Monoclonal antibodies to hepatocyte growth factor
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
REFERENCES TO RELATED APPLICATIONS
This application is a partial continuation of application for U.S. patent No. 10/825060, registered on April 15, 2004, which claimed the benefit of provisional application for U.S. patent No. 60/464061, registered on April 18, 2003, which are included in the present description in its entirety as a reference for all purposes.
The technical FIELD TO WHICH the INVENTION RELATES.
The present invention relates mainly to a combination of monoclonal antibodies and recombinant DNA for the development of new biologically active compounds and, more specifically, for example, to obtain monoclonal antibodies that bind to growth factor hepatocyte and neutralize it.
BACKGROUND of INVENTION
Hepaticotomy factor human growth (HGF) is a multifunctional heterodimeric polypeptide produced by cells of the mesenchyme. It has been shown that HGF stimulates angiogenesis, morphogenesis and metagenes, as well as the growth and proliferation of cells of different types (Bussolino et al., J. Cell Biol. 119: 629, 1992; Zarnegar and Michalopoulos, J. Cell Biol. 129: 1177, 1995; Matsumoto et al., Ciba, Found. Symp. 212: 198, 1997; Birchmeier and Gherardi, Trends Cell. Biol. 8: 404, 1998; Xin et al., Am. J. Pathol. 158: 1111, 2001). The pleiotropic nature of the activity of HGF mediated by its receptor, a transmembrane receptor encoded by protooncogenes cMet. Was Asano, in addition to the regulation of a large number of normal cellular functions of HGF and its receptor c-Met is involved in the initiation, invasion and metastasis of tumors (Jeffers et al., J. Mol. Med. 74: 505, 1996; Comoglio and Trusolino, J. Clin. Invest. 109: 857, 2002). HGF/cMet are joint expression, often overexpression in different solid human tumors, including tumors arising from lung, colon, rectum, stomach, kidney, ovary, skin, multiple myeloma and thyroid tissues (Prat et al., Int. J. Cancer 49: 323, 1991; Chan et al., Oncogene 2: 593, 1988; Weidner et al., Am. J. Respir. Cell. Mol. Biol. 8: 229, 1993; Dercksen et al., Blood 99: 1405, 2002). HGF acts as an autocrine (Rong et al., Proc. Natl. Acad. Sci. USA 91: 4371, 1994; Koochekpour et al., Cancer Res. 57: 5391, 1997) and paracrine growth factor (Weidner et al., Am. J. Respir. Cell. Mol. Biol. 8: 229, 1993) and anti-apoptotic regulator (Gao et al., J. Biol. Chem. 276: 47257, 2001) for these tumors.
HGF is a protein the size of 102 kDa, with similarity in sequence and structure with plasminogen and other enzymes of the blood coagulation system (Nakamura et al., Nature 342: 440, 1989; Weidner et al., Am. J. Respir. Cell. Mol. Biol. 8: 229, 1993; each of the works is fully included in the present description by reference) (Fig 1). Human HGF is synthesized as a precursor of 728 amino acids (preproHGF), which undergoes cleavage within the cell as inactive single-chain form (proHGF) (Nakamura et al., Nature 342: 440, 1989; Rosen et al., J. Cell Biol. 127: 1783, 1994). When extracellular secretion of proHGF split with the formation of the active heterodimeric molecule by a disulfide bond, consisting of α-subunit and β-subunit (Nakamura et al., Nature 342: 440, 1989; Naldini et al., EMBO J. 11: 4825, 1992). the α-subunit contains 440 residues (size 69 kDa, glycosylation) and consists of N-terminal hairpin domain and Kringle 4 ("kringle"domains. β-subunit contains 234 residues (34 kDa) and includes domain-like domain serine protease, which has lost its proteolytic activity. Splitting HGF is required for activation of the receptor, but not for binding of the receptor (Hartmann et al., Proc. Natl. Acad. Sci. USA 89: 11574, 1992; Lokker et al., J. Biol. Chem. 268: 17145, 1992). HGF contains 4 alleged site of N-glycosylation, 1 - α-subunit and 3 - β-subunit. HGF has 2 unique binding site with specific cells: binding site with high affinity (Kd=2×10-10M) for cMet receptor and the binding site with low affinity (Kd=2×10-9M) for heparansulfate proteoglycans (HSPG), which are present on the cell surface and in the extracellular matrix (Naldini et al., Oncogene 6: 501, 1991; Bardelli et al., J. Biotechnol. 37: 109, 1994; Sakata et al., J. Biol. Chem., 272; 9457, 1997). NK2 (protein, including the N-end and first two Kringle domain of the α-subunit) sufficient to allow binding to cMet and activation of the signalling cascade for transfer, however, for the mitogenic response required protein full length (Weidner et al., Am. J. Respir. Cell. Mol. Biol. 8: 229, 1993). HSPG binds to HGF due to the interaction with the N-end of HGF (Aoyama, et al., Biochm. 36: 10268, 1997; Sakata et al., J. Biol. Chem. 272: 9457, 1997). The proposed role for HSPG interaction-HGF includes increasing the bioavailability of HGF, its biological activity and oligomerization HGF (Bardelli et al., J. Biotechnol. 37: 109, 1994; Zioncheck et al., J. Biol. Chem. 270: 16871, 1995).
cMet belongs to class IV family of receptor protein tyrosine kinase. Gene cMet full length was cloned and identified as the cMet protooncogen (Cooper et al., Nature 311: 29; Park et al., Proc. Natl. Acad. Sci. USA 84: 6379, 1987). The cMet receptor initially synthesized as a single chain, partly glycopyranose predecessor, p170(MET)(1) (Park et al., Proc. Natl. Acad. Sci. USA 84: 6379, 1987; Giordano et al., Nature 339: 155, 1989; Giordano et al., Oncogene 4: 1383, 1989; Bardelli et al., J. Biotechnol. 37: 109, 1994). Subsequently, during the glycosylation of the protein undergoes proteolytic cleavage with the formation of the Mature heterodimeric protein with a size of 190 kDa (1385 amino acids)consisting of α-subunit size of 50 kDa (residues 1-307) and β-subunit size of 145 kDa. The cytoplasmic tyrosine kinase domain in the β-subunit is involved in the signal transmission.
It was used several strategies to obtain molecules exerting an antagonistic effect on the interaction of HGF/cMet, which included the use of: truncated HGF proteins, such as NK1 (N-terminal domain plus Kringle domain 1; Lokker et al., J. Biol. Chem. 268: 1745, 1993), NK2 (N-terminal domain plus Kringle domains 1 and 2; Chan et al., Science 254: 1382, 191) and NK4 (N-terminal domain and four Kringle domain; Kuba et al., Cancer Res. 60: 6737, 2000), anti-cMet Mat (Dodge, Master's Thesis, San Francisco State University, 1998) and anti-HGF Mat (Cao et al., Proc. Natl. Acad. Sci. USA 98: 7443, 2001, this work is fully incorporated into the present description by reference).
NK1 and NK2 can effectively compete with HGF for binding to its receptor, but has been shown to exhibit partial agonistic activity in vitro (Cioce et al., J. Biol. Chem. 271: 13110; Schwall et al., J. Cell. Biol. 133: 709, 1996), and is not desirable purely antagonistic activity. Recently, Cuba et al. (Kuba et al., Cancer Res. 60: 6737, 2000) showed that NK4 can partially inhibit primary growth (figure 2) and metastasis of murine lung tumor LLC on the model of "Nude" mice (nude) by continuous infusion NK4. The fact that NK4 need to enter continuously to achieve partial inhibition of growth of primary tumors presumably indicates a short half NK4 molecules and/or loss of activity. In comparison with the use of NK4 strategy based on the use of antibodies has several advantages that are defined favorable pharmacokinetics and the possibility of obtaining antibodies with higher activity.
In another approach Dodge (Dodge, Master's Thesis, San Francisco State University, 1998) received monoclonal anti-cMet antibodies with antagonistic activity (Mat). One monoclonal antibody 5D5, demonstrated powerful antagonists aktivnosti, according to ELISA, but induced a proliferative response in cMet-expressing BAF-3 cells, mainly due to the dimerization of membrane receptors. Prat et al. (Prat et al., J. Cell Sci. 111: 237, 1998) also reported the existence of such agonists activity in anti-cMet Mat. Zaccolo et al. (Zaccolo et al., Eur. J. Immunol. 27: 618, 1997) used the methods of phage display technique for obtaining human Fab fragments against mouse and human hepatocites growth factor. These Fab fragments had no effect on the activity of HGF when used separately. However, when one of the Fab fragments against human HGF was combined with the antibody which itself binds to the Fab fragment, there was increased activity of HGF, Biotest.
