Human monoclonal antibodies to CTLA-4
19 claims: 10 independent, 9 dependent
- 1Kröfur 1. Mennskt einklóna mótefni sem binst CTLA-4, eða mótefnavaka bindibroti þar af, þar sem þungkeðjan inniheldur amínósýruröðina með raðarnr:9 eða amínósýruröð sem er að minnsta kosti 90% eins og hún og léttkeðjan inniheldur amínósýruröðina með raðarnr: 22 eða amínósýruröð sem er að minnsta kosti 90% eins og hún, þar sem mótefnið eða brotið hefur eftirfarandi eiginleika: (a) bindisækni fyrir CTLA-4 uppá 10' 8 9 M eða meira;(b) hindrar bindingu á milli CTLA-4 og B7-1 með IC 50 uppá 100 nM eða lægra;(c) hindrar bindingu á milli CTLA-4 og B7-2 með IC 50 uppá 100 nM eða lægra;og (d) eykur frumuboðaframleiðslu i greiningu á mennskum T-frumum um 500 pg/mL eða meira.
- 2Mótefnið eða mótefnavaka bindibrotið þar af í samræmi við kröfu 1, þar sem þungkeðju amínósýruröðin inniheldur CDR1, CDR2 og CDR3 amínósýruraðirnar úr mótefni 11.2.1 eins og sýnt er á mynd 2.
- 3Mótefnið eða mótefnavaka bindibrotið þar af í samræmi við kröfu 1 eða 2, þar sem léttkeðju amínósýruröðin inniheldur CDR1, CDR2 og CDR3 amínósýruraðirnar úr mótefni 11.2.1 eins og sýnt er á mynd 5.
- 4Mótefnið eða mótefnavaka bindibrotið þar af í samræmi við hverja sem er af kröfum 1 til 3, þarsem þungkeðjan inniheldur amínósýruröðina úr mótefni 11.2.1 eins og sýnt er á mynd 2.
- 5Mótefnið eða mótefnavaka bindibrotið þar af i samræmi við hverja sem er af kröfum 1 til 4, par sem léttkeðjan inniheldur amínósýruröðina með raðarnr:22.
- 6Mennskt einklóna mótefni sem binst CTLA-4, par sem þungkeðjan inniheldur amínósýruröðina úr mótefni 11.2.1 eins og sýnt er á mynd 2 og léttkeðjan inniheldur amínósýruröðina úr mótefni 11.2.1 eins og sýnt er á mynd 5.
- 7Mennskt einklóna mótefni sem binst CTLA-4, par sem pungkeðjan inniheldur amínósýruröðina með raðarnr:70 og léttkeðjan inniheldur amínósýruröðina með raðarnr: 71.
- 8Mennskt einklóna mótefni sem binst CTLA-4, eða mótefnavaka bindibrot par af, par sem þungkeðjan í fyrrnefndu mótefni inniheldur amínósýruröðina með raðarnr:5 og léttkeðjan í fyrrnefndu mótefni inniheldur amínósýruröðina með raðarnr: 18.
- 9Mennskt einklóna mótefni sem binst CTLA-4, eða mótefnavaka bindibrot þar af, þar sem þungkeðjan ί fyrmefndu mótefni inniheldur amínósýruröðina með raðarnr:10 og léttkeðjan í fyrmefndu mótefni inniheldur amínósýruröðina með raðarnr: 23.
- 10Lyfjasamsetning sem samanstendur af mótefninu eða mótefnavaka bindibroti í samræmi við hverja sem er af kröfum 1 til 9 og lyfjafræðilega haefu burflarefni.
- 11Frumulina sem framleiflir mótefnið eða mótefnavaka bindibrot ί samraemi vifl hverja sem er af krflfum 1 til 9.
- 12Frumulínan úr krflfu 11 sem er spendýrs frumulina.
- 13Frumulinan úr krflfu 12 sem er CHO frumulina efla NSO frumulina.
- 14Kjarnsýra sem kóðar létt- og/efla þungkeðjuna í mótefninu eða mótefnavaka bindibroti eins og skilgreint er i hverri sem er af krflfum 1 til 9.
- 15Lyfjasamsetning sem samanstendur af mfltefninu efla mfltefnavaka bindibroti i samraemi vifl hverja sem er af krflfum 1 til 9 og lyfjafræðilega hasfu burflarefni, til meflhflndlunar á krabbameini.
- 16Aflferfl til framleiflslu á mennska CTLA-4 mfltefninu efla mfltefnavaka bindibroti i samræmi vifl hverja sem er af krflfum 1 til 9 sem felst í því afl tjá fyrmefnt mfltefni efla mfltefnavaka bindibrot í hýsilfrumulínu og endurheimta fyrmefnt mfltefni efla mfltefnavaka bindibrot.
- 17Aflferflin í samræmi vifl krflfu 16, þar sem fyrmefnd hýsilfrumulína er spendýrs frumulina.
- 18Aflferflin i samraemi vifl krflfu 17, þar sem fyrmefnd spendýrs frumulina er CHO frumulina efla NSO frumulina.
- 19Mfltefnifl efla mfltefnavaka bindibrotið i samraemi vifl hverja sem er af krflfum 1 til 9 til notkunar i meflhflndlun á krabbameini.
Independent claims19
586 paragraphs in 39 sections, as filed
Description
BACKGROUND OF THE INVENTION
1. Cross-reference ί related applications
This application requires priority over the U.S. Provisional License Application, Serial No. 60 / 113,647, which was registered on December 23, 1998, and its publication is hereby incorporated in full here.
2. A summary of the invention in accordance with this invention is administered completely human mononucleotide against human cytotoxic T-lymphocytes antigen 4 (CTLA-4). Co-ordinator libraries and amino acid residues containing heavy and light chain immunoglobulin molecules, especially adjacent heavy and light chain chains that complement the CDR, especially from FR1 and / or CDR1 through CDR3 and / or FR4, are provided.
3. Background of Technology
Control of immune response Patients would prefer appropriate treatment for many human diseases that could lead to specific activity that is rare in the use of drug-based drugs. Both boost and response response to the immune system would paint possible. The role of T-cells and B-cells has been studied in detail and identified in conjunction with the control of immune response. Based on these studies, the role of T-cells, in many cases, appears to be of paramount importance in the prevention and management of diseases.
T-cells have very complex systems for the power of their interactions. Interactions between the T-cells are used for the diffusion of fluctuations and soluble factors for the process. The pervasive effect of any particular signal that may have on the immune response is usually variable and depends on certain factors, receptions and benefit receptions that occur throughout the history of the process. The process of power down response is as important as the power of power. Primary education for T-cellup is a single reaction to force preventing immune response to certain metabolites. Dissolving metabolic pathways, such as secretion of suppressive cells, are also disturbed.
Activation of T-cells does not require complete control of the antigenic contamination (T-cell proliferation), but also signals from coagulation of supernatants such as CD28. The links for CD28 are the B7-1 (CD80) and B7-2 (CD86) proteins that express the antigen-inducing cells such as angiotensin, activators B / cells enhancing the integrity of T-cell C28 enhancer CTLA-4 to power deliver a coordinating signal. The role of co-morbidity was studied in experimental allergic cerebrospinal meningitis (EAE) by Perrin et al., Immunol. Res. 14: 189-99 (1995). EAE is an autoimmune disorder, which is induced by Th1 cells directed against a myelinogen molecule that provides a model in the liver to investigate the role of B7-mediated coordination in the development of a pathogenic immune response. Using a soluble fusion protein link for the B7 receptors, and also monoclonal antibodies that are specific to either CD80 or CD86, Perrin et al. suggest that the B7 synergy plays an important role in determining the outcome of clinical disease in EAE.
The interaction between B7 and CD28 is one of several coordination mechanisms that appear to be sufficient to trigger development and proliferation of antigen-specific T-cells. Lack of synergy, and concomitant lack of IL-2 production, prevents subsequent propagation of the T-cells and induces an irreversible condition called "inactivity". A number of viruses and tumors can block activation and proliferation of T-cells, resulting in insufficient activity or immunity in the host's immune system against the infected or transformed cells. Among the number of potential T-cellular disorders, inactivity may be at least partly responsible for the host's deficiency in cleansing the pathogenic or tumorigenic cells.
The use of the B7 protein to mediate anti-tumor resistance has been described in Chen et al., Cell 71: 1093-1102 (1992), and Townsend and Allison, Science 259: 368 (1993). Schwartz, Cell 71: 1065 (1992), takes over the role of CD28, CTLA-4, and B7 IL-2 production and immunotherapy. Harding et al., Nature 356: 607-609 (1994) demonstrate that CD28 mediated signals co-interact with T cells in mice and prevent the induction of inactivity in T cell clones. See also U.S. Pat. 5,434,131, 5,770,197, and 5,773,253, and international patent applications no. WO 93/00431, WO 95/01994, WO 95/03408, WO 95/24217, and WO 95/33770.
From the foregoing, it was clear that T cells need two types of antigens-inducing cell (APC) signals for activation and subsequent response responses. First, antigenspecific signals are induced by interaction between TCR on T cell and MHC molecular peptide on APC. Then, the antibody-dependent signal that is mediated by the interaction on CD28 with members of the B7 family (B7-1 (CD80) or B7-2 (CD86)). Exactly where CTLA-4 fits into the immune response environment was initially undecided. The CTLA-4 from mice was first identified and cloned by Brunet et al., Nature 328: 267270 (1987), as part of the search for molecules that are expressed in cytotoxic T lymphocytes. Human CTLA-4 was identified and cloned shortly by Dariavach et al., Eur. J. Immunol. 18: 1901-1905 (1988). Mice and human CTLA-4 molecules have approximately 76% of total serotoninity and approach to absolute sequence identity in their peripheral regions (Dariavach et al., Eur. J. Immunol. 18: 1901-1905 (1988)). CTLA-4 is a member of the immunoglobulin (Ig) transfection of proteins. Ig transfection is a group of the protein that shares key factors in either variable (V) or fixed (C) regions of Ig molecules. The Ig Ig members include, but are not limited to, the immunoglobulin itself, the major classes of fibrous molecules (MHCs) (ie, MHCs of Classes I and II), and TCR molecules. Ig transfection is a group of the protein that shares key factors in either variable (V) or fixed (C) regions of Ig molecules. The Ig Ig members include, but are not limited to, the immunoglobulin itself, the major classes of fibrous molecules (MHCs) (ie, MHCs of Classes I and II), and TCR molecules. Ig transfection is a group of the protein that shares key factors in either variable (V) or fixed (C) regions of Ig molecules. The Ig Ig members include, but are not limited to, the immunoglobulin itself, the major classes of fibrous molecules (MHCs) (ie, MHCs of Classes I and II), and TCR molecules.
In 1991, Linsley et al., J. Exp. Med. 174: 561-569 (1991) suggests that CTLA-4 was another receptor for B7. In a similar manner, Harper et al., J. Immunol. 147: 1037-44 (1991), suggest that the CTLA-4 and CD28 molecules are closely related in both mice and humans in terms of sequence, expression, gene structure, and color placement. See also Balzano et al., Int. J. Cancer Suppl. 7: 28-32 (1992). Further evidence of this role came up with functional research. For example, Lenschow et al., Science 257: 789-792 (1992) demonstrated that CTLA-4-Ig induced long-term survival of pancreatic transplantation. Freeman et al., Science 262: 907-909 (1993), investigated the role of CTLA-4 ί B7 impaired mice with inadequate B7. A study of the links for CTLA-4 is described in Lenschow et al., PNAS 90: 11054-11058 (1993). Linsley et al., Science 257: 792-795 (1992), describe immunosuppressive organism with soluble form of CTLA-4. Linsley et al., J. Exp. Med. 176: 1595-604 (1992), immunoglobulin-derived antibodies immunized with CTLA-4 and non-cross-reactive to CD28, concluded that CTLA-4 was associated with CD28 on activated T-lymphocytes and co-administration of T-cell adhesion and activation with B7. Kuchroo et al., Cell 80: 707-18 (1995), demonstrated that the B7-1 and B7-2 coagulation molecules activated the Th1 / Th2 development process in different ways. Yi-qun et al., Int. Immunol. 8: 37-44 (1996), demonstrated that there are different requirements for the B7 family members' mergers with a rest against newly activated memory T cells for soluble recall antigens. See also de Boer et al., Eur. J.Immunol. 23: 3120-5 (1993). extracted antibodies immunized with CTLA-4 and non-cross-reactive to CD28, concluding that CTLA-4 was associated with CD28 on activated T-lymphocytes and co-administration of T-cell adhesion and activation with B7. Kuchroo et al., Cell 80: 707-18 (1995), demonstrated that the B7-1 and B7-2 coagulation molecules activated the Th1 / Th2 development process in different ways. Yi-qun et al., Int. Immunol. 8: 37-44 (1996), demonstrated that there are different requirements for the B7 family members' mergers with a rest against newly activated memory T cells for soluble recall antigens. See also de Boer et al., Eur. J.Immunol. 23: 3120-5 (1993). extracted antibodies immunized with CTLA-4 and non-cross-reactive to CD28, concluding that CTLA-4 was associated with CD28 on activated T-lymphocytes and co-administration of T-cell adhesion and activation with B7. Kuchroo et al., Cell 80: 707-18 (1995), demonstrated that the B7-1 and B7-2 coagulation molecules activated the Th1 / Th2 development process in different ways. Yi-qun et al., Int. Immunol. 8: 37-44 (1996), demonstrated that there are different requirements for the B7 family members' mergers with a rest against newly activated memory T cells for soluble recall antigens. See also de Boer et al., Eur. J.Immunol. 23: 3120-5 (1993). demonstrated that the B7-1 and B7-2 synergy molecules activate the Th1 / Th2 development process in different ways. Yi-qun et al., Int. Immunol. 8: 37-44 (1996), demonstrated that there are different requirements for the B7 family members' mergers with a rest against newly activated memory T cells for soluble recall antigens. See also de Boer et al., Eur. J.Immunol. 23: 3120-5 (1993). demonstrated that the B7-1 and B7-2 synergy molecules activate the Th1 / Th2 development process in different ways. Yi-qun et al., Int. Immunol. 8: 37-44 (1996), demonstrated that there are different requirements for the B7 family members' mergers with a rest against newly activated memory T cells for soluble recall antigens. See also de Boer et al., Eur. J.Immunol. 23: 3120-5 (1993).
A variety of groups made different or different from weAdverse / Interactions Interactions for CTLA-4 compared to CD28 and even added a third B7 complex identified by BB1 antibodies. See, for example, Hathcock et al., Science 262: 905-7 (1993), Freeman et al., Science 262: 907-9 (1993), Freeman et al., J. Exp. Med. 178: 2185-92 (1993), Lenschowo.fi., Proc. Natl. Acad. Sci. USA 90: 11054-8 (1993), Razi-Wolf et al., Proc. Natl. Acad. Sci. USA 90: 11182-6 (1993), and Boussiotis et al., Proc. Natl. Acad. Sci. USA 90: 11059-63 (1993). But, see, Freeman et al., J. Immunol. 161: 2708-15 (1998), which finds that BB1 antibodies binds to a molecule similar to CD74 cellular surface form and, therefore, binds BB1 mAb to a protein that differs from B7-1, and this subject is also present on the B7-1 protein.
Beginning in 1993 and peaked in 1995, researchers began to further enhance the role of CTLA-4 in T-cell stimulation. First, with the use of monoclonal antibodies to CTLA-4, Walunas et al., Immunity 1: 405-13 (1994), proved that CTLA-4 may act as a negative control for T-cell activation. After that, Waterhouse et al., Science 270: 985-988 (1995) demonstrated that mice with CTLA-4 lacked T-cell cells with elevated activation marks in their lymph nodes and spleen. The lymphocytes also entered the liver, heart, lung and pancreatic tissue, and the serum immunoglobulin serum increased and their T-cells proliferated immediately and extensively when stimulated by the T-cell receptor, however, they were susceptible to cell death was induced by the cross-coupling of the Fas receptor and by gamma radiation. Waterhouse et al. concluded that CTLA-4 acts as a negative control for T-cell activation and is important for the management of lymphocytes. In a review in the same edition, Allison and Krummel discussed Science 270: 932-933 (1995), Waterhouse, et al. as evidence that CTLA-4 acts to reverse T cell response or has a barrier marker in T-cell activation and development. Tivol et al., Immunity 3: 541-7 (1995), also produced mice with CTLA-4 deficiency and demonstrated that such mice rapidly developed lymphocyte proliferative disease by multilateral lymphocytic and tissue destruction, with particularly severe myocarditis and pancreatitis. They concluded that CTLA-4 played a key role in downward T-cell activation and maintain immune metabolism. Also, Kummel and Allison, J. Exp. Med. 182: 459-65 (1995), further disclose that CD28 and CTLA-4 have a similar effect on T cell responsiveness upon stimulation. They produced antibodies to CTLA-4 and investigated the effect of its binding to CTLA-4 in systems that utilized very pure T-cells. In their report, they showed that the presence of a small amount of B7-2 on new T-cells may partially inhibit T cell proliferation, and this barrier was mediated by CTLA-4 interaction. Cross-linking of CTLA-4 together with TCR and CD28 inhibits strong propagation and IL-2 secretion of T cells. Finally, the results showed that CD28 and CTLA-4 yield reverse signs that appear to be aggregated by the T cell in antigen response determination. Thus, they concluded that the outcome of T-cell antigen response is controlled by CD28 promoters, and also CTLA-4 barriers. See also Kummel et al., Int. Immunol. 8: 519-23 (1996), and U.S. Pat. 5,811,097, and International patent application no. WO 97/20574.
Various other attempts have been made that shed light on the efficacy of CTLA-4 above. For example, Walunas et al., J. Exp. Med. 183: 2541-50 (1996), using the anti-CTLA-4 antibody, suggested that CTLA-4 signals do not control cell counts or response to IL-2, but inhibit CD28-dependent IL-2 proliferation. Also, Perrin et al., J. Immunol. 157: 1333-6 (1996), demonstrated the effect of anti-CTLA-4 antibodies in experimental allergic cerebrovascular disease (EAE), eluding the disease and increasing mortality. Disease stimulation was associated with increased proliferation of hepatotoxic cytoplasmic TNF-alpha, IFN-gamma and lt-2. Pannig concluded that CTLA-4 manages the extent of the self-esteem response, EASE, and reduces the proliferation of inflammatory cells and signs of clinical disease. See also Hurwitz et al., J. Neuroimmunol. 73: 57-62 (1997), and Cepero et al., J. Exp. Med. 188:
Power also includes Blair et al., J. Immunol. 160: 12-5 (1998), efficacy (oily CTLA-4 monoclonal antibodies (mAbs) on human CD4 + T cells in vivo. Their nephropathy showed a power of some CTLA-4 mAbs to inhibit CD4 + proliferation response in white and cytochrome change from GO in G1. The inhibitory effect of CTLA-4 was within 4 hours at a time when the cellular surface of CTLA-4 expression was indistinguishable. Other CTLA-4 mAbs, on the other hand, had no detectable inhibitory effect, indicating force binding on mAbs vifl CTLA-4 alone was insufficient to transmit downtime on the T-cell responses. It was interesting that while IL-2 production was closed, anti-CTLA-4 mAbs allowed the expression and expression of the cell cation gene bcl-X (L) . According to this observation, cells remained viable and controlled cell death was not diagnosed by CTLA-4 connection.
In association with inactivity, Perez et al., Immunity 6: 411-7 (1997) demonstrated that the induction of T-cell inactivation was inhibited by blocking CTLA-4 and withdrew the conclusion that antigen detection of T-cells are determined by the interaction of CD28 or CTLA-4 on the T-cells with B7 molecules. Also, Van Parijs et al., J. Exp. Med. 186: 1119-28 (1997), investigated the role of leukocyte 12 and co-existed in T-cell inactivity in the liver and found that by inhibiting CTLA-4 binding to inhibition of inactivity, T cell proliferation was blocked and not fully supported Th1 separation. However, T-cells that came into contact with antiviral antibodies in the presence of both IL-12 and anti-CTLA-4 antibodies were not inactivated, and acted like T-cells that have come into contact with immunogenic antigen. These findings indicated that two processes contribute to the induction of inactivity in the liver: CTLA-4 binding, which inhibits the blockage of T-cell proliferation, and the absence of the primary inflammatory cell line, IL-12, which prevents the separation of T cells in Th1 activation cells. The composition of IL-12 and anti-CTLA-4 antibodies was sufficient to change normal antidiabetic stimulation to immunogenic stimulation.
