Group of 4-1BB monoclonal antibodies and medical application thereof
阅读说明：本技术 一组4-1bb单克隆抗体及其医药用途 (Group of 4-1BB monoclonal antibodies and medical application thereof ) 是由 孙锴 邱均专 孙自勇 王振生 周漫 陈均勇 孙键 区日山 于 2020-12-24 设计创作，主要内容包括：本发明属于肿瘤治疗和分子免疫学领域；具体涉及一组抗4-1BB的单克隆抗体及其医药用途。本发明通过杂交瘤技术获得了一组在激活T细胞方面有优异效果的抗4-1BB单克隆抗体,并成功地对其进行了人源化改造。所述抗体在制备用于激活和调节4-1BB的作用与水平以及显著增强机体免疫力的相关药物,尤其是治疗癌症相关药物方面表现出巨大的应用前景。(The invention belongs to the field of tumor therapy and molecular immunology; in particular to a group of monoclonal antibodies resisting 4-1BB and medical application thereof. The invention obtains a group of anti-4-1 BB monoclonal antibodies with excellent effect on activating T cells by a hybridoma technology, and successfully carries out humanized modification on the antibodies. The antibody has great application prospect in the preparation of related medicines for activating and regulating the action and level of 4-1BB and remarkably enhancing the immunity of the organism, particularly in the aspect of medicines related to cancer treatment.)
1. A set of 4-1BB monoclonal antibodies, or antigen-binding fragments thereof, comprising a heavy chain and a light chain, wherein the amino acid sequence of CDR1 of the heavy chain is selected from the group consisting of SEQ ID NO: 17. 23, 29, 35, 41, 47, 53, 59; the amino acid sequence of CDR2 of the heavy chain is selected from SEQ ID NO: 18. 24, 30, 36, 42, 48, 54, 60; the amino acid sequence of CDR3 of the heavy chain is selected from SEQ ID NO: 19. 25, 31, 37, 43, 49, 55, 61; the CDR1 amino acid sequence of the light chain is selected from SEQ ID NO: 20. 26, 32, 38, 44, 50, 56, 62; the amino acid sequence of CDR2 of the light chain is selected from SEQ ID NO: 21. 27, 33, 39, 45, 51, 57, 63; the amino acid sequence of CDR3 of the light chain is selected from SEQ ID NO: 22. 28, 34, 40, 46, 52, 58, 64; wherein the heavy and light chains of the antigen-binding fragment comprise amino acid sequences spanning CDR1 to CDR3 of the heavy and light chains, respectively, of the antibody.
2. The panel of 4-1BB monoclonal antibodies or antigen-binding fragments thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 1. 3, 5, 7, 9, 11, 13, 15; the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 2. 4, 6, 8,10, 12, 14, 16.
3. The panel of 4-1BB monoclonal antibodies or antigen-binding fragments thereof of claim 2, wherein the heavy and light chains are humanized; the amino acid sequence of the humanized heavy chain variable region is selected from SEQ ID NO: 65. 67, 69, 71, 73; the amino acid sequence of the humanized light chain variable region is selected from SEQ ID NO: 66. 68, 70, 72, 74.
4. The panel of 4-1BB monoclonal antibodies or antigen binding fragments thereof of claim 3, wherein potential glycosylation, isomerization, and deamidation sites in the humanized antibody sequence are analyzed and mutagenized; the 53 th Asp (D), the 54 th Ser (S) of the hu10B2 heavy chain, the 27 th Asn (N) of the hu10B2 light chain, the 27 th Ser (S) of the hu10B2 light chain, the 61 st Asp (D) of the hu3A11 light chain and the 62 th Ser (S) are point-mutated.
5. Use of a panel of 4-1BB monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1-4 in the preparation of a medicament comprising: a drug that binds 4-1BB, a drug that modulates 4-1BB activity or 4-1BB levels, a drug that modulates 4-1BB in immune activation of the body, a drug that activates T lymphocytes, or a drug that increases IL-2 expression in T lymphocytes.
6. A monoclonal antibody conjugate comprising a monoclonal antibody according to any one of claims 1-4, wherein the monoclonal antibody is the panel of 4-1BB monoclonal antibodies or antigen-binding fragments thereof, and a conjugate moiety selected from one or more of a radionuclide, a drug, a toxin, a cytokine receptor fragment, an enzyme, fluorescein, and biotin.
7. Use of a monoclonal antibody conjugate as claimed in claim 6 for the preparation of a medicament comprising: a drug that blocks binding of 4-1BB to a 4-1BB ligand, a drug that modulates 4-1BB activity or 4-1BB level, a drug that modulates immune activation of 4-1BB to the body, a drug that activates T lymphocytes, or a drug that increases IL-2 expression in T lymphocytes.
8. Use of a monoclonal antibody conjugate as claimed in claim 6 for the preparation of a medicament for the prophylactic and/or therapeutic and/or adjunctive treatment of tumours.
9. Use of a nucleic acid molecule, an expression vector, a host cell expressing a panel of 4-1BB monoclonal antibodies or antigen binding fragments thereof according to any one of claims 1-4 in the preparation of a medicament comprising: a drug that binds 4-1BB, a drug that modulates 4-1BB activity or 4-1BB levels, a drug that modulates 4-1BB in immune activation of the body, a drug that activates T lymphocytes, or a drug that increases IL-2 expression in T lymphocytes.