Kao et al. (Cao et al., Proc. Natl. Acad. Sci. USA 98: 7443, 2001) showed that the introduction of a mixture of three anti-HGF Mat that were selected for their ability to inhibit scattering activity of HGF in vitro, leads to inhibition of growth of human tumors on the model of "Nude" mice with xenograft (figure 3). They concluded that the three Mat that can recognize three different binding site for HGF required for inhibition of biological activity of HGF in vivo: two Mat inhibit the HGF binding to cMet and one Mat inhibits HGF binding to heparin. However, for commercial applications and for regulation of narak is ichno to create a drug uniting three new Mat, in particular, due to the fact that must be proven clinical activity independently of each antibody.
Thus, there is a need for a single monoclonal antibody capable of blocking the biological activity of HGF in vitro and in vivo. The present invention meets this and other needs.
A BRIEF DESCRIPTION of the INVENTION
In one embodiment, the present invention relates to neutralizing MAB against hepatocites growth factor human (HGF). The specified antibody inhibits at least one and preferably several or all of the biological activity of HGF, including binding to its receptor cMet, inducing dispersion of cells, such as cells of the kidney of the dog Madin-Darby, induction of proliferation of epithelial cells of the monkey 4MRr-5 and/or hepatocytes and/or HUVEC and induction of angiogenesis. Anti-HGF Mat may inhibit such activity, if it is used as a sole agent. Preferably the anti-HGF antibody inhibits, most preferably completely inhibited the growth of xenograft tumors of the mouse. Preferably, the Mat of the present invention is a chimeric, humanitariannet, human-like or human antibody. Examples of such antibodies include L2G7 and chimeric and humanized army. The invention also relates to cell lines producing such antibodies. In another embodiment describes a pharmaceutical composition comprising a neutralizing anti-HGF antibody, such as chimeric or humanitariannet L2G7 antibody. In the third embodiment, the pharmaceutical composition is administered to a patient for treatment of cancer or other diseases.
BRIEF DESCRIPTION of DRAWINGS
Figure 1. Schematic illustration of the structure of HGF and cMet.
Figure 2. Graph showing that NK4 partially inhibits primary growth in a murine lung tumor LLC "Nude" mice (see Kuba et al., Cancer Res. 60: 6737, 2000). NK4 is administered by continuous infusion for 14 days starting from the 4th day after subcutaneous (s/C) implantation of the tumor "Nude" mice.
Figure 3. Graph showing that a mixture of three anti-HGF Mat is required for inhibiting the growth of tumor cells of the human brain U-118 "Nude" mice (see Cao et al., Proc. Natl. Acad. Sci. USA 98: 7443, 2001). Tumor cells U-118 is injected s/C "Nude" mice. Starting with the first day of the anti-HGF Mat a-1, -5 and -7 or Mat 7-2 and -3 is administered at a dose of 200 μg/injection twice a week for 10 weeks.
Figure 4. Determination of binding epitopes mat L1H4, L2C7, L2G7 using the mechanism of competitive binding in the context of the methodology ELISA. On the tablet layer recombinant HGF (rHGF), block separated milk and incubated with suboptimal concentrations of biotinyl the skilled Mat in the presence of 100-fold excess unlabeled Mat. Binding of biotinylated Mat expose adding HRP-streptavidin.
Figure 5. Binding of anti-HGF Mat with rHGF determined by direct binding of HGF by ELISA method. On the tablet layer H1-F11 supernatant containing rHGF, block 2% separated milk and incubated with the Mat, then add HRP-GoMIgG (as described in the examples section).
6. The ability of anti-HGF Mat to catch rHGF-Flag in the solution. Anti-HGF Mat catch on tablets for ELISA coated with goat artemisinin IgG. Next, tablets, block 2% separated milk and incubated with rHGF-Flag, then add HRP-M2 anti-Flag Mat (as described in the examples section).
7. Inhibition of binding of rHGF-Flag with cMet-Fc with anti-HGF Mat in a method ELISA with capture. cMet-Fc included in the tablet for ELISA coated with goat artemisinin IgG, incubated with HGF-Flag, which was previously preincubation with/without the Mat. Associated rHGF-Flag detected by adding HRP-M2 anti-Flag Mat (as described in the examples section).
Fig. Neutralization of HGF - induced scattering of MDCK anti-HGF Mat L2G7. (A) Control without any treatment. (C) rHGF+IgG. (C) rHGF+Mat L2G7. MDCK cells incubated with culture supernatant H1-F11 at dilution 1:20 (≈3 mg/ml HGF) in the presence of 10 μg/ml Mat. The photos were taken at 100 times magnification.
Fig.9. Inhibition of HGF-induced proliferation of Mv I LU cell monoclone is determined as being the L2G7 antibody. Fold molar excess of the Mat relative to HGF is shown on the horizontal axis, while the vertical axis shows the level of inclusion in the pulse/min ×10-2. Presents the data obtained from the analysis in the triple repetition.
Figure 10. Inhibition of HGF-induced proliferation of HUVEC monoclonal antibody L2G7 and control mouse antibody (mIgG). Presents the data obtained from the analysis in the triple repetition.
11. The effect on HGF-induced proliferation of tumor cells of the colon HCT 116 L2G7 antibody and L1H4. Presents the data obtained from the analysis in the triple repetition.
Fig. The effect of processing Mat L2G7 or PBS (control) on the growth of tumors U-118 in groups of mice NIH III Beige/Nude (n=6). Arrows indicate the start time of injection. (A) tumor Size relative to the day of tumor implantation. (C) tumor Weight by the end of the experiment.
DETAILED description of the INVENTION
The present invention relates to neutralizing anti-HGF monoclonal antibody, containing their pharmaceutical compositions and to methods of their use for the treatment of disease.
Antibodies are large, complex molecules (molecular weight ≈150000 or including approximately 1320 amino acids) with complex internal structure. The molecule of the natural antibody contains two identical pairs on peptidic circuits, where each pair contains one light chain and one heavy chain. Each light and each heavy chain, in turn, consists of two parts: variable ("V") of the site involved in binding to the target antigen, and a constant ("C") section, which interacts with other components of the immune system. Variable areas of light and heavy chains are added together in the spatial three-dimensional structure with the formation of the variable segment that binds to the antigen (e.g., to a receptor on the cell surface). Within each of the variable segment of the light and heavy chains have three short segment (including an average of 10 amino acids in length), called hypervariable sites (CDR). Six CDRs in the variable domain antibodies (three from the light chain and three from the heavy chain) are added together in a three-dimensional spatial structure with the formation of the actual binding site of the antibody to capture the antigen target. The position and length CDR precisely defined (Kabat, E. et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1983, 1987). Part of the variable segment not contained in the CDR, is called frame region, which forms the environment for CDR.
Monoclonal antibody is a single molecular species of antibody and therefore does not include polyclonal ant the body, formed by the injection of an animal (such as a rodent, rabbit or goat) antigen with subsequent selection of the serum from the animal. Humanitariannet antibody is an antibody obtained by genetic manipulation of the antibody (monoclonal), in which the CDR of a mouse antibody (donor antibody, which can also be taken from rat, hamster, or other similar type of animal) prizeplay human antibody ("acceptor antibody"). Humanized antibodies can also be obtained on the basis of the CDR of mouse antibodies, which are smaller than the full CDR (see, e.g., Pascalis et al., J. Immunol. 169: 3076, 2002). Thus, humanitariannet antibody is an antibody containing CDRs from the donor antibody, and frame the scope of the variable segment and constant areas of human antibodies. So, in the typical case humanitariannet antibody contains (i) a light chain comprising three CDRs from a mouse antibody, for example, L2G7, frame the scope of the variable segment of the human antibody and a constant region from a human antibody, and (ii) a heavy chain comprising three CDRs from a mouse antibody, for example, L2G7, frame the scope of the variable segment of the human antibody and a constant region from a human antibody. In addition, to maintain Finney activity can be used at least one of two additional structural elements. Cm. U.S. patents NoNo. 5530101 and 5585089 included in the present description by reference, which provides detailed instructions on how to design humanized antibodies.
In the case of the first structural element of a frame region of the variable segment of the heavy chain gumanitarnogo antibody is chosen so as to achieve the maximum sequence identity (65% to 95%) with frame area of the variable segment of the heavy chain donor antibodies by appropriate selection of the acceptor antibody among the set of known human antibodies. The sequence identity is determined in the matching process to compare sequences according to Kabat numbering (Kabat). In the case of the second structural element used in the construction gumanitarnogo antibodies, selected amino acids in the framework region of the acceptor human antibody (outside CDR)are replaced by the corresponding amino acids from the donor antibody in accordance with the established rules. Specifically, amino acids, subject to substitution in the frame region, chosen on the basis of their ability to interact with the CDR. For example, the substituted amino acids can join CDR in the donor sequence of the antibody or to be at a distance of 4-6 angstroms from CDR in g is monitorowania the antibody according to the measurement results in 3-dimensional space.