In connection with infections, McCoy et al., J. Exp. Med. 186: 183-7 (1997) demonstrated that anti-CTLA-4 antibodies boosted a rapid and rapid T-cell immune response to Nippostrongylus brasiliensis, which leads to a major decrease in the number of adult worms and early onset of proliferation of parasites. See also Murphy et al., J. Immunol. 161: 41534160 (1998) (Leishmania Donovani).
In conjunction with vascular cancer, Kwon et al., PNAS USA 94: 8099-103 (1997) found a similar prostate cancer model, and investigated two different transfusions designed to induce prostate cancer response with increased T-cell coagulation: (i) the condition of bone coagulation of prostate cancer cells that were altered to express the B7-1 link and (ii) in the liver antigen-mediated blockade of T-cell CTLA-4, which prevents T-cell downturn. Patients in the liver were shown antibody-mediated blockade of T-cell CTLA-4 increased prostate cancer response. Also, Yang et al., Cancer Res. 57: 4036-41 (1997), investigated whether the blockade of CTLA-4 activity led to enhancement of tumorigenic T-cell response at different levels of tumor growth. Based on the experimental and survival results, they obtained the CTLA-4 blockade in tumor-induced tumors, inducing tumorigenic T-cell response, but the expression of such potentiating effects was limited to the early stage of tumor growth in their model. Furthermore, investigate Hurwitz et al., Proc. Natl. Acad. Sci. USA 95: 10067-71 (1998), the induction of T cell proliferative tumorigenic response dependent on T-cell receptor binding of a large fibrosisable complex / antigen and also a CD28 connection of B7. Certain tumors, such as the SM1 breast cancer, were resistant to anti-CTLA-4 immune therapy. Thus, with the use of a combination of CTLA-4 blockade and a vaccine consisting of SM1 cells expressing coronary-lymphocyte coagulation activation factor, parental SM1 tumor response was observed, despite the inaction of each treatment alone. This combination treatment resulted in prolonged resistance to SM1 and was dependent on both CD4 (+) and CD8 (+) T cells. The findings indicated that CTLA-4 blockade acts at the level of host-derived antigen-generating cell.
In conjunction with diabetes, Luhder et al. J. Exp. Med. 187: 427-32 (1998), anti-CTLA-4 mAb in TCR-genetically modified mouse model of diabetes at different disease levels. They found that binding of CTLA-4 at the point when potentially diabetic T-cells are first activated is an important event; If binding is allowed, invasion occurs in the islands, but is rather harmless in a month. If not, erythema is much more difficult, and diabetes is accompanied quickly.
In connection with vaccine immunization, Horspool et al., J. Immunol. 160: 270614 (1998), that whole anti-CTLA-4 mAb but non Fab fractures bore the primary respiratory response to pCIA / beta gal without affecting reuptake indicating that CTLA-4 activation inhibited Ab production than not T-cell contamination. Blocking of the links for CD28 and CTLA-4, CD80 (B7-1) and CD86 (B7-2), revealed distinct and desired activity. Blocking of CD80 with initial immune system completely deleted the primary and increased Ab responses, while blocking CD86 inhibited primary but no additional responses. Concomitant blockade of CD80 + CD86 was less effective in suppressing the AB responses than either alone. Enhancement of coagulation by co-injection of plasmids expressing B7 enhanced CTL response but not Ab response, and without indicating Th1 in Th2 interrogation. These discoveries indicate a complex and distinctive role for CD28,
(in conjunction with the rejection of allogeneic transplantation, Markees et al., J. Clin. Invest. 101: 2446-55 (1998), in a mouse model of dermal transplantation, approved originally from the presence of IFN-gamma, CTLA-4, and CD4 (+ ) T-cells. The addition of anti-CTLA-4 or anti-IFN-gamma mAb to the protocol was associated with instantaneous transplant rejection, while the and-IL-4 mAb had no effect.
in connection with the role of CTLA-4 in connection with CD28 produced Fallarino et al., J. Exp. Med. 188: 205-10 (1998), TCR-genetically modified / rearranged activation gene 2-impaired / CD28-wild or CD28-impaired mice immunized with antigen expressing an antigen. Sensitive T-cells from both types of mice produced cellular expression and propagated as response to stimulant cells lacking B7 expression. However, while the response to CD28 + / + T cells increased with B7-1 synergy, the response to CD28 - / - T cells was significantly inhibited. This barrier was reversed by monoclonal antibodies to B7-1 or CTLA-4. Thus, CTLA-4 can effectively inhibit T-cell activation in the absence of CD28, indicating that the antagonist of TCR mediated marker is sufficient to explain the inhibitory effect of CTLA-4. Also investigate Lin et al., Exp. Med. 188: 199-204 (1998), rejection of cardiac transplant in CD28-impaired mice. H-2 (q) hearts were digested in allogeneic wild or CD28-damaged mice (H-2 (b)). transplant rejection was delayed in CD28-reduced mice relative to wild-type mice. Treatment of wild-type CTLA-4 immunoglobulin (Ig), or with anti-B7-1 plus anti-B7-2 mAbs, significantly prolonged transplant proliferation. In contrast, treatment of CD28-reduced mice induced CTLA-4-Ig, anti-B7-1 plus anti-B7-2 mAbs, or blocking anti-CTLA-4 mAb acceleration of transplantation. This increased rate of transplant rejection was associated with severe cell monovalent cell proliferation and increased levels of IFN-gamma and IL-6 transcriptions in non-treated wild-type CTLA-4-Ig or anti-CTLA-4 mAb-treated CD28-impaired mice. Thus, the negative management role of CTLA-4 exceeds its potential capability to prevent CD28 activation by link competition.
Also, further identification of the expression on CTLA-4 has been investigated. For example, Alegre et al., J. Immunol. 157: 4762-70 (1996), so that the surface of CTLA-4 is inverted rapidly, which can explain a small amount of expression that is commonly detected on the cell surface. They concluded that both CD2g and IL-2 play an important role in boosting CTLA-4 expression. In addition, cellular surface contraction appeared to be the CTLA-4 predominantly controlled by its rapid penetration. Also, Castan et al., Immunology 90: 265-71 (1997), based on immunosuppressive analysis of CTLA-4 expression on site, suggested that CT cells positive CT cells could be important for immunosuppression .
Accordingly, in view of the wide and important role of CTLA-4 in the immune response, it would be desirable to produce CTLA-4 antibodies that can be effectively used in immunotherapy. Furthermore, it would be desirable to produce antibodies to CTLA-4 that can be used in persistent diseases, as there is a need for repeated donors of the antibodies.
A brief description of the picture
Figure 1 represents a class of nucleic acids and amino acids of heavy chain and capillary light chain immunoglobulin molecules 4.1.1 (Figure 1A), 4.8.1 (Figure 1B), 4.14.3 (Figure 1C), 6.1.1 (Figure 1D), 3.1.1 Figure 1D), 4.10.2 (Figure 1F), 2.1.3 (Figure 1G), 4.13.1 (Figure 1H), 11.2.1 (Figure 11 In accordance with the invention), 11.6.1 (Figure 1J), In combination to the invention 11.7.1 (Figure 1K), 12.3.1.1 (Figure 1L), and 12.9.1.1 (Figure 1M).
Figure 2 gives a serial comparison between the predicted heavy chains of amino acid residues from clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1. ,
12.3.1.1, and 12.9.1.1 and the kimline DP-50 (3-33) amino acid residue. Variability between the DP-50 acne line and the sequence in the clones is indicated by boldness. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the shaded antibodies.
Figure 3 gives a serial comparison between the predicted heavy chain amino acid sequence from clone 2.1.3 and the chimeric DP-65 (4-31) amino acid residue. The variability between the DP-65 and the sequence in the clone is indicated by boldness. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 4 gives a serial comparison between the predicted capillaries of the light chain amino acid residue from clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 4.10.2, and 4.13.1, and the cytokine A27 amino acid residue. The variability between the A27 cell line and the clone sequence is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined. Gaps seen in CDR1 in the claw
4.8.1.4.14.3, and 6.1.1 are indicated by "0".
Figure 5 gives a serial comparison between the predicted capillaries of the light chain amino acid site from clones 3.1.1, 11.2.1, 11.6.1, and 11.7.1 and the germline 012 amino acid residue. The variability between 012 kimline lines and the sequence in the clone is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 6 gives a serial comparison between the predicted capillaries of the light chain amino acid sequence from clone 2.1.3 and the line A10 / A26 amino acid sequence. The variability between the A10 / A26 acne line and the clone sequence is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 7 gives a serial comparison between the predicted capillaries of the light chain amino acid sequence from clone 12.3.1 and the line A17 amino acid residue. The variability between the A17 cytomegal line and the sequence in the clone is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 8 gives a sequencing between the predicted capillaries of the light chain amino acid sequence from clone 12.9.1 and the line A3 / A19 amino acid residue. The variability between the A3 / A19 cytokine line and the clone sequence is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 9 summarizes N-terminal amino acids sites produced by direct protein sequencing on the heavy and light chains of the antibodies.
Figure 10 provides certain additional identifiable information about certain antibodies. Figure 10A summarizes data related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2,
4.14.3 and 6.1.1. Data-related strengths, electrophoresis (IEF), SDS-PAGE, size-selective chromatography, liquid analysis / mass analysis (LCMS), mass analysis (MALDI), light chain N-sequence. Additional detailed information related to IEF is given on Figure 10B; related to SDS-PAGE are given at 10C; and the SEC on the 4.1.1 antibody line at 10D.
Figure 11 shows the expression of B7-1 pg B7-2 on Rajifrumum using anti-CD80-PE and anti-CD86-PE mAbs.
Figure 12 shows the potentiogenic enhancement of IL-2 residues in the T-lymphoma / Raji assay induced by anti-CTLA-4 blocking antibodies (BNI3, 4.1.1,
4.8.1, and 6.1.1).
Figure 13 shows the potent enhancement of IFN-γ survival in the T-lymphoma / Raji assay induced by anti-CTLA-4 blocking antibodies (BNI3, 4.1.1,
4.8.1, and 6.1.1) (same T-cell donor).
Figure 14 shows the mefial enhancement of IL-2 survival in T cells from 6 donors induced by anti-CTLA-4 blocking antibodies in the Τ-lymphoma / Raji assay.
Figure 15 shows the mefial enhancement of IFN-γ survival I T cells from 6 donors induced by anti-CTLA-4 blocking antibodies in the T-cell / Raji assay.
Figure 16 shows the enhancement of IL-2 residues I hPBMC from 5 donors induced by anti-CTLA-4 blocking mAbs as measured 72 hours after SEA activation.
Figure 17 shows the improvement of IL-2 survival in whole blood from 3 donors induced by anti-CTLA-4 blocking mAbs as measured 72 and 96 hours post SEA activation.
Figure 18 shows the barrier of tumor growth with anti-mouse CTLA-4 antibodies in the mouse fibrosis tumor model.
Figure 19 shows the enhancement of IL-2 proliferation induced by anti-CTLA-4 antibodies (4.1.1 and 11.2.1) 72 hours of T-lymphoma / Raji and supernatants (whole blood and peripheral blood monocytes from the 6 donors) assays.
Figure 20 shows dose-dependent enhancement of IL-2 proliferation induced by anti-CTLA-4 antibodies (4.1.1 and 11.2.1) 72 hours of Τ-tumor cell / Raji analysis.
Figure 21 shows dose-dependent enhancement of IL-2 proliferation induced by anti-CTLA-4 antibodies (4.1.1 and 11.2.1) 72 hours of supernatant whole blood analysis stimulating 100 ng / mL of supernatants.
Figure 22 provides a class of additional nucleic acids and amino acids from the following anti-CTLA-4 antibody chains: full length 4.1.1 heavy chain (cDNA 22 (a), gene 22 (b), and amino acid 22 (c)), full-length non-glycosylated 4.1. 1 heavy chain (cDNA 22 (d) and amino acid 22 (e)), 4.1.1 light chain (cDNA 22 (f) and amino acid 22 (g)), full length 4.8.1 heavy chain (cDNA 22 (h) and amino acid 22 4.8.1 light chain (cDNA 22 (j) and amino acid 22 (k)), full length 6.1.1 heavy chain (cDNA 22 (1) and amino acid 22 (m)), 6.1.1 light chain (cDNA 22 n) and amino acid 22 (o)), full length 11.2.1 heavy chain (cDNA 22 (p) and amino acid 22 (q)), and 11.2.1 light chain (cDNA 22 (r) and amino acid 22 (s)).
SUMMARY OF THE INVENTION In accordance with this invention, an antibody capable of binding CTLA-4, which is further preferred in the claims, is given.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT according to this invention, is administered completely human mononucleotide against human CTLA-4. Coding for nucleic acids and amino acid residues containing heavy and light chain immunoglobulin molecules, especially rows corresponding to synoptic heavy and light chained from FR1 and CDR1 through CDR3 and FR4. Protein cells expressing immunoglobulin monovalent monoclonal antibodies are also given.
Definitions
Unless otherwise defined herein, the scientific and technical names used in the present invention have the meanings usually understood by those skilled in the art. Furthermore, unless otherwise required by context, the name of the singular must include the plural and the name of the plural to include the singular. Generally, scholarly names are used in relation to, and methods of, cellular and tissue culture, molecular biology, and protein and oligonucleotide chemistry, and factor relationships described herein in those well-known and commonly used in the art. Standard methods are used for rearranged DNA, oligonucleotides, and tissue culture and conversion (eg, electrogenation, liposome induction). Enzymes and cleaning methods are carried out according to the manufacturer's description or as commonly done in the art or as described in the above. The aforementioned methods and processes are usually performed according to conventional methods well known in the art and as described in various general and specific references referred to and discussed in this specification, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference. The scientific names used in conjunction with the experimental and processes for chemical analysis, organic synthesis, and physiological and pharmacological chemistry described herein are those well known and commonly used in the art. Staflafflar proliferation drugs are metabolites for chemical metabolism, metabolism, drug proliferation, assembly, and dialysis, and metabolism in patients.
As used in accordance with this publication of power, the following terms, unless otherwise stated, have the following meaning:
Heitifl "isolation of polynucleotide" as used herein refers to the gene complex of genetic engineering, cDNA, enhancing synthetic origin enhances any combination thereof, because of its origin, the "isolated polynucleotide" (1) is not associated with the further enhancing part of (3) is not naturally occurring as part of a larger population. (2) An integral part of nature, which is an integral part of nature.
Heitifl "isolating protein" referred to in the majority of proteins of cDNA, recombinant RNA, enhancing synthetic origin enhances any pair of pairs, pairs due to their origin, enhancing derivative origin, "isolated protein" (1) is not related to the detected species in nature, (2) free of other proteins of the same origin, eg free of mouse protein, (3) expressed by cell of another species, or (4) not occurring in nature.
The term "polypeptide" as used herein generally refers to the original protein, fractures, or analogues of the polypeptide sequence. Therefore, the original proteins, fragments, and analogous species of the polypeptide sequence. Preferred polypeptides according to the invention contain human heavy chain immunoglobulin molecules and human capillaries of the light chain immunoglobulin molecules depicted in Figure 1, as well as antibody molecules formed by compositions consisting of the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the light chain immunoglobulin molecules, and vice versa, as well as fragments and analogues thereof.
The term "occurs in nature," as used herein, refers to the fact that a phenomenon can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including such viruses) that can be isolated from a source in nature and which have not been intentionally changed by a person in a laboratory or otherwise occur in nature.
The term "effective link<sup>11</sup> as used herein, shows the status of elements that are described as being linked to allow them to function as expected. A reference sequence that is "operably linked" to the coding sequence is linked so that expression on the coding sequence is obtained under conditions that are compatible with the reference sequence.
The term "reference order<sup>11</sup> as used herein, refers to the polynomials necessary to execute the expression and the processing of the codecs to which they are related. The nature of such reference rates varies depending on the host species; Thus, in the distribution nuclei, reference ranks usually include a control region, ribosome binding site, and transcriptional arrest sequence; In general terms, in general, thus contain the control rows and the transcriptional sequence. The term "sequencing order" is intended to cover, at least, all aspects of each presence necessary for expression and processing, and can also cover other aspects of each presence, such as prerogatives and mergers.
The term "polynucleotide" as referred to herein means a polymeric form of nuclei of at least 10 bases in length, either ribosaccharide or deoxygen or altered form of any type of nucleus. The term encompasses single and double-form DNA.
The term "oligonucleotides", as referred to herein, refers to the nuclei occurring in nature, and altered nuclei that are linked by oligonucleotides that occur in nature and which do not occur in nature. Fibers are the subset of polynomials generally in length of 200 bases or less. Preferably the oligonucleotides are 10 to 60 bases in length and preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. The oligonucleotides are usually single-stranded , for example, for detectors, although the oligonucleotides can be two-fold, for example, for use in the structure of a mutant gene. The oligonucleotides can be either single or multiple oligonucleotides.
The term "naturally occurring nuclei" referred to herein refers to deoxyribosaccharide and ribosaccharide. The term "modified nuclei" referred to herein extends to the nucleus with substituted or substituted sugar groups and the like. The term "oligonucleotide link" referred to herein refers to oligonucleotides such as phosphorothioate, phosphorodithioate, phosphoroselateate, phosphorodisoleate, phosphoranothioate, phosphoraniladate, phosphoramidate, and the like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14: 9081 ( 1986); Stec et al., J. Am. Chem. Soc. 106: 6077 (1984); Stein et al., Nucl. Acids Res. 16: 3209 (1988); Zon et al., Anti- Cancer Drug Design 6: 539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, bis. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990), which disclosures are hereby incorporated by reference. Fibers can contain an analytical marker if desired.
For example, 85% homogeneity at 85% of the amino acids is identical when the two pairs are set for maximum match. Harvesting (in one of the two comparable two pairs) is allowed in the matching of matching; The shorter lengths of these are the preferred pairs of which 2 are less preferred. Preferably, two protein radicals (such as polypeptides derived from peaks of at least 30 amino acids in length) are homogeneous, as this term is used herein, if pairs have comparative scores of more than 5 (in standard units) pairs of The note is a program ALIGN with mutations of the data format and a cache of 6 or higher. See Dayhoff, MO, in the Atlas of Protein Sequence and Structure, bis. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Appendix 2 at this volume, bis. 1-10. The rows of two pairs of pairs are fairly homogeneous if the amino acids of peerra are more than equal to 50% as the peaks are optimized using ALIGN application time. Heitid "corresponds to" is used here for the translation of multicolor root as homogenous (peas, if not only prerally linked), all of the parts of the polymorphic multicolored red, as in polynucleotide redone, is like vidmidunar polynuclear root. However, the term "mdtsvarandi" by "referring here to the puzzle of the corresponding red, are homogeneous all parts of the vidmidun polynucleotide sequence. For clarification, the kernel root "TATAC" responds to the "TATAC" radar screen, and is the corresponding reference root "GTATA". Heitid "corresponds to" is used here for the translation of multicolor root as homogenous (peas, if not only prerally linked), all of the parts of the polymorphic multicolored red, as in polynucleotide redone, is like vidmidunar polynuclear root. However, the term "mdtsvarandi" by "referring here to the puzzle of the corresponding red, are homogeneous all parts of the vidmidun polynucleotide sequence. For clarification, the kernel root "TATAC" responds to the "TATAC" radar screen, and is the corresponding reference root "GTATA". Heitid "corresponds to" is used here for the translation of multicolor root as homogenous (peas, if not only prerally linked), all of the parts of the polymorphic multicolored red, as in polynucleotide redone, is like vidmidunar polynuclear root. However, the term "mdtsvarandi" by "referring here to the puzzle of the corresponding red, are homogeneous all parts of the vidmidun polynucleotide sequence. For clarification, the kernel root "TATAC" responds to the "TATAC" radar screen, and is the corresponding reference root "GTATA". Note here to the puzzle of the corresponding red is homogenous of all parts of the vidmidun polynucleotide sequence. For clarification, the kernel root "TATAC" responds to the "TATAC" radar screen, and is the corresponding reference root "GTATA". Note here to the puzzle of the corresponding red is homogenous of all parts of the vidmidun polynucleotide sequence. For clarification, the kernel root "TATAC" responds to the "TATAC" radar screen, and is the corresponding reference root "GTATA".
