell disorders, which may include some malignant conditions that affect B-cells (for example, B-cell lymphoma), but that should not be considered as limited thereby, and it is implied that they include autoimmune diseases and, in particular, diseases, disorders and conditions that are characterized by production of autoantibodies.
The autoantibodies are antibodies that react with self-antigens. Autoantibodies identified in several autoimmune diseases (i.e., the disease, disorder or condition in which the host's immune system causes an unwanted immune response directed against himself), while they are involved in the disease activity. Modern facilities for the treatment of these autoimmune diseases are IMMUNOSUPRESSIVE medicines that require continuous injection, lack the specificity and cause significant side effects. New approaches that may prevent production of autoantibodies with minimal toxicity, will be directed to the unmet health needs in relation to the spectrum of diseases that affect many people. Protein binding domain-immunoglobulin according to the present invention is designed for better penetration in lymphoid tissue. The depletion of the population of B-lymphocytes interrupts the cycle of production of autoantibodies and allows the immune system to recover, since the new B-lymphocytes are produced from precursors in the bone marrow.
Identified a number of diseases, which according to non-limiting theory assumes curative effect in the treatment depletion of the population of B-cells; a brief description of several examples of these diseases are listed below.
Autoimmune disease of the thyroid gland includes graves ' disease and Hashimoto's thyroiditis. Only in the United States has a population of approximately 20 million people have some form of autoimmune disease of the thyroid gland. Autoimmune thyroid disease occurs due to production of autoantibodies, which either stimulate the thyroid gland, causing gipertireoidizmom (graves disease), or destroy the thyroid gland causing hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid gland called autoantibodies, which bind and activate the receptor tireotropina (TSH). Destruction of the thyroid gland called autoantibodies that react with other antigens of the thyroid gland.
Currently therapy of graves ' disease includes surgery, treatment with radioactive iodine or protivotarannymi drug medium is you. Extensive use of radioactive iodine as protivomaskitnye drugs have significant side effects and a high probability of relapse. Surgery is reserved for patients with large goiter or in the case when very rapid normalization of thyroid function. There is no way of therapy, the target of which is the production of autoantibodies responsible for the stimulation of the TSH receptor. Modern therapy thyroiditis Hashimoto uses levothyroxine sodium, and therapy is usually done during life, due to the low probability of remission. Shown suppressive therapy to reduce goiter with Hashimoto thyroiditis, but no known methods of treatment, which reduce the production of autoantibodies that specifically affect the mechanism of the disease.
Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints, leading to tumors, pain and loss of function. RA estimates that affects 2.5 million people in the United States. The cause of RA is a combination of events, including an initial infection or damage, abnormal immune response, and genetic factors. Although RA are self-reactive T-cells and B-cells for the diagnosis of RA have high levels of antibodies, which are collected in the joints, called the by rheumatoid factor. Modern treatment of RA includes many medicines to suppress the pain and slowing the progression of the disease. Not open methods of therapy that can cure a disease. Medications include nonsteroidal anti-inflammatory drugs (NSAIDs) and disease modifying antirheumatoid drugs (DMARDs). NSAID effective in early disease, but cannot prevent the progression of joint destruction and pain in severe RA. Like NSAIDs and DMARDs are associated with significant side effects. Only one new DMARDs, leflunomid, was approved in the last 10 years. Leflunomid inhibit production of autoantibodies, reduces inflammation and slows the progression of RA. However, this drug also causes severe side effects, including nausea, diarrhea, hair loss, skin rashes and liver damage.
Systemic lupus erythematosus (SLE) is an autoimmune disease caused by recurrent damage blood vessels in various organs, including the kidneys, skin and joints. SLE affects more than 500,000 people in the United States. In patients with SLE incorrect interaction between T-cells and B-cells leads to the production of autoantibodies that attack the cell nucleus. Antibodies include antibodies against double-stranded DNA and the t-Sm antibodies. Approximately half of the patients with SLE also detect autoantibodies that bind phospholipids and responsible for damage to the blood vessels and the low number of blood cells. Immune complexes accumulate in the kidneys, blood vessels and joints of patients with SLE, where they cause inflammation and tissue damage. Not found the treatment of SLE, which cure the disease. For therapy use NSAIDs and DMARDs depending on the severity of the disease. Plasmapheresis with replacement plasma to remove autoantibodies can cause a temporary improvement in patients with SLE. It is generally accepted that for SLE responsible autoantibodies, thus, new methods of therapy, which depletes B-cell line, allowing the immune system to recover, since the new B-cells are formed from precursors that give hope for a long-term beneficial effects in patients with SLE.
Sjogren syndrome is an autoimmune disease characterized by destruction of moisturizing glands. Sjogren syndrome is one of the most prevalent autoimmune diseases, affecting up to 4 million people in the United States. About half of people with Sjogren syndrome also have the disease of connective tissue, such as rheumatoid arthritis, while the other half has a primary Sjogren syndrome without the other with Westwego autoimmune diseases. In patients with Sjogren syndrome often present autoantibodies, including antinuclear antibodies, rheumatoid factor, antipodes and antibodies against muscarinic receptor. Conventional therapy includes corticosteroids.
Immune thrombocytopenic purpura (ITP) is caused by autoantibodies that bind to platelets and cause their destruction. Some cases of ITP is caused by drugs, while others are associated with infection, pregnancy, or an autoimmune disease such as SLE. About half of all cases identified as "idiopathic", which means the cause is unknown. Treatment of ITP is determined by the severity of symptoms. In some cases, therapy is not required. In most cases, use IMMUNOSUPRESSIVE drugs, including corticosteroids or intravenous infusion of immunoglobulin to Deplete the population of T-cells. Another method of treatment, which usually leads to an increase in the number of platelets, is the removal of the spleen, the organ that destroys antibody coated platelets. More effective IMMUNOSUPRESSIVE drugs, including cyclosporine, cyclophosphamide, or azathioprine use for patients in severe cases. Removal of autoantibodies by means of passing plasma of the patient through a column of protein A used in the AC is estwe second-line treatment of patients with severe form of the disease.
Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation of the Central nervous system and destruction of myelin, which insulates nerve cell fibers in the brain, spinal cord and body. Although the cause of MS is unknown, it is widely believed that the primary contribution to the pathogenesis of the disease contribute to autoimmune T cells. However, in the cerebrospinal fluid of patients with MS present high levels of antibodies, and some theories predict that B-cell response, resulting in production of antibodies, it is important for mediating the disease. No methods have been developed for the treatment of patients with MS based on the depletion of B-cells. MS is incurable. Modern therapy is a corticosteroid, which can reduce the duration and severity of attacks, but do not affect the course of MS over time. Recently approved treatments for MS biotechnology derived interferon (IFN).
Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disease characterized by weakness of the group of voluntary muscles. MG affects about 40,000 people in the United States. MG is caused by autoantibodies that bind to the receptors of acetylcholine, expressed at neuromuscular junctions. Autoantibodies reduce or block of acetylcholine the e receptors, preventing the transmission of signals from nerves to muscles. MG incurable. Conventional treatments include immunosuppression with corticosteroids, cyclosporine, cyclophosphamide, or azathioprine. Surgical removal of the thymus is often used to dull the autoimmune response. Plasmapheresis is used to reduce the levels of antibodies in the blood, is effective in MG, but short-lived, because the IgG production continues. Plasmapheresis is usually prescribed in severe muscle weakness before the operation.
Psoriasis affects approximately five million people. Autoimmune inflammation in the skin. Psoriasis associated with arthritis in 30% of cases (psoriatic arthritis). Many methods of therapy, including steroids, UV light, retinoids, derivatives of vitamin D, cyclosporine, methotrexate.
Scleroderma is a chronic autoimmune disease of the connective tissue, which is also known as systemic sclerosis. Scleroderma characterized by overproduce collagen, leading to thickening of the skin. Approximately 300,000 people in the United States have scleroderma.
Inflammatory bowel disease, including Crohn's disease and ulcerative colitis are autoimmune diseases of the digestive system.
This invention also relates to constructs coding for fused proteins binding domain-immunoglobulin, is, in particular, to methods for introducing recombinant constructs encoding such proteins that can be expressed, for example, in the form of fragments, analogs and derivatives of such polypeptides. The terms "fragment", "derivative" or "analog" when referring to the fused polypeptides or fused proteins binding domain-immunoglobulin belong to any fused to the polypeptide or fused to a protein binding domain-immunoglobulin, which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes the precursor protein, which can be activated by removal of part of the protein precursor to obtaining active fused polypeptide binding domain-immunoglobulin.
The fragment, derivative or analog is fused polypeptide or fused protein binding domain-immunoglobulin, including fused polypeptides or fused proteins binding domain-immunoglobulin encoded described in this description of DNA, may be (i) a fragment, derivative or analog, in which one or more amino acid residues replaced by conservative or non-conservative amino acid residue (preferably a conservative amino acid residue)and such substituted amino acid residue may be encoded or not encoded using the genetic code, or (ii) a fragment, derivative or analogue in which one or more amino acid residues include the replacement group, or (iii) a fragment, derivative or analog, in which additional amino acids are fused with the merged polypeptide binding domain-immunoglobulin, including amino acids, which are used for the detection or specific functional changes sequence fused polypeptide binding domain-immunoglobulin or sequence propelca. It is believed that such fragments, derivatives and analogues understandable to experts in this field, based on the instructions given in this description.
The polypeptides according to this invention include fused polypeptides and fused proteins binding domain-immunoglobulin having the amino acid sequence of the polypeptide binding domain that is identical or similar to sequences known in this field, or their fragments or parts. For example, in the illustration, and without limiting the extracellular domain of CD154 molecule person suggest to use according to this invention, as are polypeptides having at least 70% similarity (preferably 70% identity) and more preferably 90% similarity (more preferably a 90% identity) with the specified polypeptide, and more preferably 95% similarity is the your (even more preferably 95% identity) with the specified polypeptides and portions of such polypeptides, at the same time such part of the fused polypeptide binding domain-immunoglobulin usually contain at least 30 amino acids and more preferably at least 50 amino acids.
As is known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequences and conservative amino acid substitutions in the sequence of the polypeptide with the sequence of the second polypeptide. Fragments or portions of nucleic acids encoding the polypeptides according to this invention, can be used to synthesize full-sized nucleic acid according to this invention. Used in this sense,"% identity" refers to the percentage of identical amino acids located in the relevant provisions of amino acid residues when comparing two or more polypeptides or analysis of their sequence using the BLAST algorithm for spaces (for example, Altschul et al., 1997 Nucl. Ac. Res. 25: 3389), which assigns weights to gaps in the sequence and erroneous pairings in sequence in accordance with the weighting coefficient default provided in the database of the National Institutes of Health/NCBI (Bethesda, MD; see www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast).
The term "isolated" means that the material extracted from the WWTP is th original environment (for example, natural environment, if it is a substance of natural origin). For example, nucleic acid or polypeptide of natural origin present in a living organism, is not highlighted, but the same nucleic acid or polypeptide, separated from some or all together existing in the natural system of substances, are highlighted. Such nucleic acids may be part of a vector and/or such nucleic acids or polypeptides could be part of the composition and being selected as such vector or composition is not part of its natural environment.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes the preceding and following the coding region, "the leader and the end part", as well as insertion sequences (introns) between individual coding sections (exons).
As discussed herein, the invention relates to fused proteins binding domain-immunoglobulin encoded by nucleic acids that have a sequence encoding a binding domain fused in frame with an additional sequence that encodes a domain of immunoglobulin, to ensure the expression of the polypeptide sequence of the binding domain fused with additional functionality the school polypeptide sequence, which makes it possible, for example, in the illustration, and without limitation, registration, functional change, separation and/or purification of fused protein. Such fused proteins can provide the possibility of functional changes binding domain due to the additional content received from immunoglobulin polypeptide sequences that affect the behavior of the merged product, for example (and as described above) by reducing the availability of sulfhydryl groups to participate in the formation of disulfide bonds and by adding the ability to enhance ADCC and/or CDC.