Technical Field
The invention belongs to the field of tumor immunotherapy and molecular immunology, and relates to a 4-1BB antibody and application thereof. In particular, the invention relates to a plurality of monoclonal antibodies to 4-1 BB.
Technical Field
4-1BB, also known as CD137, belongs to the tumor necrosis factor receptor superfamily (TNFRSF9), a receptor with synergistic activation (Vinay, et al, (2012), mol. Initially, 4-1BB was demonstrated to be expressed on activated T lymphocytes, but not on quiescent T lymphocytes (Kwon, et al., (1989), Proc. Natl. Acad. Sci. USA 86: 1963-; later, expression was found on some non-T lymphocytes, such as monocytes, centrogranulocytes, B cells, NK cells and NKT cells (Pollok, et al., (1993), J.Immunol.150:771 781; Vinay, et al., (1998), Sem.Immunol.10: 481-9; Vinay et al., (2012), PLoS One 7: e 50272; Melero, et al., (1998), Cell Immunol 190: 167-72). The ligand of 4-1BB is CD137L (or 4-1BBL), a transmembrane protein molecule of the TNF family, expressed on the surface of activated macrophages, B cells and dendritic cells (Alderson, et al., (1994), Eur.J.Immunol.24: 2219-2227; Goodwin, et al., (1993), Eur.J.Immunol.23: 2631-2641; Pollok, et al., (1994), Eur.J.Immunol.24: 367-374; Croft, et al., (2003), Nat.Rev.Immunol.3: 609-20).
4-1BB is a multifunctional molecule, and cross-linked 4-1BB ligand or 4-1 BB-elicited antibody can rapidly activate 4-1BB on the surface of T cells, thereby promoting the proliferation and activation of T cells (Vinay, et al., (2006), J.mol.Med.84: 726-. 4-1BB activates transcription factors NF-. kappa.B and MAP kinase by recruiting TNFR-related factor 1(TRAF1) and related factor 2(TRAF2) and further induces signaling of cells (Jang et al., (1998), Biochemical & Biophysical Research Communications 242: 613-20; Sabbagh, et al., (2008), J.Immunol.180: 8093-. 4-1BB is capable of inducing transcription of factors such as the anti-apoptotic Bcl-2 family members Bcl-XL, Bcl-2 and Bfl1, and blocking Apoptosis (AICD) induced by T cell activation (Lee, et al., (2003), Cellular Immunol.223: 143-50; Lee, et al., (2002), J.Immunol.169: 4882-. 4-1BB also stimulates T cells to produce type I cytokines such as IL-2, IFN-. gamma.and TNF-. alpha.and the like, and reduces the production of the immunomodulatory factors IL-4 and TGF-. beta.by synergistic interaction with a costimulatory molecule of CD 28. 4-1BB breaks the anergy of Cytotoxic T Lymphocytes (CTL) by inducing TH1 type effector T cells to differentiate. 4-1BB may also promote the proliferation, survival and cytokine secretion of B cells by interacting with its ligands (Zhang, et al., (2010), J.Immunol.184: 787-. Furthermore, 4-1BB also enhances NK cell killing (Kohrt, et al., (2012), J.Clin.invest.122: 1066-.
Due to its multiple immunoregulatory functions, 4-1BB is a hot target for current tumor therapy. Melero et al first reported that 4-1BB antibody was able to eliminate tumors in mice, including Ag104A sarcoma and P815 mastocytoma, which lack immunogenicity (Melero, et al, (1997), nat. Med.3: 682-5). Vinay et al reported that 4-1 BB-activated antibodies had good anti-tumor effects in tumor models such as MCA205 sarcoma, MC38 colon cancer, GL261 glioma, TC1 ovarian cancer, J558 myeloma and a549 (Vinay DS, et al, (2012), mol. Treatment of tumors with 4-1BB antibodies relies primarily on CD8+ T cells (Melero, et al., (1997), nat. Med.3: 682-5; Wilcox, et al., (2002), J Clin Invest 109: 651-9), and involvement of NK cells or dendritic cells is also required in certain models (Melero, et al., (1998), Cell Immunol.190: 167-72; Murillo, et al., (2009), Eur. J. Immunol.39: 2424-36). The 4-1BB antibody enables mice to develop a persistent immune memory response to tumors (Yonezawa, et al., (2016), Chin Clin Oncol 5: 5-11). Treatment with 4-1BB antibody alone failed to eliminate tumor cells lacking immunogenicity, such as C3 tumor and B16/D5 melanoma (Kimet, al., (2001), Cancer Res.61: 2031-7; Wilcox, et al., (2002), J.Clin.invest.109: 651-9). Combination therapy can increase the therapeutic effect of 4-1BB antibody, for example, the combination of 4-1BB antibody with a therapeutic means such as polypeptide vaccine (Bartkowiak, et al., (2015), Proc. Natl. Acad. Sci. USA 112: E5290-E5299), dendritic cell vaccine (Ito, et al, (2004), Cancer Res.64: 8411-. Furthermore, 4-1BB antibodies have synergistic effects with other immune checkpoint antibodies in tumor therapy, including CTLA-4 antibodies (Kocak, et al., (2006), Cancer Res.66: 7276-.