The following names are used to describe the radical compounds between the two more polycyclic amino acids: "vidmidunarröd", "comparator window", "radarsamsemd", "percentage of radarsamism", and "substantive synonym." "Reference sequence" is defined as a rod note as a basis for racial dialogue; epithelial redness may be a subset of a staerri rod, for example, as part of full-length cDNA gene sequences administered in sequential order or may contain a whole cDNA or gene sequence. Generally, the reference sequence is at least 18 nuclei or 6 amino acids in length, often at least 24 nuclei or 8 amino acids in length, and often at least 48 nuclei or 16 amino acids in length. Since two polynucleotide or amino acid residues can each (1) contain a sequence (i.e., part of the whole polynucleotide or amino acid sequence) which is similar between the two molecules, and (2) may further contain a sequence different between the polynucleotides of the amino acid residues; a series of comparisons between two (more) molecules is done by comparing the two of the two molecules into a "comparing window" for the purpose of comparing the local area of series. "Comparison window", as used by the user, refers to the conceptual part have at least 18 contiguous nucleotide positions of 6 amino acids in which the polynucleotide sequence of the amino acid sequence can be compared to a reference sequence of at least 18 contiguous nuclei of 6 amino acid residues and wherein the portion of the polynucleotide sequence in the comparator can contain additions, deletions, substitutions, and similar (i.e., hatch) of 20 percent efia less compared to the reference sequence (which does not include additions or deletions) for the best comparison of the two pairs. The best compilation of ranks to compare comparison windows can verify execution by local uniforms from Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), by homogeneous comparative algorithm from Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), using the probability method from Person and Lipman, Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), with computer games on these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, version 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector software packages), and the best compilation (ie,
The term "sequences" means two polynucleotide amino acid residues are identical (ie, based on the nucleotide-to-nucleotide residual residues) in the compilation window. The term "percentage of electromagnetic compatibility" is calculated by comparison with the best comparison on the two-row comparison window, determine the number of positions where the same nucleic acid base (eg, A, T, C, G, U, efia I) efia residues occurs in both rows to give a number of consolidated positions, dividing the masses of consolidated positions with the total number of positions in the compilation window (ie, the window size), and multiply the result by 100 to give the percentage of electromagnetic compatibility. Heitifi "substantive togetherness" as used herein denotes the symptoms of polycystic or amino acid-derived, wherein the polynucleotide or amino acid contains a sequence having at least 85 percent electromagnetic compatibility, preferably having at least 90 to 95 percent electromagnetic compatibility, more common to at least 99 percent electromagnetic compatibility, compared to the reference sequence in the comparator window at least 18 nucleic acid (6 amino acid) positions, often in a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of electromagnetic compatibility is calculated by comparing the reference sequence to the order that can be decomposed or add-ons that are a total of 20 percent or less of the control order in the comparator window. The reference sequence may be a subset of a larger order. compared to the control sequence in the comparator window of at least 18 nucleic acid (6 amino acid) positions, often in a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of electromagnetic compatibility is calculated by compare the row of orders to the row that may contain declines or additions that are a total of 20 percent or less of the reference row in the comparator window. The reference sequence may be a subset of a larger order. compared to the control sequence in the comparator window of at least 18 nucleic acid (6 amino acid) positions, often in a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of electromagnetic compatibility is calculated by compare the row of orders to the row that may contain declines or additions that are a total of 20 percent or less of the reference row in the comparator window. The reference sequence may be a subset of a larger order.
As used herein, the other twenty conventional amino acids follow the abbreviations of conventional use. See: Immunology - A Synthesis (2nd Edition, ES Golub and DR Gren, Editor, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Enantiomers (e.g., D-amino acids) of the twenty conventional amino acids, non-natural amino acids such as α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids may also be suitable components for the polypeptide of this invention. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-Ν, Ν, Ν-trimethyllysine, ε-Δ-acetyllysine, O-phosphoserine, N-acetylsiline, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine , σ-Ν-methylarginine, and other similar amino acids and imino acids (for example, 4-hydroxyproline).
Similarly, unless stated otherwise, the left end of single-stranded polynomial ranks is 5 'end; reference is made to the left of the twisted polynomial series as the 5 'direction. Reference is made to the 5 'to 3' addition of RNA transcripts in images as the transcriptional pointers; the DNA sequences of RNA and 5 'in the 5' end of the RNA transcript are referred to as "upper rows", I refer to the sequences of the DNA sequence that are the same as RNA and which are 3 'in 3 'end of the RNA transcript as "lower rows<sup>11</sup>.
As used for polypeptide, the term "substantive cohesion" means that two peptide rhythms, when compared best, like the GAP or BESTFIT applications using automatic bulkheads, share at least 80 percent of the sequences, preferably that it is at least 90 percent of the series, more preferably at least 95 percent of the series, and preferably at least 99 percent of the series. Preferably, the difference in residual positions that are not the same is the persistence of amino acid shifts. Heldin amino acid shifts show for interchangeability residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; A group of amino acids having amine-containing side chains are asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred amino acid replacement groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartin, and asparagine-glutamine. and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred amino acid replacement groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartin, and asparagine-glutamine. and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred amino acid replacement groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartin, and asparagine-glutamine.
As discussed herein, it is contemplated that minor changes in the amino acid sequences of antibodies or immunoglobulin molecules are included in this invention, provided that the changes in the amino acid sequence maintain at least 90%. Particular attention is given to the hardened amino acid shifts. Heldin exchange is the ones that occur within the family of amino acids that are related to their side chains. Genocoded amino acids are generally divided into families: (1) acid = aspartate, glutamate; (2) alkaline = lysine, arginine, histidine; (3) nonchalated = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) unlabelled polygons = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Preferred families are: serine and threonine are aliphatic-hydroxy polyacrylamide; asparagine and glutamine are an amid-containing family; alanine, valine, leucine, and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is realistic to expect isolated isolation of leucine with isoleucine or valine, glutamate aspartate, serine serine, or similar to amino acid with structural amino acid will not significantly affect the binding or properties of the molecule that comes out, especially if the exchange does not include amino acid within the framework set. Whether amino acid exchange leads to active peptide can easily be determined by measuring the specific activity of the polypeptide derivative. Measurements are described in detail here. Violations of antibodies or immunoglobulin molecules can easily be produced by those of ordinary skill in the art. Preferred amino and carboxy ends of fragments or analogues are near the range of functional areas. A building and functional area can be identified by comparing the data of the nucleic and / or amino acid residues to official series or private-line serial databases. It is preferable that computerized comparative methods be used to identify sequences or predicted protein sites that are present in other proteins with known construction and / or activity. Methods for identifying protein sequences that fall into the known three-dimensional structure are known. Bowie et al., Science 253: 164 (1991). Thus, the aforementioned example illustrates that those skilled in the art can identify arithmetic and building structures that can be used to define building and functional areas according to the invention. It is preferable that computerized comparative methods be used to identify sequences or predicted protein sites that are present in other proteins with known construction and / or activity. Methods for identifying protein sequences that fall into the known three-dimensional structure are known. Bowie et al., Science 253: 164 (1991). Thus, the aforementioned example illustrates that those skilled in the art can identify arithmetic and building structures that can be used to define building and functional areas according to the invention. It is preferable that computerized comparative methods be used to identify sequences or predicted protein sites that are present in other proteins with known construction and / or activity. Methods for identifying protein sequences that fall into the known three-dimensional structure are known. Bowie et al., Science 253: 164 (1991). Thus, the aforementioned example illustrates that those skilled in the art can identify arithmetic and building structures that can be used to define building and functional areas according to the invention.
Preferred amino acid shifts are those which: (1) decrease protein solubility, (2) decrease oxidative sensitivity, (3) change binding affinity for the formation of protein complexes, (4) change binding affinity, and (5) provide or modify other pharmacoactive or functional properties accordingly. Equilibrium can cover various mutants of a sequence other than the sequence of peptides that occur in nature. For example, one or more amino acid shifts (preferably hardened amino acid shifts) may occur in the sequence occurring in nature (preferably in the part of the polypeptide outside the region (s) that form the intermediate molecule linkage). Heldin amino acid shifts should not change the substantive structural characteristics of the mother-in-law (eg, substitute amino acids should not tend to break a helix that occurs in the mother's array, or damaging other types of twin buildings that characterize the mother row). Examples of polypeptides of two-dimensional and three-dimensional structures known in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York. (1984)); Introduction to Protein Structure (C: Branden and J. Tooze, Editor, Garland Publishing, New York, NY (1991)): and, Thornton et al., Nature 354: 105 (1991), each incorporated herein with reference.
The term "polypeptide bridge as used herein refers to a polypeptide having an amino-terminal and / or carboxy-end precipitation, but since the residues of the amino acid sequence are identical to the corresponding positions in the sequence that occurs in the derivative nature, for example, a full-length DNA sequence. Typically, fragments of at least 5, 6, 8 or 10 amino acids are in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length, and even more preferably to be at least 70 amino acids in length.
"Antibody" or "antibody peptide" refers to a healthy antibody, or bindibrots thereof that compete with the brain's antibody for specific binding. Binder fragments are produced by recombinant RNA methods, or with enzyme or chemical fractions of whole antibodies. Binding widths include Fab, Fab ', F (ab')<sub>2</sub>, Fv and single-chain antibodies. It is understood that antibodies other than "double-stranded" or "double-acting" antibodies have one of their binding sites alike. Antibodies inhibit the significant adhesion of recipient receptors when excess antibodies decrease the amount of receptors bound to recipient receptors of at least 20%, 40%, 60% or 80%, and usually more than about 85% ( as recommended by a competitive binder in a test tube).
The term "vakaeining" encompasses all protein determinants that are capable of specific binding to immunoglobulin or T-cell receptors. Unit units usually consist of chemically active groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional characteristics, and also specific loading characteristics. Antibodies are said to bind antigen is selective when the cleavage is <1 μm, preferably at 100 nM and most preferably at 10 nM.
The term "intermediates" is used herein to represent a compound, a mixture of compounds, biological macromolecules, or biologically extracted.
As used herein, the term "label" or "labeled" for insertion of detectable labels, for example, by incorporating radiolabelled amino acid or attachment of a polypeptide of biotinyl polymers that can be detected by marked avidin (for example, streptavidin containing fluorescence signals or enzyme activity that can be analyzed by optical or chromatographic methods). Under certain circumstances, the mark may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: CD-ROMs or geysers (eg,<sup>3</sup>H, <sup>14</sup>C <sup>15</sup>n, <sup>35</sup>S, <sup>90</sup>Y, <sup>99</sup>Tc, <sup>111</sup>1n, <sup>125</sup>l, <sup>131</sup>l) fluorescence markers (eg, FITC, rhodamine, lanthanide phosphorus), enzyme signals (eg, piparrost peroxidase, β-galactosidase, luciferase, alkaline phosphatase), reaction markers, biotinyl groups, or predefined polypeptide compartments known by liposomes (e.g. , leucine zippers, binders for indirect antibodies, metabolites, vaccines). In some embodiments, labels are affixed with space gaps of different lengths to reduce potential sterile barrier.
The term "pharmaceutical medium or medicament, as used herein, refers to a compound or combination which is capable of inducing a desired therapeutic effect when administered properly to a patient. Other chemical names here used are used in accordance with conventional use in the art, as explained in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Editor, McGraw-Hill, San Francisco (1985)), which is incorporated here with a reference.
The term "antimicrobial agent" is used herein for displaying agents that have the function of inhibiting the development or progression of tumor growth in a person, especially malignant (cancer) lesions, such as cancer, sarcoma, lymphoma, or leukemia. Obstacle of the teleconversion is often a feature of antitumour media.
As used herein, "substantially pure" means that the type of behavior is the predominant species present (i.e., on a molecular basis, it is more than any other type of composition), and preferably a substantially pure fraction is a combination A pair of subjects will form at least about 50% (at a molecular level) of a pillar of all large molecules present. Generally, a substantially pure composition will form more than about 80 percent of all macromolecules present in the composition, the more preferred pathway is more than 85%, 90%, 95%, and 99%. Preferably, the type of purity is purified by real homogeneity (pollutants can not be analyzed by conventional methods of analysis), the combination of which actually consists of one macro molecule.
The named patient covers human beings and veterinary supplies.
Mótefnisbvaaing
It is known that the antibody base unit contains four. Each quadrilateral is made up of two pairs of polypeptide chains, each pair having one "light" (around Pil Pil 25 kDa) and one "bulging" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable range of about 1.00 to 110 more amino acids, which are primarily responsible for identifying antigen. The carboxy-end portion of each chain defines a solid region that is primarily responsible for steering. Human light chains are categorized as kappa and light chain chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody class as IgM, IgD, IgG, IgA, and IgE, as appropriate. Within the light and punch chains are variable and constant the areas associated with the "J" area contain about 12 more amino acids, A pair of pillars also contain a "D" area of about 10 more amino acids. See, generally, Fundamental Immunology, Chapter 7 (Paul, W., Ed., 2nd Edition, Raven Press, NY (1989)) (incorporated by reference whole for universal use). The variable regions of each light / heavy chainer form the antibody binding site.
Pannig has an integral IgG antibody two binders. Apart from double or double-specific antibodies, bindisetin is two identical.
The chains show all the same general structure of relatively harsh framework regions (FR) which are merged into three highly variable regions, also called additional decision areas or CDR. The two-CDR CDR of each pair is set up by the frame regions, which makes possible binding to a specific section. From N-end to C-end, both light and heavy chain regions have FR1, CDR1, FR2, CDR2, FR3, CDR3 and For4. Assignments of amino acids to each region conform to the definitions of Kabat, Sequences of Protein of Immunological Interest (National Institutes of Bethesda, MD (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989).
Double-acting or bilayer antidote is a synthetic blend antibody that has two different heavy / light chainers and two different binding sites. Double-stranded antibodies can be produced by a variety of methods including fusion of hybridomas or linkages to Fab's fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In addition, double-tailed antibodies can be formed as "double antibodies" (Holliger et al., "Diabodies": Small bivalent and bispecific antibody fragments, PNAS USA 90: 6444-6448 (1993)) or "Janusin" (Traunecker et al. "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells", EMBO J. 10: 3655-3659 (1991); and Traunecker et al., Janusin: New molecular design for bispecific reagents, Int J. Cancer, Suppl. 7: 51-52 (1992)). The production of divalent antibodies can be a relatively labor-intensive process compared with the survival of conventional antibodies, and yield and purity are generally lower for dual-specific antibodies. Tissue-resistant antibodies are not in the form of fragments with one binding kit (eg, Fab, Fab, and Fv).
Human antibodies and manifervations of antibodies
Human antibodies avoid certain problems related to antibodies that contain mouse efia rat varies and / or fixed regions. Presence such as mouse or rat-derived proteins can lead to rapid blood purification of the antibodies, which may lead to the development of an immune response to the antibody by a patient. To avoid the use of mouse or rat-derived antibodies, it has been assumed that humanized antibodies can be developed completely human antibodies with the introduction of human antibody activity in rodents, so that the rodents would produce residues of an entire human order.
Human antibodies
The ability to clone and rebuild the megabase size of human genetic sites in YAC and to implement them (the mouse acline provides a powerful approach to clearing functionalities of very large or coarse mapped genes, and also to create useful models of human diseases. Furthermore, the use of such Mechanisms for the replacement of mouse genes for human equivalence could provide a unique insight into the expression and management of human genetic products in their development, their interaction with other systems, and their involvement in the development and progression of diseases.
Important practical use of such technology is the "humanization" of the mouse immunosuppressive system. Introduction of human immunoglobulin (Ig) gene site In mice where the intrinsic Ig genes have been inactivated, it is possible to investigate the pathway underlying the programmed expression and composition of antibodies and also their role in B-cell development. Furthermore, such technology could provide a source of choice for the production of completely human monoclonal antibodies (Mabs) which is an important milestone in meeting the promise of antibody therapy for human diseases. It is expected that the absolute human antibody levels will be minimal immunogenic and allergic response, which is natural and mouse-derived Mabs, thereby enhancing the efficacy and safety of the antibodies given.The use of completely human antibodies can be expected to give a significant advantage in the treatment of persistent and recurrent diseases in humans, such as inflammation, self-esteem, and cancer that require a repeated donor of antibodies.
One approach to this goal was to construct mouse cells with impaired proliferation of mouse antibodies with large fragments of human Ig gene sequences in the hope that such mice would produce a large collection of human antibodies in the absence of mouse antibodies. Large human Ig fragments would preserve the highly variable gene diversity and also proper control of proliferation and expression of antibodies. By using the mouse system for the variety and selection of antibodies and the lack of resistance to human proteins, the recombinant human antibody library in these mouse cells should give antibodies a high susceptibility to potentially potent human subjects, including human antibody. Using the cytosynthetic technique, antigen-specific human Mabs with the desired selectivity could easily be verified and selected.
This general technology was demonstrated in the context of our survival in the first XenoMouse ™ agencies, as published in 1994. See, Green et al., Nature Genetics 7: 13-21 (1994). The XenoMouse ™ strains were infectious gers (YAC) containing 245 kb and 190 kb-size lineage fractures of the human heavy chain gene set and the capillary light chain gene set, as appropriate, containing nuclear and fixed-region transplants. Human Ig-containing YAC proved to be compatible with the mouse system for both rearrangement and expression of antibodies and were able to deliver the power of the inactivated mouse Ig gene gene. This evidence of the ability of peer to demonstrate the induction of B-cell development was demonstrated to produce an adult-like human collection of completely human antibodies, and to elicit antigen-specific human Mabs. These results also indicated that the introduction of higher proportions of human Ig genes containing more V of V, additional controls, and human Ig-trapped regions could restore almost all of the collective characteristics characteristic of human vascular transmission due to infection and immunity. Verk Green et al. was recently introduced to the introduction of more than about 80% of the human antibody library including megabase size, cell line alignment YAC fragments of the human heavy chain geneases and the capillary light chain genes, as appropriate, to produce
XenoMouse ™ mice. See, Mendez et al., Nature Genetics 15: 146-156 (1997), Green and
Jakobovits, J. Exp. Med. 188: 483-495 (1998); and U.S. Patent Application Serial No.
08 / 759,620, registered December 3, 1996, each publication is hereby incorporated by reference.
Such an approach is further discussed and disclosed in U.S. Patent Application Serial No. 07 / 466,008, filed January 12, 1990, 07 / 610,515, filed November 8, 1990, 07 / 919,297, filed July 24, 1992, 07 / 922,649, registered July 30, 1992, 08 / 031,801, registered March 15, 1993, 08 / 112,848, registered August 27, 1993, 08 / 234,145, filed April 28, 1994, 08 / 376,279, filed January 20, 1995, 08/430, 938, filed April 27, 1995, 08 / 464,584, filed June 5, 1995, 08 / 464,582, filed June 5, 1995, 08 / 463,191, registered June 5, 1995, 08 / 462,837, registered June 5, 1995, 08 / 486,853, registered June 5, 1995, 08 / 486,857, registered June 5, 1995, 08 / 486,859, registered June 5, 1995, 08 / 462,513, filed July 5, 1995 , 08 / 724,752, recorded October 2, 1996, and 08 / 759,620, filed December 3, 1996. See also Mendez et al., Nature Genetics 15: 146-156 (1997), and Green and Jakobovits, J. Exp. Med. 188: 483-495 (1998). See also European patent no. EP 0 463 151 B1, published June 12, 1996, International Patent Application No. WO 94/02602, published February 3, 1994, International Patent Application No. WO 96/34096, published October 31, 1996, and WO 98/24893, published June 11, 1998. The disclosures of each of the patents, the applications, and the references referred to above are hereby incorporated by reference in their entirety.