Modification of the polypeptide can be accomplished in any way known to specialists in this field. Preferred methods according to this invention is based on the modification of DNA that encodes a protein, and expression of the modified DNA. DNA encoding one discussed above mergers binding domain-immunoglobulin can be subjected to mutagenesis using standard techniques, including the techniques described next. For example, cysteine residues, which otherwise may contribute to the formation of multimer or maintain a particular conformation of the molecule, can be deleteroute of the polypeptide or to replace, for example, cysteine residues, which are responsible for the formation of aggregates. When the mu is the necessity of cysteine residues, that contribute to the formation of aggregates can be identified empirically by dellarovere and/or replacement of the cysteine residue and determine whether aggregates the resulting protein solutions containing physiologically acceptable buffers and salts. In addition, you can construct and use fragments of mergers binding domain-immunoglobulin. As indicated above, the described contraceptor/binding domains of the ligand to many potential mergers binding domain-immunoglobulin, so that the person skilled in the art can easily select the appropriate polypeptide domains for inclusion in the encoded data products expressing structures.
Conservative substitutions of amino acids are well known and may be made generally without altering the biological activity of the resulting molecule fused protein binding domain-immunoglobulin. Such substitutions generally carried out through mutual replacement in the group of polar residues, charged residues, hydrophobic residues, small residues, and the like. If necessary, such replacement can be determined only empirically by testing the resulting modified protein the ability to bind to corresponding receptors on the cell surface in the biological is their in vitro or contact the relevant antigens or required by target molecules.
This invention also relates to nucleic acids which hybridize to the sequences of polynucleotides encoding a protein binding domain-immunoglobulin, which are described in this description, or them with complementary sequences that easily it will be clear to the person skilled in the art, if the sequences have at least 70%, preferably at least 90% and more preferably at least 95% identity. This invention particularly relates to nucleic acids, which in conditions of high stringency hybridize with described in this description of the nucleic acids encoding the fusion binding domain-immunoglobulin. Used in this sense, the term "conditions of high stringency" means hybridization will take place only if there is at least 95% and preferably at least 97% identity between the sequences. Nucleic acids which hybridize with a given in this description of the nucleic acids encoding the fusion binding domain-immunoglobulin, in preferred variants encode polypeptides which retain essentially the same biological function or activity as fused polypeptides connecting the house is n-immunoglobulin, encoded by DNA that are listed in the references cited in this specification references.
Used in this sense, the term "gibridizatsiya" in certain rigidity is used to describe the stability of hybrids formed between two single-stranded molecules of nucleic acids. Stiffness hybridization is usually expressed in terms of ionic strength and the temperature at which the annealed and washed hybrids. Typically, "high", "moderate" and "low" stiffness covers the following conditions or equivalent conditions: high hardness: 0.1 x SSPE or SSC, 0,1% SDS, 65°C; moderate hardness: 0.2 x SSPE or SSC, 0,1% SDS, 50°C; and low hardness: 1.0 x SSPE or SSC, 0,1% SDS, 50°C. As is well known to specialists in this field, change the rigidity of the conditions of hybridization can be achieved by altering the time, temperature and/or concentration of the solutions used for the preliminary stages hybridization, hybridization and washing, and suitable conditions may also partly depend on the specific nucleotide sequences of the used probe and blokirovannom sample of the original nucleic acid. Thus, it will be clear that suitable conditions of rigidity can be easily selected without undue experimentation in that case, if the required selectivity of the probe, on the basis of its ability to hybridizat the I with one or more specific initial sequences and not gibridizatsiya with certain other of the source sequences.
Nucleic acid according to this invention, also referred to in this description polynucleotide, can be in the form of RNA or in the form DNA, and DNA includes cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded DNA may be the coding chain or non-coding (antisense) strand. The coding sequence which encodes a fused polypeptide binding domain-immunoglobulin for use according to this invention, may be identical to the coding sequence, known in the field, for any given merger binding domain-immunoglobulin or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same fused polypeptide binding domain-immunoglobulin.
Nucleic acids that encode fused polypeptides binding domain-immunoglobulin for use according to the invention, may include, but are not limited to: only the coding sequence fused polypeptide binding domain-immunoglobulin; the coding sequence fused polypeptide binding domain-immunoglobulin and additional coding sequence; the coding sequence fused in which peptide binding domain-immunoglobulin (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5'and/or 3'-side coding sequence fused polypeptide binding domain-immunoglobulin, which, for example, can include, but is not limited to, one or more regulatory sequences, nucleic acid, which may be a regulatory or regulated promoter, enhancer, another sequence regulation of transcription, a sequence that binds the repressor sequence regulation of translation or any other regulatory sequence of a nucleic acid. Thus, the term "nucleic acid encoding" or "polynucleotide encoding" protein binding domain-immunoglobulin that comprises a nucleic acid which includes only coding sequence fused polypeptide binding domain-immunoglobulin, as well as nucleic acid, which includes additional coding and/or noncoding sequence(ti).
Nucleic acids and oligonucleotides for listed in this description and application can be synthesized by any method known to specialists in this field (see, for example, WO 93/01286, the application for the grant of a U.S. patent with registration No. 07/723454; U.S. patent No. 5218088; U.S. patent No. 5175269; U.S. patent No. 5109124). The IDA is the certification of oligonucleotides and nucleic acids sequences for use in this invention includes methods, well known in this field. For example, the required properties, length and other characteristics applicable oligonucleotides are well known. In some embodiments can be constructed of synthetic oligonucleotides and nucleic acid sequences that are resistant to degradation of endogenous nucleotidase enzymes of the host cell due to the presence of links such as: phosphorothioate, methylphosphonate, sulfonic, sulfate, catiline, phosphorodithioate, phosphoamide, complex phosphoroamidite and other such communications, for which it is shown that they are suitable for applications such as antisense (see, for example, Agrwal et al., Tetrhedron Lett. 28: 3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93: 6657-6665 (1971); Stec et al., Tetrhedron Lett. 26: 2191-2194 (1985); Moody et al., Nucl. Acids Res. 12: 4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40: 137-143 (1984); Eckstein, Annu. Rev. Biochem. 54: 367-402 (1985); Eckstein, Trends Biol. Sci. 14: 97-100 (1989); Stein in: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London, pp.97-117 (1989); Jager et al., Biochemistry 27: 7237-7246 (1988)).
In one embodiment, this invention relates to a shortened components (for example, the polypeptide binding domain polypeptide hinge region, a linker and so on) for use in fused protein binding domain-immunoglobulin, and in another embodiment, the invention relates to nucleic acids coding for a protein binding domain-immuno obelin, with shorter components. Shortened molecule can be any molecule that contains a version of the molecule that is less than full size. Shorter molecules presented in this invention may include a shorter biological polymers, and in preferred embodiments of the invention such shorter molecules can be shortened molecules of nucleic acids or short peptides. Shortened nucleic acid molecule have a nucleotide sequence that is shorter than the full sequence is known or described nucleic acid molecules, such known or described the nucleic acid molecule may be of natural origin, synthetic or recombinant nucleic acid molecule provided that the person skilled in the art may regard it as a full-sized molecule. Thus, for example, shorter molecules of nucleic acids that correspond to the sequence of the gene contain less than the full length of the gene, where the gene contains the coding and non-coding sequences, promoters, enhancers and other regulatory sequences, flanking sequences, and the like, and other functional and non-functional sequences, which are considered a part of the gene. is another example of a shortened molecule of nucleic acid, which correspond to the sequence of mRNA that contain less than the full length of the mRNA transcript, which can include a variety of broadcast and non-translated region, as well as other functional and non-functional sequence.
In other preferred embodiments, the truncated molecules are polypeptides that contain amino acid sequence shorter than the full sequence of a particular protein or polypeptide component. In used in this description of the meaning of "deletion" has its generally accepted meaning, which is clear to a person skilled in the art, and may refer to molecules that are missing one or more parts of a sequence, either from the terminal or from reconcavo region compared with the corresponding full length molecule, for example, as in the case described in this description of short molecules. Shorter molecules that are linear biological polymers, such as molecules of nucleic acids or polypeptides may have one or more deletions or terminal region of the molecule, or deletions in reconcavo region of the molecule, with such deletions can be deleted 1-1500 continuously following each other nucleotide or amino acid residues, preferably 1-500 continuously following each d the natives nucleotide or amino acid residues and more preferably 1-300 continuously following each other nucleotide or amino acid residues. In some particularly preferred embodiments, the truncated molecule nucleic acid can have a deletion 270-330 continuously consecutive nucleotides. In certain other particularly preferred embodiments, the truncated polypeptide molecules may have a deletion 80-140 continuously consecutive amino acids.
This invention also relates to variants specified in this description of the nucleic acids that encode fragments, analogs and/or derivatives fused protein binding domain-immunoglobulin. Variants of nucleic acids encoding the fusion binding domain-immunoglobulin may be allelic variants of the nucleic acids of natural origin, or variants of unnatural origin. As you know, in this area of the allelic variant is an alternate form of the nucleic acid sequence, which may have at least one substitution, deletion or adding one or more nucleotides, any of which does not substantially alter the function of the encoded fused polypeptide binding domain-immunoglobulin.
Variant and derivative merge binding domain-immunoglobulin can be obtained by mutations of nucleotide sequences coding for fused polypeptides binding domain-immunoglobu is in. Changes to the native amino acid sequence can be accomplished in any of several standard methods. Mutations can be introduced in a particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites, providing ligation to fragments of the native sequence. After ligating the resulting reconstructed sequence encodes analog having the desired insertion, substitution or deletion of amino acids.
Alternatively, you can use the site-specific mutagenesis on the basis of oligonucleotides to obtain an altered gene, in which the predefined codons can be altered by substitutions, deletions or insertions. Typical ways to implement such changes described by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37: 73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering. Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad Sci. USA 82: 488, 1985); Kunkel et al. (Methods in Enzymol. 154: 367, 1987) and in U.S. patent No. 4518584 and 4737462.
As an example, the modification of DNA can be performed using site-specific mutagenesis of DNA that encodes a protein, along with application methods DNA amplification using primers to introduce and amplify alterations in the DNA matrix, such as splicing by extending p is retribalize sites by PCR (SOE). Site-specific mutagenesis is typically performed with the use of phage vector, which has single - and double-stranded forms, such as vectors on the basis of phage M13, which are well known and commercially available. You can use other suitable vectors which contain replication to begin single-stranded phage (see, for example, Veira et al., Meth. Enzymol. 15: 3, 1987). In General, site-specific mutagenesis is performed by obtaining a single-stranded vector that encodes the protein of interest (for example, all or part of this fused protein binding domain-immunoglobulin). Oligonucleotide primer that contains the desired mutation in the region of homology with the DNA in single-stranded vector, annealed with the vector and then adding a DNA polymerase such as DNA polymerase I of E. coli (fragment maple), which uses double-stranded region as a primer to obtain heteroduplex, in which one strand encodes the modified sequence and the other of the source sequence. Heteroduplex injected into appropriate bacterial cells and select the clones that contain the desired mutation. The resulting modified DNA molecules can recombinante to Express in appropriate cell hosts, to obtain a modified protein.
This image is giving also covers the equivalent DNA constructs, that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity. For example, and as discussed above, the sequence encoding the Cys residues that are not desirable or are not essential for biological activity, can be changed on the basis of deletions of residues Cys or replaced by other amino acids, preventing the formation of inappropriate intramolecular disulfide bridges upon renaturation.
Organisms-hosts include such organisms, which may be recombinant products merged products binding domain-immunoglobulin encoded by recombinant constructions according to this invention, such as bacteria (for example E. coli), yeast (such as Saccharomyces cerevisiae and Pichia pastoris), insect cells and mammals, including expression in vitro and in vivo. Thus, organisms-owners may enable organisms to design, reproduction, expression or other stages of the production of vaccines given in this description; the owners also include constituents who are immune responses that described above. Currently, the preferred organisms hosts are bacterial strains E.coli, inbred whether the AI mice and cell line of mice, and human cells in the body and in the form of cell lines.
Construction of DNA encoding the desired fusion binding domain-immunoglobulin is injected into a plasmid for expression in an appropriate host. In preferred embodiments, the host is a bacterial host. The sequence encoding the ligand or binding domain nucleic acid, preferably optimize against codons for expression in a particular host. Thus, if, for example, the merger of binding domain-immunoglobulin Express in bacteria, the codons can be optimized for use by bacteria. In the case of small coding regions of the gene can be synthesized in the form of individual oligonucleotide. For larger proteins, you can use the splicing of different oligonucleotides, mutagenesis or other methods known to experts in this field. Nucleotide sequences in the plasmids, which are regulatory regions such as promoters and operators, functionally associated with each other for transcription. A nucleotide sequence encoding a protein binding domain-immunoglobulin may also include DNA encoding a secretion signal, with the resulting peptide is a protein precursor. The resulting protestirovannyx can be extracted from periplasmatic space or environment for fermentation.