Two 4-1BB antibodies (Urelumab and Utomillab) are currently in clinical trials. Urelumab shows antitumor activity in the treatment of melanoma, renal and ovarian cancer patients (Sznol, et al., (2008), j.clin.oncol.26(supp15):3007), but dose-dependent hepatotoxic side effects occur in some patients. Compared with Urelumab, Utomillumab has good safety, but the antitumor effect is relatively weak. Therefore, the development of a safer and more effective 4-1BB antibody would have important value for the clinical treatment of tumors.
Disclosure of Invention
The invention adopts hybridoma technology to obtain a group of 4-1BB monoclonal antibodies, and successfully carries out humanized transformation on the monoclonal antibodies. The antibodies can remarkably promote the activation of T cells and the secretion of cytokines under the crosslinking action of Fc gamma R II (CD 32).
The technical scheme of the invention is as follows:
the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-human 4-1BB hybridoma antibody 1A2 are respectively SEQ ID NO: 1. 2; the amino acid sequences of the heavy chain and light chain variable regions of 8H8 are respectively SEQ ID NO: 3. 4; 10B2 heavy and light chain variable region amino acid sequences are SEQ ID NOs: 5. 6; the amino acid sequences of the heavy chain and light chain variable regions of 3F10 are respectively SEQ ID NO:7, 8; the amino acid sequences of the heavy chain and light chain variable regions of 8E9 are respectively SEQ ID NO: 9. 10; 8A8 heavy and light chain variable region amino acid sequences are SEQ ID NOs: 11. 12; the amino acid sequences of the heavy chain and light chain variable regions of 2F10 are SEQ ID NOs: 13. 14; the amino acid sequences of the heavy chain and light chain variable regions of 3a11 are SEQ ID NOs: 15. 16. The CDR region amino acid sequences of these antibodies are: 1A2 heavy chain CDR1, CDR2, CDR3 amino acid sequences are SEQ ID NO: 17. 18 and 19, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 20. 21, 22; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of 8H8 are respectively SEQ ID NO: 23. 24, 25, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 26. 27, 28; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of 10B2 are respectively SEQ ID NO: 29. 30, 31, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 32. 33, 34; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of 3F10 are respectively SEQ ID NO: 35. 36 and 37, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 38. 39, 40; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of 8E9 are respectively SEQ ID NO: 41. 42, 43; the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 44. 45, 46; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of 8A8 are respectively SEQ ID NO: 47. 48 and 49, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 50. 51, 52; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of 2F10 are respectively SEQ ID NO: 53. 54 and 55, and the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 56. 57, 58; 3A11 heavy chain CDR1, CDR2, CDR3 amino acid sequences are respectively SEQ ID NO: 59. 60 and 61, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO: 62. 63, 64.
Further, the anti-human 4-1BB antibody or fragment is modified to become a humanized antibody.
An isolated nucleic acid molecule: encoding the above-mentioned antibody or functional fragment.
The amino acid sequence of the heavy chain variable region of the anti-human 4-1BB humanized antibody 8H8 is SEQ ID NO: 65, the light chain variable region amino acid sequence of which is SEQ ID NO: 66; the amino acid sequence of the heavy chain variable region of the humanized antibody 10B2 is SEQ ID NO: 67, the light chain variable region amino acid sequence of which is SEQ ID NO: 68; the amino acid sequence of the heavy chain variable region of the humanized antibody 3A11 is SEQ ID NO: 69, the light chain variable region amino acid sequence of which is SEQ ID NO: 70; the amino acid sequence of the heavy chain variable region of humanized antibody 1A2 is SEQ ID NO:71, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 72; the amino acid sequence of the heavy chain variable region of the humanized antibody 2F10 is SEQ ID NO: 73, having the amino acid sequence of the light chain variable region of SEQ ID NO: 74.
an expression vector comprising a nucleic acid molecule expressing the antibody.
Pharmaceutical composition comprising the above antibody or functional fragment thereof, and a pharmaceutically acceptable carrier
The antibody or functional fragment thereof, nucleic acid molecules, expression vectors, host cells and pharmaceutical compositions are used for preparing medicines for influencing 4-1BB immune function.
The invention has the following beneficial effects: the invention adopts a mammalian cell expression system to prepare a recombinant 4-1BB as an antigen immune mouse, and obtains hybridoma cells after fusion of mouse spleen cells and myeloma cells. After a large number of hybridoma cells are cloned and screened for many times, some monoclonal hybridoma cell strains are obtained. These hybridoma cell lines can produce monoclonal antibodies (FIG. 1 and FIG. 2) specifically binding to 4-1BB, effectively block the binding of 4-1BB and 4-1BB ligand (FIG. 3), activate NF- κ B signals (FIG. 4 and FIG. 5), promote the production of cytokine IL-2 by Jurkat (FIG. 6 and FIG. 7), and promote the secretion of cytokine IFN- γ by human CD4+ T cells (FIG. 8). The genes encoding the light and heavy Chain variable regions of the antibody were cloned by RT-PCR (Reverse Transcription-Polymerase Chain amplification), and the humanized antibody was constructed by the complementary determinant-grafting (CDR-grafting) method. In vitro functional assays indicated that these humanized 4-1BB antibodies specifically bind to 4-1BB protein (fig. 9, fig. 10), activate NF- κ B signaling pathway under Fc γ RII (CD32) -mediated cross-linking (fig. 11), promote cytokine IL-2 secretion by Jurkat (fig. 12), stimulate IFN- γ secretion by human peripheral blood lymphocytes (PBMCs), and that the stimulatory activity was significantly higher than that of the positive control antibody (fig. 13). The sequence of the positive control antibody in this patent is derived from patent EP2614082B1(MOR7480.1, Utomilumab). The experimental results show that the monoclonal antibody or the antigen binding fragment thereof, or the conjugate containing the monoclonal antibody or the antigen binding fragment thereof, has good application prospect in the preparation of drugs for stimulating the activity of T lymphocytes and promoting the T lymphocytes to express IL-2 and IFN-gamma, and drugs for preventing and treating or adjuvant treating tumors.