In another approach, others, including GenPharm International, Inc., have used a "microgenetic" approach. In the transgenic approach, it is cited for an external Ig gene site by the inclusion of parts (individual genes) from the Ig gene set. Thus, one or more V<sub>H</sub> gene, one or more D<sub>H</sub> gene, one or more J<sub>H</sub> gene, mu fixed area, and other fixed area (preferably a fixed area) formed in construction for installation in animals. This approach is described in U.S. Pat. 5,545,807 to Surani et al .; and U.S. Patents No. 5,545,806, 5,625,825, 5,625,126, 5,633; 425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318 each to Lonberg and Kay, U.S. Pat. 5,591,669 to Krimpenfort and Berns; U.S. Patents No. 5,612,205, 5,721,367, 5,789,215 to Bems et al .; and Bandarfsku patent no. 5,643,763 to Choi and Dunn, and GenPharm International U.S. Patent Application Serial No. 07 / 574,748, filed August 29, 1990, 07 / 575,962, filed August 31, 1990, 07 / 810,279, filed December 17, 1991, 07 / 853,408, registered March 18, 1992, 07 / 904,068, registered 23 June, 1992, 07 / 990,860, registered December 16, 1992, 08 / 053,131, registered April 26, 1993, 08 / 096,762, filed July 22, 1993, 08 / 155,301, registered November 18, 1993, 08 / 161,739, filed December 3, 1993, 08 / 165,699, filed December 10, 1993, and 08 / 209,741 , listed March 9, 1994, disclosure of which is hereby incorporated by reference. See also European patent no. 0 546 073 B1; and Universal Patent Applications No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 , disclosure of each is hereby incorporated by reference in its entirety. See also Tayloro .fi., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole. 739, registered December 3, 1993, 08 / 165,699, filed December 10, 1993, and 08 / 209,741, filed March 9, 1994, disclosure of which is hereby incorporated by reference. See also European patent no. 0 546 073 B1; and Universal Patent Applications No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 , disclosure of each is hereby incorporated by reference in its entirety. See also Tayloro .fi., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole. 739, registered December 3, 1993, 08 / 165,699, filed December 10, 1993, and 08 / 209,741, filed March 9, 1994, disclosure of which is hereby incorporated by reference. See also European patent no. 0 546 073 B1; and Universal Patent Applications No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 , disclosure of each is hereby incorporated by reference in its entirety. See also Tayloro .fi., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole. See also European patent no. 0 546 073 B1; and Universal Patent Applications No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 , disclosure of each is hereby incorporated by reference in its entirety. See also Tayloro .fi., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole. See also European patent no. 0 546 073 B1; and Universal Patent Applications No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 , disclosure of each is hereby incorporated by reference in its entirety. See also Tayloro .fi., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole. Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole. Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al., 1994; Tayloro.fi., 1994; Tuaillon et al., 1995; and Fishwild et al., 1996, disclosure of each is hereby incorporated by reference as a whole.
The inventors of Surani, etc., referred to above and designated by the MRC, produced genetically engineered mouse that has Ig gene site using the microgenetic approach. The inventors of the GenPharm International work referred to above, Lonberg and Kay, who led the lead of the present inventors, enabled the inactivation of the intestinal mucosa genes associated with the material doubling of Surani's work, etc.
The advantage of the small-scale approach is how fast you can produce buildings, including parts of the Ig gene set and introduced into animals. Correspondingly, however, there is a significant disadvantage of the short-term approach, theoretically, the insufficient diversity is introduced by the inclusion of a small number of V, D, and J genes. Indeed, apparently publishing the work supports these concerns. Apparently, B-cell development and antibody production in animals produced using the microgenetic approach has been reduced. Therefore, studies around this invention have consistently been directed towards the introduction of large portions of the Ig gene set for more variety and to reorganize the immune system of the animals.
Human anti-mouse antibody (HAMA) response has led the industry to produce hybrid or other humanized antibodies. Although blending antibodies have a human fixed region and mouse variable region, it is expected that certain human anti-blendings of antibody (HACA) response will be observed, especially in the prolonged or multidose use of the antibody. Thus, it would be desirable to give completely human antibodies to CTLA-4 to eliminate concerns and / or effects of HAMA or HACA response.
Manngerving and display methods
As discussed above in connection with the proliferation of human antibodies, there are advantages to proliferating immunogens that are less immunogenic. This can be achieved in some ways by means of manners and methods of publishing using vigorous collections. Understand that mouse antibodies or antibodies from multiple species can be manipulated or immunized using methods well known in the art. See, for example, Winter and Harris, Immunol Today 14: 43-46 (1993), and Wright et al., Crit. Reviews in Immunol. 12: 125-168, (1992). In addition, the antibody constructs the interest of the DNA molecule to replace CH1, CH2, CH3, the hemorrhoids, and / or enhance the framework for the corresponding human order (see WO 92/02190, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761 , 5,693,792, 5,714,350, and 5,777,085). Also, the use of the IgD cDNA for the construction of blendings of immunosuppliers is known in the art (Liu et al., PNAS 84: 3439 (1987), and J. Immunol. 139: 3521 (1987)). mRNA is isolated from primary cell proliferation enhancing another cell that produces the metabolites and utility to the proliferation of cDNA. The power of cDNA may be enhanced by the use of selective viscosity (U.S. Pat. Nos. 4,683,195 and 4,683,202). Otherwise, a collection is made and it is screened to isolate the order of interest. The DNA sequence encoding the variable region of the antibody is then linked to human vascular fixed region. You can find the rows of human solid region genes in Kabat et al. (1991), Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242. It is easy to get a human C region gene from known clover. The selection of the samastasis will be guided by the desired function, such as magnitude binding, or activity in antibody-dependent cytotoxicity. Preferred isotopes are lgG1, IgG2, IgG3 and IgG4. Particularly preferred isotopes for antibodies in the invention are IgG2 and IgG4. You canuse either of human light chain fasting areas, kappa or lambda. The blending humanized antibody is then expressed by conventional methods.
You can produce antibody fragments, such as Fv, F (ab ')<sub>2</sub> and Fab by splitting the whole protein, for example, with protein or chemical cleavage. Alternatively, a designed stiff gene. For example, a blendingsgen encoding part of F (abj<sub>2</sub> The fragment contains DNA generators encoding the CH1 region and the Hector region of the H cell, followed by the translation-stop symbols to give the amplified molecule.
In one approach, force can use coordinates that code heavy and light chain J areas to power designing oligonucleotides for use as a view to force implement a useful scaling set in the J region for the subsequent connection of V region regions in human C region regions. It is important to change the C region of cDNA with the expression of endogenous mutations in order to place a coding set in the analogous position of the human sequence.
Expression vectors include plasmids, intervertebrates, cosmics, YAC, EBV-derived sections, and progeny. A useful vector is the one that encodes the brainwashing human human CH promoting CL immunoglobulin sequence, including vascular sclerosis kits, which is constructed as easily as possible, the force introduces and expresses the vph of VH enhancing the VL sequence. In such a way, the connection usually takes place between the linkage set-up J region and the linkage set set in front of the human C region, and also in the junctions within the human CH symbol. Polyadenylation and transcription capabilities are stacked at the original chromosome sites below the coding regions. The blendings can be associated with the antibody that emits a strong stimulator, which is linked to the selected LTR virus, for example, SV-40 early, (Okayama et al., Mol. Cell, Bio 3: 280 (1983)), Rous sarkmeins virus LTR (Gorman et al., PNA S. 79: 6777 (1982)); and Moloney mouse leukemia virus LTR (Grosschedl et al., Cell 41: 885 (1985)); original Ig steering, etc.
Furthermore, the proliferation of human metabolites enhances antibodies from other types of manifestation-enhancing bronchospheres, interactions, limitations, colonoscopy, xenophobia, ribosomalysis, and excessive proliferation, using power-efficient techniques that are well known in the art. forces make the molecules that are undergoing further development, such as affinity development, couples who are well-behaved are well-known. Wright and Harris, supra., Hanes and Plucthau, PNAS USA 94: 4937-4942 (1997) (riposom publication); Parmley and Smith, Gene 73: 305-318 (1988) (gerilveirubirting); Scott,
TIBS 17: 241-245 (1992); Cwirla et al., PNAS USA 87: 6378-6382 (1990); Russel et al., Nucl. Acids Research 21: 1081-1085 (1993); Hoganboom et al. Immunol. Reviews 130: 43-68 (1992), Chiswell and McCafferty, TIBTECH 10: 80-84 (1992); and U.S. Pat. 5,733,743. If delivery methods are used to produce non-human antibodies, you can modify a pannig antibody as described above.
By using these methods, antibodies to cells expressing CTLA-4, CTLA-4 itself, forms of CTLA-4, vaccine units, and peptide pairs (see, e.g., U.S. Patent No. 5,703,057) which can be used as follows, as described above, for the functions described above.
Additional conditions for treatment trials
As understood, it is generally not desirable to kill cells expressing CTLA-4. Rather, it is usually possible to prevent simply CTLA-4 binding at the link to a mild T-cellular nephropathy. One of the adal reactions after each of the target kill cells is with magnetic binding and participation in CDC. Fixed immunity to antibodies plays an important role in the ability of mdtefnis to quantify and participate in CDC. Therefore, it is usually preferred to specify whether or not the ability to quantify it. In the case of this invention, generally, as mentioned above, generally, it is not preferable to use antibodies that kill the cells. There are a number of mttefm groups that are capable of magnitude binding and CDC, par with without limitation the following: mouse IgM, mouse lgG2a, mouse lgG2b, mouse lgG3, human IgM, human lgG1, and human lgG3. Peir antibodies not included, without limitation, human lgG2 and human lgG4.
Separate the products that are produced by the purse do not initially have the desired detergent as desired, but rather, the protein produced with a catalytic enzyme that is capable of exchanging the homologous antibody to the antibody by using conventional behaviors who are well-known ί profession. Pannig behaviors cover the use of direct rearrangement pathways (see, for example, U.S. Patent No. 4,816,397), cell-cellular merging behaviors (see, e.g., U.S. Patent No. 08 / 730,639, filed October 11, 1996), the medal of others.
In the cell-cellular fusion procedure, multiple myeloma is produced by the other molecules that have a depleted chain of asymptomatic nucleus, and the other myeloma of the other cells is produced with the light chain. Pannig cells can almost always be associated with and the cellular which expresses a complete sense of self-esteem.
By way of example, the majority of the CTLA-4 antibodies discussed herein are human anti-CTLA-4 IgG2 receptors. A pair of binding proteins have a desirable binding to the CTLA-4 molecule, which is readily possible on a single-acting vector of any type of protein to produce human lgG4 samsaetugerd, for example, while the same variable region is still vidhaldid (which defines the specificity of the antibody and part of its affinity).
In summary, when potential antibodies are produced that have theoretically "structural" features as discussed above, they can usually be provided with at least certain additional "functional" properties that are evident in the application of cytotoxicity.
Design and production of other agents
As discussed above, the activity of the antibodies of the invention can be altered by isotransplantation of IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM for various therapeutic uses.
In conjunction with the production of developed therapeutic antigens, where magnetic binding is a desirable feature, it may be possible to avoid the porosity level for cellular eradication using the use of dysentery, immune, or radiolabels, for example.
In conjunction with double-stranded antibodies, double-specific antibodies can be produced containing (i) two antibodies, one with specificity against CTLA-4 and another against another molecule that are interconnected, (ii) one single-chain antibody specific to CTLA- 4 and another chain specific to another molecule, or (iii) one chain of antibodies that has specificity against CTLA-4 and the other molecule. Thus, double-specific antibodies can be prepared using methods well known, for example, in conjunction with (i) and (ii) see, for example, Fanger et al., Immunol. Methods 4: 72-81 (1994), and Wright and Harris, supra. ', And in relation to (iii) see, for example, Trauneckero.fi., Int. J. Cancer (Appendix) 7: 51-52 (1992).
Additionally, "antibody molecule" (III et al., "Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions", Protein Eng. 10: 949-57 (1997)) can also be produced. , "small molecules" (Martin et al., "The affinity selection of a mini-polypeptide inhibitor of human interleukin-6", EMBO J. 13: 5303-9 (1994.)), "double antibodies" (Holliger et al. , "Diabodies": Small bivalent and bispecific antibody fragments, PNAS USA 90: 6444-6448 (1993)), and Janusin (Traunecker et al., "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells, EMBO J 10: 3655-3659 (1991), and Traunecker et al., Janusin: New molecular design for bispecific reagents, Int. J. Cancer, vidauki 7: 51-52 (1992)). .
In conjunction with immunosuppressants, it is possible to modify the agents to act as immunosuppressants using methods well known in the art. See, for example, Vitetta, Immunol. Today 14: 252 (1993). See also U.S. Pat. 5,194,594. In conjunction with the production of radiolabelled content, it is also possible to reproduce the generated information by using behaviors that are well-known. See, for example, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, ed., Lippincott Raven (1996)). See also U.S. Pat. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each of the immunosuppressive and radiolabelled molecules would be useful for killing cells expressing CTLA-4, and specifically those cells where the antibodies of the invention are effective.
Therapeutic combinations
It will be appreciated that administration of therapeutic units according to the invention will be given with suitable excipients, excipients, and other agents incorporated into combinations to provide improved transmission, dialysis, stamina, and the like. A number of appropriate combinations can be found in the prescription booklet, which all pharmacists know: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975)), especially section 87 by Blaug, Seymour, therein. Pessar combinations include, for example, powder, paste, ointment, gel, wax, oils, lipids, lipids (cationic and anionic) containing carriers (such as Lipofectin ™), conjugated DNA, anhydrous absorption paste, oil-in-water and water-in-oil emulsion, carbon black emulsion (polyethylene glycol of different molecular weight), semi-solid gel, and semi-solid mixtures containing carbon wax. Any of the foregoing compositions may be useful in the treatments and treatments according to this invention, provided that the active substance in the composition is not inactivated by the composition and that the composition is physiologically compatible and tolerable delivery route. See also, Powell, et al., "Compendium of excipients for parenteral formulations", PDA J. Pharm. Sci. Technol. 52: 238-311 (1998), and the references for more information related to auxiliaries and excipients well-known by pharmacologists.
Development of antibodies
It is preferable to produce antibodies according to the invention by the use of a GM mouse in which a large portion of the genetic linkage that produces human antibodies has been introduced but has been inactivated in the production of intrinsic mouse antibodies. Thus, mice are capable of producing human immunoglobulin molecules and antibodies and are impaired in the production of mouse immunoglobulin molecules and antibodies. Methods used to achieve the same are disclosed in the patents, applications, and references shown in the background herein. Specifically, however, an optimal manifestation of genetically engineered production of mice and antibodies from peam is disclosed in U.S. Patent Application Ser. 08 / 759,620, registered December 3, 1996, disclosure of which is hereby incorporated by reference. See also Mendez et al., Nature Genetics 15:
With the use of pannig technology, we have produced completely human monoclonal antibodies against the number of antigens. basically immune to XenoMouse ™ lines of mice with an antigen of interest, restores viable cells (such as B cells) from the murine expressing antibodies, link pannig regenerated cells to the merged cell line to produce immortal cellular cellular cellular cellularity, cell lines are screened and selected for the cellular immunodeficiency cell line which produce specific antibodies that are specific to the interest of the cytotoxic agent. The use of these methods of combining this invention to the production of antibodies specific to CTLA-4. Herein we describe the production of many cytosuppressive cells as proliferative compounds that are specific to CTLA-4.
The antibodies obtained from the cytosynthesis cell discussed herein are referred to as 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1. , 11.7.1, 12.3.1.1, and 12.9.1.1. Each of the polypeptides produced by the above-mentioned cell lines is either completely human or other human IgG4, but also in human body. In general, the compounds of the invention have a high degree of affinity, typically have a value of about 10 '<sup>9</sup> for about a car 10 "<sup>11</sup> M, when it is recommended whether or not a phytophilic phase of the solution phase.
As can be understood, it is possible to express synthetic compounds in accordance with this invention in the cellular domain of the cytotoxic cell. Assays that encode the cDNA efia gene linkage for a particular polypeptide can verify the use of a mammalian host cell for conversion to suitable mammalian mammalian cells. Transformation can verify postpopular peripheral neuropathy which introduces a pharyngeal cell into a host cell, optionally linked to the virus, into the virus of the virus, and introduces the host cell with the virus (efia vigranum), with the incidence of residual hepatocellular infected cells in the art, as is typical of U.S. Pat. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (each patent is hereby incorporated herein by reference). The utilization quota used is dependent on the host that transforms the image.
Mammalian cell lines available as host cells for expression are well known in the art and include many immortal cell cultures available from the American Type Culture Collection (ATCC), including non-limiting vifi, chronic hamster ovary (CHO) cells, NSO<sub>0</sub> cells, HeLa cells, childhood kidney (BHK) cells, pancreatic cells (COS), human hepatocellular cancer cells (eg, Hep G2), and ankle of the fifth cell. Non-mammalian cells, including others, but not limited to bacteria, yeast, insects, and pesticides, can also verify the use of microbiological sources. Sequential mutation of the antibody CH2 subtype to eliminate glycosylation can be verified to prevent any changes in the immunogenic, pharmacological and / or functional roles resulting from non-human glycosylation. The expression methods are chosen by determining which system produces the most expression and produces antibodies with undefined CTLA-4 binding properties.
Furthermore, expression of the antibodies of the invention (or other pairs thereof) may be promoted from cell line proliferation using a number of known afiferfiums. For example, glutamine ligase and DHFR gene expression systems are common approaches to enhance expression under certain circumstances. High-quality cell clones can be identified using conventional methods, such as limited dilution cloning and microdrop technology. The GS system is discussed in the entirety of Part I in connection with European Patent No. 0 216 846, 0 256 055, and 0 323 997, and European patent application no. 89303964.4.
Alternatively, the present invention may be derived from genetic modification with the production of a mammal or plant that is genetically modified for the immunoglobulin heavy and light chain of interest and the retention of the antibody in a recoverable form from a pair. In the context of the GMO in mammals, the potential for proliferation of antibodies in, and recovery from, milk, goat, cow, and other mammalian animals. See, for example, U.S. Pat. 5,827,690, 5,756,687, 5,750,172, and 5,741,957.
Antibodies according to this invention have verified identified constructively and electrically. In the context of the antibody structure, verified the amino acid sequences of the heavy and lightweight chains based on the cDNA sequence obtained by RT-PCR on the hybridomas. See Examples 3 and 4 and pictures 1-8. N-terminal sequencing of the antibodies was also performed to confirm the results discussed in Examples 3 and
4. See Example 5 and Figure 9. Response rate analyzes of the antibodies were performed in order to determine the result. See Section 2. Further, antibodies were detected by electrophoresis (IEF), SDS-PAGE reduction scale, size-selective chromatography, quantum analysis / mass analysis, and mass spectrometry, and antibody production of the hybridomas was evaluated. See example 6 and Figure 10.
Thus, in the context of a functional assay of antibodies according to this invention, antibodies were found to be potent inhibitors of CTLA-4 and its binding to the compounds of the B7 molecule of molecules. For example, the afi antibody of this invention was shown to inhibit CTLA-4 binding to either B7-1 or B7-2. See Example 7. Indeed, many of the antibodies according to the invention have nanomols and subnovaols IC<sub>50 </sub>value for the prevention of CTLA-4 binding of V7 B7-1 and B7-2. Furthermore, the antibodies of the invention have excellent selectivity for CTLA-4 combined with CD28, CD44, B7-2 inhibitors. See example 8. Valvisi is a ratio that reflects the amount of precursor binding on the molecule of the first molecule by comparing the binding of the molecule to the latter, and possibly by molecules. Herein, we show the selectivity to the amount of precursor binding of the invention from the invention CTLA-4 together with the binding of the molecule to other molecules such as CD28, CD44, B7-2. The selection values of antibodies of the invention of more than 500: 1 are common. Also, the verifi cation has demonstrated the presence of antibodies from the invention inducing the expression of certain cytobodies (such as IL-2 and IFN-γ) with raectufium T cells ί T-germ cells. See Examples 9 and 10 and Figures 12-17. Furthermore, it is expected that antibodies from the invention will inhibit tumor growth in tumorigenic tumors. The designs of these models are discussed in Examples 11 and 12.