In preferred embodiments, the plasmid DNA also contain the sequence termination of transcription. Used in this sense, the scope of the termination of transcription" is a sequence that signals termination of transcription. Full transcription terminator can be obtained from a gene encoding a protein, which may be the same or may be different from the gene that encodes the fusion binding domain-immunoglobulin, or from the source of the promoter. The transcription terminators are optional components expressing systems according to the invention, but are used in preferred embodiments.
Used in this invention plasmids include a promoter in functional connection with a DNA that encodes a protein of interest or polypeptide, and are designed for protein expression in a suitable host, as described above (for example, bacteria, mouse or human), depending on the desired application plasmids (for example, vaccine, containing the sequence encoding the fusion binding domain-immunoglobulin). Suitable promoters for expression of proteins and polypeptides according to the invention are widely available and well known in this field. Preferred inducible promoters or constitutive promoters, which links the Ana with the regulatory regions. Such promoters include, but are not limited, the promoter of phage T7 and other phage promoters, such T7, such as promoters T3, T5 and SP6 promoters trp, lpp and lac, such as lacUV5, from E.coli; P10 or polyhedrin gene promoter expressing system baculovirus/insect cell (see, for example, U.S. patent No. 5243041, 5242687, 5266317, 4745051 and 5169784) and inducible promoters other expressing eukaryotic systems. For the expression of proteins such promoters inserted into the plasmid in functional connection with the regulatory region, such as the lac-operon.
Preferred area of promoters represent areas that are inducible and functional in E. coli. Examples of suitable inducible promoters and promoter regions include, but are not limited to: the operator of the E. coli lac, answering isopropyl-β-D-thiogalactopyranoside (IPTG; see Nakamura et al., Cell 18: 1109-1117, 1979); adjustable metal promoter elements of metallothionein responsible for the induction of a heavy metal (e.g. zinc) (see, for example, U.S. patent No. 4870009, Evans et al.); the promoter of phage T7lac answering IPTG (see, for example, U.S. patent No. 4952496 and Studier et al., Meth. Enzymol. 185: 60-89, 1990) and the TAC promoter.
Plasmids can optionally include a gene or genes of breeding marker that is functional in the host. Gene breeding marker includes any gene which definition is employed, the phenotype of the bacteria, which allows you to identify and selectively growing the transformed bacterial cells among the vast majority of untransformed cells. Suitable genes of breeding marker for bacterial hosts include, for example, the gene for resistance to ampicillin (Ampr), a gene of resistance to tetracycline (Tcr) and the gene for resistance to kanamycin (Kanr). Currently preferred is a gene for resistance to kanamycin.
The plasmids may also include DNA encoding a secretion signal functionally related protein. Suitable for use signals secretion widely available and well known in this field. You can use the prokaryotic and the eukaryotic secretion signals, functional in E. coli. Preferred currently secretion signals include, but are not limited to, signals encoded by the following genes of E.coli: ompA, ompT, ompF, ompC, beta-lactamase and alkaline phosphatase, and the like (von Heijne, J. Mol. Biol. 184: 99-105, 1985). In addition, you can use the signal secretion of bacterial gene pelB (Lei et al., J. Bacteriol. 169: 4379, 1987), a signal secretion of phoA and cek2, functional in a cell of an insect. The most preferred secretion signal is a signal secretion of the E. coli ompA. You can also use other prokaryotic and eukaryotic secretion signals, zvezdnye specialists in this field (see, for example, von Heijne, J. Mol. Biol. 184: 99-105, 1985). Using the methods described in the present description, a person skilled in the art can manipulate the secretion signals that are functional or yeast cells, insect cells, or mammalian cells to secrete proteins from these cells.
Preferred plasmids for transformation of E. coli cells include expressing the pET vectors (e.g., pET-11a, pET-12a-c, pET-15b; see U.S. patent No. 4952496; available from Novagen, Madison, WI). Other preferred plasmids include plasmid pKK, in particular pKK 223-3, which contains the tac promoter (Brosius et al., Proc. Natl. Acad. Sci. 81: 6929, 1984; Ausubel et al., Current Protocols in Molecular Biology; U.S. patent No. 5122463, 5173403, 5187153, 5204254, 5212058, 5212286, 5215907, 5220013, 5223483 and 5229279). Plasmid pKK modified by replacement of the gene of resistance to ampicillin resistance gene for kanamycin (available from Pharmacia; obtained from pUC4K, see, e.g., Vieira et al. (Gene 19: 259-268, 1982; and U.S. patent No. 4719179). Baculovirus vectors, such as pBlueBac (also called pJVETL and its derivatives), in particular, pBlueBac III (see, for example, U.S. patent No. 5278050, 5244805, 5243041, 5242687, 5266317, 4745051 and 5169784; available from Invitrogen, San Diego) can also be used for expression of polypeptides in insect cells. Other plasmids include plasmids pIN IIIompA (see U.S. patent No. 4575013; see also Duffaud et al., Meth. Enz. 153: 492-507, 1987), such as pIN-IIIompA2.
Before occhialino DNA molecule is replicated in bacterial cells, preferably in E. coli. Preferred DNA molecule also includes bacterial replication to begin to ensure the preservation of DNA molecules in bacteria from generation to generation. Thus, it is possible to obtain large quantities of DNA through replication in bacteria. Preferred bacterial beginning of replication include, but are not limited, early replication fl-ori and col E1. Preferred hosts contain chromosomal copies of the DNA that encodes RNA polymerase T7, functionally associated with inducible promoter, such as the lacUV promoter (see U.S. patent No. 4952496). Such hosts include, but are not limited, lysogeny strains of E. coli HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). It is preferable strain BL21(DE3). Strains pLys give low levels of T7 lysozyme, a natural inhibitor of RNA polymerase T7.
Presents the DNA molecule may also contain a gene encoding protein-repressor. Protein-repressor is able to repress transcription from a promoter that contains a sequence of nucleotides that binds to a protein-repressor. The promoter may be derepression by changing physiological conditions in the cell. For example, the change can be done by adding to the growing environment of the molecule, which inhibits the ability to interact with the operator or with the regulatory be the kami or other areas of DNA, or by changing the temperature of the growth medium. Preferred proteins repressor substances include, but are not limited to, the lacI repressor of E. coli sensitive to IPTG induction, the temperature-sensitive λ repressor cI857, and the like. It is preferable for the lacI repressor of E. coli.
In General, recombinant constructs according to this invention will also contain the elements necessary for transcription and translation. In particular, such elements are preferred in cases where recombinant expressing the construct containing the nucleic acid sequence encoding the fused protein binding domain-immunoglobulin, is intended for expression in a cell-the owner or the body. In some embodiments of the present invention is preferred for the type of cells or specific cell type expression of the gene encoding the fusion binding domain-immunoglobulin, can be achieved by placing the gene under the control of a promoter. The choice of promoter will depend on the type of cells that you want to transform, and the degree or type the desired control. Promoters can be constitutive or active and may also be specific to a cell type, tissue-specific, a-specific cells, specific for the event, temporarily-specific or inducible. Preferably the mi are the promoters, specific to cell type, and promoters that are specific for a particular event. Examples of constitutive or non-specific promoters include the early SV40 promoter (U.S. patent No. 5118627), late SV40 promoter (U.S. patent No. 5118627), the early promoter of the gene of CMV (U.S. patent No. 5168062) and adenoviral promoter. In addition to viral promoters in the context of the present invention is also applicable to cellular promoters. In particular, the applicable cellular promoters of the so-called genes household. Preferred viral promoters, as a rule, they are stronger promoter than cellular promoters. Promoter region identified genes in many eukaryotes, including higher eukaryotes, so that the experts in this field can easily choose suitable promoters for use in particular host.
You can also use inducible promoters. These promoters include MMTV LTR (PCT WO 91/13160)induced by dexamethasone; the promoter metallothionein induced by heavy metals; and promoters with elements of the response to the camp-induced camp. When using an inducible promoter sequence, a nucleic acid encoding a protein binding domain-immunoglobulin, can be delivered into the cell by means of expressing design according to this invention, and it can in order to be at rest until the addition of the inductor. This provides the time control production of the gene product.
Specific to event promoters active or stimulated only in the case when a particular event occurs, such as tumors or viral infection. LTR HIV is a well-known example of a promoter that is specific for the event. The promoter is inactive if not present gene product tat, which appears when a viral infection. Some promoters that are specific for a particular event, are also tissue-specific.
In addition, it is possible to use promoters, regulation which is consistent with the specific cell genome. For example, promoters of genes that are consistently expressed, can be used if necessary expression of a specific gene encoding a protein binding domain-immunoglobulin, together with the expression of one or more endogenous or introduced exogenous genes. The specified type of promoter is particularly suitable in the case where the known expression pattern of a gene related to the induction of immune responses in specific tissues of the immune system, in order to activate or otherwise to attract specific immunocompetent cells in such tissue to participate in an immune response.
In addition to the promoter, can be embedded in d is rezerwuj sequence, negative regulators or the tissue-specific silencers to reduce non-specific expression of the genes encoding protein binding domain-immunoglobulin, in certain situations, such as, for example, the situation where the owner temporarily weaken the immune system, which is part of the treatment. In the promotor region you can embed various repressive elements. Repression of transcription does not depend on the orientation of the repressive elements or distance from the promoter. One type of repressive sequence is the sequence insulator. Such sequences inhibit transcription (Dunaway et al., Mol. Cell. Biol. 17: 182-9, 1997; Gdula et al., Proc. Natl. Acad. Sci. USA 93: 9378-83, 1996, Chan et al., J. Virol. 70: 5312-28, 1996; Scott and Geyer, EMBO J. 14: 6258-67, 1995; Kalos and Fournier, Mol. Cell. Biol. 15: 198-207, 1995; Chung et al., Cell 74: 505-14, 1993) and will suppress background transcription.
Repressive elements are also identified in the promoter regions of genes collagen type II (cartilage), cholineacetyltransferase, albumin (Hu et al., J. Cell Growth Differ. 3(9): 577-588, 1992), phosphoglycerate (PGK-2) (Misuno et al., Gene 119 (2): 293-297, 1992) and in the gene for 6-phosphofructo-2-kinase/fructose-2,6-biphosphate (Lemaigre et al., Mol. Cell Biol. 11 (2): 1099-1106). In addition, identified a negative regulatory element Tse-1 the number of genes that are specific for the liver, and it has been shown that it blocks the induction of gene activation in hepatocytes, posredovanju element in response to camp (CRE) (Boshart et al., Cell 61 (5): 905-916, 1990).
In preferred embodiments, the design includes elements that enhance expression of the desired product. Such elements include internal binding sites of the ribosome (IRES; Wang and Siddiqui, Curr. Top. Environ. Immunol. 203: 99, 1995; Ehrenfeld and Semler, Curr. Top. Environ. Immunol. 203: 65, 1995; Rees et al., Biotechniques 20: 102, 1996; Sugimoto et al., Biotechnology 12: 694, 1994). IRES increase the efficiency of translation. Other sequences may also increase the expression. In the case of some gene sequence, particularly at the 5'end of the inhibit transcription and/or translation. These sequences are palindromes that can form patterns studs. As a rule, any such sequence in the nucleic acid to be taken, deleteroute. To confirm or determine the sequence influences the expression, analyze the levels of expression of a transcript or translated product. The levels of transcripts can be analyzed by any known method, including Northern blot hybridization, protecting the probe from RNase and the like. Levels of the protein can be analyzed by any known method, including ELISA, Western blot, immunocytochemical or other well known methods.
In design, encoding a protein binding domain-immunoglobulin according to the invention can on the process other elements. In preferred embodiments, the construct contains a sequence of transcription termination, including a polyadenylation sequence, the donor and acceptor splicing sites and enhancer. You can also include other elements suitable for the expression and maintenance of structures in mammalian cells or other eukaryotic cells (e.g., the beginning of replication). As designs are usually obtained in bacterial cells include items that are necessary or that enhance reproduction in bacteria. Such items include replication to begin, breeding marker and the like.
As shown in this description, it is possible to provide an additional level of regulation of expression of nucleic acids encoding the fused protein binding domain-immunoglobulin delivered into cells via structures according to the invention, by simultaneous delivery of two or more differentially regulated structures of nucleic acids. The use of this approach on the basis of different structures of nucleic acids may provide an opportunity for coordinated regulation of immune response, such as spatial-temporal coordination, which depends on the type of cells and/or the presence of any other expressed the encoded component. Specifications the leaves in this area will be clear, what different levels of regulated gene expression can be achieved in a similar manner by selecting appropriate regulatory sequences, including, but not limited specified, the promoters, enhancers and other well-known regulatory elements of genes.