Drawings
FIG. 1: determining EC50 binding of 4-1BB hybridoma antibody to 4-1BB-hFc protein using ELSIA;
FIG. 2: measuring EC50 binding of 4-1BB hybridoma antibodies to Jurkat-4-1BB cells using FACS;
FIG. 3: measuring the IC50 of the 4-1BB hybridoma antibody blocking the binding of 4-1BB ligand to Jurkat-4-1BB cells using FACS;
FIG. 4: activation of NF- κ B signaling pathway by 4-1BB hybridoma antibody in association with 293T-CD32a cells;
FIG. 5: activation of the NF-. kappa.B signaling pathway by 293T-CD32B cell cross-linking with 4-1BB hybridoma antibody;
FIG. 6: stimulation of IL-2 secretion by Jurkat-4-1BB by the 4-1BB hybridoma antibody in combination with 293T-CD32a cells;
FIG. 7: stimulation of IL-2 secretion by Jurkat-4-1BB by the 4-1BB hybridoma antibody in combination with 293T-CD32b cells;
FIG. 8: stimulation of IFN- γ secretion by human CD4+ T cells by 4-1BB hybridoma antibodies;
FIG. 9: determining the binding of the humanized 4-1BB antibody to the 4-1BB protein by ELISA 50;
FIG. 10: measuring EC50 binding of humanized 4-1BB antibody to Jurkat-4-1BB cells using FACS;
FIG. 11: activation of NF-kB signaling pathway by the 4-1BB humanized antibody in combination with 293T-CD32B cells;
FIGS. 12A-B: stimulation of IL-2 secretion by humanized 4-1BB antibody, panel A being uncrosslinked and panel B being crosslinked, by crosslinking 293T-CD32B on Jurkat-4-1 BB;
FIG. 13: stimulation of IFN-gamma secretion from human PBMC by humanized 4-1BB antibody;
Detailed Description
Example 1
Preparation of 4-1BB hybridoma antibody
A fusion protein (4-1BB-mFc) of an extracellular region of human 4-1BB and mouse Fc is used as an antigen, and after full emulsification with an equal volume of complete Freund's adjuvant (Sigma, Cat No: F5581), Balb/c mice (purchased from Showa-derived (Suzhou) New drug research center, Ltd.) with the age of 6-8 weeks are immunized subcutaneously, and the antigen immunization amount is 50 mu g/mouse. Mice were then immunized subcutaneously three times every 2 weeks after sufficient emulsification with the same dose of antigen in incomplete Freund's adjuvant (Sigma, Cat No: F5506). After three immunizations, the serum titer of the mice was measured, and 3 days before the fusion, the mice were boosted by the abdominal cavity. Mouse spleen cells were mixed with SP2/0 cells at a ratio of 4:1 using PEG Hybri-Max (Sigma, Cat No:7181) as a fusogenic agent. The fused cells were added to a 96-well plate (1X 10)5Cells/well) containing 0.1mL of 1 XHAT (Invitrogen, Cat No:21060-017) medium per well. 0.1mL of HT (Invitrogen, Cat No:11067-030) medium was added on day 3, the medium in the 96-well plate was aspirated off on day 7, and 0.2mL of fresh HT medium was added. On day 9, supernatants were harvested for ELISA and FACS detection.
A96-well ELISA plate (Corning, Cat No:9018) was coated with 4-1BB-ECD-hFc, left overnight at room temperature, washed 3 times with washing buffer (PBS + 0.05% Tween20), and then incubated for 1 hour with blocking buffer (PBS + 1% BSA (Sigma, Cat No: V90093)); washing the 96-well plate for 3 times; adding hybridoma supernatant, incubating for 1 hour, and washing for 3 times; mu.L of a goat anti-mouse IgG secondary antibody (Thermo, Cat No:31432) diluted 1:400 times was added to each well, incubated at room temperature for 1 hour, and washed 3 times; 100. mu.L of TMB (Cat No: ES-002) was added to each well for color development for 3 minutes, and 100. mu.L/well of stop buffer (2N H)2SO4) The reaction was stopped and the OD450 signal of each sample was measured using a Tecan Spark plate reader.
Mu.l of hybridoma supernatant positive in the above ELISA assay was mixed with 50. mu.l of Jurkat-4-1BB cells (1X 10)5Cells/well) were added to a U-bottomed 96-well plate and incubated for 1 hour, washed 2 times by centrifugation with FACS buffer (PBS + 3% FCS), and 1: 400-fold diluted was addedThe released PE-labeled goat anti-mouse (Biolegend, Cat No:405307) secondary antibody was incubated for 30 minutes, washed with FACS buffer, and PE signal of Jurkat-4-1BB cells was detected by a BD C6 flow cytometer.