A chimeric antibody is an antibody in which the variable area mouse antibody (or antibodies from other rodent) combined with a constant area of a human antibody; the design of such antibodies by methods of genetic engineering is well known in this field. Such antibodies retain the binding specificity characteristic of murine antibodies, while approximately 2/3 they are human antibodies. Share not human sequences available in murine, chimeric or humanitariannet the antibody, suggesting that the immunogenicity of chimeric antibodies will be characterized by the average value between the values for murine and humanized antibodies. Other types of genetically engineered antibody, which may have reduced immunogenicity relative to mouse antibodies include human antibodies, obtained using the methods of phage display (Dower et al., WO 91/17271; McCafferty et al., WO 92/001047; Winter, WO 92/20791; and Winter, FEBS Lett. 23:92, 1998, each of these works is included in the present description by reference) or using transgenic animals (Lonberg et al., WO 93/12227; Kucherlapati, WO 91/10741, each of these works is included in the present description by reference).
In the context of the present description, the term "human-like" with respect to the antibodies which have relates to the Mat, in which a substantial portion of the amino acid sequence in one or both circuits (in particular, about 50% or more) comes from a human immunoglobulin genes. In this regard, human-like antibodies include, without limitation, chimeric, humanized and human antibodies. In the context of the present description, the term "reduced immunogenicity" as applied to the antibody is seen as expected significantly less immunogenic than murine antibodies, with the introduction of such antibodies to the people. Such antibodies comprise chimeric, humanized and human antibodies, and antibodies, obtained by substitution of specific amino acids in the murine antibodies that may be involved in b - or T-cell epitopes, for example, exposed residues (Padlan, Mol. Immunol. 28: 489, 1991). In the context of the present description, the term "genetically engineered" when referring to an antibody refers to this antibody, for which genes have been constructed or put into non-natural environment (e.g., human genes in mouse or bacteriophage) using recombinant DNA techniques and which will not enable mouse Mat obtained using conventional hybridoma methods.
Epitope Mat represents a portion of the antigen with which the Mat is associated. Two antibodies svyazivaytes is one epitope or an overlapping epitope, if each of them competitively inhibits (blocks) the binding of another antibody of the pair of antibodies to the antigen. So, 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits the binding of another antibody of at least 50%but preferably 75%, 90% or even 99% according to the results of measurements in the test for competitive inhibition compared to control, non-competing antibody (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990, this work is incorporated into this description by reference). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody, reduce or eliminate the binding of another antibody. Two antibodies have overlapping epitopes, if some amino acid mutations that reduce or eliminate binding of one antibody, reduce or eliminate the binding of another antibody.
2. Neutralizing anti-HGF antibodies
It is believed that a monoclonal antibody (MAB)that binds to HGF, will neutralize HGF or may be expelled if the binding is partially or completely inhibits one or more of the biological activities of HGF (for example, when HGF is used as a single agent). Among the biological properties of HGF, caloriemate to inhibit neutralizing antibody includes the ability of HGF to contact cMet receptor, causing the proliferation of some cell lines, such as cells of the kidney of the dog Madin-Darby (MDCK); to stimulate proliferation of some cells (i.e. to be mitogenic), including hepatocytes, epithelial cells of the monkey 4MBr-5 cells of various human cancers; or to stimulate angiogenesis, for example, the results of measuring the proliferation of epithelial cells of vessels (HUVEC), or by forming a tube, or induction of blood vessels, when applied to chorioallantoic membrane of the chicken embryo (CAM). Antibodies according to the present invention preferably associated with human HGF, for example with the protein encoded by the sequence deposited in Genbank under number D90334 (corresponding to the information included in this description by reference).
Neutralizing Mat of the present invention at a concentration of, for example,, 0,01, 0,1, 0,5, 1, 2, 5, 10, 20 or 50 µg/ml to inhibit the biological function of HGF (e.g., stimulation of proliferation or scattering) by about 50%but preferably 75%, more preferably 90%, 95% or even 99% and most preferably 100% (essentially completely), the evaluation on the methods of analysis described in the examples or known in this field. Inhibition is considered as complete if preventatively is within the errors for the negative control without HGF. In a typical case, the degree of inhibition was measured using the number of HGF, sufficient for stimulation of biological activity or 0.05, 0,1, 0,5, 1, 3 or 10 μg/ml Preferably achieved at least 50%, 75%, 90% or 95% or essentially complete inhibition in the presence of molar ratio of antibody to HGF 0.5-fold, 1-fold, 2-fold, 3-fold, 5-fold or 10-fold the quantity. Preferably the Mat is to neutralize, for example to inhibit the biological activity, in case of its use as a single agent, but it may be necessary joint action 2 Mat for inhibition. Most preferably, the Mat will neutralize only one of several of the biological activities listed above; in the context of the present description anti-HGF Mat, which is used as the sole agent neutralizes all kinds of biological activity of HGF is called "fully neutralized", and such Mat are the most preferred. The Mat according to the present invention preferably are specific for HGF, which means that they are not linked or associated in a substantially lesser extent (for example, with a value of Kandlower , at least 10 times) with proteins close to HGF, such as fibroblast growth factor (FGF) and factor the OST endothelial cells of blood vessels (VEGF). Preferred antibodies are not agonists activity against HGF. This means that antibodies block the interaction of HGF with cMet without stimulation directly to cells containing HGF. The Mat according to the present invention typically have an affinity binding (Kandfor HGF, equal to at least 107M-1but preferably 108M-1or higher, and most preferably 109M-1or higher or even 1010M-1or higher.
The Mat according to the present invention include anti-HGF antibodies in their natural tetramer form (2 light chains and 2 heavy chains) and can refer to any of the known isotypes IgG, IgA, IgM, IgD and IgE, and their subtypes, such as human IgG1, IgG2, IgG3, IgG4 and mouse IgG1, IgG2a, IgG2b and IgG3. It should also be understood that the Mat according to the present invention include antibody fragments, such as Fv, Fab and F(ab')2; bifunctional hybrid antibodies (see, e.g., Lanzavecchia et al., Eur. J. Immunol. 17: 105, 1987), single-chain antibodies (Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879, 1988; Bird et al. Science 242: 423, 1988) and antibodies with modified constant plots (see, for example, U.S. patent No. 5 624 821). The Mat may also be of animal origin (for example, can be obtained from mouse, hamster or chicken), or they can be created by genetic engineering methods. Mat rodents can be obtained from the mill is artimi methods known in this field, which include multiple immunization HGF in the corresponding Freund in/b or/or pads with subsequent selection of cells from the spleen or lymph nodes and merge them with the appropriate immortalizing cell line, followed by a selection of hybridomas that produce the antibody to bind to the HGF, in particular, as described in the examples. Chimeric and humanized antibodies produced by known methods mentioned above, represent a preferred aspect of the present invention. Human antibodies obtained, for example, using phage display or transgenic mice, are also preferred (see, for example. Dower et al., McCafferty et al., Winter, Londberg et al., Kucherpalati, supra). In a broader aspect of antibodies, human-like, with reduced immunogenicity, and antibodies obtained by genetic methods, in accordance with this description by definition, all are considered as preferred.
Neutralizing anti-HGF Mat L1H4, L2C7 and L2G7, described below, represent embodiments of the present invention, where L2G7 is a preferred example. Neutralizing Mat with the same or overlapping epitope as any of these Mat, for example, as L2G7, belong to another variant implementation of the ia of the invention. Mat (including chimeric, humanized and human antibodies), which compete with L2G7 for binding to HGF and neutralize HGF, the results of one and preferably all of the tests in vitro or in vivo, in the present description, are also preferred. The Mat, which are 90%, 95%, 99% or 100% identical (when the determination by comparing the sequences of the antibodies in accordance with rule Kabata), with L2G7 amino acid sequence, at least in CDR included in the present invention. Preferably such antibodies differ from L2G7 on a small number of functionally non-consecutive amino acid substitutions (e.g., conservative substitutions), deletions or insertions. Preferably such antibodies retain the functional properties of L2G7, these antibodies neutralize HGF, the results of one and preferably all of the tests in vitro or in vivo, in the present description. For the purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids may be grouped as follows: Group I (hydrophobic side chains): norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing the orientation the chain): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions include substitution between amino acids within the same class. Non-conservative substitutions are substitution of one representative class representative of another class.
Native Mat according to the present invention can be obtained from the hybrid. Mat resulting from genetic manipulation, can be expressed using many well-known in this field techniques. For example, the genes encoding the V regions of their light and heavy chains, can be synthesized on the basis of overlapping oligonucleotides and then integrated together With lots of expression vectors (e.g., commercially available from Invitrogen)that provide the necessary regulatory areas, such as promoters, enhancers, poly-A sites and the like, Preferably using a promoter-enhancer of CMV. The expression vectors can be transliterowany using various known methods such as a method based on the use of lipofection, and electroporation, in many cell lines mammals, such as SNO or neproducyruth myeloma, including Sp2/0 and NSO, and the cells expressing the antibodies are selected by selection using the appropriate antibiotic. See, for example, Pat is NT USA No. 5530101. Large quantities of antibodies can be obtained by growing cells in commercially available bioreactors.