This invention also relates to vectors and to constructs derived from known vectors that include nucleic acid according to this invention, and in particular to "recombinant expressing constructs"that include any nucleic acids encoding the fused protein and the polypeptide binding domain-immunoglobulin according to the invention, which is shown above; the cells of the host, which are genetically engineered with vectors and/or constructs according to the invention, and to methods of introducing expressing constructs containing nucleic acid sequences encoding such fused polypeptides and fused proteins binding domain-immunoglobulin according to the invention, or their fragments or variants, methods based on recombination. Slit proteins binding domain-immunoglobulin can Express virtually any cell host under the control of appropriate promoters, depending on the nature of the design (e.g., type of promoter, which is described above) and on the nature of the desired to etki host (for example, whether it is a terminal postmitotic differentiated or actively dividing; for example, whether expressing the construct in the cell-master in the form of episome or integrated into the genome of the host cell). Appropriate cloning and expressing vectors for use in prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989); as noted above, in particularly preferred embodiments of the invention, recombinant expression is carried out in mammalian cells, which were transliterowany or transformed with recombinant expressing the design according to this invention.
Usually the design is obtained from plasmid vectors. The preferred design is a modified vector pNASS (Clontech, Palo Alto, CA), which has a nucleic acid sequence encoding a gene of resistance to ampicillin, a polyadenylation signal and site of the T7 promoter. Other suitable expressing vectors mammals are well known (see, for example, Ausubel et al., 1995; Sambrook et al., above; see also, e.g., directories, Invitrogen, San Diego, CA; Novagen, Madison, WI; Pharmacia, Piscataway, NJ; and others). You can get the currently favored designs that contain a sequence encoding dihydrotetrazolo (DHFR) under suitable regulatory control, to provide higher levels of production fused protein binding domain-immunoglobulin, these levels are the result of gene amplification after application of the appropriate agent selection (e.g., methotrexate).
Generally, recombinant expressing the vectors will contain the start replication and breeding markers, allowing to transform a cell-master, and a promoter derived from a highly expressed gene to direct transcription located below the structural sequence described above. Assemble heterologous structural sequence in appropriate phase with sequences of initiation and termination of translation. Thus, for example, presented in this description of a nucleic acid encoding a protein binding domain-immunoglobulin, you can include any of a variety of constructions expressing vectors as recombinant expressing constructs for expression of the fused polypeptide binding domain-immunoglobulin in the cell host. In some preferred embodiments, the design included in the composition that is administered in vivo. Such vectors and designs include chromosomal, achromosome and synthetic DNA sequences, e.g., derivatives of SV40; bacteria the performance communications plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and ragovoy DNA, DNA virus, such as the smallpox virus, adenovirus, poxvirus birds and the virus bulbar palsy or deficient in replication of retroviruses, which are described below. However, to obtain a recombinant expressing the constructs you can use any other vector, and in preferred embodiments, this vector is capable of replication and will be viable in the host.
The appropriate sequence(ti) DNA can be embedded into a vector in a variety of ways. In General, the DNA sequence is inserted into the corresponding site(you) restriction endonucleases ways that are known in this field. Standard methods of cloning, isolation, amplification and purification of DNA, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various methods of separation are well known and widely used by specialists in this field means. A number of standard methods are described, for example, in Ausubel et al. (1993, Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. and John Wiley and Sons, Inc., Boston, MA); Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY); Glover (Ed.) in 1985, DNA Cloning Vol.I and II, IRL Press, Oxford, UK); Hames and Higgins (Eds.), (1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK); and in other history is literature.
The DNA sequence in expressing the vector is functionally connected, at least one suitable sequence expression regulation (for example, a constitutive promoter or regulated promoter)to direct mRNA synthesis. Typical examples of such sequences regulating the expression include the promoters of eukaryotic cells or their viruses, which are described above. The promoter can be selected from any desired gene using CAT vectors (chloramphenicolchloramphenicol) or other vectors from breeding markers. Eukaryotic promoters include pretani the CMV promoter, the promoter timedancing HSV, early and late SV40 promoter, retrovirus LTR and the promoter metallothionein-I mouse. The selection of the appropriate vector and promoter is well-known specialist in this field, and gaining some particularly preferred recombinant expressing constructs containing at least one promoter or regulated promoter functionally linked to a nucleic acid that encodes a fused polypeptide binding domain-immunoglobulin given in this description.
Transcription of DNA encoding the polypeptides according to this invention, higher eukaryotes can be increased by embedding in a vector sequence of the enhancer. The enhancers is the tsya CIS-acting elements of DNA, generally, about 10 to 300 BP that act on a promoter, enhancing its transcription. Examples include the SV40 enhancer at the end of the beginning replication, BP 100 to 270, early enhancer promoter and cytomegalovirus enhancer of virus polyoma at the end of the beginning replication and adenovirus enhancers.
As specified in this description in some embodiments, the vector may be a viral vector such as a retroviral vector (Miller et al., 1989 BioTechniques 7: 980; Coffin and Varmus, Retroviruses 1996, Cold Spring Harbor Laboratory Press, NY). For example, retroviruses, which can be obtained retroviral plasmid vectors include, but are not limited, leukosis virus of mice, Malone, virus, spleen necrosis, retroviruses such as rous sarcoma virus, virus, Harvey sarcoma virus leukemia birds, leukosis virus monkey Gibbon, human immunodeficiency virus, adenovirus, virus, myeloproliferative sarcoma virus tumor of the breast.
Retroviruses are RNA viruses that can replicate and integrate into the genome of the host cell by means of an intermediate product DNA. Specified intermediate product DNA or provirus can be stably integrated into the DNA of the host cell. According to some variants of the present invention, the vaccine may contain retrovirus, which introduced foreign gene, which encodes courtn the th protein, instead of the normal retroviral RNA. When retroviral RNA enters the cell host during infection, the foreign gene is also introduced into the cell and can then integrate into the DNA of the host cell, as if he is part of the retroviral genome. The expression of specified foreign gene in the host leads to the expression of a foreign protein.
Most retroviral vector systems that have been developed for gene therapy based on retroviruses of mice. Such retroviruses exist in two forms, as free viral particles, called virions, or in the form of proviruses integrated into the DNA of the host cell. Firiona form of the virus contains structural and enzymatic proteins of retrovirus (including the reverse transcriptase enzyme), two RNA copies of the viral genome and part of the plasma membrane of cells of the source containing the viral envelope glycoproteins. The retroviral genome is organised into four main areas: long terminal repeat (LTR), which contains CIS-acting elements necessary for the initiation and termination of transcription, and is set as 5'-and 3'-side coding genes, and three gene encoding gag, pol and env. These three gene gag, pol and env encode, respectively, the internal viral structure, enzyme proteins such as integrase) and the glycoprotein shell (called the ' gp70 and p15e), which gives the infectivity and limits the specificity of the virus with respect to the host, as well as peptide "R" with an undefined function.
Developed individual line packing of cells and cell line producing vectors, in connection with the security in respect of the application of retroviruses, including their use in vaccines, as described in this invention. Briefly, in this method, two components are used, the retroviral vector and the line of packing cells (PCL). The retroviral vector contains long terminal repeats (LTR), alien DNA that you want to migrate, and packing sequence (y). Specified retroviral vector will not be reproduced by itself, because the genes that encode structural proteins and membrane proteins, are not included in the vector genome. PCL contains the genes encoding the proteins gag, pol and env, but does not contain the packaging signal "y". Thus, the very PCL can only form an empty viral particles. In the specified General way, the retroviral vector is introduced into PCL, creating, thus, the cell line producing the vector (VCL). Specified VCL produces particles of virions containing only the genome of the retroviral vector (alien), and therefore it was considered safe retroviral vector for therapeutic applications.
Construction of retroviral vector" refers to a con is e.g., which in preferred embodiments of the invention capable of directing the expression of interest sequence(TEI) or gene(s), such as nucleic acid sequence encoding the fusion binding domain-immunoglobulin. Briefly, the design of retroviral vectors should include the 5'LTR, a tRNA binding site, a packaging signal, the beginning of the synthesis of the second DNA strand and the 3'LTR. In vector structure you can include a wide variety of heterologous sequences, including, for example, sequences that encode a protein (e.g., cytotoxic protein associated with a disease antigen, supporting the immune molecule or substituted gene) or which are suitable in the form of the molecule (e.g., a ribozyme or antisense sequence).
Construction of retroviral vector according to this invention can easily be constructed from a wide variety of retroviruses, including for example, retroviruses type B, C and D, as well as spumaviruses and lentiviruses (see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses can be easily obtained from depositories or collections such as the American type culture collection ("ATCC"; Rockville, Maryland), or select from known sources using public methods. Any of the above retroviruses which can easily be used in order to assemble or construct retroviral constructs vector packing cells or cells of producers according to this invention, on the basis of descriptions and standard methods of recombination (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 82: 488, 1985).
Suitable promoters for use in viral vectors typically may include, but are not limited to, the retroviral LTR; the SV40 promoter and the promoter of the human cytomegalovirus (CMV), described in Miller, et al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited specified, the promoters of histone, pol III, and β-actin). Other viral promoters that can be used include, but are not limited to, adenoviral promoters, promoters timedancing (TK) and the promoters of parvovirus B19. The choice of a suitable promoter will be obvious to a person skilled in this field, based on the instructions given in this description, and can be accessed from a number of regulated promoters or promoters, which are described above.
As described above, the retroviral plasmid vector used for transduction lines packing of cells to form a cell line-producers. Examples of packing cells, which can transpirirovat the ü, include, but are not limited to, cell line PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN, which is described in Miller, Human Gene Therapy, 1: 5-14 (1990). Packing cells can be transducible vector using any method known in this field. Such methods include, but are not limited to, electroporation, the use of liposomes and precipitation CaPO4. Alternative, the retroviral plasmid vector may be encapsulated in a liposome or associated with the lipid and then introduced into the host.
Cell line-producers generates infectious retroviral vector particles which contain the sequence(ti) a nucleic acid encoding a fused polypeptide or fused proteins binding domain-immunoglobulin. Then such retroviral vector particles can be used to transducible eukaryotic cells, either in vitro or in vivo. Transduced eukaryotic cells will Express the sequence(ti) a nucleic acid that encodes a fused polypeptide or protein binding domain-immunoglobulin. Eukaryotic cells that can be transducible include, but are not limited to, embryonic stem cells and hematopoietic stem cells, hepatocytes, fibroblasts, circulating peripheral mononuclear and poly is oheaders blood cells, including myelomonocytic cells, lymphocytes, cultured myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoid and reticuloendothelial cells of lymph nodes and spleen, keratinocytes, endothelial cells, and bronchial epithelial cells.
In another example variant of the invention, which uses a viral vector to get expressing design merge binding domain-immunoglobulin, in one preferred embodiment, cells are the owners of transduced by the recombinant viral construct, directing the expression of the fused polypeptide or fused protein binding domain-immunoglobulin may produce viral particles containing expressed fused polypeptides or fused proteins binding domain-immunoglobulin obtained from parts of the membrane of the host cell, captured virus particles during active viral replication (bazinga).
In another aspect this invention relates to a cell host containing the above described recombinant constructs expressing the fusion binding domain-immunoglobulin. Cells are the owners of genetically design (transducers, transform or transferout) vector and/or expressing constructs according to this invention, which may, for example, predstavljati a cloning vector, a Shuttle vector or expressing the construct. Vector or design, for example, may be in the form of a plasmid, a viral particle, a phage, etc. Designed cell host can be cultured in conventional nutrient media modified so that they are suitable for activating promoters, selecting transformants or amplification of individual genes, such as genes encoding fused polypeptides binding domain-immunoglobulin or fused proteins binding domain-immunoglobulin. The cultivation conditions specific host cells selected for expression, such as temperature, pH and the like, will be apparent to a person skilled in this field.
The host-cell may be a cell of higher eukaryotes, such as mammal cells, or cell of lower eukaryotes, such as yeast cell, or the host-cell can be a prokaryotic cell such as a bacterial cell. Typical examples of appropriate host cells according to this invention include, but are not limited to, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; cells of fungi, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO cells, COS or 293; adenoviruses; plant cells, or any suitable cell already adapted to reproduce in vitro or introduced in Kul is ur de novo. It is assumed that the experts in this field are free to choose a suitable host, based on the instructions given in this description.