The results shown in accordance with this invention indicate that antibodies of this invention have certain properties that can render these antibodies more effective than current therapeutic antagonists against CTLA-4.
Particularly, 4.1.1, 4.8.1. and 6.1.1 the antibodies have very moral properties. Their structural properties, roles, efficacy, give the assumptions that aufivelda design may be chosen for additional antibodies of the same molecules as discussed above. Such assumptions cover only one more of the following:
Being able to compete for the binding of CTLA-4 to one more of the antibodies of the invention;
Similar binding techniques for CTLA-4 and one more of the antibodies of the invention; Binding technology for CTLA-4 is approximately 10 "<sup>9</sup> M efia more and preferably it is about 10 '<sup>1θ</sup> M efia more.
Do not cross-react with lower mammalian CTLA-4, including, preferably, mouse, rat, or rabbit, and preferably murine rat CTLA-4;
Cross-reactive to primates of CTLA-4, including, preferably, synomologus and resus CTLA-4;
Selection for CTLA-4 rather than CD28, B7-2, CD44 or hlgG1 of at least about 100: 1 and more preferably about 300, 400, more preferably.
IC<sub>50</sub> In the blood, the CTLA-4 binding of B7-2 is approximately 100 nM lower and preferably 5, 4, 3, 2, 1, 0.5, and 0.38 nM lower.
IC<sub>50</sub> In the blood, the CTLA-4 binding of B7-1 to about 100 nM is lower and preferably 5, 4, 3, 2, 1, 0.5, 0.51 nM lower;
Enhancement of primary production In one more measure, more experimental measurements, for example:
Enhancement of IL-2 survival in the T-cytotoxicity / Raji diagnosis of about 500 pg / mL more preferably 750, 1000, 1500, 2000, 3000, 3846 pg / mL more;
Enhancement of the IFN-γ survival in the T-germ cell / Raji diagnosis of about 500 μg / ml more preferably 750, 1000, 1233 μg / mL more,
Enhancement of IL-2 survival in the hPBMC of whole blood supernatant analysis of about 500 pg / ml more preferably 750, 1000, 1200, more preferably 1511 pg / ml. In other words, it is desirable that IL-2 output is enhanced by approximately 30, 35, 40, 45, 50 present levels more quantitative criteria in the analysis.
Antibodies (or molecules designed or constructed therefrom) having one or more of these properties are expected to have similar activity to the antibodies described in this invention.
Preferred functionalities discussed above may often result from the binding and blockade of CTLA-4 by molecules (peas, antibodies, antibody fragments, peptides, or small molecules) similar to antibodies of the invention (peas, binding to the same or similar compartment of the CTLA-4 molecule).
The molecule can either be administered directly (peas, direct donation to the patient on a pannig molecule). Or, alternatively, the molecule may be "administered" indirectly (peas, peptide or similar that induces a patient's immune response (similar to a vaccine) pair whose immune response reaches the production of antibodies that bind to the same or similar unit or antibody or fraction produced on site after administration of genes encoding pannig antibodies or fragments of pairs that bind to the same or similar compartment). Thus, it will be understood that the CTLA-4 receptor component of which the antibodies of the invention bind may be useful in connection with the production and / or the design of therapeutic agents in In accordance with the invention, in drug design are negative information. Ilka often useful (peas, the fact that antagonism that binds to CTLA-4 does not seem to bind a vaccine that acts as a barrier to CTLA-4 is beneficial). Thus, the unit of vaccine of the invention that binds to non-desired activity may also be useful. In a comparative sense, also contemplated in accordance with this invention are molecules (and specific agents) that bind to a multiple enhancing subject matter and subject matter of the invention.
In the context of the invention, the characteristic force of the invention and the pacemaker paws are conceived. In accordance with the present invention, we have carried out some preliminary pivotal mapping studies of certain antibodies according to the invention, and especially the 4.1.1 metabolite and the 11.2.1 metabolite of the invention.
As the first step of the construction of BIAcore competitive antigens to power, there is a significant map of binding between certain antibodies of the invention in relation to the ability of the peer to force to compete for the binding of CTLA-4. For this purpose, CTLA-4 was a bundle of BIAcore flares and the first metabolite, in terms of saturation conditions, was the basis of this and competing for the follow-up second-level metabolic binding of CTLA-4 was measured. This power factor allows power to generate a large map that could be used by families of groups i.
With these factors, the desired wave power could be categorized as a specific factor, because of the following factors:
<td>Category</td><td>Mfltefni</td><td>Competition for CTLA-4 binding</td>
<td rowspan="2">A</td><td>BO1M *</td><td rowspan="2">Cross-competing forces want to know whether or not annafl; cross-competing fan category B; any cross-contest fan of category D.</td>
<td>BO2M **</td>
<td rowspan="2">B</td><td>4.1.1</td><td rowspan="2">Cross-competing forces want to know whether or not annafl; cross-competing fan category A, C and D.</td>
<td>4.13.1</td>
(Continued)
<td>Category</td><td>antibodies</td><td>Competition for CTLA-4 binding</td>
<td rowspan="6">C</td><td>6.1.1</td><td rowspan="6">Cross-compete with each other, cross-compete with class B and class D.</td>
<td>3.1.1</td>
<td>4.8.1</td>
<td>11.2.1</td>
<td>11.6.1</td>
<td>11.7.1</td>
<td>D</td><td>4.14.3</td><td>Cross-compete with classes C and B; any cross-contest with class A.</td>
<td rowspan="2">E</td><td>4.9.1</td><td rowspan="2">BNI3 blocks 4.9.1 binding to CTLA-4, but not vice versa.</td>
<td>BNI3 ***</td>
<td colspan="3">(*) (**) Available from Biostride. (***) Available from Pharmingen.</td>
As a next step, we tried to determine if the antibodies knew a linear cavity of CTLA-4 in reducing and non-reducing conditions on protein thrips. We found that none of the 4.1.1, 3.1.1, 11.7.1, 11.6.1, or 11.2.1 antibodies appeared to recognize a reduced form of CTLA-4 on the protein pressures. Accordingly, it seemed likely that the vaccine of each of these antibodies would not be a linear vacuum, but it would be likely that a collapse of the structure could be damaged by deteriorating conditions.
Therefore, we tried to determine if we could learn about residues within the CTLA-4 molecule that are important for binding antibodies from the invention. One way we used was to execute the rate of reactivity at the rate between human CTLA-4 and two highly-successful primates of CTLA-4 molecules (synomogiogus and silkworm CTLA-4). BIAcore studies showed that the 4.1.1 antibody binds human, synomomogogus, and silk CTLA-4 at the same rate. However, with respect to the rate of affinity (affinity), the 4.1.1 antibody had the highest (lowest) rate of humans, higher rate of synomoglovir, and much higher rate of silk loss. 11.2.1 The antibody of the invention, on the other hand, binds human, synomomogogus, and silkworm CTLA-4 at approximately the same rate and has approximately the same relative to-velocity for each of the three.
In order to further investigate the vaccine unit of classes B and C, the antibodies from the invention bind to certain cytotoxic mutation studies. Silkiapa CTLA-4 has two important changes in residues 105 and 106 relative to human CTLA-4. Such a difference is leucine in the methionine change in residue 105 and glycine in serine change in residue 106. Accordingly, mutant mutant to cDNA encoding human CTLA-4 to encode mutant CTLA-4 having the L105M and G106S changes. Asylum replacement mutagenic CTLA -4 did not bind the B7-2-IgG1 fusion protein. Furthermore, no binding to the 11.2.1 antibody was obtained. However, such a molecule was significantly inhibited by its ability to bind the 4.1.1 antibody (similar to silk lobe). Next mutated to cDNA coding for CTLA-4 silk screen to create mutant CTLA-4 silk screen that has an S106G change. Such a change led to the reconstruction of a stable link between the 4.1.1 antibody and the silk lip of the CTLA-4 mutant. In addition, the cDNA mutant encodes the CTLA-4 silk screen to create mutant CTLA-4 silk screen that has an M105L change. Such a change partially reconfirmed the relationship between 4.1.1 of the antibody and mutant CTLA-4.
All Class B through D D antibodies appear to have similar functional properties and appear to have the potential to act as strong anti-CTLA-4 therapeutic agents. Furthermore, all the molecules show a particular cross-competition in their binding to CTLA-4. However, as will be apparent from the discussion above, all the molecules in the different classes appear to bind separate discontinuous units on CTLA-4.
From the foregoing, it will be understood that the vaccine data discussed above indicate that antibodies (or other molecules, as discussed above) that cross-compete with antibodies of the invention will definitely have a particular therapeutic potential in combination with this invention. Furthermore, antibodies (or other molecules, as discussed above) are expected to cross-compete against antibodies from the invention (i.e., cross-compete with class B, C and / or D antibodies) will probably have certain additional treatment options in accordance with this invention. In addition, antibodies (or other molecules, as discussed above) are expected to cross-compete with antibodies from the invention (i.e., cross-compete with class B, C and / or D antibodies) and which (i) are not reduced in their binding to CTLA-4 silk screen (similar to 11.2. 1 antibody) or (ii) are reduced. In their binding to the CTLA-4 silk screen (similar to the 4.1.1 antibody), there will be certain additional treatment options in accordance with this invention. Antibodies (or other molecules, as discussed above) that compete with classes A and E, may also have certain treatment options.
DÆIVII
The following examples, including the experiments carried out and the results obtained, are given in the sole discretion and are not to be construed as limiting for this invention.
DÆM11
Proliferation of cytotoxicity that induces anti-CTLA-4 antibodies
Antibodies from the invention were produced, selected, and analyzed in combination with this case.
Antibody Developmental Antibodies: Three separate immunogens were produced for immunization of the XenoMouse ™ mice: (i) CTLA-4-IgG fusion protein, (ii) CTLA-4 peptide, and (iii) 300.19 mouse lymphoma cells implanted with CTLA-4 mutants Y201
V) which is expressed unconditionally on the cell surface.
(i) CTLA-4-IgG1 fusion protein:
Bygqinq tiáninqarviqurs:
cDNA coding for mature extracellular regions on CTLA-4 was chain synthesized from human cDNA library (Clontech) using visions designed for display sequences (Eur. J. Immunol. 18: 1901-1905 (1998)). The fracture was targeted subcloned into pSR5, a Sindbid viral expression plasmid (InVitrogen), between the human oncostatin M marker peptide and the human IgG gamma 1 (lgG1) CH1 / CH2 / CH3 regions. The fusion protein does not contain an area of brains but contains cysteine 120 in the extracellular region of CTLA-4 to form a double-sided joint. The resulting vector was called CTLA-4-lgG1 / pSR5. The brain CTLA-4-IgG1 cDNA in vigurns was a sequence confirmed in both factors. The amino acid sequence for the CTLA-4-Ig protein is shown below.
OM-CTLA-4-IgG1 fusion protein:
mgvlltortllslvlallfpsmasmamhvaopavvlassrgiasfvc
EYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICT
GTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY vidpepcpdsdlegapsvflfppkpkdtlmisrtpevtcvwdvshedpe
VKITWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPTPKKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Underlined: tag peptide.
Bold: CTLA-4 extracellular area.
The cDNA for mature extracellular region in CD28 was chained from human lymphocytes (Clontech) and then subcloned into pCDM8 (J. Immunol. 151: 5261-71 (1993)) to produce a human lgG1 fusion protein containing beta thrombin cleavage and hemorrhoids. Silkiapa, synamologus, and resus CTLA-4 were cloned from mRNA isolated from PHA PBMC using standardized chain regimens. Radiation showed the resus and synamologous amino acid sequences were identical with three changes from the mature human CTLA-4 extracellular region (S13N, 117T and L105M). Silkiapa CTLA-4 showed ten amino acid alterations from the mature human CTLA-4 extracellular region (V21A, V33I, A41T, A51G, 54I, S71F, Q75K, T88M, L105M and G106S). Septic mutation was used to make single site mutations on all amino acids that were different in the CTLA-4 silk screen to map amino acids that were important for the antagonism of the antibodies to human CTLA-4-IgG. Mutations of human and silk patches CTLA-4-IgG for unit dose mapping were created by Matchmaker's cytotoxic mutagenicity (Promega). IgG fusion proteins were prepared by short-term induction of COS7 cells and purified using standard protein A methods. Mutant CTLA-4-IgG proteins were evaluated for binding to antibody with antibody expression and using BIAcore analysis. Mutations of human and silk patches CTLA-4-IgG for unit dose mapping were created by Matchmaker's cytotoxic mutagenicity (Promega). IgG fusion proteins were prepared by short-term induction of COS7 cells and purified using standard protein A methods. Mutant CTLA-4-IgG proteins were evaluated for binding to antibody with antibody expression and using BIAcore analysis. Mutations of human and silk patches CTLA-4-IgG for unit dose mapping were created by Matchmaker's cytotoxic mutagenicity (Promega). IgG fusion proteins were prepared by short-term induction of COS7 cells and purified using standard protein A methods. Mutant CTLA-4-IgG proteins were evaluated for binding to antibody with antibody expression and using BIAcore analysis.
Bacterial protein synthesis / purification:
The recombinant serotonin virus was created by electrically (Gibco) childhood renal cells with SP6 in vitro, transcribed CTLA-4-lgG1 / pSR5 mRNA and DH-26S helps mRNA, as described in In Vitrogen. Forty-eight hours later, recombinant virus was harvested and titrated to achieve the maximum protein expression in Chinese hamster ovary (CHO-K1) cells. CHO-K1 cells were cultured in suspension of DMEM / F12 (Gibco) containing 10% heat-inactivated embryonic acid (Gibco), non-essential amino acids (Gibco), 4 mM glutamine (Gibco), pensiline / streptomycin (Gibco), 10 mM HEPES pH 7.5 (Gibco). To produce CTLA-4-IgG, CHO-K1 cells were resuspended at 1x10<sup>7</sup> cells / mL in DMEM / F12 and cultured with meningitis virus for one hour at room temperature. Cells were then diluted to 1x10<sup>6</sup>/ mL in DMEM / F12 containing 1% bovine fetal serum that had been emptied by nautical IgG using Protein A Sepharose (Pharmacia), non-essential amino acids, 4 mM glutamine, 12.5 mM HEPES, pH 7.5, and penicillin / streptomycin. Forty-eight hours post-infection, cell pellets were created and the condenser medium was excised and supplemented with whole protease inhibitors (Boehringer Mannheim), pH adjusted to 7.5, and filtered 0.2μm (Nalgene). FPLC (Pharmacia) was used to affinity purify the fusion protein using 5 ml Protein A HiTrap column (Pharmacia) at 10 mL / min flow rate. The column was washed with 30 volumes of PBS eluted with 0.1 M glycine / HCl, pH 2.8 at 1 mL / min. Violations (1 mL) were immediately neutralized to pH 7.5 with Tris, pH 9. The fragments containing CTLA-4-IgG1 were identified by SDS-PAGE and then pooled using Centriplus 50 (Amicon) before they were placed on Sepharose 200 column (Pharmacia) at 1 mL / min using PBS as a laser . Breaches containing CTLA-4-IgG1 were assembled, sterile 0.2 μg (Millipore) divided into doses and frozen at -80 ° C. CD44-lgG1 was expressed and purified using the same methods. CD28-IgG was purified from conditional medium from short-term introduced COS7 cells.
Identification of CTLA-4-IgG1:
Purified CTLA-4-IgG1 was transferred as a single band on SDS-PAGE using Coomassie Coloring (Novex). In non-decreasing conditions, CTLA-4-IgG1 disambiguation (100 kDa) was reduced to 50 kDa monolithy when treated with 50 mM DTT. Amino acid sequencing of purified CTLA-4-IgG1 in solution confirmed the N-terminus of CTLA-4 (MHVAQPAWLAS); and that the onkostatin-M marker peptide was cleaved from the mature fusion protein.
CTLA-4-IgG1 barked resistant (B7-1-IgG) in a potentiated manner, and the binding was blocked by Hamster-Human-CTLA-4 Antibodies (BNI3: Pharmingen). Death-purified CTLA-4-IgG was intact and amplified with OD280 using 1.4 as an anesthetic factor. The yield of purified CTLA-4-IgG was between 0.5-3 mg / liter of CHO-K1 cells.
(ii) CTLA-4 peptides:
The following CTLA-4 peptide was prepared as described below:
NH<sub>2</sub>: MHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVT
EVCAATYMMGNÉLTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICK
VELMYPPPYYLGIGNGTQIYVIDPEPC-CONHz
Abbreviations / Materials:
NMP, N-Methylpyrrolidinone; TFE, 2,2,2-Trifluoroethanol; DCM, Dichloromethane; FMOC, Fluorenyl Methoxycarbonyl. all reagents were obtained from Perkin Elmer, with the following exceptions: TFE, Aldrich Chemical; FMOC-PAL-PEG resin, Perseptive Biosystems. Fmoc-Arg (PMC) -OH, FMOC-Asn (Trt) -OH, FMOC-Asp (tBu) -OH, FMOC-Cys (Trt) -OH, FMOC-Glu (tBu) -OH, FMOC-Gln ) -OH, FMOC-His (Boc) -OH, FMOC-Lys (BOC) -OH, FMOC-Ser (tBu) -OH, FMOC-Thr (tBu) -OH and FMOC-Tyr (tBu) -OH were used for those amino acids that needed side chain protection groups.
Peptide Synthesis:
Peptide synthesis was performed on Perkin-Elmer 431a, prepared by a response response to UV absorption at 301 nm (Perkin-Elmer Model 759A nemi). The peptide sequence was assembled on FMOC-PAL-PEG resin using a conditional double linker. The forced double bond was performed in circles 10, 11, 18, 19, 20 and 28 through 33. The resin was washed with 50% DCM and TFE mixture at the end of each asylation cycle followed by the heating of unreacted amino groups with acetic anhydride in NMP . Resin was removed from the reaction tank after ring 49 was completed and the remainder continued to the end. Peptide cleavage from the resin was performed using Reagent K (King et al., International
Journal of Protein and Peptide Research 36: 255-266 (1990)) for 6 hours on 415 mg resin which gave 186 mg of crude CTLA-4 peptide.
Agenkenninq peptide:
mg doses of the raw CTLA-4 peptide were dissolved in 5 mL of 6 M guanidine-HCl / 100 mM K<sub>2</sub>PO<sub>3</sub> at pH 6.4 and eluted on the Pharmacia Hi Load Superdex 75 16/60 column (16 mm x 600 mm, 120 mL volume) with 2 M guanidine-HCl / 100 mM K<sub>2</sub>PO<sub>3</sub> at pH 6.4 at 2 mL / min for 180 minutes collecting 5 mL fragments. The fractures were analyzed by loading 1.7 pL fragments on NuPAGE Laemmli gel, run with MES runway samples and examined by Daichii silver staining protocol. The fragments exhibiting molecular weight of 12 kDa, as judged by molecular weight standards, were combined and stored at 4 ° C. The combined fragments were detected by UV and gel electrophoresis. Amino acid analysis was performed by absorbing 100 microlitre samples in ProSorb capsules (absorbed on PVDF membrane) and washing to remove the dummy salts. Track analysis was performed on Applied Biosystems 420. The expected N-end sequence (MHVAQPAVVLA) was observed. Antibody demonstrated that the peptide was known by BNI3 anti-human CTLA-4 (Pharmingen).<sub>2</sub>O at 4 ° C for 9 days with stirring. All contents of the membrane bag were freeze-dried in powder.