After the expression of Mat or other antibiotics according to the present invention can be purified in accordance with known standard procedures, such as microfiltration, ultrafiltration, affinity chromatography with protein a or G, gel chromatography, anion exchange chromatography, cation exchange chromatography and/or other forms of affinity chromatography, based on organic dyes or the like are Preferred essentially pure antibodies that 90 or 95% are homogeneous, and the most preferred antibodies with homogeneity 98% or 99% for their pharmaceutical applications.
3. Therapeutic methods
In a preferred embodiment, the present invention relates to pharmaceutical compositions containing the antibodies described in the present description. Thus, antibodies can be used in the production of drugs for the treatment of diseases. Pharmaceutical compositions (e.g., drugs) antibodies contain the Mat in a physiologically acceptable carrier, optionally in the presence of fillers or stabilizers, in the form of dried or aqueous solutions. Acceptable carriers, excipients or stabilizers are netoc the ranks for recipients in the used doses and concentrations, and include buffers, such as phosphate, citrate or acetate, at pH in the typical case, from 5.0 to 8.0, and most often at pH from 6.0 to 7.0; salts such as sodium chloride, potassium chloride, etc. to maintain isotonicity; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, such as Polysorbate 80, amino acids, carbohydrates, chelating agents, sugar and other standard ingredients known to specialists in this field (Remington''s Pharmaceutical Science 16thedition, Osol, A. Ed. 1980). The Mat is typically present in concentrations of from 1 to 100 mg/ml, for example 10 mg/ml
Antibodies according to the present invention are essentially clean and free from unwanted contamination. This means that the antibody is typically clean at least 50 wt.% (weight/weight), and essentially free from interfering proteins and contaminants. Preferably the antibodies are clean at least 90, 95 wt.% or 99 wt.%. Pharmaceutical compositions for parenteral administration are generally sterile, essentially isotonic and received in accordance with the requirements of the production processes of the FDA or similar institutions.
In another preferred embodiment, the present invention relates to a method of treating a patient using anti-HGF within the pharmaceutical is eskay composition. Mat, made in the form of pharmaceutical compositions can be administered to the patient in any acceptable way, particularly parenterally, by infusion or bolus injection, subcutaneously or intramuscularly. Intravenous infusion can be carried out within 15 minutes, but more often within 30 minutes or within 1, 2 or even 3 hours. The Mat can also be injected directly into the affected area (e.g., tumor) or can be encapsulated inside agents, carriers, such as liposomes. Enter the dose should be sufficient to ease the condition to be treated ("therapeutically effective dose"), and may be from 0.1 to 5 mg/kg body weight, for example 1, 2, 3, or 4 mg/kg, for example 50, 100, 200, 500, or 1000 mg/kg May be also fixed standard dose, such as 50, 100, 200, 500, or 1000 mg/kg, or injected dose can be selected with reference to the square of the patient's body, for example, 100 mg/m2. Usually for cancer treatment is administered 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), but may be 10, 20 or more doses. The Mat can be entered daily, biweekly, weekly, every other week, monthly, or at some other interval, depending on, for example, from the half-life of the Mat in 1 week, 2 weeks, 4 weeks, 8 weeks, 3-6 months or longer. It is also possible repeated courses of treatment, as in the example, in the case of chronic administration. The dosage and intervals between doses that may facilitate or at least partially stop the symptoms of the disease (biochemical, histologic and/or clinical), including its complications and intermediate phenotypes in development of the disease, is considered therapeutically effective mode.
The pharmaceutical compositions according to the present invention can also be used for prophylaxis in the case of a patient with cancer risk. Such patients include people with a genetic predisposition to cancer, patients exposed to carcinogenic agents, such as radiation or toxins, as well as patients who had previously undergone treatment for cancer or have the risk of its recurrence. Preventive dose is a quantity which is sufficient to eliminate or reduce the risk, reduce the severity, or delay the symptoms of the disease, including biochemical, histologic and/or clinical symptoms of the disease, its complications and intermediate phenotypes manifested in the development of the disease. Introduction the pharmaceutical composition in amounts and at intervals effective to achieve one or more of these effects is considered as preventive effect is wny mode.
Diseases that are characterized by a special sensitivity to treatment with anti-HGF Mat of the present invention include solid tumors, which are known, or will need to angiogenesis or having Association with elevated levels of HGF, for example ovarian cancer, breast cancer, lung cancer (small cell or not small cell lung), cancer of the colon, prostate cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, head and neck cancer, melanoma, sarcomas, and brain tumors (e.g., glioblastoma) in adults and children. This treatment can also be carried out in patients with leukemia and lymphoma. In a preferred embodiment, the anti-HGF Mat can be entered together with other anticancer agents (e.g. before, during or after such therapy). For example, anti-HGF Mat can be administered together with one or more chemotherapeutic drugs known to specialists in the field of Oncology, such as, for example, Taxol (paclitaxel) or its derivatives, platinum compounds, such as carboplatin or cisplatin, anthracycline, such as doxorubicin, alkylating agents such as cyclophosphamide, antimetabolites such as 5-fluorouracil, or etoposide. Anti-HGF Mat can be administered in combination with two or more agents in a standard chemotherapy regimen, t the com as for example, Taxol and carboplatin, for example, in breast cancer and ovarian cancer. Other agents with which anti-HGF Mat can be entered include biological active compounds such as monoclonal antibodies, including Herceptin™ (Herceptin™) against the HER2 antigen, Avastin™ (Avastin™) against VEGF, or antibodies against the EGF receptor, as well as small molecule drugs with antiangiogenic activity or antagonistic activity against EGF receptor. In addition, anti-HGF Mat can be used in conjunction with radiation therapy or with surgical methods.
Treatment (for example, in the course of standard chemotherapy, including anti-HGF monoclonal antibody, can increase the average time of survival without progression or overall survival time of patients with these tumors (e.g., ovarian, breast, lung, pancreas, brain and colon, especially in the case of recurrent or refractory tumors) at least 30% or 40%but preferably 50%, 60%-70% or even 100% or more, compared to the same treatment (e.g. chemotherapy)but without anti-HGF MABs. In addition or alternatively, the treatment (e.g. chemotherapy), including anti-HGF Mat, can increase the rate of complete response, partial response or speed of the objective (olego + partial) in patients with these tumors (e.g., ovarian, breast, lung, pancreas, brain and colon, especially in the case of recurrent or refractory tumors) at least 30% or 40%but preferably 50%, 60%-70% or even 100% or more, compared to the same treatment (e.g. chemotherapy), but without anti-HGF MABs. Optional this treatment can inhibit the invasion of tumors or their metastases.
In the typical case in a clinical trial (for example, phase II, phase II/III or phase III trials) indicated the average time of survival without disease progression and/or response in patients undergoing chemotherapy plus anti-HGF Mat, relative to the control group of patients undergoing only chemotherapy (or plus placebo)will be statistically significant, for example, at the level of p=0.05, or 0.01 or even 0.001 in. For each specialist in this area it is obvious that the rate of complete and partial response determined by objective criteria used in clinical trials for cancer, for example in accordance with the criteria listed or approved by the National cancer Institute (National Cancer Institute) and/or control of the United States regulating the use of food and medicinal products (Food and Drug Administration).
4. Other methods
Anti-HGF Mat according to the present invention m is may also find use in diagnostic methods, forecasting and laboratory methods. They can be used to determine the level of HGF in tumor or in the bloodstream of a patient with a tumor and therefore can be used for and the direction of the treatment of the tumor. For example, a tumor associated with high levels of HGF, will be particularly sensitive to treatment with anti-HGF MABs. In specific embodiments, the implementation of the present invention, the Mat can be used in the analysis by the method of ELISA or radioimmunoassay test to determine the level of HGF, for example in biopsy specimens of the tumor, or in serum or supernatant from the environment, or in HGF-secreting cells in culture. The use of two anti-HGF Mat, communicating with different epitopes (i.e., not competing for binding), will be particularly useful when developing sensitive variant ELISA "sandwich"method for detection of HGF. For carrying out different tests HGF can be tagged with fluorescent molecules, spin-labeled molecules, enzymes or radioactive isotopes and can be offered in the form of a set that includes all the necessary reagents to perform the test on HGF. In other embodiments, the anti-HGF Mat is mainly used for purification of HGF, for example, by affinity chromatography.