Various systems based on mammalian cell cultures can also be used to Express the recombinant protein. Examples expressing systems mammals include lines of fibroblasts monkey kidney COS-7, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of Express compatible vector, for example, cell line C127, 3T3, CHO, HeLa and BHK. Expressing the vectors mammals will contain the start of replication, a suitable promoter and enhancer, and also any necessary binding sites of the ribosome, the site of polyadenylation, donor and acceptor splicing sites, sequence termination of transcription and 5'-flanking retranscribing sequence, for example, the sequence given in the description relating to the receipt of constructs expressing the fusion binding domain-immunoglobulin. DNA sequences obtained from splicing sites and the SV40 polyadenylation, can be used to obtain the required retranscribing genetic elements. The introduction of design in the cell-master carried out in various ways which will be familiar to specialists in this field, including, but neogranichena specified, for example, transfection using calcium phosphate, transfection mediated by DEAE-dextran, or electroporation (Davis et al., 1986, Basic Methods in Molecular Biology).
Slit proteins binding domain-immunoglobulin according to this invention can be prepared in pharmaceutical compositions for administration according to well known methods. Pharmaceutical compositions typically contain one or more recombinant expressing designs and/or products of expression of these structures in combination with a pharmaceutically acceptable carrier, excipient or diluent. Such media should be non-toxic in relation to the recipient when used doses and concentrations. In the case of compositions based on nucleic acids or in the case of compositions containing the products of recombinant expression constructs according to this invention, should be introduced approximately from 0.01 μg/kg to 100 mg/kg of body weight, usually intradermal, subcutaneous, intramuscular or by intravenous or other routes. The preferred dose is from about 1 μg/kg to about 1 mg/kg, particularly preferred is from about 5 μg/kg 200 μg/kg of Specialists in this field will understand that the number and frequency of introduction will depend on the response of the host. "Pharmaceutically acceptable carriers" for therapeutic Ave the changes are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.Gennaro edit. 1985). For example, you can use sterile saline or phosphate-saline buffer at physiological pH. The pharmaceutical compositions can contain preservatives, stabilizers, dyes and even corrigentov. For example, as preservatives may be added sodium benzoate, sorbic acid and esters of para-hydroxybenzoic acid. Ibid, 1449. Additionally, you can use antioxidants and suspendresume agents. Ibid.
"Pharmaceutically acceptable salt" refers to salts of the compounds according to this invention, the resulting combination of such compounds and organic and inorganic acid (acidic additive salts) or organic or inorganic bases (basically additive salt). Compounds according to this invention can be used either in the form of free base or in the form of salts, it is assumed that both forms are included in the scope of this invention.
Pharmaceutical compositions that contain one or more constructs encoding a protein binding domain-immunoglobulin (or their expression products), can be in any form that allows you to enter the composition to the patient. For example, the composition may be in the form of solid, liquid or gas (air is Sol). Typical routes of administration include, without limitation, oral, local, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal or intranasal. The term "parenteral" as used in this description, the meaning includes subcutaneous injections, intravenous, intramuscular, vnutrigrudne, vnutrikavernozno, intrathecal, intra, intraurethral injection or infusion. The pharmaceutical composition is prepared so that it is allowed inside the active ingredients to be bioavailable upon introduction of the composition to the patient. Songs that will enter the patient, have the form of one or more dosage units, in this case, for example, a tablet may be a single unit dosing, and a container containing one or more compounds according to the invention in aerosol form may contain a large number of dosage units.
In the case of oral administration may be excipient and/or binder. Examples are sucrose, kaolin, glycerin, dextrin, starch, sodium alginate, carboxymethylcellulose and ethylcellulose. May be present coloring agents and/or corrigentov. You can use the covering shell.
The composition may be in the form of a liquid, for example, elixir, syrup, solution, emulsion or WM is ansii. As two examples, the fluid may be used for oral administration or delivery by injection. Preferred compositions, if they are intended for oral administration, contain in addition to one or more structures or expressed product of the merger of the binding domain-immunoglobulin one or more sweeteners, preservatives, dyes/color enhancers and taste. In a composition intended for administration by injection may be included one or more surfactants, preservatives, humectants, dispersing agents, suspendida agents, buffers, stabilizers and agents for isotonicity.
Liquid pharmaceutical composition used in this invention, either in the form of solution, suspension, or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological solution, ringer's solution, isotonic solution of sodium chloride, fatty oils such as synthetic mono or diglycerides which may serve as a solvent or suspiciouse medium, polyethylene glycols, glycerine, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or IU Ilmarinen; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting toychest, such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or vials with multiple doses, made of glass or plastic. Preferred auxiliary agent is saline. Injectable pharmaceutical compositions are preferably sterile.
Also desirable may be included in the preparation of other components, such as carriers for delivery, including, but not limited specified, aluminium salts, the emulsion type water in oil, biodegradable oil carriers, emulsions of the type oil-in-water, biodegradable microcapsules, and liposomes. Examples of immunostimulatory substances (adjuvants) for use in such media include N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), lipopolysaccharide (LPS), glucan, IL-12, GM-CSF, IFN-γ and IL-15.
Although the pharmaceutical compositions according to this invention can use any suitable carrier known to specialists in this field, the media type will depend on the route of administration and whether long visual the discussion. For parenteral administration, such as subcutaneous injection, the carrier preferably contains water, saline, alcohol, a fat, a wax or a buffer. For oral administration, you can use any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, glucose, sucrose, and magnesium carbonate. Also as carriers for the pharmaceutical compositions according to this invention can be used biodegradable microspheres (e.g., polylactic-galactic). Suitable biodegradable microspheres described, for example, in U.S. patent No. 4897268 and 5075109. In this regard, preferably, the microsphere was more than about 25 microns.
Pharmaceutical compositions may also contain diluents, such as buffers, antioxidants such as ascorbic acid, polypeptides of low molecular weight (less than approximately 10 residues), proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Examples of suitable diluents are neutral buffered salt solution or saline mixed with nonspecific serum albumin. Preferably the product is prepared in the form of freeze-dried using what W as diluents suitable solutions of excipients (for example, sucrose).
As described above, the present invention includes compositions capable of delivering the nucleic acid molecule encoding the fused protein binding domain-immunoglobulin. Such compositions include recombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698 and WO 94/03622), adenovirus (see Berkner, Biotechniques 6: 616-627, 1988; Li et al., Hum. Gene Ther. 4: 403-409, 1993; Vincent et al., Nat. Genet. 5: 130-134, 1993, and Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219, 1994), poxvirus (see U.S. patent No. 4769330; U.S. patent No. 5017487 and WO 89/01973)), recombinant expressing the structure of nucleic acids in complex with poly-molecule (see WO 93/03709) and nucleic acids associated with liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84: 7851, 1987). In some embodiments, the DNA may be associated with killed or inaktivirovannye adenovirus (see Curiel et al., Hum. Gene Ther. 3: 147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89: 6094, 1992). Other suitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem. 264: 16985-16987, 1989) and combination lipid-DNA (see Felgner et al., Proc. Natl. Acad Sci. USA 84: 7413-7417, 1989).
In addition to direct methods, in vivo, can be used ex vivo methods in which cells are removed from the host, modify and placed in the same or other animal host. Obviously, you can use any of the above compositions for the introduction of a fused protein binding domain-immun is a globulin or molecules of nucleic acids, encoding a protein binding domain-immunoglobulin, in the cells of the tissue in ex vivo. Protocols viral, physical and chemical methods of capture are well known in this field.
Thus, this invention is applicable for the treatment of patients with B-cell disorder or malignant condition, or for processing cultures of cells derived from a patient. Used in this sense, the term "patient" refers to any warm-blooded animal, preferably a human. The patient may suffer from cancer, such as B-cell lymphoma, or can be healthy (i.e. no detectable disease or infection). "Cell culture" is any drug that can be processed ex vivo, for example, a product containing immunocompetent cells or selected cells of the immune system (including, but not limited specified, T-cells, macrophages, monocytes, B-cells and dendritic cells). These cells can be distinguished by any of many methods known to experts in this field (e.g., centrifugation in density gradient ficol-hipac). Cells can be (but not necessarily) isolated from the patient, the affected B-cell disorder or malignancy, and can be re-introduced to the patient after treatment.
A liquid composition intended for either parenteral, either for oral administration should contain such a number of designs that encodes a protein binding domain-immunoglobulin, or downregulation of the product so that you can get the right dose. Usually, this amount is at least 0.01% wt. construction merge binding domain-immunoglobulin or expressed product in the composition. If the composition is intended for oral administration, such amounts may vary from 0.1 to about 70% by weight of the composition. Preferred oral compositions contain from about 4% to 50% of the structure of the merger binding domain-immunoglobulin or expressed product(s). Preferred compositions and preparations are prepared so that a parenteral dosage form contains from 0.01 to 1% active ingredient by weight.
The pharmaceutical composition may be intended for local administration, and in this case, it is convenient carrier may contain a solution, emulsion, ointment or gel base. The basis of, for example, may contain one or more of the following components: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents, such as water and alcohol, and emulsifiers and stabilizers. In the pharmaceutical compositions for local injection can present the SQL thickeners. In the case of the planned TRANS-dermal composition can include a transdermal patch or a device for iontophoresis. Local composition may contain a concentration of merge constructs binding domain-immunoglobulin or expressed product from about 0.1 to 10% wt./about. (mass per unit volume).
The composition may be intended for rectal administration, for example, in the form of a suppository, which will melt in the rectum and release the drug. Composition for rectal injection may contain oil-based as a suitable non-irritating to excipient. Such bases include, without limitation lanolin, cocoa butter and polyethylene glycol.
In the methods according to the invention constructs encoding the fusion binding domain-immunoglobulin, or downregulation of the product(you), you can enter using the tab(Doc), granules(zero), composition(s) slow-release, patch(Rey) or composition(s) quick release.
The following examples are offered to illustrate but not to limit.
CLONING of the VARIABLE REGIONS of 2H7 AND the DESIGN AND sequencing of 2H7scFv-Ig
This example illustrates the cloning of cDNA molecules that encode the variable regions of the heavy the th chain and light chain of monoclonal antibody 2H7. This example also demonstrates the design, sequencing and expression of 2H7scFv-Ig.
Cell hybridoma expressing monoclonal antibody 2H7 that specific binds to CD20, was used as the starting material. Before collecting cells hybridoma maintained in logarithmic growth phase in a few days in RPMI medium 1640 (Life Technologies, Gaithersburg, MD) supplemented with glutamine, pyruvate, non-essential amino acids DMEM and penicillin-streptomycin. Cells were besieged from culture medium by centrifugation and used 2×107cells to obtain RNA. RNA from producing 2H7 cells hybridoma was isolated using a kit for isolation of total RNA Pharmingen (San Diego, CA) (No. in catalogue 45520K) according to the manufacturer's instructions attached to the kit. One microgram (1 μg) of total RNA was used as a matrix to obtain cDNA using reverse transcription. Mixed RNA and 300 ng of random primers and denaturiruet at 72°C for 10 minutes before adding the enzyme. Reverse transcriptase Superscript II (Life Technologies) was added to the RNA plus a mixture of primers in a total volume of 25 μl in the presence of 5X buffer for the synthesis of the second chain and 0.1 M DTT, supplied with the enzyme. The reverse transcription reaction was allowed to proceed at 42°C for one hour.
2H7-cDNA created in about ethno-transcriptase reaction with random primers, and primers specific for the V-region, used to using PCR to amplify variable regions of the light and heavy chains of the antibody 2H7. Primers specific for the V region was designed, based on the published sequence (GenBank, Depository and non M17954 for VLand M17953 for VH). Designed two variable chains with a compatible terminal sequences, so you can collect the scFv legirovaniem two V-regions after amplification and cleavage with enzymes.
Peptide linker (gly4ser)3that must have been built between the two V-regions, was built by adding additional nucleotides to the antisense primer for VL2H7. Also built in restriction site Sac I at the junction of the two V-regions. Sense primer used for amplification of VL2H7, which contained the restriction site HindIII and leader peptide of light chain, represented the 5'-gtc aag ctt gcc gcc atg gat ttt caa gtg cag att ttt cag c-3' (SEQ ID NO: 23). Antisense primer was a 5'-gtc gtc gag ctc cca cct cct cca gat cca cca ccg ccc gag cca ccg cca cct ttc agc tcc agc ttg gtc cc-3' (SEQ ID NO: 24). Frame read V-area is specified as shown in bold, underlined codon. Sites Hind III and SacI shown underlined shown italicized sequences.