(iii) 300.19 cells introduced by CTLA-4 (Y201V)
Full length CTLA-4 cDNA was chained from human mouse cDNA library (Stratagene) and subcloned into plRESneo (Clontech). Mutagenicity of CTLA-4, which resulted in unconditional cell surface expression, was introduced using the MatchMaker Mutagenesis System (Promega). The mutation of tyrosine, Y201 in the selection inhibits binding of the Adaptin protein, AP50, which is responsible for the rapid incorporation of CTLA-4 (Chuang et al., J. Immunol. 159: 144-151 (1997)). Berry-free 300,000 mouse lymphoma cells were cultured in RPMI-1640 containing 10% bovine fetal serum, non-essential amino acids, pensiline / streptomycin, 2 mM glutamine, 12.5 mM HEPES, pH 7.5, and 25 μM beta-mercaptoethanol. Cells were electrophoresed (3x10® / 0.4 mL serumless RPMI) in 1 mL chamber with 20 μg of CTLA-4-Y201V / plRESneo using 200V / 1180uF (Gibco CellPorator). Cells were incubated for 10 minutes and then suspended in 8 mL of pre-filled whole RPMI medium. At 48 hours, cells were diluted to 0.5 x 10<sup>6</sup>/ mL in a whole RPMI medium containing 1 mg / mL of G418 (Gibco). Vulnerable cells were resistant and demonstrated that they expressed CTLA-4 on the cell surface using the BNI3 antibody associated with algal mortality (Pharmingen). High-expression cells were isolated by sterile classification.
Immunization and production of primary cells: XenoMouse mice (8 to 10 weeks old) were immune (i) subcutaneous in the base of the trunk with 1 x 10<sup>7</sup> 300.19 cells introduced to express CTLA-4 as described above, resuspended in phosphate-doped saline (PBS) with complete Freund's adjuvant, or (ii) subcutaneously in the base of the trunk with (a) 10 μg of CTLA- 4 fusion protein or (b) 10 μg of CTLA-4 peptide, immersed in whole Freund's adjuvant. In each case, the dose of repeated commodity was boosted four times in non-complete Freund's adjuvant. Four days prior to fusion, mice received final injection of the antigen or cells in PBS. Miltitis and / or lymphocytes from immune mice were associated with mouse non-secretion of the multiple myeloma P3 cell line and were undergoing HAT selection as previously described (Galfre, G. and Milstein, C., "Preparation of monoclonal antibodies: strategies and procedures ", Methods Enzymol. 73: 3-46 (1981)).<sub>2</sub>K or lgG<sub>4</sub>K (as reported below) antibodies was recovered.
ELISU analysis: ELISA analysis for antigen-specific antigen-specific antibodies in mouse serum and IUD of primary cells was performed as described (Coligan et al., Unit 2.1, Enzyme-linked immunosorbent assays, I Current Protocols in Immunology ( 1994)) using pvl using CTLA-4-Ig fusion protein to capture the antibodies. For animals immunized with the CTLA-4-Ig fusion protein, we extrapolate for non-specific reactivity against the human Ig part of the fusion protein. This comes with a pvl using ELÍSU plates coated with human lgG1 as a negative benchmark for selectivity.
The following ELISA analysis is the following:
ELÍSU plates are coated with 100 μl / well of the antigen in the platelet coating (0.1 M carbonate, pH 9.6, and NaHCO<sub>3</sub> (MW 84) 8.4 g / L). Plug-in is the sieve of a 4 ° C crossover. After cultivation, skin scintillation is removed and the platelet is blocked with 200 pL / well of blocking buffer (0.5% BSA, 0.1% Tween-20, 0.01% Timerosal I 1 x PBS) and cultured vial room temperature for 1 hour. For a good many reasons, the plates are stored in a refrigerator with a blocking pad and a plate lid. Blockard duodenum is a scarlet and 50 pL / well of a hybridoma supernatant, a serum enhancer cell proliferative superficial (positive criterion) and a HAT mutagenic inhibitor blockade (negative criterion) is added. The plates are grown at room temperature for 2 hours. After cultivation, the plate is washed with washing cloth (1 x PBS). The detection antibody (peas, mouse anti-human lgG2-HRP (SB, # 9070-05) for lgG2 enhancement of mouse anti-human lgG4-HRP (SB # 9200-05) for lgG4 antibodies) is added for 100 pL / well (mouse antihuman lgG2-HRP @ 1: 2000 mouse mice anti-human lgG4-HRP @ 1: 1000 (whether diluted in blockade)). Pluturnareu range of room temperature for 1 hour and the filter is washed with washing cloth. Pvl next is 100 pL / well of fresh impregnation solution (10 mL of reagent buffer, 5 mg of OPD (o-Phenylenediamine; Sigma, Cat.No.-7288), and 10 pL of 30% H<sub>2</sub>O<sub>2</sub> (Sigma)) added to the wells. Plplt is allowed force induced I 10-20 minutes, pair to negative reference wells correctly start power display color. The pulp is 100 pL / well of stubborn solution (2 MH<sub>2</sub>SO<sub>4</sub>) are added and the plots are read on an ELISA-record reader with a wavelength of 490 nm.
Determination of Extreme Human Mabs with BIAcore:
Sickness measurement of purified human monoclonal antibodies, Fab fragments, or cytotoxicity cytosynthesis flasks was performed using the BIAcore 2000 device using general methods extracted by the manufacturer.
Immunoassay analysis of the antibodies was performed using an antigen that had been arrested on the low density nematode. Three surfaces of the BIAcore nematode were assayed with the CTLA-4-Ig fusion protein at densities ranging from about 390-900 using CTLA-4-Ig fusion protein at 20 or 50 μg / mL in 10 mM sodium acetate at pH 5 , 0 using the amine-cluster assembly from the manufacturer's analogue (BIAcore, Inc.). The fourth surface of the BIAcore nematic layer was confirmed by lgG1 (900 RU) and was used as a negative reference surface for non-specific binding. Reaction rate analysis was performed at a flow rate of 25 or 50 microns per minute and cleavage (kd or k<sub>off</sub>) and connection (ka or k<sub>on</sub>) faster was determined using the software from the manufacturer (BLA evaluation 3.0) that allows the aggregators of set calculations.
EXAMPLE 2
The affinity measurements of anti-CTLA-4 antibodies in the following table are given affinity measurements for certain of the antibodies selected in this way:
TABLE I
<td></td><td colspan="5">Solid Phase (from BIAcore)</td>
<td>cell</td><td>On-faster</td><td>From speed</td><td>Connections-</td><td>Klofnunar-</td><td>surface</td>
<td>hybrid</td><td>Ka</td><td>K<sub>d</sub></td><td>constant</td><td>constant</td><td>density</td>
<td></td><td>(M<sup>1</sup>S '<sup>1</sup>x10<sup>6</sup>)</td><td>(S'fylO ·<sup>4</sup>)</td><td>KA (1 / M) = k<sub>a</sub>/ k<sub>d</sub>x10<sup>1</sup>°</td><td>KD (Μ) = k<sub>d</sub>/ k<sub>a</sub>x10 '<sup>1</sup>°</td><td>[RU]</td>
<td>Moab01</td><td>0.68</td><td>1.01</td><td>0.67</td><td>1.48</td><td>878.7</td>
<td></td><td>0.70</td><td>4.66</td><td>0.15</td><td>6.68</td><td>504.5</td>
<td></td><td>0.77</td><td>6.49</td><td>0.19</td><td>8.41</td><td>457.2</td>
<td></td><td>0.60</td><td>3.08</td><td>0.20</td><td>5.11</td><td>397.8</td>
<td>4.1.1</td><td>1.85</td><td>0.72</td><td>2.58</td><td>0.39</td><td>878.7</td>
<td></td><td>1.88</td><td>1.21</td><td>1.55</td><td>0.64</td><td>504.5</td>
<td></td><td>1.73</td><td>1.54</td><td>1.13</td><td>0.88</td><td>457.2</td>
<td></td><td>1.86</td><td>1.47</td><td>1.26</td><td>0.79</td><td>397.8</td>
(Continued)
<td></td><td colspan="5">Solid Phase (from BIAcore)</td>
<td>cell</td><td>On-faster</td><td>From speed</td><td>Connections-</td><td>Klofnunar-</td><td>surface</td>
<td>hybrid</td><td>K<sub>a</sub></td><td>K<sub>d</sub></td><td>constant</td><td>constant</td><td>density</td>
<td></td><td>(M<sup>1</sup>S '<sup>1</sup>x10<sup>6</sup>)</td><td>(S'xW<sup>4</sup>)</td><td>KA (1 / M) = k<sub>a</sub>/ k<sub>d</sub>x10<sup>1</sup>°</td><td>KD (M) = k<sub>d</sub>/ k<sub>a</sub>x10 '<sup>1</sup>°</td><td>[RU]</td>
<td>4.8.1</td><td>0.32</td><td>0.07</td><td>4.46</td><td>0.22</td><td>878.7</td>
<td></td><td>0.31</td><td>0.23</td><td>1.33</td><td>0.75</td><td>504.5</td>
<td></td><td>0.28</td><td>0.06</td><td>4.82</td><td>0.21</td><td>397.8</td>
<td>4.14.3</td><td>2.81</td><td>3.04</td><td>0.92</td><td>1.08</td><td>878.7</td>
<td></td><td>2.88</td><td>3.97</td><td>0.73</td><td>1.38</td><td>504.5</td>
<td></td><td>2.84</td><td>6.66</td><td>0.43</td><td>2.35</td><td>457.2</td>
<td></td><td>3.17</td><td>5.03</td><td>0.63</td><td>1.58</td><td>397.8</td>
<td>6.1.1</td><td>0.43</td><td>0.35</td><td>1.21</td><td>0.83</td><td>878.7</td>
<td></td><td>0.46</td><td>0.90</td><td>0.51</td><td>1.98</td><td>504.5</td>
<td></td><td>0.31</td><td>0.51</td><td>0.61</td><td>1.63</td><td>457.2</td>
<td></td><td>0.45</td><td>0.79</td><td>0.57</td><td>1.76</td><td>397.8</td>
<td>3.1.1</td><td>1.04</td><td>0.96</td><td>1.07</td><td>0.93</td><td>878.7</td>
<td></td><td>0.95</td><td>1.72</td><td>0.55</td><td>1.82</td><td>504.5</td>
<td></td><td>0.73</td><td>1.65</td><td>0.44</td><td>2.27</td><td>457.2</td>
<td></td><td>0.91</td><td>2.07</td><td>0.44</td><td>2.28</td><td>397.8</td>
<td>4.9.1</td><td>1.55</td><td>13.80</td><td>0.11</td><td>8.94</td><td>878.7</td>
<td></td><td>1.43</td><td>19.00</td><td>0.08</td><td>13.20</td><td>504.5</td>
<td></td><td>1.35</td><td>20.50</td><td>0.07</td><td>15.20</td><td>397.8</td>
<td>4.10.2</td><td>1.00</td><td>2.53</td><td>0.39</td><td>2.54</td><td>878.7</td>
<td></td><td>0.94</td><td>4.30</td><td>0.22</td><td>4.55</td><td>504.5</td>
<td></td><td>0.70</td><td>5.05</td><td>0.14</td><td>7.21</td><td>457.2</td>
<td></td><td>1.00</td><td>5.24</td><td>0.19</td><td>5.25</td><td>397.8</td>
<td>2.1.3</td><td>1.24</td><td>9.59</td><td>0.13</td><td>7.72</td><td>878.7</td>
<td></td><td>1.17</td><td>13.10</td><td>0.09</td><td>11.20</td><td>504.5</td>
<td></td><td>1.11</td><td>13.00</td><td>0.09</td><td>11.70</td><td>397.8</td>
<td>4.13.1</td><td>1.22</td><td>5.83</td><td>0.21</td><td>4.78</td><td>878.7</td>
<td></td><td>1.29</td><td>6.65</td><td>0.19</td><td>5.17</td><td>504.5</td>
<td></td><td>1.23</td><td>7.25</td><td>0.17</td><td>5.88</td><td>397.8</td>
As can be seen, antibodies produced according to the invention will have high affinity and binding properties.
EXAMPLE 3
Construction of anti-CTLA-4 antibodies that are produced in combination with the invention
The following discussion provides structural information related to antibodies produced in accordance with the invention.
In order to detect the construction of antibodies produced by the invention, we cloned a gene encoding a heavy and light chain fragmented from a particular nucleotide gene. Recalculation and sequencing were achieved as follows:
Poly (A)<sup>+</sup> mRNA was isolated from approximately 2 X 10<sup>5</sup> cytotoxic cells derived from immune XenoMouse mice using a Fast-Track console (Invitrogen). The production of randomized cDNA was followed by chain regression. Human V<sub>H</sub> or human v<sub>K</sub> family-specific variable region indicators (Marks et al., "Oligonucleotide primers for polymerase chain reaction amplication of human immunoglobulin variable genes and design of family-specific oligonucleotide probes", Eur. J. Immunol. 21: 985-991 (1991)) or general human V<sub>H</sub> viscosity, MG-30 (CAGGTGCAGCT-GGAGCAGTCIGG) was used with combinations specific to the human Cy2 solid region (MG-40d; 5'-GCTGAGGGAGTAGAGTCCTGAGGA-3 ') or Ck solid region (ήκΡ2; as previously described in Green et al., 1994). Rows of human Mabs-derived heavy and capillary light chain transcripts from primary nuclei were obtained by direct sequencing of chain retardation products produced from poly (A)<sup>+</sup> mRNA using the indicators described above. Chain lining products were also cloned into pCRII using a TA clone assembly (Invitrogen) and both components were sequenced using Prism litaloka sequencing and ABI 377 sequencing devices. All rows were analyzed by comparison with the "V BASE sequence directory" (Tomlinson et al., MRC Center for Protein Engineering, Cambridge, UK) using MacVector and Geneworks software applications.
Furthermore, each of the antibodies 4.1.1, 4.8.1, 11.2.1, and 6.1.1 were subjected to full-length DNA sequencing. For such sequencing, poly (A)<sup>+</sup> mRNA isolated from approximately 4 X 10<sup>6</sup> cytotoxic cells using the mRNA Direct complex (Dynal). RNA was transcribed by using the oligo-dT (18) and the Advantage RT / PCR group (Clonetech). The V base was used to design the predictor signals that started at the ATG initial site in the heavy chain DP50 gene (5-TATCTAAGCTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3 ') and the stop sign in the lgG2 fixed region (5'-TTCTCTGATCAGAATTCCTATCA-TTTACCCGGAGACAGGGAGAGCT-3'). Choices of the Kozak series (ACCGCCACC) were added 5 'to the ATG initial set. The same method was used to designate the ATG initiation site of the kappa chain A27 gene (5'-TCTTCAAGCTTGCCCGGGCCCGCCACCATGGAAACCCCAGCG-CAG-3 ') and the stop sign on the cardiac fixed region (5'-TTCTTTGATCAGAATTCTCACT-AACACTCTCCCCTGTTGAAGC-3'). 012 cDNA was cloned using a reference for the ATG initial set (5'-TCTTCAAGCTTGCCCGGGCCCGCCACCATGGACATGAGGGTCCCC-GCT-3) and the capability of the permanent region cessation code above. Heavy chain cDNAs were also cloned as a genome construct with cytoplasmic mutation to add Nehl's end to the variable J region and subcloned Nhel fragment containing the gene lgG2 CH1 / heme / CH2 / CH3 regions. The point mutation to create a Nehl set does not change the amino acid sequence from the line of lineage. The indicators were used to amplify cDNA using the Advantage High Fidelity PCR Component (Clonetech). The sequence of the chain assignment was obtained by direct sequencing using litaloka sequencing and ABI sequencing devices. The chain data product was cloned into pEE glutamin lipase mammalian expression vector (Lonza) and three clones were sequenced to confirm physical mutations. For each clone, the sequence was confirmed for both factors in at least three instances. Olycosylated 4.1.1 antibodies were produced by the cytotoxic mutation of N294Q mutation in the CH2 region. Racial antibodies were produced by transient induction of Cos7 cells in IgG-reduced FCS and purified using standard Protein A Sepharose methods. Stable introductions were produced by electrolyte on mouse NSO cells and selection in glutamine-free medium. Assay 4.1.1 with or without glycosylation showed similar selectivity and affinity in CTLA-4 in the ELISA and BIAcore experiments. For each clone, the sequence was confirmed for both factors in at least three instances. Olycosylated 4.1.1 antibodies were produced by the cytotoxic mutation of N294Q mutation in the CH2 region. Racial antibodies were produced by transient induction of Cos7 cells in IgG-reduced FCS and purified using standard Protein A Sepharose methods. Stable introductions were produced by electrolyte on mouse NSO cells and selection in glutamine-free medium. Assay 4.1.1 with or without glycosylation showed similar selectivity and affinity in CTLA-4 in the ELISA and BIAcore experiments. For each clone, the sequence was confirmed for both factors in at least three instances. Olycosylated 4.1.1 antibodies were produced by the cytotoxic mutation of N294Q mutation in the CH2 region. Racial antibodies were produced by transient induction of Cos7 cells in IgG-reduced FCS and purified using standard Protein A Sepharose methods. Stable introductions were produced by electrolyte on mouse NSO cells and selection in glutamine-free medium. Assay 4.1.1 with or without glycosylation showed similar selectivity and affinity in CTLA-4 in the ELISA and BIAcore experiments. Racial antibodies were produced by transient induction of Cos7 cells in IgG-reduced FCS and purified using standard Protein A Sepharose methods. Stable introductions were produced by electrolyte on mouse NSO cells and selection in glutamine-free medium. Assay 4.1.1 with or without glycosylation showed similar selectivity and affinity in CTLA-4 in the ELISA and BIAcore experiments. Racial antibodies were produced by transient induction of Cos7 cells in IgG-reduced FCS and purified using standard Protein A Sepharose methods. Stable introductions were produced by electrolyte on mouse NSO cells and selection in glutamine-free medium. Assay 4.1.1 with or without glycosylation showed similar selectivity and affinity in CTLA-4 in the ELISA and BIAcore experiments.
Genanotkunarqreiningar
The following table depicts the gene utilization seen by selected cytokine receptor antibodies in accordance with the invention:
TABLE II
<td colspan="7">Thunq and low-key power consumption</td>
<td rowspan="2">clone</td><td colspan="3">heavy Chain</td><td></td><td colspan="2">Kappa light chain</td>
<td>VH</td><td>D</td><td>JH</td><td></td><td>VK</td><td>JK</td>
<td>4.1.1</td><td>DP-50</td><td>DIR3 DIR3</td><td>JH4</td><td></td><td>A27</td><td>JK1</td>
<td>4.8.1</td><td>DP-50</td><td>7-27</td><td>JH4</td><td></td><td>A27</td><td>JK4</td>
<td>4.14.3</td><td>DP-50</td><td>7-27</td><td>JH4</td><td></td><td>A27</td><td>JK4 JK3</td>
<td>6.1.1</td><td>DP-50</td><td>DIR5 or DIR5rc</td><td>JH4</td><td></td><td>A27</td><td>JK3</td>
<td>3.1.1</td><td>DP-50</td><td>3-3</td><td>JH6</td><td></td><td>012</td><td>JK3</td>
<td>4.10.2</td><td>DP-50</td><td>7-27</td><td>JH4</td><td></td><td>A27</td><td>JK3</td>
<td>2.1.3</td><td>DP-65</td><td>1-26</td><td>JH6</td><td></td><td>A10 / A26</td><td>JK4</td>
<td>4.13.1</td><td>DP-50</td><td>7-27</td><td>JH4</td><td></td><td>A27</td><td>JK3</td>
<td>11.2.1</td><td>DP-50</td><td>D1-26</td><td>JH6</td><td></td><td>012</td><td>JK3</td>
<td>11.6.1</td><td>DP-50</td><td>D2-2 or D4</td><td>JH6</td><td></td><td>012</td><td>JK3</td>
<td>11.7.1</td><td>DP-50</td><td>D3-22 or D21-9</td><td>JH4</td><td></td><td>012</td><td>JK3</td>
<td>12.3.1.1</td><td>DP-50</td><td>D3-3 or DX4</td><td>JH6</td><td></td><td>A17</td><td>JK1</td>
<td>12.9.1.1</td><td>DP-50</td><td>D6-19</td><td>JH4</td><td></td><td>A3 / A19</td><td>JK4</td>
<td>4.9.1</td><td>DP-47</td><td>5-24 and / or 6-19</td><td>JH4</td><td></td><td>L5</td><td>JK1</td>
As demonstrated, antibodies were produced with a strong tendency for utilization of the DP-50 heavy chain variable region. Reference is also made to the DP-50 genre as V<sub>H</sub> 3-33 family history. Only one antibody that was valid on the basis of CTLA-4 binding and transient activity assays showed a heavy chain gene utilization other than DP-50. That clone, 2.1.3, uses a DP-65 heavy chain variable region and is lgG4 isotope. Reference is also made to the DP-65 genre as V<sub>H</sub> 4-31 family gene. On the other hand, Clause 4.9.1, which has a DP-47 heavy chain variable region, inhibits CTLA-4 but does not inhibit binding to B7-1 or B7-2. In XenoMouse mice there are more than 30 distinct active heavy chain variable genes to produce multiple species. Hneigf gives a preferable binding modulus of the antibody-antigen interaction with respect to the combined properties of the binding of the antigen and activity.