1. Getting anti-HGF Mat
To get the Mat that will tie the interference with human HGF and block its activity, first used the expression system of the mammal for the production of recombinant human HGF (rHGF). cDNA encoding the recombinant human HGF (rHGF) or rHGF-FLAG peptide (in which 8 amino acid residues attached to the C-end HGF), was introduced in pIND-the inducible expression vector (No et al., Proc. Natl. Acad. Sci. USA, 93:3346, 1996). These cDNA was transfusional cells fibroblasts human kidney EcR-293 (Invitrogen) using the transfection reagent Fugene (Roche). Stable cell lines, H1-F11 and 24.1, secreting HGF and HGF-Flag, respectively, were selected in the presence of 600 μg/ml G418 and 400 μg/ml of Zeocin (Zeocin) (Invitrogen). In cells H1-F11 and 24.1 induced secretion of HGF and HGF-Flag by treatment with 4 μm of ponasterone A (Ponasteron A) (Invitrogen) for 4-5 days in serum free DMEM containing glutamine and antibiotics. After removal of aggregates by centrifugation at 15,000 rpm for 30 minutes at 4°C, HGF, secreted into the culture supernatant was concentrated approximately 100-fold using a membrane cartridge ultrafiltration filter, shut-MV 50000 [amicon Centriprep YM-50 and then filter microcon YM-50 (Millipore)]. This concentrated culture supernatant H1F11 contains about 100 μg/ml HGF and about 120 μg/ml bovine serum albumin.
Balb/c mice were immunized in the tip of each hind paws >10 times at intervals of one week is about 1-2 µg purified rHGF (Pepto Tech) or 1-2 µg rHGF plus 1-2 µg BSA (concentrated supernatant H1-F11) resuspending in MDL-TDM (Ribi Immunochem. Research). Three days after the last boosting injections of cells popliteal lymph node was merged with myeloma cells of mice, P3X63AgU.1 (ATCC CRL1597) using 35% polyethylene glycol. Hybridoma were selected in medium HAT described in the literature methodology (Chuntharapai and Kim, J. Immunol. 163:766, 1997, this work is incorporated into this description by reference). Ten days after the merger supernatant hybridoma culture was fractionally by direct binding of HGF in the framework of ELISA and also by the method of ELISA capture HGF-Flag. The last test can be used to further confirm the specificity of the selected anti-HGF Mat using a direct binding of HGF by ELISA method and to select a Mat that can communicate with HGF in the solution phase. Then was determined by blocking the activity of the selected Mat by detecting the binding of HGF-Flag/cMet-Fc method ELISA test and dispersion. MDCK described in the literature methodology (Jeffers et al., Proc. Natl. Acad. Sci. USA 95:14417, 1998). Selected hybridoma twice cloned using the method of limiting dilutions. The isotype Mat was determined using kit ittipiboon (Zymed). Based on the selected Mat received ascites and purified them using the cleaning kit ImmunoPure (A/G) IgG (Pierce). Also received biotinylated Mat using EZ-sulfo-NHS-LC-Biotin, is accordance with the recommendations of Pierce. Each of these tests will be described hereinafter in more detail. For direct binding of HGF in the framework of the ELISA on microtiter plates (Maxisorb; Nunk) was layered on 50 μg/well of cultural supernatants H1-F11 containing rHGF, diluted with PBS in a ratio of HGF:PBS = 1:2, and was kept over night at 4°C. After washing tablet nonspecific binding sites were blocked by PBS containing 2% separated milk for 1 hour at room temperature (RT). After washing tablets in each well was added for 1 hour at 50 μg/well of purified Mat or cultural supernatants hybrid. Next, the tablets after washing incubated made with 50 ál/well of 1 μg/ml goat artemisinin IgG, conjugated with horseradish peroxidase (HRP-GαMIgG, Cappel) for 1 hour. Bound HRP-GαMIgG was detected by adding substrate tetramethylbenzidine (Sigma). The reaction was stopped by adding 1N H2SO4and then in the tablets was determined by the absorbance at 450 nm using a device for analyzing ELISA plates. Washing was performed 3 times in wash buffer (PBS containing 0.05% tween-20).
In the analysis according to the method of capture HGF-Flag method ELISA on microtiter tablets were layered on 50 μg/well of 2 μg/ml goat antibodies specific for the Fc portion of mouse IgG (GαMIgG-Fc in PBS overnight at ambient temperature the re 4°C and blocked with 2% separated milk for 1 hour at RT. After washing the plates were incubated with purified Mat or cultural supernatants hybrid, made of 50 μg/well, for 1 hour. Then the tablets after washing incubated with 50 µl/well of culture supernatant 24/1 cells containing rHGF-Flag. After washing the plates were incubated with 50 μg/ml HRP-M2 anti-Flag Mat (Invitrogen) in the presence of 15 μg/ml mouse IgG. Related HRP-anti-Flag M2 was detected by adding substrate, as described above. Washing was performed 3 times using wash buffer.
At least three Mat, designated as L1H4, L2C7 and L2G7 obtained from hybridomas generated by immunization of Balb/c mice in the concentrated culture supernatant H1-F11, as described above, demonstrated binding of both direct binding rHGF method is ELISA, and in the analysis of capture HGF-Flag ELISA method, which were selected for further studies. These hybridoma twice cloned received in mice ascites by standard techniques and purified Mat using a column with protein G/A. Isotypes were determined using a kit ittipiboon (Zymed Lab). Hybridoma L2G7 was deposited on April 29, 2003 in the American type culture Collection (American Type Culture Collection, P.O. Box 1549 Manassas, VA 20108), under the number stored in ATSC MOUTH-5162, in accordance with the Budapest Treaty. This Deposit shall be kept in his personal collection, and will be replaced as soon as unsustainable or destructive mutations over a period of time, equal to at least five years after the last request for the grant of a sample obtained in the collection for at least thirty years from the date of Deposit or during the period of validity of the related patent, in accordance with the longest of these periods. All restrictions on availability for General use will be permanently revoked the patent has been granted.
After selection of one archetypal antibodies against human HGF, for example, L2G7, which has desirable properties according to the present description on the neutralization of HGF in vitro and/or inhibition (e.g., full) tumor growth in vivo, then proceeded directly to the products of other Mat with similar properties when used for this purpose the known methods. For example, mice can be immunized HGF by the method described above, can then be obtained hybridoma and educated Mat subjected to screening to analyze their ability to compete with archetypal Mat for binding to HGF. An alternative method of Jespers et al. (Jespers et al., Biotechnology 12:899, 1994), which is included in the present description by reference, can be used for breeding Mat having the same epitope and therefore the same properties with the properties of archetypal Mat, for example L2G7. Using phage method, the first heavy chain archetypal antibodies is pariwat with a repertoire of light chains (preferably a human) for selection of HGF-binding Mat and then a new light chain pair with a repertoire of heavy chain (preferably a human) for selection of HGF-binding the Mat having the same epitope as archetypal Mat.
2. Characterization of anti-HGF Mat in vitro
The binding epitopes of the antibodies initially investigated in the method of competitive binding in the framework of the ELISA, which used a 100-fold excess of the Mat to achieve competition for binding to the same or other biotinylated Mat in the ELISA method, the binding of HGF. Figure 4 shows that binding of anti-HGF Mat, L1H4 and L2G7 inhibited only by themselves, which indicates that they recognize a unique epitope. Linking L2C7 inhibited L2G7, but not L1H4. This allows us to suppose that L2C7 epitope overlaps with the epitope L2G7, but not with L1H4. However L2C7 did not show inhibitory ability against L2G7 that indicates that L2C7 and L2G7 epitopes overlap, but are distinct differences, and/or the affinity L2C7 much lower than the affinity of L2G7. Epitopes L1H4, L2C7 and L2G7 designated as a, b and C respectively.
The relative ability to bind three anti-HGF Mat was determined using purified antibodies in a direct ELISA method of binding HGF, which rHGF initially associated with the tablet. In this test L2C7 and L2G7 contacted better than L1H4 (figure 5). Determined the ability of the Mat to contact rHGF-Flag in the solution using the ELISA method with capture HGF-Flag. All three Mat was able to capture rHGF-Flag in the solution phase, but Mat L2G7 was more the effect is positive, than others (6). The results suggest that the Mat L2G7 has the highest binding affinity to HGF among the three Mat.