Domain VHamplified b is h leader peptide, but
includes a 5'-restriction site SacI to merge with VLand BclI restriction site at the 3'-end to merge with different tails, including the Fc domain of human IgG1 and shortened forms of CD40 ligand, CD154. Sense primer was a 5'-gct gct gag ctc tca ggc tta tct ACA gca agt ctg g-3' (SEQ ID NO: 25). The SacI site is indicated by italicized and underlined, and the frame readout of the codon for the first amino acid domain VHindicated in bold, underlined font. Antisense primer was a 5'-gtt gtc tga tca gag acg gtg acc gtg gtc cc-3' (SEQ ID NO: 26). The BclI site is indicated by italics, underlined, and the last serine sequence of domain VHindicated in bold, underlined font.
The Assembly of scFv-Ig was performed by embedding HindIII-BclI fragment 2H7scFv in pUC19 containing the hinge, CH2 and CH3-region of human IgG1, which was digested with restriction enzymes HindIII and BclI. After ligation the ligation products were transformed bacteria DH5α. Organized the screening of positive clones in relation to precisely embedded fragments using SacI site in connection VL-VH2H7 as a diagnostic site. cDNA 2H7scFv-Ig were subjected to cycle sequencing in thermocycler PE 9700, using a program designed for 25 cycles with denaturation at 96°C for 10 seconds, annealing at 50°C for 30 seconds and elongation at 72°C for 4 minutes. Primers for sequani the Finance were forward and reverse primers pUC and internal primer, which annealed to the CH2 domain of human rights in part of the constant region of IgG. The sequencing reaction was performed using the mixture for sequencing with terminators labeled with Big dye Dye (PE Applied Biosystems, Foster City, CA)according to the manufacturer's instructions. Then, the samples were purified using Centrisep columns (No. in catalogue CS-901, Princeton Separations, Adelphia, N.J.), eluate was dried in a vacuum dryer Savant, was denaturiruet in the reagent for the suppression of the matrix (PE-ABI) and analyzed in the genetic analyzer ABI 310 (PE Applied Biosystems). Sequence editing, broadcast and analyzed using Vector Nti version 6.0 (Informax, North Bethesda, MD). The figure 1 shows the cDNA and designed amino acid sequence design 2H7scFv-Ig.
EXPRESSION of 2H7-ScFv-Ig IN STABLE CELL LINES CHO
This example illustrates the expression of 2H7scFv-Ig in eukaryotic cell line and characterization downregulation of 2H7scFv-Ig using SDS-PAG and functional analyses, including ADCC and complement fixation.
HindIII-XbaI fragment 2H7scFv-Ig (~1,6 TPN) with the correct sequence was built in expressing vector mammals pD18, and DNA of positive clones was amplified using sets to obtain the QIAGEN plasmid (QIAGEN, Valencia, CA). Then the recombinant plasmid DNA (100 μg) was linearizable little plot by splitting AscI, cleaned and phenol extraction and resuspendable in the medium for tissue culture Excell 302 (No. in catalogue 14312-79P, JRH Biosciences, Lenexa, KS). Cells for transfection, cells CHO DG44, maintained in logarithmic growth phase and collected 107cells for each transfection reaction. The linearized DNA was added to the CHO cells in a total volume of 0.8 ml for electroporation.
Stable production fused protein 2H7scFv-Ig (SEQ ID NO: 10) was achieved by electroporation of breeding, capable of amplification of plasmids pD18 containing cDNA 2H7scFv-Ig under the control of the CMV promoter in the cells of the Chinese hamster ovary (CHO). Cassette expressing 2H7, subclinically below the CMV promoter in the vector multiple cloning site in the form of a HindIII-XbaI fragment length ~1,6 TPN pD18 Vector is a modified version of pcDNA3, coding breeding marker DHFR with weak promoter to increase the selection pressure for plasmid. Plasmid DNA was obtained using a set of Qiagen maxiprep, and purified plasmid was linearizable in a unique AscI site before phenol extraction and precipitation with ethanol. DNA salmon sperm (Sigma-Aldrich, St. Louis, MO) was added as a DNA carrier and 100 μg of plasmid DNA and the DNA of the medium used for transfection of 107cells CHO DG44 by electroporation. Cells were grown to logarithmic phase in the environment Excell 302 (JRH Biosciences)containing glutamine (4 mm), pyruvate, recombinant insulin, penicillin-streptomycin and non-essential amino acids X DMEM (all from Life Technologies, Gaithersburg, Maryland), hereafter referred to as "complete medium Excell 302". Environment for nitrostilbene cells also contained HT (diluted from 100X solution gipoksantina and thymidine) (Life Technologies). Environment for transfection in terms of selection contained various levels of methotrexate (Sigma-Aldrich) as the selectivity of the agent in the range from 50 nm to 5 μm. Electroporation was carried out at 275 volts, 950 F. Transfitsirovannykh cells allowed to recover overnight in selective medium before seeding on the selective medium in 96-well tablets with a flat bottom (Costar) at different serial dilutions in the range of 125 cells/well up to 2000 cells/well. Culture medium for cloning cells was complete environment Excell 302 containing 100 nm methotrexate. After sufficient growth of the clones were screened serial dilutions of nadeshiko culture from the mother of the holes in relation to the binding transfitsirovannykh cells CD20-CHO. Clones with the highest production of the fused protein was replicated in bottles KZT25, and then Kzt75 to obtain the appropriate number of cells for freezing and for large-scale obtain 2H7scFv-Ig. Then increase the levels of production in cultures of the three clones with progressive amplification in culture medium containing methotrexate. At each subsequent passarello full environment Excell 302 contained increased concentrations of methotrexate, so could survive only cells with amplified DHFR-plasmid.
Nadeshiko cells SNO expressing 2H7scFv-Ig, were collected, filtered through Express PES filters, 0.2 µm (Nalgene, Rochester, NY) and passed through a column of protein A-agarose (IPA 300 crosslinked agarose) (Repligen, Needham, MA). The column was washed in PBS and then bound protein was suirable using 0.1 M citrate buffer, pH 3.0. Fractions were collected and suirvey protein was neutralized using 1M Tris, pH 8.0, before dialysis overnight in PBS. The concentration of purified 2H7scFv-Ig (SEQ ID NO: 15) was determined by absorption at 280 nm. The extinction coefficient 1,77 was determined using service program for protein analysis in the software package Vector Nti version 6.0 (Informax, North Bethesda, MD). This program uses data on amino acid composition for the calculation of extinction coefficients.
The levels of production of 2H7scFv-Ig transfitsirovannykh stable cells SNO were analyzed by flow cytometry. Purified 2H7scFv-Ig cells SNO gave the opportunity to contact the cells of Cho, who expressed CD20 (CD20-CHO) and were analyzed by flow cytometry using conjugated with fluorescein second reagent against human IgG (numbers in the directory N and N, CalTag, Burlingame, CA). In figure 2 (top) shows a standard curve obtained by titration of the binding of 2H7scFv-Ig with CD20-CHO. Each time it is ncentratio 2H7scFv-Ig shows the average brightness signal of fluorescein in linear units. Then adosados collected from T-flasks containing stable cell clones of Cho, expressing the 2H7scFv-Ig, were given the opportunity to contact CD20 CHO and binding was analyzed by flow cytometry. Measured fluorescein signal generated by the 2H7scFv-Ig, in nadeshiko, and the concentration of 2H7scFv-Ig in nadeshiko was calculated on the basis of the standard curve (figure 2, bottom).
Purified 2H7scFv-Ig (SEQ ID NO:15) were analyzed by electrophoresis in SDS-polyacrylamide gels. Samples of 2H7scFv-Ig, peeled independent runs through a column of protein a-agarose was boiled in sample buffer with SDS without restoring disulfide bonds and put on 10% Tris-BIS gels with SDS (No. in catalogue NP0301, Novex, Carlsbad, CA). Twenty micrograms of each treated batch was applied to the gels. After the electrophoresis the proteins were visualized by staining Kumasi blue (Pierce Gel Code Blue Stain Reagent, No. in catalogue 24590, Pierce, Rockford, IL), and the dye was removed in distilled water. In the same gel included markers of molecular weight (pre-colored Kaleidoscope standards, no directory 161-0324, Bio-Rad, Hercules, CA). The results are presented in figure 3. Numbers above the lanes marked independent party cleanup. Molecular weight markers size kilodalton indicated on the left side of the figure. Further experiments using alternative terms of cooking the samples showed recovery of the disulfide bonds by boiling in sample buffer with SDS containing DTT or 2-mercaptoethanol, causes aggregation of 2H7scFv-Ig.
You can control a number of other immunological parameters, using standard assays that are well known in the field. Analyses include, for example, the analysis of mediated antibody-dependent cell cytotoxicity (ADCC), analysis of secondary humoral responses in vitro, analysis of various subpopulations of peripheral blood cells or mononuclear lymphoid cells flowing immunochemiluminometric using well-established systems of marker genes, immunohistochemistry or other appropriate analyses. These and other assays can, for example, be found in Rose et al. (Eds.), Manual of Clinical Laboratory Immunology, 5-th Ed., 1997 American Society of Microbiology, Washington, DC.
The ability of 2H7scFv-Ig to kill CD20-positive cells in the presence of complement was tested with B-cell lines Ramos and Bjab. The analysis used the rabbit complement (Pel-Freez, Rogers, AK) at final dilution of 1/10. Purified 2H7scFv-Ig was incubated with B-cells and complement for 45 minutes at 37°C with subsequent counting of live and dead cells by eliminating staining Trifanova blue. The results in figure 4A show that in the presence of rabbit complement 2H7scFv-Ig was lizirovat B-cells expressing CD0.
The ability of 2H7scFv-Ig to kill CD20-positive cells in the presence of mononuclear cells in peripheral blood (PBMC) were tested by measuring the release of51Cr of labeled Bjab cells in 4-hour analysis using the ratio of PBMC to Bjab cells 100:1. The results, shown in figure 4B show that 2H7scFv-Ig can mediate antibody-dependent cellular cytotoxicity (ADCC), since the release of51Cr was higher in the presence of PBMC and 2H7scFv-Ig together than in the presence of either PBMC or 2H7scFv-Ig.
The EFFECT of SIMULTANEOUS LIGATION of CD20 And CD40 ON the GROWTH of NORMAL B-CELLS AND ON the EXPRESSION of CD95 AND the INDUCTION of APOPTOSIS
This example illustrates the impact of cross-linking of CD20 and CD40, expressed on the cell surface in cell proliferation.
Resting in the high density B cells were isolated from almond-shaped glands person in a stepwise gradient pergola and T-cells were removed using E-rosethorne. The proliferation of resting tonsillar B-cells with high density was measured by the absorption of3[H]-thymidine for the last 12 hours of a 4-day experiment. Proliferation was measured in cultures in four repetitions, calculating averages and standard deviations, as shown. Mouse MAB against CD20 human, 1F5 (anti-CD20), used separately or together with anti-Ishenim ĸ Mat, 187.1 (anti-CD20XL). Activation of CD40 was performed using soluble CD154 person, fused with murine CD8 (CD154) (Hollenbaugh et al., EMBO J. 11: 4212-21 (1992)), and CD40 crosslinking was carried out using anti-mouse CD8 Mat, 53-6 (CD154XL). This method also allows the simultaneous crosslinking of CD20 and CD40 on the cell surface. The results are shown in figure 5.
Evaluated the impact of cross-linking of CD20 and CD40 on the cells Ramos, line B-cell lymphoma. The Ramos cells were analyzed in relation to the expression of CD95 (Fas) and determined the percentage of apoptosis in eighteen hours after treatment (without goat anti-mouse IgG (GAM)and/or staple (+GAM) using mouse Mat, which is specific binds CD20 (1F5) and CD40 (G28-5). Control cells were treated with control antibody nesvezhego isotype (64.1), it is specific to CD3.
Treated Ramos cells were harvested, incubated with FITZ-anti-CD95 and were analyzed by flow cytometry to determine the relative level of expression of Fas on the cell surface after crosslinking of CD20 or CD40. The data shown on the graph as the average fluorescence of the cells after processing these stimuli (figure 6A).
Treated Ramos cells from the same experiment were collected and measured the binding of annexin V to show the percentage of apoptosis in the treated cultures. Apoptosis was measured by binding of annexin V over 18 cha is s after crosslinking of CD20 and CD40 using 1F5 and G28-5, followed by sewing the GAM.