Screening 4-1BB excitation antibody by reporter gene method. Add 50. mu.l (5X 10) to 96-well plates4Cell/well) Jurkat-4-1BB-NFkB-luc cells and 50. mu.l of hybridoma supernatant positive in the above ELISA assay, 293T-CD32a or 293T-CD32b cells (2X 10)4Cells/well), mixed and incubated at 37 ℃ for 4 hours. 25 μ l of Bright Glo (Promega, Cat No: E2620) was added and incubated at room temperature for 5 minutes, and the chemiluminescent signal of each sample was measured using a Tecan Spark plate reader.
The hybridoma capable of stimulating the 4-1BB reporter gene signal is subcloned by limiting dilution method, and then the detection and screening by ELISA and FACS method are repeated to obtain the positive hybridoma monoclonal. The positive monoclonal hybridomas were cultured in 50mL serum-free medium (Invitrogen, Cat No:12045-076) for 8-9 days, and the supernatant was collected by centrifugation. Monoclonal antibodies were purified by Protein A affinity chromatography, the purified antibody samples were concentrated by exchange in an ultrafiltration centrifuge tube (Millipore, Cat No: ACS500024), the Protein concentration was determined by the BCA method, and the endotoxin content of the antibody samples was determined by the use of a chelate reagent (Limulus tridentate Biotech Co., Ltd., Xiamen). Purified antibody samples were tested by ELISA and FACS for binding to 4-1BB, blocking of 4-1BB binding to its ligand (4-1BBL), and for their activity in activating the 4-1BB reporter gene, as shown in tables 1-5 and FIGS. 1-5. Candidate 4-1BB hybridoma antibodies are capable of binding to 4-1BB protein, inhibiting the binding of 4-1BB to its ligand, 4-1BBL, and activating downstream signaling pathways.
TABLE 1 determination of the binding of hybridoma antibodies to human 4-1BB by ELISA
TABLE 2 measurement of binding of hybridoma antibodies to human 4-1BB by FACS
TABLE 3 measurement of blocking of 4-1BB binding to its ligand, 4-1BBL, by hybridoma antibodies using FACS
TABLE 4 determination of the effect of hybridoma antibodies and 293T-CD32a cells on NF- κ B by reporter Gene method
TABLE 5 determination of the effect of hybridoma antibodies and 293T-CD32B cells on NF- κ B by reporter Gene method
Example 2:
effect of 4-1BB hybridoma antibody on cytokine secretion by T lymphocyte line Jurkat-4-1BB
In a 96-well plate, 50. mu.L of 2X 10 was added per well6Jurka-4-1BB cells, 50. mu.l 4X 105293T-OKT3 cells/ml and 50. mu.l 4X 105293T-CD32a or 293T-CD32b cells in mL and 50. mu.L of 4-1BB antibody at various concentrations (starting at 20. mu.g/mL, 10-fold serial dilutions), 96-well cell culture plates were placed at 37 ℃ in 5% CO2Incubate in incubator for 48 hours, collect supernatant, and use IL-2ELISA kit (R)&D Systems, Cat No: DY202), selected 4-1BB antibody was able to significantly activate the expression of IL-2, as shown in FIGS. 6 and 7.
Example 3
Effect of 4-1BB hybridoma antibodies on cytokine secretion by human CD4+ T cells and PBMC cells
Lymphocyte cell-separating medium Histopaque (Sigma, Cat No:1077-1) was added to a 50mL sterile centrifuge tube, followed by an equal volume of fresh blood and centrifugation at 1500rpm for 30min at room temperature. The sample is divided into four layers in a centrifuge tube, and the four layers are a plasma layer, a leucocyte layer, a lymphocyte separation solution and a red blood cell layer from top to bottom. The middle buffy coat was collected into a new centrifuge tube, washed by mixing with 5 volumes of washing buffer (PBS + 3% FBS), centrifuged at 1500rpm for 10min, washed 3 times in total, resuspended cells in washing buffer and counted. CD4+ T cells were isolated from PBMCs using CD4+ T cell isolation reagents according to the instructions. One day in advance, 2. mu.g/mL of Goat anti-mouse Fc (Jackson, Cat: 115-. The next day, after washing with PBS, Blocking buffer (PBS + 2% BSA) was added, incubated at 37 ℃ for 60 minutes, washed with PBS, added with PBS containing 40ng/mL OKT3(eBioscience, Cat No:16-0037-85), incubated at 37 ℃ for 90 minutes, and washed with PBS. After counting CD4+ T cells, CD4+ T cells were resuspended with complete medium RPMI1640+ 10% FCS (1X 10)6cells/mL). In 96-well plates, 100. mu.L of CD4+ T cells and 100. mu.L of 4-1BB antibody at various concentrations (starting at 20. mu.g/mL, 10-fold serial dilutions) were added to each well, and the 96-well cell culture plates were incubated at 37 ℃ in a 5% CO2 incubator for 48-72 hours to collect the supernatant. Using IFN-gamma ELISA kit (R)&D Systems, Cat No: DY285) to detect the concentration of the cytokine. As shown in FIG. 8, 4-1BB antibodies 1A2,2F10,3A11,3F10,8A8,8E9,8H8, and 10B2 listed in the present invention have stronger IFN-. gamma.secretion activity from CD4+ T cells than the positive control antibody Utomillumab.