Stflkkbreytingargreining
As will delimit the understanding, the gene use analysis provides a comprehensive overview of the complexity structure. Couples whose B cells ί XenoMouse animals proliferate VDJ heavy-chain VJ coat light-chain transcript in a randomized manner, derived from derived pathways, including the following without limitation, physical supersonic mutations, n-additions, and CDR3 prolongations. See, daemis, Mendez et al., Nature Genetics 15: 146-156 (1997), and U.S. Patent Numbers. 08 / 759,620, shrinking December 3, 1996. According to the invention, for proliferation, further metabolic sites were predicted amino acid residues of the antibodies produced from the cDNA obtained from the clones. Additionally, N-terminal amino acids were obtained in protein sequencing.
Figure 1 gives a nucleotide and predicted amino acid sequence of the lung and capillary light chains from clones 4.1.1 (Figure 1A), 4.8.1 (Figure 1B), 4.14.3 (Figure 1C), 6.1.1 (Figure 1D), 3.1.1 (Figure 1D), 4.10.2 (Figure 1F), 2.1.3 (Figure 1G), 4.13.1 (Figure 1H), 11.2.1 (Figure 11), 11.6.1 (Figure 1J), 11.7.1 Figure 1K), 12.3.1.1 (Figure 1L), and 12.9.1.1 (Figure 1M). In Figs. 1A, 1B, and 1D, the length of the cells of the antibodies 4.1.1, 4.8.1, and 6.1.1 with full length cloning of the cDNA was obtained as described above. On the flip charts, the tag peptide sequence (enhancing the bases of the other one) is given by fatigue, and the luminous cells used for the 5 'chain reference reaction are sub-charts.
Figure 2 gives a correlation between predicted heavy chains of amino acid residues from clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1. , 12.3.1.1, and 12.9.1.1 and the kimline DP-50 (3-33) amino acid residue. The variability between the DP-50 chime line and the sequence in the clones is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the shaded antibodies.
Figure 3 gives a correlation between the predicted chain of chains of the amino acid site from clone 2.1.3 and the chimeric DP-65 (4-31) amino acid residue. The variability between the DP-65 line of lines and the sequence in the clone is indicated by fatigue. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 4 provides a regeneration between the predicted capillaries of the light chain amino acid residue from clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 4.10.2, and 4.13.1 and the A3 amino acid sequence line. The variability between the A27 acne line and the clone sequence is indicated by boldness. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined. Destruction seen in CDR1 in clones 4.8.1, 4.14.3, and 6.1.1 is indicated by "0".
Figure 5 gives a correlation between the predicted capillaries of the light chain amino acid sequence from clones 3.1.1, 11.2.1, 11.6.1, and 11.7.1 and the germline 012 amino acid residue. The variability between 012 kim lines and the clone sequence is indicated by bold. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 6 gives a correlation between the predicted capillary light chain amino acid sequence from clone 2.1.3 and the A10 / A26 amino acid sequence line. The variability between the A10 / A26 acne line and the clone sequence is indicated by boldness. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 7 gives a correlation between predicted capillaries of the light chain amino acid site from clonin 12.3.1 and the line A17 amino acid sequences. The variability between the A17 and the sequence of the clone is indicated by boldness. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 8 gives a correlation between the predicted capillaries of the light chain amino acid site from clone 12.9.1 and the line A3 / A19 amino acid residue. The variability between the A3 / A19 line of lines and the sequence of the clone is indicated by boldness. The figure also shows the position of the CDR1, CDR2, and CDR3 sequences in the antibody as underlined.
Figure 22 provides a class of additional nuclei and amino acid sites of the following anti-CTLA-4 antibody chains:
4.1.1:
full lengths 4.1.1 heavy chain (cDNA 22 (a), gene 22 (b), and amino acid 22 (c)); full length non-glycosylated 4.1.1 heavy chain (cDNA 22 (d) and amino acid 22 (e));
4.1.1 light chain (cDNA 22 (f) and amino acid 22 (g));
4.8.1:
full length 4.8.1 heavy chain (cDNA 22 (h) and amino acid 22 (i));
4.8.1 light chain (cDNA 22 (j) and amino acid 22 (k));
6.1.1:
full lengths 6.1.1 heavy chain (cDNA 22 (1) and amino acid 22 (m));
6.1.1 light chain (cDNA22 (n) and amino acid 22 (o));
11.2.1:
full length 11.2.1 heavy chain (cDNA 22 (p) and amino acid 22 (g)); and
11.2.1 light chain (cDNA 22 (r) and amino acid 22 (s)).
Markup patterns are shown in bold and large text. The full-length open-frame frames 4.1.1 of the DNA sequence (Figure 22 (b)) are underlined. And, the mutations introduced to make the non-glycosylated 4.1.1 heavy chain and the release (N294Q) are shown by double underline and bold text (cDNA (Figure 22 (b)) and amino acid (Figure 22 (c)).
Example 4
Analysis of low and light chain amino acid metabolism
Figure 2, which gives a serial comparison, predicts the heavy chain amino acid residues from clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1 and the kimline DP-50 (3-33) amino acid residue, an interesting pattern appears. In addition to the fact that the PD-50 heavy chain inhibitor in the majority of clones is a relatively limited hyperchromic change in the antibodies relative to the chimpanzee DP-50 gene. For example, clones 3.1.1 and 11.2.1 have no mutations. Furthermore, the mutations in the other clones are generally subject to changes in the metabolism of amino acids with similar properties to the amino acids in the germ line. Mutations within many of the CDR1 and CDR2 sequences are especially hereditary in nature. Three of the heavy chains shown in Figures 2, 4.10.2, 4.13.1, and 4.14.3, are clearly derived from one reposition event (ie, derived from one end center) and are almost identical in sequence. If these three are considered as single sequences, among the 10 different antibodies containing the DP-50 heavy chain, CDR1 and CDR2 contain 3 positions where the non-polar residue is replaced by another non-polar residue, 12 where the polar unladen residue is replaced by other polar unloaded residues, and 1 in which the polar loaded residual is replaced by another polar charged residual. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers. derived from one end center) and are almost identical in sequence. If these three are considered as single sequences, among the 10 different antibodies containing the DP-50 heavy chain, CDR1 and CDR2 contain 3 positions where the non-polar residue is replaced by another non-polar residue, 12 where the polar unladen residue is replaced by other polar unloaded residues, and 1 in which the polar loaded residual is replaced by another polar charged residual. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers. derived from one end center) and are almost identical in sequence. If these three are considered as single sequences, among the 10 different antibodies containing the DP-50 heavy chain, CDR1 and CDR2 contain 3 positions where the non-polar residue is replaced by another non-polar residue, 12 where the polar unladen residue is replaced by other polar unloaded residues, and 1 in which the polar loaded residual is replaced by another polar charged residual. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers. among the 10 different antibodies containing the DP-50 heavy chain, CDR1 and CDR2 positions 3 positions where the polar residue is replaced by another non-polar residue, 12 where the polar unladen residue is replaced by another polar unladen residue, and 1 there as a polarized loaded residue is replaced by another polar loaded residue. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers. among the 10 different antibodies containing the DP-50 heavy chain, CDR1 and CDR2 positions 3 positions where the polar residue is replaced by another non-polar residue, 12 where the polar unladen residue is replaced by another polar unladen residue, and 1 there as a polarized loaded residue is replaced by another polar loaded residue. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers. and 1 in which the polar loaded residual is replaced by another polar loaded residue. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers. and 1 in which the polar loaded residual is replaced by another polar loaded residue. Furthermore, two positions are pairs of which two residues that are very similar to structural, glycine and alanine, are replaced by each other. The only mutations that do not completely override turn to 3 polarized polarized residues for polar unladen residues and one shift of uncharted leftovers for polar leavers.
The light chains in these antibodies are derived from 5 different Vk genes. The A27 gene is most common and is the source of 6 different light chains. Comparing these 6 ranks reveals two interesting points. First, three of them, 4.8.1.4.14.3, and 6.1.1, contain deletions on one or two residues in CDR1, which is a rare event. Secondly, there are high levels of prejudice against the serotype serine in CDR3, so that the serine has been replaced in each order. This indicates that serine in this position is incompatible with CTLA-4 binding.
It will be understood that many of the amino acid shifts identified above are in close proximity to or within the CDR. Such a replacement would seem to have some effect on the binding of the antibody to the CTLA-4 molecule. Furthermore, such a change could have a significant effect on the affinity of the antibodies.
EXAMPLE 5
N-terminal amino acid sequencing of antibodies according to the invention
In order to further confirm the composition and structure of the antibodies according to the invention, identified above, sequenced by certain of the antibodies using Perkin-Elmer sequences. Both heavy and cardiovascular light chains in the antibodies were isolated and purified by the use of anatomic gel electrophoresis and electrophoresis methods and then rabbit straight as described in Example 6. The majority of the heavy chains of the sequences were blocked on their aminoids. Therefore, antibodies were first treated with pyroglutamate aminopeptidase and subsequently sequenced.
The results of this experiment are shown in Figure 9. Figure 9 also gives the molecular weight on the heavy and light chains as determined by mass analysis (MALDI).
EXAMPLE 6
Additional identification of antibodies
Figure 10 provides certain additional identification information for certain antibodies. A picture is compiled data related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.14.3, and 6.1.1. The following data are given: intensity, electrophoresis (IEF), SDS-PAGE, size-dependent chromatography, FACS, mass analysis (MALDI), and light chain N-terminals.
Generally, the data was created as follows:
Materials and methods
Protein stability was determined at 280 nm from UV screening (200-350 nm), with 1.58 absorbent units at 280 nm equal to 1 mg / mL.
SDS-PAGE was developed using the Novex NuPAGE electrode system with 10% NuPAGE gel and MES runtime. Samples were prepared by diluting 3: 1 with 4x NuPAGE viscera (+/- beta-mercaptoethanol), heated and ~ 5μg of protein were added to the gel. The gel was then stained with Brilliant Blue R Coloring Solution (Sigma, Cat # B-6529) and the measurement of molecular size was then done by wearing a colored bond with Perfect Protein Markers (Novagen, Cat # 69149-3).
For N-end sequencing, samples were run as above on NuPAGE gel, transferred to Pro Blot (Applied Biosystems), then stained with Coomassie
Blue R-250. The colored ribbons were cut off and subjected to sequencing using automatic Edman degradation on the Applied Biosystems 494 Precise HT Serial Detector.
Battery fusion (IEF) was performed using Pharmacia IEF 3-9 Phast Gels (Cat # 17-0543-01). Samples were diluted in 10% glycerol to 0.8 mg / mL and 1 μL was loaded onto the gel and then silver-colored. The evaluation was done by comparing the colored bands to a wide range (pH 3-10) of IEF standards (Pharmacia, Cat, # 17-0471-01).
High-resolution chromatography (SEC) was performed in phosphate-buffered saline (PBS) on the Pharmacia SMART system using the Superdex 75 PC 3.2 / 30 column. Molecular size estimation was done by comparing the retention time peak to the retention time of the gel.
For FACS studies, human peripheral T-cells were prepared and stimulated for 48 hours. T-cells were washed once, resuspended ί FACS dúa at 1 x 10<sup>6 </sup>cells / 100 pL and coloring for CD3 surface expression with 10 pL of anti-CD3-FITC (Immunotech, Marseille, France) for 30 minutes at room temperature. Cells were washed twice, then attached, Fixed and Perm, Caltag, and color cells for intracellular CTLA-4 expression with 10 pL of anti-CD152-PE (Pharmingen). Fluid cell measurement was performed using Becton Dickinson FACSort. Quarters were set by analysis of the relevant cytotoxic reference target (Caltag).
As discussed above, anti-CTLA-4 antibodies have been shown to have a certain potent immunosuppressive activity. The following experiments were performed to determine if antibodies according to this invention thus had activity. In general, experiments were designed to evaluate the ability of the antibodies to inhibit the interaction between CTLA-4 and B7 molecules, preferably between CTLA-4 and B7 molecules and CD28, and promote proliferation of T-cell cytokines, in number, but non limiting factor IL-2 and / enhancing IFN-γ expression. Further, a study of cross-reactivity antibodies from the invention was found in certain human tissues and CTLA-4 molecules of several species (e.g., mouse and primate).
EXAMPLE 7
ELÍSA Competition: Obstruction of CTLA-4 / B7-1 or B7-2 Adverse Drug Reaction with the Invention of the Invention
An analysis was performed to determine whether antibodies in accordance with this invention possess a force inhibiting the binding of CTLA-4 to either B7-1 enhancing B7-2. As understood, it would be expected that antibodies from the present invention capable of inhibiting the binding of CTLA-4 to the B7 molecule would be a candidate for immunodeficiency of CTLA-4 cytokines. The analysis included the following materials and uses:
Materials and Methods nM B7-1-Ig (G1) or B7-2-Ig (G1) (Repligen, Inc., Needham, MA) In Dulbecco's
PBS was coated on 96-well MaxiSorp pulp (Nunc, Denmark, # 439454) and cultured with
4 ° C overnight. On day 2, B7-Ig was removed and pituitary gland blocked with 1% BSA plus 0.05%
Tween-20 in D-PBS for two hours. Plates were washed 3X with washing cloth (0.05%
Tween-20 in D-PBS). Antibodies in potent experimental concentrations and CTLA-4-Ig (G4) (0.3 nM final potency) (Repligen, Inc., Needham, MA) were premixed for 15 minutes and then added to the B7-lg coated plate (60 μL total volume) and grown at room temperature for 1.5 hours. Plates were washed 3X and 50 pL of 1 to 1000 dilutions of HRP-labeled mouse humanity lgG4 mite (Zymed, San Francisco, CA, # 05-3820) were added and grown at room temperature for one hour. Peptides were purged 3X and 50 pL of TMB Microwell peroxidase reagent (Kirkegaard & Perry, Gaithersburg, MD, # 50-76-04) were added and incubated at room temperature for 20 minutes, and the sifian was 50 pL of 1N H<sub>2</sub>SO<sub>4</sub> bait out in the pads. Poultry was read 450 nm by using Molecular Devices plfitulesara (Sunnyvale, CA). Oil samples were tested in duplicate. The maximum mark was determined as CTLA-415 μg binding in the absence of experimental antibody. Non-specific binding was defined as absorption in the absence of CTLA-4-Ig and experimental compound.
The results of the analysis are given in Tables IIIA and IIIB. Table IIIA shows the results for a range of mitics. In FIG. IIIB, there are shown a number of pairs that are bored together with a 4.1.1 microfibre 11.2.1 synthesized from the invention in a gradual experiment.
TABLE IIIA
<td>Klfin CTLA-4-Ig</td><td>isotype</td><td>CTLA-4 / B7.2 Assembly. ELISA IC50 (nM)</td><td>CTLA-4 / B7.1 Assembly. Elisa IC50 (nM)</td>
<td>CT3.1.1</td><td>IgG2</td><td>0.45 ± 0.07 (n = 3)</td><td>0.63 ± 0.10 (n = 2)</td>
<td>CT4.1.1</td><td>IgG2</td><td>0.38 ± 0.06 (n = 3)</td><td>0.50 ± 0.05 (n = 2)</td>
<td>CT4.8.1</td><td>IgG2</td><td>0.57 ± 0.03 (n = 3)</td><td>0.17 ± 0.28 (n = 2)</td>
<td>CT4.9.1</td><td>IgG2</td><td>Non-competition (n = 3)</td><td>Non-competition (n = 2)</td>
<td>CT4.10.2</td><td>IgG2</td><td>1.50 ± 0.37 (n = 3)</td><td>3.39 ± 0.31 (n = 2)</td>
<td>CT4.13.1</td><td>IgG2</td><td>0.49 ± 0.05 (n = 3)</td><td>0.98 ± 0.11 (n = 2)</td>
<td>CT4.14.3</td><td>IgG2</td><td>0.69 ± 0.11 (n = 3)</td><td>1.04 ± 0.15 (n = 2)</td>
<td>CT6.1.1</td><td>IgG2</td><td>0.39 ± 0.06 (n = 3)</td><td>0.67 ± 0.07 (n = 2)</td>
TABLE IIIB
<td>Klfin CTLA-4-Ig</td><td>isotype</td><td>CTLA-4 / B7.2 Assembly. ELTs IC50 (nM)</td><td>CTLA-4 / B7.1 Assembly. ELISA IC50 (nM)</td>
<td>CT4.1.1</td><td>IgG2</td><td>0.55 ± 0.08 (n = 4)</td><td>0.87 ± 0.14 (n = 2)</td>
<td>CT11.2.1</td><td>IgG2</td><td>0.56 ± 0.05 (n = 4)</td><td>0.81 ± 0.24 (n = 2)</td>
EXAMPLE 8
Selective ratios of antibodies of the invention with respect to CTLA-4 against either
CD28 or B7-2
Another experimental assay was performed to determine selective antibodies from the invention with respect to CTLA-4 versus either CD28 or B7-2. The following materials and methods were used in the experiments:
CTLA-4 Selective ELISA: Materials and Methods
96-well Fluor-NUNC Plate (Nunc, Cat. No. 475515) was coated with four antigen: CTLA-4 / Ig, CD44 / Ig, CD28 / Ig, and B7-2 / Ig (Internal Immunogens) . The antigens were plated overnight at + 4 ° C at 1 μg / mL 100 μL / well in 0.1 M sodium bicarbonate buffer, pH 9.6. The plate was then washed with PBST (PBS + 0.1% Tween-20) three times using a NUNC plate dishwasher. Platelet was blocked with PBST + 0.5% BSA at 150 pL / well. The plate was grown at room temperature for 1 hour, then washed with PBST three times. Next, the anti-CTLA-4 antibodies from the invention were diluted to a block at 1 μg / mL and added to the plate. The plate was grown at room temperature for 1 hour and then washed with PBST three times. The wells containing the antibodies from the invention were then treated with 100 μL / well of anti-human lgG2-HRP (Southern Biotech Cat No.9070-05) at 1: 4000 dilution in a block. Also, one series was treated with anti-human IgG (Jackson Cat No. 209-035-088) to standardize for platelet coating. This antibody was diluted 1: 5000 in a block and added to 100 pL / well. Also, one series was treated with anti-human CTLA-4-HRP (Pharmingen Cat No. 345815 / Custom HRP conjugated) as a positive benchmark. This antibody was used at 0.05 μg / mL diluted in a block. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time. 9070-05) at 1: 4000 dilution in block. Also, one series was treated with anti-human IgG (Jackson Cat No. 209-035-088) to standardize for platelet coating. This antibody was diluted 1: 5000 in a block and added to 100 pL / well. Also, one series was treated with anti-human CTLA-4-HRP (Pharmingen Cat No. 345815 / Custom HRP conjugated) as a positive benchmark. This antibody was used at 0.05 μg / mL diluted in a block. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time. 9070-05) at 1: 4000 dilution in block. Also, one series was treated with anti-human IgG (Jackson Cat No. 209-035-088) to standardize for platelet coating. This antibody was diluted 1: 5000 in a block and added to 100 pL / well. Also, one series was treated with anti-human CTLA-4-HRP (Pharmingen Cat No. 345815 / Custom HRP conjugated) as a positive benchmark. This antibody was used at 0.05 μg / mL diluted in a block. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time. This antibody was diluted 1: 5000 in a block and added to 100 pL / well. Also, one series was treated with anti-human CTLA-4-HRP (Pharmingen Cat No. 345815 / Custom HRP conjugated) as a positive benchmark. This antibody was used at 0.05 μg / mL diluted in a block. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time. This antibody was diluted 1: 5000 in a block and added to 100 pL / well. Also, one series was treated with anti-human CTLA-4-HRP (Pharmingen Cat No. 345815 / Custom HRP conjugated) as a positive benchmark. This antibody was used at 0.05 μg / mL diluted in a block. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time. The plate was grown at room temperature for 1 hour and then washed with PBST three times. LBA Emission Detergent (Pierce) was added at 100 pL / well and the plate was grown on a plate shaker for 5 min. The plate was then read using an icing camera for 2 minutes. exposure time.