The binding of annexin V was measured using a set of FITZ-annexin V (No. in catalogue PN-IM2376, Immunotech, Marseille, France). It is known that binding of annexin V is an early event in the promotion of cell apoptosis. Apoptosis or programmed cell death is a process characterized by a cascade of catabolic reactions leading to cell death as a result of suicide. In the early phase of apoptosis before the cells change morphology and DNA hydrolysis, the integrity of cell membranes is maintained, but the cells lose the asymmetry of phospholipids in their membranes, bringing to the surface of the cell negatively charged phospholipids, such as phosphatidylserine. Annexin V, a protein that binds calcium and phospholipids, preferably with high affinity associated with phosphatidylserine. Results showing the impact of cross-linking of both CD20 and CD40 on the expression of the receptor (CD95), shown in figure 6A. The impact of cross-linking of both CD20 and CD40 on the binding of annexin V to cells shown in figure 6B.
DESIGN AND CHARACTERIZATION of FUSED PROTEINS 2H7ScFv-CD154
To design a molecule that can bind CD20 and CD40, cDNA encoding 2H7scFv was merged with cDNA encoding CD154, CD40 ligand. cDNA 2H7scFv encoded HindIII-BclI fragment was extracted from the design 2H7scFv-Ig and was built in pD18 vector with BamHI-XbaI-Fraga is entom cDNA, encoding the extracellular domain of human CD154. The extracellular domain is encoded carboxyl end of CD154, like other membrane proteins, type II.
The extracellular domain of human CD154 amplified in PCR using cDNA obtained by using random primers and RNA from T-lymphocytes human activated PHA (phytohemagglutinin). Sets of primers consisted of two different 5'- or sense primer, which created the connection mergers in two different positions in the extracellular domain of CD154. Designed two different connections merge, the result of which was a short or abbreviated form (form S4), containing 108 amino acids (Glu)-261 (Leu)+(Glu), and long or full form (form L2), containing 48 amino acids (Arg)-261 (Leu)+(Glu) of the extracellular domain of CD154, which was designed in the form of a BamHI-XbaI fragments. Sense primer, which merges two different truncated extracellular domain 2H7scFv, contains a BamHI site for cloning. Sense primer for the form S4 CD154 cDNA denoted by SEQ ID NO: 11 or CD154BAM108 and encodes a 34-Mer sequence 5'-gtt gtc gga tcc aga aaa cag ctt tga aat gca a-3', while the antisense primer is designated SEQ ID NO: 12 or CD154XBA and encodes a 44-Mer sequence 5'-gtt gtt tct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3'.
Oligonucleotide primers used for amplification of the long form (L2) of the extracellular domain of CD154, the code is the dominant amino acids 48 (Arg)-261 (Leu)+(Glu), represented the following primers: Sense primer, designated CD154 BAM48 (SEQ ID NO: 13), encodes a 35-Mer with the following sequence: 5'-gtt gtc gga tcc aag aag gtt gga caa gat aga ag-3'. Antisense primer, designated CD154XBA (SEQ ID NO:__), encodes a 44-Mer: 5'-gtt gtt tct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3'. Other reaction conditions of PCR were identical to the conditions used for amplification of 2H7scFv (see example 1). The PCR fragments were purified using kits for rapid PCR (QIAGEN, San Diego, CA), was suirable 30 μl ddH2O and were digested with restriction endonucleases BamHI and XbaI (Roche) in a reaction volume of 40 μl at 37°C for 3 hours. The fragments were purified in the gel, purified using sets QIAEX according to the manufacturer's instructions (QIAGEN) and ligated with HindIII-BclI fragment N in expressing vector pD18, split HindIII+XbaI. The reaction mixture for ligation was used to transform chemically competent bacteria D5-alpha and bacteria were sown on LB-cups containing 100 μg/ml ampicillin. Transformants were grown overnight at 37°C and selected colonies were used for inoculation of 3 ml of liquid medium in the broth, Luria containing 100 μg/ml ampicillin. Organized the screening of clones after receiving miniplate (QIAGEN) for embedding 2H7scFv and a fragment of the extracellular domain of CD154.
Construction of cDNA 2H7scFv-CD154 were subjected to cyclic sequencing in thermocycler PE 700, using the program, designed for 25 cycles, which includes denaturation at 96°C, 10 seconds, annealing at 50°C for 5 seconds and changed at 60°C for 4 minutes. Used for sequencing primers were direct primer pD18 (SEQ ID NO: 30: 5'-gtctatataagcagagctctggc-3') and reverse primer pD18 (SEQ ID NO: 31: 5'-cgaggctgatcagcgagctctagca-3'). In addition, used an internal primer, which had homology with the sequence of human CD154 (SEQ ID NO: 32: 5'-ccgcaatttgaggattctgatcacc-3'). The reaction mixture contained 3.2 pmol primers, 200 ng of template DNA and 8 μl of the mixture for sequencing. The sequencing reaction was performed using the mixture for sequencing with terminators labeled with Big dye Dye (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Then, the samples were purified using Centrisep columns (Princeton Separations, Adelphia, NJ). Eluate was dried in speed vacuum dryer Savant, was denaturiruet in 20 ál for the suppression of the matrix (ABI) at 95°C for 2 minutes and were analyzed genetic analyzer ABI 310 (PE Applied Biosystems). Sequence editing, broadcast and analyzed using Vector Nti version 6.0 (Informax, North Bethesda, MD). The cDNA sequence and the calculated amino acid sequence of 2H7scFv-CD154 L2 are presented in figure 7A, and the cDNA sequence and the calculated amino acid sequence of 2H7scFv-CD154 S4 is redstavleny in figure 7B.
Binding activity of fused proteins 2H7scFv-CD154 (SEQ ID N0:33 and 34) simultaneously with CD20 and CD40 was determined by flow cytometry. In the analysis used cellular targets SNO, which Express CD20. After a 45-minute incubation of cells CD20-CHO with nagasadori from cells transfected with a plasmid expressing the 2H7scFv-CD154 cells, CD20-CHO washed twice and incubated with conjugated with Biotin fused protein CD40-Ig in PBS/2% FBS. After 45 min, the cells were twice washed and incubated labeled with phycoerythrin (PE) - streptavidin at a dilution of 1:100 in PBS/2% FBS (Molecular Probes, Eugene OR). After an additional 30 minute incubation, the cells were washed 2X and were analyzed by flow cytometry. The results show that the molecule 2H7scFv-CDl54 able to contact CD20 on the cell surface and capture from solution CD40, conjugated with Biotin (figure 8).
To determine the effect of 2H7scFv-CDl54 on the growth and viability of B-lymphoma and lymphoblastoid cell lines, cells were incubated with 2H7scFv-CDl54 L2 (SEQ ID NO:33) for 12 hours and then evaluated the binding of annexin V Binding of annexin V was measured using a set of FITZ-annexin V (Immunotech, Marseille, France, No. in catalogue PN-IM2376). Line b-cells were incubated in cultures of 1 ml with dilution of concentrated cialisovernight of nadeshiko of cells expressing the secreted form fused proteins 2H7scFv-CDl54. The result is s presented in figure 9.
The growth rate of the line B-lymphoid cells Ramos in the presence of 2H7scFv-CDl54 was assessed by the uptake of3H-thymidine during the last 6 hours of a 24-hour cultivation. The influence of 2H7scFv-CDl54 on cell proliferation are shown in figure 10.
Design and characterization of derivatives of antibodies CytoxB
Antibodies CytoxB was obtained from a polypeptide 2H7scFv-IgG. 2H7scFv (see example 1) was associated with the Fc domain of human IgG1 via a modified hinge domain (see figure 11). Residues of cysteine in the hinge region were replaced by serine residues site-specific mutagenesis, and other methods known in this field. Mutant hinge poured or Fc-domain of wild-type to create one design, marked CytoB-MHWTG1C or was merged with mutant Fc domain (CytoxBMHMG1C), which had additional mutations are introduced into the CH2 domain. Amino acid residues in the CH2, which are involved in effector function, shown in figure 11. Mutations in one or more of these residues can reduce the FcR binding and mediating effector functions. In this example, the leucine residue 234, which, as is well known in this area, important for binding of the Fc receptor were subjected to matirovanie in fused protein 2H7scFv, CytoxB[MG1H/MG1C]. In another design of the hinge region of human IgG1 was replaced as part of the IgA hinge of a man who had merged with the human is m Fc-domain of wild-type (CytoxB-IgAHWTHG1C) (see figure 11). The indicated mutant hinge region were allowed to Express the mixture of Monomeric and dimeric molecules that retain the functional properties of the CH2 and CH3 domains of IgG1 man. Designed synthetic recombinant expressing cassettes these cDNA molecules and expressed polypeptides in cells CHODG44 according to the methods described in example 2.
Purified derivative molecules fused protein CytoxB-scFvIg analyzed in SDS-page according to the methods described in example 2. Polyacrylamide gel was broken up and non reducing conditions. Each gel was applied a different set of molecular weight markers, pre-painted markers BioRad (BioRad, Hercules, CA) and molecular weight markers Novex Multimark. Picture of migration of different designs and rituximab presented in figure 12.
The ability of various derivative molecules CytoxB-scFvIg to mediate ADCC was measured using as a target cell B-lymphoma Bjab and fresh human PBMC as effector cells (see example 2). Relationship effector to target varied as follows: 70:1, 35:1 and 18:1, and the number of Bjab cells per well remained constant, and the number of PBMC varied. Bjab cells were labeled for 2 hours51Cr and carried the aliquot at a density of cells 5×104cells/well in each well of 96-l the night of tablets with a flat bottom. Added purified fused proteins or rituximab at a concentration of 10 mg/ml of various dilutions of PBMC. Spontaneous release was measured without the addition of PBMC or fused protein and the maximum release was measured using the detergent (1% NP-40) into the appropriate wells. The reaction mixture was incubated for 4 hours and collected 100 ál nadeshiko culture in Lumaplate (Packard Instruments) and allowed to dry overnight before counting the release pulse/min the Results are shown in figure 13.
Also measured is dependent on complement cytotoxic (CDC) activity of derivatives CytoxB. The reaction was carried out as described in example 2. The results are presented in figure 14 as the percentage of dead cells to total cells for each concentration of the fused protein.
IN VIVO STUDIES ON MONKEYS
Initial in vivo studies derived CytoxB performed in primates other than humans. Figure 15 shows data describing the half-life of CytoxB in the serum of monkeys. The measurements were carried out in serum samples obtained from two different macaques (J99231 and K99334) after each macaque was injected dose of 6 mg/kg on days indicated by arrows. For each sample the current level 2H7scFvIg was assessed by comparison with a standard curve obtained by binding of purified fused protein Cytox-(MHWTG1C)-Ig cells CD20-CHO (see example 2). The data are given in tabular form in the bottom panel of figure 15.
Investigated the influence of the fused protein CytoxB-(MHWTG1C)Ig on the levels of circulating cells CD40+in macaques. Complete blood count was performed on each of the days indicated on figure 16. In addition, he performed an analysis of FACS (fluorescent activated cell sorter) on peripheral blood lymphocytes using is specific to CD40 antibody conjugated with fluorescein to detect B-cells in the cell population. Then used the percentage of positive cells to calculate the number of B-cells in the initial samples. The data shown graphically in thousands of B-cells per microliter of blood when measured on the indicated days after injection (figure 16).
CONSTRUCTION AND expression of the FUSED PROTEIN ANTI-CD19-scFv-Ig
Protein anti-CD19-scFv-Ig was designed, was transfusional in eukaryotic cells and expressed according to the methods described in examples 1, 2 and 5, and the standard in this area. Variable regions of the heavy chain and the variable region of the light chain was cloned from RNA isolated from cells hybridoma producing antibody HD37 that specific binds to CD19. Measured the levels of expression of HD37scFv-IgAHWTG1C and HD37scFv-IgMHWTG1C and compared with a standard curve obtained with purified HD37scFvIg. The results are shown in figure 17.
CONSTRUCTION AND expression of the FUSED PROTEIN ANTI-L6-scFv-Ig
Protein scFv-Ig was designed with the use of the variable regions derived from a MAB against carcinoma, L6. Protein was designed, was transfusional in eukaryotic cells and expressed according to the methods provided in examples 1, 2 and 5, and the standard in this area. Measured the levels of expression of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C and compared with a standard curve obtained with purified HD37scFvIg. The results are shown in figure 18.