One of the types of biological activity of HGF includes its ability to bind with its receptor cMet, so we analyzed the ability of anti-HGF Mat to inhibit the binding of HGF to cMet. For this purpose initially received cMet-Fc by transfection fibroblastic human cells 293 using cDNA encoding residues 1-929 ECD in cMet associated with the Fc part of human IgGI (residues 216-446), as described by Brand et al. (Mark et al., J. Biol. Chem. 267: 26166, 1992), in the expression vector pDisplay (Invitrogen). On microtiter tablets were layered on 50 μg/well of 2 μg/ml goat antibodies specific for the Fc portion of human IgG (GαHIgG-Fc)in PBS, incubated overnight at 4°C and blocked with 2% BSA for 1 hour at RT. After washing tablets in each well was added for 1 hour at RT in 50 μl of culture supernatant of 293 cells transfected with the cDNA cMet-Fc. After washing tablets in each well for 1 hour was added at 50 μg/ml of culture supernatant 24.1 cells containing rHGF-Flag, after pre-incubation with different concentrations of the Mat. After washing the plates were incubated with 50 μg/well of HRP-M2 anti-Flag Mat (Invitrogen). Bound HRP-anti-Flag M2 was detected by adding substrate, as bloomian above. Washing was performed 3 times with wash buffer.
In the specified test on the inhibition of binding of HGF-Flag/cMet-Fc all three antibodies showed some level of inhibition, whereas the control Ig antibody did not show such activity (Fig.7). Mat L2G7 at a concentration of ≥1 μg/ml and Mat L1H4 at a concentration of 50 μg/ml completely eliminate the binding rHGF-Flag with cMet-Fc; Mat L2C7 even at a concentration of 50 μg/ml gives only 85% inhibition. Accordingly, the Mat L2G7 much more effective in terms of inhibiting the interaction rHGF-Flag with cMet-Fc (and therefore mostly HGF to its receptor cMet), other than antibodies, which is consistent with its supposedly higher affinity for HGF.
Because protein is the receptor used in the ELISA method binding cMet-Fc/HGF-Flag, is a soluble protein of the receptor, its conformation may differ from the natural conformation of the receptor that is associated with the membrane. Furthermore, in addition to binding to cMet HGF binds to HSPG and it is known that the interaction of HSPG-HGF promotes different kinds of activity of HGF. So, Mat, blocking the interaction of HGF with soluble cMet, not necessarily will have the ability to neutralize the biological activity of HGF in the cells. Thus, additionally it is essential to confirm the blocking activity Mat in selected biological systems. It is known that HGF is the fast and powerful the scattering factor. Thus, the neutralizing activity of anti-HGF Mat was also determined using cells of the kidney of the dog Madin-Darby (MDCK cells derived from ATSS) in the test for scattering (Jeffers et al., Proc. Natl. Acad. Sci. USA 95: 14417, 1998). MDCK cells grown in DMEM with the addition of 5% PBS, placed on the tablets of 103cells/well in the presence of specified concentrations of rGHF, in the presence or in the absence of a mate in DMEM with 5% PBS. After 2 days incubation at 37°C and in the presence of 5% CO2cells were washed in PBS and fixed in 2% formaldehyde for 10 minutes at RT. After washing in PBS, cells were stained with 0.5% crystal violet dye in 50% vol. ethanol for 10 minutes at RT. Scattering activity was determined in the study under the microscope.
Cultural supernatant H1-F11 clone secreting HGF, described above, was used as a source of HGF in the test for dispersive. Already in the dilution 1:80 culture supernatant H1-F11 induced dispersion and growth of MDCK cells. However, tests on the dispersion was performed using a dilution of 1:20 the culture supernatant. H1-F11 (approximately 3 μg/ml). Mat L2G7 even at a molar ratio of HGF/Mat 1:5, inhibited HGF-induced scattering of MDCK due to its own effect (Fig), which ultimately demonstrates that the Mat is really a neutralizing matmat L2C7 even at a concentration of 20 μg/ml showed only a partial neutralizing activity (data not shown).
Various characteristics of the three anti-HGF antibody, as defined in the above tests are shown in table 1.
|Characterization of MAB against HGF|
|Mat||The isotype||Bind the epitope||Blocking the binding of HGF/cMet-Fc||Blocking dispersion MDCK|
|L1H4||G1, k||A||Weak blocking||+|
|L2C7||G2b, k||In||Partial blocking||+/-|
|L2G7||G2a, k||Strong blocking||+++|
HGF is a representative of a family of heparin-binding growth factors, including fibroblast growth factor (FGF) and vascular endothelial growth (VEGF). HGF also has about 40% similarity in the whole sequence with plasminogen (Nakamura et al., Nature, 342: 440, 1989) and the em structure of the domain similar to that of the macrophage stimulating protein (MSF, Wang et al., Scand. J. Immunol. 56: 545, 2002). Thus, it must be defined binding specificity of anti-HGF. Binding of anti-HGF Mat with the specified HGF-related proteins (available from the company R&D) analyze using ELISA test for direct binding similar test for the analysis of HGF described above. Mat L2G7, Mat L2C7 and Mat L1H4 will be a slight contact with these proteins, demonstrating its specificity for HGF.
3. The ability of anti-HGF Mat to inhibit tumor biological activity of HGF
HGF has many kinds of biological activity that allows him to play a role in the growth and invasion of some human cancers. One such known activity of HGF is its ability to act as a powerful mitogen for hepatocytes and other epithelial cells (Rubin et al., Proc. Natl. Acad. Sci. USA, 88: 415, 1991). Thus, for additional confirmation of neutralizing activity of anti-HGF Mat determined the effects Mat on HGF-induced proliferation of epithelial cells mug-5 monkeys (ATSS) or rat hepatocytes. Hepatocytes were isolated according to the method of Harrison and Chinese (Harrison and Haynes, J. Biol. Chem. 269: 4262, 1985). Cells resuspendable in an amount of 5×104cells/well in DMEM containing 5% PBS and stimulated a given concentration is the situation of HGF with different concentrations of the Mat. After incubation for 2.5 days at 37°C with 5% CO2to determine the level of cell proliferation adding3H-thymidine for 4 hours. Next, the cells were collected using an automatic collector for collecting cells and determined the levels included3H-thymidine in a scintillation counter. If sufficient concentrations Mat L2G7 can largely or completely inhibit HGF-induced cell proliferation and Mat L2C7 and L1H4 can, at least partially, to inhibit proliferation. These antibodies can also inhibit HGF-induced proliferation of other lines of epithelial cells.
For example, it was determined the inhibitory activity of L2G7 against HGF-induced proliferation of mink lung cells Mv I Lu (Borset et al., J. Immunol. Methods 189: 59, 1996). Cells growing in DMEM containing 10% PBS, collected during the processing EDTA/trypsin. After washing the cells resuspendable in an amount of 5×104cells/ml in serum-free DMEM medium containing a given concentration of HGF (50 ng/ml) +/- different concentrations of the Mat. After incubation for 1 day at 37°C with 5% CO2to determine the level of proliferation adding 1 MX3H-thymidine for a further 24 hours. Next, the cells were collected on glass fiber filters using an automatic collector for collecting cells and Regaleali level included 3H-thymidine in a scintillation counter. Figure 9 shows that the addition of 100-fold molar concentrations Mat L2G7 completely inhibited the proliferative response Mv I Lu cells. In fact, L2G7 even with 3-fold molar ratio of the Mat to HGF showed complete inhibition, whereas control IgG showed no inhibition even at 100-fold molar excess.
It was also reported that HGF is a potent angiogenesis factor (Bussolino et al., J Cell Biol. 119: 629, 1992; Cherrington et al., Adv. Cancer Res. 79: 1, 2000), and it is believed that angiogenesis, i.e. the formation of new blood vessels, is essential for tumor growth. In this regard, the ability of anti-HGF Mat to inhibit angiogenic properties of HGF was shown in three tests: (i) proliferation of endothelial cells of blood vessels human (HUVEC), (ii) formation of HUVEC tube for; and (iii) the development of new blood vessels on chorioallantoic membrane of the chicken embryo (CAM). Since it has been shown that HGF acts synergistically with VEGF in angiogenesis (Xin et al., Am. J. Pathol. 158: 1111, 2001), these tests were conducted in the presence and in the absence of VEGF.
Test on the proliferation of HUVEC was performed according to a known procedure with a modification (Conn et al., Proc. Natl. Acad. Sci. USA 87: 1323, 1991). The HUVEC cells obtained from the company Cloneties, were grown in growth medium for endothelial cells (computers-2)containing 10% PBS plus supplements for rectangularly cells, supplied by Clonetics. Preferably, the cells used in this study were subjected to 4 to 7 passages. Cells resuspendable to achieve a concentration of 105cells/ml in medium-199 containing antibiotics, 10 mm HEPES and 10% PBS (test environment). The HUVEC cells (50 μl/well) was added to the wells of the microtiter tablet containing a suitable concentration of HGF with various concentrations of anti-HGF Mat, for 1 hour at 37°C. after the incubation of the cells for 72 hours at 37°C in a medium containing 5% CO2determined the level of cell proliferation by the number of enabled3H-thymidine for 4 hours. If sufficient concentrations Mat L2G7 and Mat L1H4 can at least partially inhibit proliferation.