Example 4
Cloning of 4-1BB antibody variable region Gene
4-1BB monoclonal hybridoma cell lines were lysed with TRIzon (Cwbiotech, Cat No: CW0580) to extract total RNA from the hybridoma cells. RNA from hybridoma cells was reverse transcribed into cDNA using HiFi Script cDNA Synthesis kit (Cwbiotech, Cat No: CW 2569). The variable region genes of the heavy and light chains of the Antibody were amplified by PCR method (Kettleborough, et al., (1993) Eur.J.Immunol.23: 206-211; Strebe, et al., (2010) Antibody Engineering 1:3-14) using the cDNA as a template and degenerate primers. After ligation of the PCR amplification products to the T/A vector, DH5a competent cells were transformed, plated and cultured overnight at 37 ℃. The monoclonal antibody is selected from the culture plate, amplified, extracted and used to determine the gene sequence of the antibody. The Complementarity Determining Regions (CDRs) and framework regions of the antibody were analyzed based on its gene sequence. The variable region gene sequences and amino acid sequences of the heavy and light chains of the antibodies are shown in Table 6.
TABLE 6.4-1BB hybridoma antibody sequence numbering
Example 5
Construction of humanized 4-1BB antibody
4-1BB hybridoma antibodies 10B2, 8H8, 3A11, 1A2 and 2F10 were humanized
Humanization of the 4-1BB antibody was performed by CDR grafting. First, the IMGT database was searched for sequences of human germline antibodies (germline antibodies) with the highest homology to the light and heavy chain variable regions of the murine antibody, respectively. The 10B2 antibody light chain variable region humanized selected germ line is IGKV2-29 x 02, and the heavy chain variable region humanized selected IGHV3-30 x 03. The 8H8 antibody light chain variable region humanized selected embryo line is IGKV4-1 x 01, and the heavy chain variable region humanized selected IGHV7-4-1 x 02. The 3A11 antibody light chain is humanized by IGKV1-39 × 01, and the heavy chain is humanized by IGHV3-15 × 01. The light chain of the 1A2 antibody is humanized by IGKV3-11 x 01, and the heavy chain is humanized by IGHV1-69-2 x 01. The light chain of the 2F10 antibody is humanized by IGKV3-11 x 01, and the heavy chain is humanized by IGHV1-69-2 x 01. The CDR regions of the murine antibody are retained and the framework region (framework) sequences of the murine antibody are replaced with the framework region sequences of the human germline antibody. Secondly, establishing a structural model of the murine antibody, comparing each different amino acid position in the structural models of the humanized antibody and the corresponding murine antibody one by one, if the adopted human amino acid sequence at a certain position of the framework region does not cause the damage or change of the space structure of the CDR region, using the human amino acid sequence at the position, or using the corresponding murine sequence (namely, carrying out reversion to the murine sequence) at the position. According to the structural simulation, partial amino acids of the humanized antibody framework region are back mutated into a murine sequence.
TABLE 7.4-1BB humanized antibody sequence numbering
The humanized heavy chain of the 10B2 antibody had Ala at position 93 back mutated to Thr. The humanized light chain of the 10B2 antibody had an Ile back-mutated to Val at position 2 and a Met back-mutated to Val at position 4. The 2 nd Val of the humanized heavy chain of the 8H8 antibody is back mutated to Ile, and the 30 th Thr is back mutated to Ser. The 4 th Met of the humanized light chain of the 8H8 antibody is back mutated into Leu, and the 68 th Gly is back mutated into Arg. The humanized heavy chain of the 3A11 antibody has 28 th Thr back mutation to Ile, 30 th Ser back mutation to Asn, 48 th Val back mutation to Ile, 49 th Gly back mutation to Ala, 93 th Thr back mutation to Asn, and 94 th Thr back mutation to Trp. The 49 th Tyr of the humanized light chain of the 3A11 antibody is back mutated into Phe, the 66 th Gly is back mutated into Ala, the 69 th Thr is back mutated into Asn, and the 71 th Phe is back mutated into Tyr. The 2 nd Val of the humanized heavy chain of the 1A2 antibody is back mutated to Ala, the 27 th Tyr is back mutated to Phe, the 28 th Thr is back mutated to Asn, the 29 th Phe is back mutated to Ile, the 30 th Thr is back mutated to Lys, the 48 th Met is back mutated to Ile, and the 67 th Val is back mutated to Ala. The 68 th Gly of the humanized light chain of the 1A2 antibody is mutated back to Arg. The humanized heavy chain of the 2F10 antibody has the 27 th Tyr back mutated to Phe, the 28 th Thr back mutated to Asn, the 29 th Phe back mutated to Ile, the 30 th Thr back mutated to Glu, the 48 th Met back mutated to Ile, the 67 th Val back mutated to Ala, and the 93 th Ala back mutated to Thr. The 68 th Gly of the humanized light chain of the 2F10 antibody is mutated back to Arg.
Nucleic acid sequences encoding the humanized antibody light and heavy chains of 10B2, 8H8, 3a11, 1a2, and 2F10 were synthesized and inserted into expression vector pcdna3.1. 200 ml 293 cells (cell density 1X 10) were co-transfected with 0.1mg each of antibody light and heavy chain expression plasmids6) After culturing for 6 days at 37 ℃ with shaking, the supernatant was collected by centrifugation, and the humanized antibody was purified by Protein A, and the purified humanized antibody was used for activity detection.