CHARACTERISTIC of VARIOUS FUSED PROTEIN scFv-Ig
Besides the already described fused protein scFv-Ig has been fused proteins G28-1 (anti-CD37)scFv-Ig, essentially as described in examples 1 and 5. Variable regions of the heavy and light chains were cloned according to methods known in this field. ADCC activity 2H7-MHWTG1C, 2H7-IgAHWTG1C, G28-1-MHWTG1C, G28-1-IgAHWTG1C, HD37-MHWTG1C and HD37-IgAHWTG1C was determined according to the methods described above (see example 2). The results are shown in figure 19. ADCC activity L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C was measured using the cell line of human lung carcinoma 2981. The results are shown in figure 20. It is known that mouse monoclonal antibody L6 shows ADCC activity.
Purified proteins were analyzed by SDS-page under reducing and non conditions. Preparation the samples and justify in the gels was carried out, essentially as described in examples 2 and 5. Results for fused proteins L6 and 2H7-scFv-Ig presented on figure 21, and the results for the fused protein G28-1 and HD37-scFv-Ig presented in figure 22.
From the above it will be understood that although this description for purposes of illustration, the specific variants of the invention may be implemented in various modifications without departing from the essence and without exceeding the scope of the invention. Thus, this invention is not limited by anything except the attached claims.
1. Protein containing, from the amino end to the carboxyl end:
(a) a polypeptide binding domain that binds to a biological molecule-target;
(b) the peptide of the hinge region of human IgG1 with one cysteine;
(c) a polypeptide constant region CH2 heavy chain immunoglobulin;
(d) a polypeptide constant region CH3 heavy chain immunoglobulin and protein, composed of a mixture of monomers and dimers is mediated antibody-dependent cell cytotoxicity, complement-dependent cytotoxicity, or both types of cytotoxicity.
2. Protein of claim 1, wherein the polypeptide binding domain is a single-chain Fv polypeptide.
3. Fused protein according to claim 2, in which the variable regions of the heavy and light chain single-chain Fv attached poly is heptenyl linker, which includes at least one polypeptide having the amino acid sequence Gly-Gly-Gly-Gly-Ser [SEQ ID NO:21].
4. A protein according to one of claims 1 to 3, where the biological molecule-target is on the surface of target cells.
5. A protein according to one of claims 1 to 3, where the protein is able to connect with b-cell targets.
6. Fused protein according to claim 5, where In the cell the target is a CD37.
7. Fused protein according to claim 5, in which the polypeptide binding domain-immunoglobulin includes single-chain Fv polypeptide G28-1.
8. Fused protein according to claim 5, where In the cell the target is a CD20.
9. A protein according to one of claims 1 to 3, where the biological molecule-target is a CD19, CD22, CD30 ligand, CD54, CD106, or interleukin-12.
10. A protein according to one of claims 1 to 3, where the biological molecule-target is a CD2, CD5, CD10, CD27, CD28, CD40, CTLA-4, 4-1BB, a ligand, 4-1BB, interferon-γ, interleukin-4 or interleukin-17.
11. A protein according to one of claims 1 to 3, where the biological molecule-target is a CD59, CD48, CD72, CD70, CD86/B7.2, CD40 ligand, CD43 or VLA-4 (α4β7).
12. A protein according to one of claims 1 to 3, where the biological molecule-target is a CD83 or DEC-205.
13. A protein according to one of claims 1 to 3, where the biological molecule-target is a HER1, HER2, HER3,, HER4, growth factor, endothelial cells of blood vessels, insulinopenia growth factor I, insulin-like growth factor II, MUC-1, NY-ESO-1, NA 17-a, mesland-A/MART-1, tyrosinase, Gp-100, MAGE, BAGE, GAGE, antigen HOM-MEL-40, encoded by the genome of SSX2, oncofetal antigen or PyLT.
14. A protein according to one of claims 1 to 3, where the biological molecule-target is a receptor, interleukin-17 receptor, epidermal growth factor, growth factor receptor endothelial cells of blood vessels, the transferrin receptor, estrogen receptor, progesterone receptor, follicle-stimulating hormone receptor, retinoic acid receptor or receptor STA-class.
15. A protein according to one of claims 1 to 14, in which the immunoglobulin-binding domain humanitarian.
16. A protein according to one of claims 1 to 15, in which the polypeptides constant regions CH2 and CH3 heavy chain immunoglobulin are polypeptides constant regions CH2 and CH3 IgG1.
17. Pharmaceutical composition for the treatment of b-cell disorders, containing a therapeutically effective amount of the fused protein according to one of claims 1 to 16 and a physiologically acceptable carrier.
18. The pharmaceutical composition according to 17, where b-cell disorder is a malignant condition or an autoimmune disease.
19. The pharmaceutical composition according p, where a malignant condition is a b-cell lymphoma.
20. The pharmaceutical composition according p, dislokatsionnoi condition is a chronic lymphocytic leukemia.
21. The pharmaceutical composition according p, where the autoimmune disease is a rheumatoid arthritis, psoriasis, systemic lupus erythematosus, myasthenia gravis, graves disease, diabetes type I, multiple sclerosis, autoimmune thyroid disease, Hashimoto's thyroiditis, Sjogren syndrome, immune thrombocytopenic purple, scleroderma, inflammatory bowel disease, Crohn's disease or ulcerative colitis.
22. A method of treating b-cell disorder, comprising the administration to a patient a therapeutically effective amount of fused protein according to one of claims 1 to 16.
23. The method according to item 22, where b-cell disorder is a malignant condition or an autoimmune disease.
24. The method according to item 23, where a malignant condition is a b - cell lymphoma.
25. The method according to item 23, where a malignant condition is a chronic lymphocytic leukemia.
26. The method according to item 23, where the autoimmune disease is a rheumatoid arthritis, psoriasis, systemic lupus erythematosus, myasthenia gravis, graves disease, diabetes type I, multiple sclerosis, autoimmune thyroid disease, Hashimoto's thyroiditis, Sjogren syndrome, immune thrombocytopenic purple, scleroderma, inflammatory bowel disease, Crohn's disease or ulcerative to the lit.
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology and biotechnology. The method prevents immune cell attachment to the other cells involving contacting of the immune cells and other cells with a composition containing Bst2 antagonist. The antagonist is specified in a group: protein Bst2, fused protein on the based thereof or on the basis of its fragments with anti-inflammatory activity or aHTH-Bst2 antibodies. There are also described: a method of inflammation reduction in a subject and a method of treatment of a disease associated connected with the inflammation, mediated by Bst2 superexpression with the use of Bst2 antagonists.
EFFECT: inventions provide the use of Bst2 protein and its proper antagonists for prevention of adhesion of immune cells, as well as said cells and immune system cells that can find further application in medicine in treating inflammatory diseases.
12 cl, 39 dwg, 42 ex
SUBSTANCE: invention represents immunogenic hybrid polypeptide for prevention and treatment of obesity. Also described is vaccine for prevention or treatment of obesity, containing immunogenic hybrid polypeptide. In addition, describes are polynucleotide, coding immunogenic hybrid polypeptide, recombinant expression vector, carrying said polynucleotide, host cell, transformed by recombinant expression vector, and method of obtaining immunogenic hybrid polypeptide by cultivation of host cell, transformed by recombinant expression vector.
EFFECT: invention can be efficiently used as means for prevention or treatment of obesity.
18 cl, 25 dwg, 15 ex
SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.
EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.
29 cl, 15 dwg, 6 tbl, 9 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: compounds, included into one of compositions represent polypeptides, which contain at least immunogenic fragment of protein CT858 or protein CT089 Chlamydia trachomatis with specific amino acid sequence, or polynucleotides, coding immunogenic fragments of said proteins. Compounds, included into other composition, represent fused polypeptides, which contain immunogenic fragment of protein CT858 or CT089 and fusion partner, selected from immunologic fusion partners, expression amplifiers, affine markers and unrelated known proteins of Chlamydia trachomatis. Said compositions can be used for stimulation of specific T-cell response to Chlamydia trachomatis, for inhibition of infection development and for treatment of infection induced by Chlamydia trachomatis, ensuring high level of immune response and therapeutic effect.
EFFECT: described are compositions based on compounds for prevention and treatment of Chlamydia infection.
11 cl, 16 dwg, 7 tbl, 13 ex
SUBSTANCE: modified virus-like particle (VLP) includes hybrid protein which consists of AP205 bacteriophage protein and any antigen. Also, a composition including VLP, being a derivative of RNA-containing AP205 bacteriophage is described. Besides, the invention describes a method for producing said VLP. Modified VLP of the present invention is applicable for producing compositions for inducing immune response for prevention or treatment of diseases, disorders, including infectious diseases, allergies, cancer and drug addiction.
EFFECT: offered group of inventions can be used in medicine, immunology, virology and molecular biology.
35 cl, 3 dwg, 1 tbl, 26 ex
SUBSTANCE: aprotinin analogue with a TFFYGGSRGKRNNFKTEEY sequence is obtained, as well as a conjugate based thereon.
EFFECT: invention enables delivery of a compound or medicinal agent through the hematoencephalic barrier in mammals.
6 cl, 9 dwg, 6 tbl, 3 ex
SUBSTANCE: invention concerns nucleic acid molecules including the glycolising fusion designs containing catalytic domain of beta-1,4-N-acetylglucosaminyl transferase III or beta-1,4-galactosyl transferase, and resident polypeptide domain of Golgi complex responsible for localisation in Golgi complex, as well as their applications in host cell glycolisation modification.
EFFECT: invention allows producing polypeptides with the improved therapeutic properties, including antibodies with higher Fc-receptor binding and enhanced effector function.
21 cl, 37 dwg, 2 tbl, 7 ex
SUBSTANCE: there is claimed isolated human antibody or its fragment, which binds to human EGFR. Antibody contains corresponding CDR areas of light and heavy chain. Its conjugate with anti-neoplastic means or marker is described. Also described are: coding nucleic acid, expression vector, recombinant cell-host for obtaining antibodies and method of inhibiting growth of tumor, expressing EGFR on the basis of antibody.
EFFECT: application of invention provides antibodies with affinity comparable or higher, than in IMC-C225, which neutralises EGFR activation, what can be applied in medicine for treatment of tumours.
36 cl, 14 dwg, 6 tbl, 13 ex
SUBSTANCE: method of producing an antigen-binding VH domain wherein an elongated CDR3 loop similar to a camel loop is absent, involves transformation of a mammal cell by a heavy-chain VH heterolocus. The locus contains a gene coding a variable area, at least containing one segment of VH gene, one segment of D gene not being camel's, one segment of J gene not being camel's, one invariable heavy chain region provided that any gene coding the invariable regions does not code a Sn1 functional domain. The segments of V, D and J genes are capable to recombination and formation of the VDJ coding sequence. The transformed cell is capable to express a heavy chain antibody only containing an antigen-binding VH domain and the invariable constant effector region without the functional domain Sn1. The cell is used for producing a transgene animal to be immunised with an antigen of concern. Further the cells or tissues expressing specific antibodies to the heavy chain antigen of concern are recovered; nucleic acid coding the VH domain of the specific heavy chain antibody only is recovered; and the specified antigen-binding VH domain is expressed. The specified VH domains can be used for production of fused proteins or binding complexes of monovalent, bivalent or polyvalent polypeptide.
EFFECT: invention allows producing antigen-specific human antibodies of any class, exhibiting high affinity.
11 cl, 26 dwg, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to obtaining genetically engineered vaccines and can be used in medicine. A recombinant gene structure containing a series of human SLC gene, antigen gene and gene Fc-fragment of IgG 1 is obtained.
EFFECT: invention considerably increases efficiency of the immune response of the body to the introduced antigen compared to existing antigen structures.
10 cl, 15 dwg, 1 tbl, 12 ex
SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.
EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.
29 cl, 15 dwg, 6 tbl, 9 ex
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
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
SUBSTANCE: invention refers to a compound presented by formula (1), to its salt or hydrate where in formula R1 represents a methylene group, R2 represents a phenyl group which can contain a substitute(s), or a heterocyclic group which can contain a substitute(s), the cycle A represents a 6- or 7-members cycle (where cycle-making atoms of the cycle A different from a sulfur atom in position 6 are carbon atoms), and R3 represents a hydrogen atom, or 1-3 equal or different substitutes used to substitute the cycle A where the possible substitutes are specified in clause 1 of the patent claim. Also, the invention refers to a pharmaceutical composition exhibiting an anticancer activity, on the basis of the compound presented by formula (1).
EFFECT: there are produced new compounds and pharmaceutical composition on their basis which can find application in medicine for cancer treatment.