Alternatively, the level of cell proliferation can be determined by known colorimetric test using MTT reagent. The HUVEC cells (104 cells/100 μl/well) were grown in serum-free medium for 24 hours and then incubated with 100 μl of 50 ng/ml HGF (i.e., the number of pre-defined as suboptimal number), with different concentrations Mat L2G7 within 72 hours. Was added to each well of MTT (20 μl/200 μl medium) for 4 hours. Then selected 100 μl medium/well and were mixed with 100 μl/well of an acidified isopropyl the CSOs of alcohol of 0.04 N HCl in isopropyl alcohol). Absorption in the wells was determined on the device for analysis by ELISA method at a wavelength of 560 nm. % of the maximum response rate was calculated as follows: [OP for HGF + Mat-treated cells - OD for untreated cells]/[OD for HGF-treated cells - OD for untreated cells]×100. Figure 10 shows that even a 2-fold molar excess Mat L2G7 largely blocked the proliferation of HUVEC in response to exposure to HGF.
Test for the formation of tubes of endothelial cells was performed as described in the literature methodology (Matsumura et al., J. Immunol. 158: 3408, 2001; Xin et al., Am. J. Pathol. 158:1111, 2001). Cells HUVEC (Clonetics) after 4-7 passages raised in an environment Clonetics EGM with the addition of 10% PBS and with the additives necessary for the growth of endothelial cells. The tablets were layered Matrigel (BD Bioscience) according to the manufacturer's instructions at 37°C for 30 minutes and then cells were sown with a density of 3×106cells/ml in a basic medium with HGF in the presence of various concentrations of anti-HGF MABs. The tube formation was assessed under a microscope at low (10×) magnification. If sufficient concentrations Mat L2G7 largely or completely inhibited HGF-induced tube formation of endothelial cells, and Mat L2C7 and L1H4 can at least partially inhibit it.
Test using chorioallantoic membrane ambr is it chicken (SAM) was performed according to the literature method (Kim et al., Nature 362: 841, 1992). The three-day chick embryos were selected from the shell and was reared in Petri dishes at 37°C and in the presence in the environment of 5% CO2. After seven days was layered on ITSELF dried methylcellulose disks containing HGF with various concentrations of anti-HGF MABs. Methylcellulose disks were prepared by mixing 5 µl of 1.5% methylcellulose in PBS with 5 ál of HGF after incubation with matchers three days examined methylcellulose disks to detect the development of blood vessels around the disks. If sufficient concentrations Mat L2G7 largely or completely inhibited the formation of blood vessel and Mat L2C7 and L1H4 can at least partially inhibit it.
It was also reported that HGF promotes tumor growth (Comoglio and Trusolino, J. Clin Invest. 109: 857, 2002). The ability of anti-HGF antibodies to inhibit the specified activity has been demonstrated in two stages. Initially inspected many lines of tumor cells for their ability to secrete HGF and proliferate in response to exposure to HGF, because HGF may be an autocrine growth factor for some of these cells. These cell lines include the number of lines of tumor cells of a person in respect of whom it is known that they Express HGF and cMet (Koochekpour et al., Cancer Res. 57: 5391, 1997; Wang et al., J. Cell. Biol. 153: 1023, 2001). Specific cell the line, to be checked include U-118 a glioma, a cancer of the colon ST, carcinoma of the lung A cells and epidermoid carcinoma A431 received, all from ADS. After the identification of such cell lines was determined the effect of anti-HGF Mat on the proliferative response of these cells to HGF using methods similar to those described above. If sufficient concentrations Mat L2G7 largely or completely inhibited HGF-induced proliferation of many of these cell lines, and Mat L2C7 and L1H4 can at least partially inhibit proliferation.
For example, the tumor cells are human NST were sown in 96-well microtiter tablets with a density of 5×103cells/well in 200 μl of DMEM plus 5% PBS. After incubation for 24 hours at 37°C and at a content of 5% CO2cells were washed in PBS and incubated with serum-free medium DMEM for 48 hours. Then cells were incubated with 100 ng/ml HGF +/- 20 µg/ml Mat in DMEM for 20 hours. As controls in the experiment included cells growing in DMEM or DMEM plus 10% PBS. At the end of incubation to determine the level of cell proliferation by the inclusion of3H-thymidine for 4 hours. Figure 11 shows the results obtained in the framework of this experiment, conducted in the triple repetition. HGF induces moderate PR is literaly NST cells, which were completely eliminated by the addition of L2G7 antibody (but with lower efficiency than the antibody L1H4).
In all of the tests described above, each of the anti-HGF antibody neutralizes or inhibits the activity, when used by themselves, without the addition of HGF antagonists, that is, when using each of them as a single agent, and additive or synergistic effects can be achieved with the introduction of antibodies in combination with other anti-HGF antibody or with other active agents.
4. The ability of anti-HGF Mat to inhibit tumor growth in vivo
The ability of anti-HGF Mat to inhibit the growth of human tumor demonstrated on models xenografts in immunodeficient mice or other rodents such as the rat. As a representative but not limiting examples of immunodeficient strains of mice that can be used, can be called "Nude" mice, such as CD-1 nude, Nu/Nu, Balb/c nude, NIH-III (NIH-bg-nu-xid BR); scid mice, such as Fox Chase SCID (C.B-17 SCID), outbred Fox Chase SCID mice and SCID Beige; mice deficient in the enzyme RAG, as well as "naked" rats. The experiments were performed according to the procedure described previously in the literature (Kim et al., Nature 362: 841, 1992, this work is incorporated into this description by reference). The tumor cells are human, grown in complete DMEM, collected in HBSS. Immunodeficient female mouse is, for example, "Nude" mice without thymus (4-6 weeks of age), in dorsal areas were injected with p/C 5×106cells in 0.2 ml HBSS. When the tumor size reached 50-100 mm3mice were grouped randomly and put in b/W the appropriate quantity Mat (typically from 0.1 to 1.0 mg, for example 0.5 mg) one, two or three times a week, in an appropriate amount, for example, 0.1 ml, for example, 1, 2, 3, or 4 weeks or during the time period of the experiment. The size of the tumors was determined in a typical case, two times per week by measuring in two dimensions [length (a) width (b)]. Tumor volume was calculated by the equation V=ab2/2 and expressed as mean values of tumor volume ± standard deviation. The number of mice in each study group was at least 3, but more often from 5 to 10, for example 7. Statistical analysis can be performed using, for example, student's t test. In one variant of this experiment introduction antibodies was started simultaneously with the injection of tumor cells or soon after the injection. The effect of antibodies can be characterized by prolongation in survival time of mice or increase the percentage of surviving mice.
Various tumor cells, which are known that they secrete or respond to input HGF used in individual expertise is the cops, for example, cells from human glioblastoma U118 and/or tumor cells of the colon of a person NST. Preferred antibodies according to the present invention, such as human-like antibodies and antibodies with reduced immunogenicity, as well as L2G7 antibody and chimeric and humanized forms and antibodies to the same epitope as L2G7, in the case of using each of them as the sole agent inhibited tumor growth by at least 25%, but at least 40% or 50% and up to 75% or 90% or even higher, or even completely inhibited tumor growth after a certain period of time, or causing the regression or disappearance of the tumor. Specified the inhibition takes place in at least one line of tumor cells, such as U118, at least in one strain of mice, such as NIH III Beige/Nude, but preferably is 2, 3, several, many, or even essentially all of HGF-expressing lines of tumor cells of a particular type (e.g., gliomas) or any other type, when tested in one or more strains of immunodeficient mice, which do not form in response to the injected antibody neutralizing antibodies. Treatment with the use of certain preferred antibodies for one or more models with xenograft results not described is consistent survival 50%, 75%, 90% or even essentially all of the mice that would otherwise have died or which would need to kill due to the growth of the tumor.
For example, such an experiment was performed using cells of glioblastoma U-118, growing in DMEM medium containing PBS and collected in HBSS. Female mice NIH III Beige/Nude (4-6 weeks of age) in the dorsal zone were injected with p/106cells in 0.2 ml HBSS. When the tumor size reached approximately 50 mm3mice were grouped randomly for 6 mice in each group and were injected in b/W 200 mcg Mat (experimental group) or PBS (control group) twice a week in a volume of 0.1 ml of the size of the tumors was determined in a typical case, two times a week by the above procedure. At the end of the experiment, tumors were excised and weighed. On Fig shown that the introduction of L2G7 completely inhibited tumor growth.
Similar experiments on the inhibition of tumor performed using anti-HGF antibody, administered in combination with one or more chemotherapeutic agents such as 5-FU (5-fluorouracil) or CPT-11 (Camptostar), which, as expected, the tumor of this type need to be revoked, in accordance with the data Askania et al. (Ashkenize et al., J. Clin Invest. 104: 155, 1999). The combination of antibody and chemotherapeutic agent may exhibit a stronger inhibition of tumor growth than either agent individually. the shown effect may be additive or synergistic and can lead to potent growth inhibition, for example 80% or 90% or more, or may even cause regression or disappearance of the tumor. Anti-HGF antibody may also be administered in combination with an antibody against the other growth factors or angiogenic factor, such as anti-VEGF, and the expectation is additive or synergistic inhibition and/or regression or disappearance of the tumor.