Example 6
Purified humanized antibody samples were tested for binding to 4-1BB by ELISA and FACS, and the activation of the NF-. kappa.B signaling pathway by the humanized 4-1BB antibody was determined by the reporter gene method. The specific procedure is as in example 1. The results of the measurement of humanized antibodies hu8H8, hu10B2, hu3A11, hu1A2 and hu2F10 are shown in tables 8 to 10 and FIGS. 9 to 11. The results show that the humanized antibody can be combined with 4-1BB, and the humanized antibody can remarkably activate an NF-kb signaling pathway under the cross-linking of 293T-CD32b cells.
TABLE 8 determination of binding of 4-1BB humanized antibody to 4-1BB by ELISA
TABLE 9 measurement of binding of 4-1BB humanized antibody to 4-1BB by FACS
TABLE 10 determination of the activation of NF-. kappa.B signalling pathway by 4-1BB humanized antibodies crosslinked with 293T-CD32B cells Using reporter genes
Example 7
Effect of 4-1BB humanized antibody on cytokine secretion by Jurkta-4-1 BB.
The 4-1BB humanized antibody was co-cultured with 293T-OKT3 and 293T-CD32b for 48 hours, and cell supernatants were collected to examine the expression level of IL-2, which was determined as described in example 2. As a result, as shown in FIG. 12A, neither the humanized 4-1BB antibody nor the positive control Utomillab activated the expression of IL-2 without crosslinking (Uncrosslinking). As shown in FIG. 12B, in the case of 293T-CD32B cross-linking, the humanized antibodies hu8H8, hu10B2, hu3A11, hu1A2 and hu2F10 have stronger IL-2 secretion promoting activity in Jurkat cells than the positive control antibody Utomillumab.
Example 8
Effect of humanized 4-1BB antibody on cytokine secretion by human CD4+ T cells and PBMCs.
4-1BB humanized antibody was co-cultured with human PBMC for 72 hours, and the supernatant was collected and assayed for IFN-. gamma.concentration in the supernatant according to example 3. As a result, as shown in FIG. 13, the humanized 4-1BB antibody can significantly promote IFN- γ secretion from PBMC, and the activity of the humanized antibodies hu10B2, hu3A11, hu1A2 and hu2F10 is significantly better than that of the positive control antibody Utomillumab.
Example 9
Affinity detection of humanized 4-1BB antibodies
Binding of each humanized antibody sample to 4-1BB was determined using Biacore. The determination method comprises the following steps: anti-human IgG was immobilized on CM5 chip in an amino-coupled manner, followed by capture of humanized antibody at a flow rate of 10. mu.L/min. The flow rate was switched to 30. mu.L/min, and different concentrations of 4-1BB antigen (100nM,50nM,25nM,12.5nM,6.25nM,3.125nM, etc.) labeled with histidine were flowed through the sample channel and the reference channel in sequence, with a binding time of 3min and a dissociation time of 10 min. Finally, the chip was regenerated with glycine buffer solution of pH 1.7.
Potential glycosylation sites, isomerization sites and deamidation sites in the sequence are analyzed and subjected to mutation modification. Point mutations were made at position 53 Asp (D) of the hu10B2 heavy chain, at position 54 Ser (S), at position 27 Asn (N) of the hu10B2 light chain, at position 27E Ser (S), and at position 61 Asp (D) of the hu3A11 light chain heavy chain, at position 62 Ser (S), and after the mutations were expressed and purified in 293F cells, and the affinity of the antibodies after the mutations was determined by Biacore. As shown in tables 11-13, the humanized antibodies hu8H8, hu10B2, hu3A11 and hu2F10 had better affinity for 4-1BB than the positive control antibody Utomillumab, while the humanized antibody hu1A2 dissociated from 4-1BB significantly slower than the positive control antibody.
TABLE 11 affinity of humanized 4-1BB antibody to 4-1BB antigen
TABLE 12 affinity of humanized 3A11 and its mutant antibodies to 4-1BB antigen.
TABLE 13 affinity of humanized 10B2 and its mutant antibodies to 4-1BB antigen.
The above examples 1-9 show that the monoclonal antibody or the antigen binding fragment thereof of the present invention, or the conjugate comprising the monoclonal antibody or the antigen binding fragment thereof of the present invention, has a good application prospect in the preparation of drugs for stimulating the activity of T lymphocytes, promoting the expression of IL-2 and IFN- γ by T lymphocytes, and drugs for preventing and treating or adjunctively treating tumors.