AGAINST CTLA-4-LG Selective Binding: Materials and Methods
M-450 Dynabeads (Dynal AS, Oslo, Norway, # 140.02) were washed 3X with Na phosphate buffer, pH 7.4, and resuspended in Na-phosphate buffer. 1.0 μg of CTLA-4-Ig (G1), 1.0 μg of CD28-Ig (G1) or 1.0 to 3.0 μg of B7-2-Ig (G1) (Repligen, Inc., Needham , MA) was bait into 100 pL of pearls and grown overnight at a rotary device at 4 ° C. On day 2, the beads were washed 3X in 1% BSA plus 0.05% Tween-20 in Dulbecco's PBS and blocked for 30 minutes. Pearls were diluted 1 to 10 with blocking pad and 25 μL of the coated beads was added to 12x75 mm polypropylene solutions. All samples were tested in duplicate. 50 pL of experimental antibody (1 μg / mL final strength) or blockade buffer was added to glass and grown for 30 minutes
Origen 1.5 Analyze the ring chain (IGEN International, Inc., Gaithersburg, MD) at room temperature, shaking at 100 rpm. 25 pL of ruthenylated mouse anti-human lgG1, IgG2 or IgG4 (Zymed, Inc., San Francisco, CA, # 05-3300, 05
3500 and 05-3800) (final concentration of 3 μg / mL in 100 μL total volume) was added to the vials. Glasses were incubated for 30 minutes at room temperature on the ring cake while shaking at 100 rpm. 200 pL of Origen analysis tubes (IGEN International, Inc., Gaithersburg, MD, # 402-050-03) were added to each glass and shaken briefly and then the glasses were considered in Origen Analyzer and ECL (electrolytic) units were determined for every glass. Sequencing factors were determined to correct for different binding of fusion proteins to Dynabeds, and ECL units were corrected for non-specific binding before selectivity ratios were calculated.
The results of the analyzes are given in Tables IVA and IVB.
TABLE IVA
<td>clone</td><td>isotype</td><td>CTLA-4 / CD28 the eldest</td><td>CTLA-4 / B7.2 Elisa</td><td>CTLA-4 / CD44 Elisa</td><td>CTLA-4 / CD28 Heiligenstadt</td><td>CTLA-4 / B7.2 Heiligenstadt</td>
<td>3.1.1</td><td>IgG2</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 2)</td><td>> 500: 1 (n = 1) 195: 1 (n = 1)</td>
<td>4.1.1</td><td>IgG2</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 2) 485: 1 (n = 1)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 1) 261: 1 (n = 1)</td><td>> 500: 1 (n = 1) 107: 1 (n = 1)</td>
<td>4.8.1</td><td>IgG2</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 2) 190: 1 (n = 1)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 2)</td><td>> 500: 1 (n = 2)</td>
<td>4.9.1</td><td>IgG2</td><td>> 500: 1 (n = 2) 244: 1 (n = 1)</td><td>> 500: 1 (n = 2) 33: 1 (n = 1)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 1)</td><td>> 500: 1 (n = 1)</td>
<td>4.10.2</td><td>IgG2</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 1)</td><td>> 500: 1 (n = 1)</td>
<td>4.13.1</td><td>IgG2</td><td>> 500: 1 (n = 2) 46: 1 (n = 1)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 1) 329.Ί (n = 1)</td><td>> 500: 1 (n = 2)</td>
<td>4.14.3</td><td>IgG2</td><td>> 500: 1 (n = 2) 80: 1 (n = 1)</td><td>> 500: 1 (n = 2) 10: 1 (n = 1)</td><td>> 500: 1 (n = 2) 126: 1 (n = 1)</td><td>> 413: 1 (n = 1)</td><td>> 234: 1 (n = 1)</td>
<td>6.1.1</td><td>IgG2</td><td>> 500: 1 (n = 2) 52: 1 (n = 1)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 2)</td><td>> 500: 1 (n = 2)</td>
TABLE IVB
<td>clone</td><td>Samsætugerö</td><td>CTLA-4 / CD26 the eldest</td><td>CTLA-4 / B7-2 Elisa</td><td>CTLA-4 / hlgG Elisa</td>
<td>4.1.1</td><td>IgG2</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 2)</td><td>> 500: 1 (n = 3)</td>
<td>11.2.1</td><td>IgG2</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td><td>> 500: 1 (n = 3)</td>
EXAMPLE 9
Human T-cellular mercury
In order to further define the efficacy of antibodies in combination with the invention to act as immunosuppressants, we developed a certain T-cell analysis to quantify the increase in
T-cell IL-2 proliferation in the blockade of CTLA-4 signals with the antibodies. The following topics and forces were used in conjunction with the experimental experiments:
Materials and methods
Newly introduced human T cells were prepared using Histopaque (Sigma, St. Louis, MO, # A-70543) and T-Kwik (Lympho Kwik, One Lambda, Canoga Park, CA, # LK-50-T) , and stimulated with PHA (1 μg / mL) (Phytohemaglutin Cleanse, Murex Diagnostics, Ltd., Dartford, England, #HA 16) in miflli (RPMI-1640 containing L-glutamine, MEM non essential amino acids, pensiline, streptomycin , 25 mM HEPES and 10% FBS) concentration of 1x10<sup>6</sup> cells / mL and cultured 37 ° C for 2 days. Cells were washed and diluted in miflli ί 2x10<sup>6</sup> cells / mL. Rajifrumur (Burkitt lymphoma, Human ATCC No.: CCL 86 Class II, American Type Culture Collection, Rockville, MD) were treated with Mitomycin C (Sigma, St. Louis, MO, # M-4287) (25 μg / mL) one hour breeze 37 ° C. The raisins were washed 4X ί PBS and resuspended fan 2x10<sup>6 </sup>cells / mL. Human T-germ cells (5x10<sup>s</sup>/ mL), Rajifrumum (5x10<sup>5</sup>/ mL) and anti-CTLA-4 molecules enhancing cytotoxic equivalent of different concentrations were charged to 96-well microplate plates and the plates were cultured for 37 ° C 72 hours. The total volume of each well was 200 pL. Seventy-one hours after flushing, the plots spun nifles and flotifl were absent and frozen for subsequent determination of IL-2 (Quantikine IL-2 ELISA Kit, R & D Systems, Minneapolis, MN, # D2050) and IFN-γ (Guantikine IFN-γ ELISA Kit , R & D Systems). An increase in cytoplasmic population was defined as the difference between cytotoxicity in cultures containing anti-CTLA-4 blocking mAb versus cytosynthesis-relevant reference antibodies. For flow cytometry experiments, Rajifrums were washed 1x with FACS duodenum (PBS containing 2% heat-activated FCS, 0.025% sodium azide).<sup>s </sup>cells / 100 pL and cultured with 10 pL of anti-CD80-PE (Becton Dickinson, Sam Jose, CA) promotes anti-CD86-PE (Pharmingen, San Diego, CA) for 30 minutes at room temperature. Cells were washed twice and resuspended 1 mL of FACS buffer. Fluid cell proliferation was performed by force using Becton Dickinson FACSort. Stufllarit labels were included in the analysis of the various cytotoxic reference targets (Caltag, Burlingame, CA).
Generally, we have developed a possible analysis that can be used for rapid determination of T-cell IL-2 boost. As understood, the stimulation of T cells depends on B7 and CD28. Moreover, washed Τ-germ cells do not detect IL-2, and β-cells do not detect IL-2 even when they are interfering with LPS enhancing PWM. However, in combination, T-germ cells grown with Rajifrumum can model B7, CTLA-4, and CD28 marker cells, and the effect can be estimated by the effects of these.
Figure 11 shows the expression of B7-1 pg B7-2 on Rajifrums using andCD80-PE and anti-CD86-PE mAbs using flow cytometry (FACS) as described in Example 6.
Figure 12 shows the potent enhancement of IL-2 survival in the T-lymphoma / Raji assay induced by CTLA-4 blocking antibodies (BNI3 (Pharmingen), and 4.1.1.4.8.1, and 6.1.1, the antibodies of the invention).
Figure 13 shows the potent enhancement of the IFN-γ survival in the T-lymphoma / Raji assay induced by CTLA-4 blocking antibodies (BNI3 (Pharmingen), and 4.1.1.4.8.1, and 6.1.1 antibodies of the invention) ( same T-cell donor).
Figure 14 shows the mefial enhancement of IL-2 survival I T cells from 6 donors induced by CTLA-4 blocking antibodies in the T-cell / Raji assay. Ahugavert is aware that mAb-ifi, CT4.9.1, binds to CTGA-4 vivo but does not block B7 binding. Pannig, simply grandmother CTLA-4 is not enough In itself, an effective antibody of the invention is given.
Figure 15 shows the mefial enhancement of IFN-γ survival in T cells from 6 donors induced by CTLA-4 blocking antibodies in the Τ-lymphoma / Raji assay.
Figure 19 shows the comparison between 4.1.1 and 11.2.1 of the antibodies of the invention in terms of the 30 μg / mL 72 hours T-germ / Raji assay, as described in Example 9, and the supernatant assay described in Example 10 .
Figure 20 shows the potency of enhancement of IL-2 survival in the T-lymphoma / Raji assay produced by 4.1.1 and 11.2.1 of the CTLA-4 antibodies of the invention.
The following table IVC provides information related to a significant increase and increased increase in the cellular response I Raji and SEA analysis of the invention. Each of the experiments taken in the results is based on an antibody at a dose of 30 pg / mL and measured for 72 hours. The number of donors as users are in the experiments and also the responses are shown.
TABLE IVC
<td>Analysis</td><td>mAb</td><td>Frumubofii</td><td>Mefial increase pg / mL</td><td>SEM</td><td>soaring increase pg / mL</td><td>n</td><td>responses gift</td>
<td>T blasts / Raji</td><td>4.1.1</td><td>IL-2</td><td>3329</td><td>408</td><td>0 to 8861</td><td>42</td><td>19 of 21</td>
<td>T-kim cell / Raji</td><td>4.1.1</td><td>IFN-γ</td><td>3630</td><td>980</td><td>600 to 13939</td><td>17</td><td>13 of 13</td>
<td>T-klmfrumu / Raji</td><td>11.2.1</td><td>IL-2</td><td>3509</td><td>488</td><td>369 to 6424</td><td>18</td><td>14 of 14</td>
<td>SEA (PBMC)</td><td>4.1.1</td><td>IL-2</td><td>2800</td><td>312</td><td>330 to 6699</td><td>42</td><td>17 of 17</td>
(Continued)
<td>Analysis</td><td>mAb</td><td>Frumubofli</td><td>Meflal increase pg / mL</td><td>SEM</td><td>soaring increase pg / mL</td><td>n</td><td>Svflrun gift</td>
<td>SEA (PBMC)</td><td>11.2.1</td><td>IL-2</td><td>2438</td><td>366</td><td>147 to 8360</td><td>25</td><td>15 of 15</td>
<td>SEA (Whole Blood)</td><td>4.1.1</td><td>IL-2</td><td>6089</td><td>665</td><td>-168 to 18417</td><td>46</td><td>15 of 17</td>
<td>SEA (Whole Blood)</td><td>11.2.1</td><td>IL-2</td><td>6935</td><td>700</td><td>-111 to 11803</td><td>25</td><td>12 of 14</td>
EXAMPLE 10
Human T-cell Signal
We developed other cellular assays to amplify the increase in T-cell IL-2 boost by blockade of CTLA-4 signals by the antibodies. The following materials and methods were used in conjunction with the experiments:
Materials and methods
Human PBMC were produced by the use of Accuspin. Cytoplankton plates were coated with anti-CD3 metabolites (Leu4, Becton Dickinson) (60 ng / mL) and raspberries for 2 hours at 37 ° C. hPBMC was added to the wells of 200,000 cells in each well. Staphylococcus Entertoxin A (SEA) (Sigma) was dispensed into the wells of 100 ng / mL. Flow rates were carried out in the wells, usually at 30 μg / mL. Cells were stimulated in 48, 72 boost 96 hours. There were spun nifles at the desired point point, and the fleet was removed from the wells. Pvl naest was a nice shotgun for IL-2 proliferation with the use of ELIS (R & D Systems).
Results from these experiments are shown in Figures 16, 17, and 21. In Figure 16, the induction of IL-2 proliferation of I hPBMC from 5 donors was measured 72 hours after flowering. Figure 17 shows results from whole blood measurements, which analyzes the difference between the induction of IL-2 proliferation in blood 3 donor as measured 72 and 96 hours post stimulation.
In Figure 21, the increase in IL-2 proliferation in whole blood donor 2 is as recommended 72 hours after stimulation.
EXAMPLE 11
Tumor dandelion
We have created an animal model model for the detection of an organism of anti-mouse CTLA-4 antibodies to inhibit tumor growth. In the model, the mouse mammalian cells are grown, and the animals are treated with anti-mouse CTLA-4 antibodies. The materials and methods used to create the model are given below:
Materials and methods
Female A / J mice (6-8 weeks old) were injected subcutaneously on the back of the neck with 0.2 mL of Sa1N tumor cells (1x10<sup>6</sup>) (Basque, 1995). The mouse mouse CTLA-4 or isotopic-equivalent reference antibody (Pharmingen, San Diego, CA, 200 μg / animal) was injected into the abdomen days 0, 4, 7 and 14 after tumor injection. Tumor measurements were performed over the weeks 3-4, experiments using the Starred SPC Plus electric meter (Athol, MA) and tumor size were expressed as the surface area covered by tumor growth (nm<sup>2</sup>).
Figure 18 shows the barrier to tumor growth with anti-mouse CTLA-4 antibodies in mouse-fibrosis tumor model. As shown in Figure 18, animals treated with anti-CTLA-4 reduction in tumor growth were compared to animals treated with cytosynthetic reference antibodies. Accordingly, anti-mouse CTLA-4 mAbs are capable of inhibiting the growth of fibrosis in a mouse tumor model.
Crosslinked antibodies with CTLA-4 mice are expected to behave similar to the model. However, of the antibodies from the invention that have been screened for cross-reactivity, one is cross-reactive to murine CTLA-4.
EXAMPLE 12
Tummy animal model
In order to further investigate antibody activity in accordance with the invention, a non-homogeneous SCID mouse model was designed to test the eradication of tumors and metastases derived from them. In the model, SCBD mice are administered with human transplanted human T cells, which are donated to non-small cell lung cancer (NSCC) or colon cancer (CC) cells derived from a patient. (thrombosis is produced in SCID mouse pancreas. The tumors are allowed to grow and afterwards they are removed. The mice develop human-like tumors and liver mesothelioma. Such a model is described in Bumpers et al., J. Surgical Res., 61: 282 -288 (1996).
Antibodies from the invention are expected to inhibit the growth of tumors formed by mice.
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Contents39
48 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7 Sheet 8 Sheet 9 Sheet 10 Sheet 11 Sheet 12 Sheet 13 Sheet 14 Sheet 15 Sheet 16 Sheet 17 Sheet 18 Sheet 19 Sheet 20 Sheet 21 Sheet 22 Sheet 23 Sheet 24 Sheet 25 Sheet 26 Sheet 27 Sheet 28 Sheet 29 Sheet 30 Sheet 31 Sheet 32 Sheet 33 Sheet 34 Sheet 35 Sheet 36 Sheet 37 Sheet 38 Sheet 39 Sheet 40 Sheet 41 Sheet 42 Sheet 43 Sheet 44 Sheet 45 Sheet 46 Sheet 47 Sheet 48
115 members in 41 offices
Priority claims7
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|---|---|---|---|
| 11364798 | United States of America | P | |
| 11364798 | United States of America | P | |
| 9930895 | United States of America | W | |
| 9930895 | United States of America | W | |
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| US19980113647P | – | – | – |
| WO1999US30895 | – | – | – |
Members115
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|---|---|---|---|
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| CA2619682A1 | Canada | A1 | |
| WO0037504A2 | World Intellectual Property Organization (WIPO) | A2 | |
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| WO0037504A3 | World Intellectual Property Organization (WIPO) | A3 | |
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| NO20013147D0 | Norway | D0 | |
| NO20013147L | Norway | L | |
| NO20120398L | Norway | L | |
| AP2001002213A0 | African Regional Intellectual Property Organization (ARIPO) | A0 | |
| EP1141028A2 | European Patent Office (EPO) | A2 | |
| CZ20012349A3 | Czechia | A3 | |
| ID29991A | Indonesia | A | |
| KR20010099899A | Republic of Korea | A | |
| BR9916853A | Brazil | A | |
| SK9142001A3 | Slovakia | A3 | |
| TR2001001831T2 | Türkiye | T2 | |
| TR200101831T2 | Türkiye | T2 | |
| EA200100698A1 | Eurasian Patent Organization (EAPO) | A1 | |
| CN1328571A | China | A | |
| HU0104604A2 | Hungary | A2 | |
| HU1300750D0 | Hungary | D0 | |
| HUP0104604A2 | Hungary | A2 | |
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| IL143797A0 | Israel | A0 | |
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| TR2002000735T2 | Türkiye | T2 | |
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| EP1141028B1 | European Patent Office (EPO) | B1 | |
| AT458008T | Austria | T | |
| ATE458008T1 | Austria | T1 | |
| DE69942037D1 | Germany | D1 | |
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| PT1141028E | Portugal | E | |
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| HU0104604A3 | Hungary | A3 | |
| HUP0104604A3 | Hungary | A3 | |
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| EE05627B1 | Estonia | B1 | |
| SK288057B6 | Slovakia | B6 | |
| EP2112166A3 | European Patent Office (EPO) | A3 | |
| CZ303703B6 | Czechia | B6 | |
| HRP20010551B1 | Croatia | B1 | |
| PL214003B1 | Poland | B1 | |
| US8491895B2 | United States of America | B2 | |
| HRP20130077A2 | Croatia | A2 | |
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Numbers
- Publication
- 2798
- Publication, DOCDB
- 2798
- Publication, EPODOC
- IS2798B
- Application
- 5974
- Application, DOCDB
- 5974
- Application, EPODOC
- IS20010005974
Titles2
- Icelandic
- Einræktuð mótefni úr mönnum gegn CTLA-4
- English
- Cloned human antibodies to CTLA-4
Classification
- CPC, 8
- C07K16/2818
- C07K16/28
- C07K2317/21
- A61P29/00
- A61P35/00
- A61P37/00
- A61P37/02
- A61P37/04
- IPC, 23
- A01H5 00
- A01K67 027
- C07K16 28
- A61K39 395
- A61P29 00
- A61P35 00
- A61P37 02
- A61P37 04
- C07K16 00
- C12N1 15
- C12N1 19
- C12N1 21
- C12N5 071
- C12N5 10
- C12N15 02
- C12N15 09
- C12P21 08
- C12Q1 02
- G01N33 15
- G01N33 50
- G01N33 53
- G01N33 577
- G01N33 68