Although the present invention has been described with reference to some preferred variations in its implementation, it should be understood that there may be introduced various modifications without departure from the essence of the invention. Unless it follows otherwise from the context, any stage, any item, option, feature, or aspect of the present invention can be used in conjunction with any other.
All cited in the description of the publications, patents and patent applications included in the present invention fully to the extent that each individual publication/ patent and patent application is specifically and individually indicated for full inclusion as a reference for all purposes.
1. Chimeric or humanitariannet monoclonal L2G7 antibody against growth factor hepatocyte, where L2G7 is a murine antibody produced by hybridomas ATS MOUTH-5162.
2. Cell line producing a monoclonal antibody according to claim 1.
3. Hybridoma deposited under the nom is the rum ATS MOUTH-5162, which produces the L2G7 antibody.
4. L2G7 antibody against growth factor hepatocyte produced by hybridomas according to claim 3.
5. Pharmaceutical composition for the treatment of diseases mediated by growth factor hepatocyte containing a therapeutically effective amount of the antibody according to claim 1.
6. A method of treating cancer in a patient, providing for the introduction to the patient the antibody of claim 1.
7. The method according to claim 6, characterized in that said cancer is a glioblastoma.
SUBSTANCE: invention concerns area of medicine and concerns the therapeutic agent for treatment of diseases related to children's chronic arthritic diseases, for example, actually children's chronic arthritic diseases, Still's disease and similar to it, including an antagonist of the interleukin-6 IL-6 receptor as an active ingredient, humanised antibody PM-1. Advantage of the invention consists in efficiency increase.
EFFECT: efficiency increase.
5 cl, 4 ex
FIELD: biotechnology, immunology.
SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.
EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.
26 cl, 16 dwg, 1 tbl, 8 ex
FIELD: medicine, biotechnology.
SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.
EFFECT: valuable medicinal properties of antibodies.
24 cl, 16 dwg, 5 tbl, 20 ex
FIELD: medicine, analytical immunology.
SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.
EFFECT: improved assay method.
3 tbl, 5 ex
SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.
EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.
10 cl, 21 dwg, 11 ex
SUBSTANCE: present invention relates to biotechnology and immunology. Proposed here is a polynucleotide, encoding a cyclic single-stranded tri-specific antibody. The antibody is directed against human ovarian carcinoma in vitro, has mass of approximately 84 kD and consists of three components: an antibody against human ovarian carcinoma cells, anti-CD3 antibody and anti-CD28 antibody, which are joined together by peptide interlinks such that, they form a cyclic antibody. Invented is an expression vector, containing a coding polynucleotide and versions of E.coli host cell based on the polynucleotide and expression vector.
EFFECT: use of the invention provides for a stable antibody molecule, optimum for activation of T-cells, which can be used in curing human ovarian carcinoma.
8 cl, 12 dwg
SUBSTANCE: versions of the bond intended for linkage with the external domain B (ED-B) of a fibronectin are offered. The bond includes an antigen-binding fragment of one-chained antibody L19 and a cysteinum-containing linker for hanging of a radioactive label. Versions of a pharmaceutical composition for diagnostics and treatment of angiogenic diseases on the basis of the specified bond are opened. Application of bond for linkage with radioactive bond is described. The method of reception of bond in eucariotic cells is opened, including in Pichia pastoris and a set for reception is radioactive labelled agents on the basis of bond.
EFFECT: high-avid bond accumulation in solid tumours.
23 cl, 4 dwg, 5 tbl, 15 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.
EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.
22 cl, 13 dwg, 8 tbl
FIELD: medicine, microbiology.
SUBSTANCE: invention concerns biotechnology. It is described bispecific antibody which binds also the factor of blood coagulation IX or the activated factor of blood coagulation IX, and the factor of blood coagulation X, and functionally replaces the factor of blood coagulation VIII or the activated factor of blood coagulation VIII which strengthens enzymatic reaction. The pharmaceutical composition containing the described antibody is revealed. The present invention can be used as an alternative agent for functional replacement of cofactor which strengthens enzymatic reaction.
EFFECT: creation of bispecific antibody which can replace functional proteins, strengthens enzymatic reaction.
14 cl, 18 dwg, 37 ex
SUBSTANCE: invention relates to biotechnology and immunology. Claimed is therapeutically active fused protein with reduced immunogenicity. Protein consists of two proteins derived from human proteins connected through the fusion region. Connective region, which covers or surrounds fusion region within the limits from 1 to 25 amino acid residues, contains modification, which removes T-cell epitope, in norm absent in humans. Claimed is application of fused protein for obtaining pharmaceutical composition for tumour treatment. Claimed is nucleic acid coding fused protein. Method of reduction of fused protein immunogenicity by introduction of substitutes of corresponding amino acids is described. Application of the invention allows reducing ability of connective epitope of therapeutically active fused protein to bind with molecules of the main complex of hystocompatibility (MHC) of class II, which finally reduces interaction of epitope with receptors of T-cells and can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.
EFFECT: reduction of interaction of epitope with receptors of T-cells, which can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.
23 cl, 12 ex
SUBSTANCE: versions of the molecule binding CD45RO and CD45RB, and the anti-CD45RO and anti-CD45RB antibody are invented. In one of versions, the said molecule contains at least one antigen-binding site and includes the subsequently located hypervariable sites CDR1, CDR2 and CDR3. The molecule represents the humanised or monoclonal antibody. CDR1 has the amino acid sequence NYIIH, CDR2 has the amino acid sequence YFNPYNHGTKYNEKFKG and CDR3 has the amino acid sequence SGPYAWFDT. The molecule can additionally contain the subsequently located hypervariable sites CDR1', CDR2' and CDR3'. CDR1' has the amino acid sequence RASQNIGTSIQ, CDR2' has the amino acid sequence SSSESIS and CDR3' has the amino acid sequence QQSNTWPFT. In another version, the molecule contains both heavy and light chains where the amino acid sequences contain the corresponding CDR. The versions of the corresponding coding polynucleotide are disclosed; expression vector and based on it expression system. The host cell is disclosed basing on the expression system. The application of the molecule in treatment of autoimmune diseases, graft rejection, psoriasis, intestine inflammatory disease and allergy is described. The pharmaceutical composition for the said application is disclosed.
EFFECT: enables immunosuppressant induction; inhibiting T-cell response and primary lymphocyte response in mixed lymphocyte culture (MLC); prolongs survival period in mice with severe combined immunodeficiency SCID.
20 cl, 5 dwg, 2 tbl, 8 ex
FIELD: biotechnology, immunology.
SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.
EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.
26 cl, 16 dwg, 1 tbl, 8 ex
FIELD: biotechnology, genetic engineering.
SUBSTANCE: invention describes recombinant plasmid DNAs constructed in vitro that comprise artificial genes for light and heavy chains of full-scale human antibody prepared by genetic engineering methods. These genes are created on basis of variable fragments of light and heavy chains of recombinant antibody 1F4 and constant human genes IgG1, cytomegalovirus promoter and polyadenylation site BGH. Plasmids provide biosynthesis of recombinant full-scale human antibodies of class IgG1 in mammalian cells. These antibodies interact specifically with smallpox vaccine virus. The affinity constant for prepared recombinant antibodies is 3.54 x 109 ± 0.38 x 109 M-1. Plasmids are used by combined transfection of human cells HEK 293T. Prepared full-scale recombinant antibody against protein of size 27 kDa of smallpox virus vaccine can be used as a base for creature of pharmaceutical preparations used for diagnosis of some post-vaccine complications caused by smallpox virus vaccine. Also, preparations will comprise decreased therapeutic doses of immunoglobulins that will provide minimal undesirable immune response in patients after administration of the preparation.
EFFECT: valuable medicinal properties of plasmid DNA.
4 cl, 7 dwg, 6 ex
FIELD: biotechnology, immunology.
SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.
EFFECT: valuable biological and medicinal properties of antibodies.
53 cl, 4 tbl, 10 dwg, 2 ex
FIELD: chemistry, medicine.
SUBSTANCE: novel antibodies and fragments of human antibodies are bound with GDF-8 in a specific way and inhibit its activity in vitro and/or in vivo. On the basis of said invention pharmaceutical composition is created, which can be used for diagnostics, prevention or treatment of degenerative dysfunctions of muscle or bone or disorders of insulin metabolism.
EFFECT: extending range of arsenal of technical means used in treatment of diseases related to muscular, bone tissue or insulin metabolism.