SEQUENCE LISTING
<110> east China Biotechnology (Suzhou) Ltd
<120> a group of 4-1BB monoclonal antibodies and medical uses thereof
<130> 2020
<160> 74
<170> PatentIn version 3.5
<210> 1
<211> 118
<212> PRT
<213> Artificial
<223> 1A2 heavy chain variable region amino acid sequence
<400> 1
Glu Ala Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met Asn Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr Ala Pro Gln Phe
50 55 60
Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Asn
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Tyr Gly Ser Asn Phe Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ala Leu Thr Val Ser Ser
115
<210> 2
<211> 111
<212> PRT
<213> Artificial
<223> 1A2 light chain variable region amino acid sequence
<400> 2
Glu Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Val Asp Asn Tyr
20 25 30
Asp Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Arg Ala Ser Asn Leu Glu Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Val
85 90 95
Glu Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Val Glu Leu Lys
100 105 110
<210> 3
<211> 117
<212> PRT
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<223> 8H8 heavy chain variable region amino acid sequence
<400> 3
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Glu Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Val Leu Thr Met Val Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 4
<211> 111
<212> PRT
<213> Artificial
<223> 8H8 light chain variable region amino acid sequence
<400> 4
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ile Ile Asp Gly Ser
20 25 30
Gly Asn Ser Phe Val His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Thr Ser Thr Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Ser Cys Leu Gln Thr Val
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 5
<211> 127
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<223> 10B2 heavy chain variable region amino acid sequence
<400> 5
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe
20 25 30
Gly Leu His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Asp Ser Asn Thr Ile Tyr Tyr Ala Asp Thr Met
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Asp Pro Ala Tyr Tyr Gly Tyr Gly Gly Arg Phe Val Tyr
100 105 110
Ala Leu Asp Phe Trp Gly Gln Gly Thr Ser Val Ala Val Ser Ser
115 120 125
<210> 6
<211> 112
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<223> 10B2 light chain variable region amino acid sequence
<400> 6
Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Phe Gly
1 5 10 15
Asp Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser
20 25 30
Tyr Gly His Thr Tyr Leu Ser Trp Tyr Leu His Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gly Ile Ser Ile Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Thr Val Lys Pro Glu Asp Leu Gly Met Tyr Tyr Cys Leu Gln Gly
85 90 95
Thr His Gln Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 7
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<223> 3F10 heavy chain variable region amino acid sequence
<400> 7
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Phe Gly Phe Thr Phe Thr His His
20 25 30
His Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Tyr Ser Ile Tyr Asn Gln Lys Phe
50 55 60
Glu Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Leu Gly Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 8
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<212> PRT
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<223> 3F10 light chain variable region amino acid sequence
<400> 8
Asp Ile Val Met Thr Gln Ser Thr Ala Ile Met Ser Ala Ser Leu Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Thr Ser Ser
20 25 30
Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Ile Trp
35 40 45
Ile Ser Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Asn Pro
85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 9
<211> 120
<212> PRT
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<223> 8E9 heavy chain variable region amino acid sequence
<400> 9
Gln Ala Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Trp Met His Trp Met Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Asn Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Asp Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Arg Tyr Tyr Gly Tyr Asp Gly Ile Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 10
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<212> PRT
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<223> 8E9 light chain variable region amino acid sequence
<400> 10
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Val Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp His Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 11
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<223> 8A8 heavy chain variable region amino acid sequence
<400> 11
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Ala Ile Trp Ser Gly Gly Asn Thr Asp Tyr Ser Ala Ala Phe Met
50 55 60
Ser Arg Val Thr Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Asn Pro Tyr Tyr Thr Asn Val Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 12
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<223> 8A8 light chain variable region amino acid sequence
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Gly Asn Phe
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Gly Glu Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 13
<211> 118
<212> PRT
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<223> 2F10 heavy chain variable region amino acid sequence
<400> 13
Gln Val Gln Leu Gln Gln Ser Gly Ala Ala Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Thr
20 25 30
Tyr Leu Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Lys Ile Tyr Pro Ala Asn Gly Asp Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Glu Thr Pro Ser Asn Lys Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Gly Ser Asn Phe Phe Asp Cys Trp Gly Gln Gly Thr
100 105 110
Ser Leu Thr Val Ser Ser
115
<210> 14
<211> 111
<212> PRT
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<223> 2F10 light chain variable region amino acid sequence
<400> 14
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ala Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Asp Asn Ser Phe Met His Trp Tyr Gln Gln Lys Val Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Leu Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ile
100 105 110
<210> 15
<211> 115
<212> PRT
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<223> 3A11 heavy chain variable region amino acid sequence
<400> 15
Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Asn Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asn Thr Asn
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ala Arg Ile Arg Ser Lys Ile Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Gly Met Tyr
85 90 95
Tyr Cys Asn Trp Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
<210> 16
<211> 107
<212> PRT
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<223> 3A11 light chain variable region amino acid sequence
<400> 16
Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
1 5 10 15
Gly Arg Val Thr Val Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Phe Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Ala Ser Gly Asn Asp Tyr Thr Leu Thr Ile Thr Ser Leu Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Ile Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 17
<211> 5
<212> PRT
<213> Artificial
<223> 1A2 heavy chain CDR1 region amino acid sequence
<400> 17
Asp Thr Tyr Met Asn
1 5
<210> 18
<211> 17
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<223> 1A2 heavy chain CDR2 region amino acid sequence
<400> 18
Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr Ala Pro Gln Phe Gln
1 5 10 15
Asp
<210> 19
<211> 9
<212> PRT
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<223> 1A2 heavy chain CDR3 region amino acid sequence
<400> 19
Ser Tyr Gly Ser Asn Phe Phe Asp Tyr
1 5
<210> 20
<211> 15
<212> PRT
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<223> 1A2 light chain CDR1 region amino acid sequence
<400> 20
Lys Ser Ser Gln Ser Val Asp Asn Tyr Asp Asn Ser Phe Met His
1 5 10 15
<210> 21
<211> 7
<212> PRT
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<223> 1A2 light chain CDR2 region amino acid sequence
<400> 21
Arg Ala Ser Asn Leu Glu Thr
1 5
<210> 22
<211> 9
<212> PRT
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<223> 1A2 light chain CDR3 region amino acid sequence
<400> 22
Gln Gln Ser Val Glu Asn Pro Phe Thr
1 5
<210> 23
<211> 5
<212> PRT
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<223> 8H8 heavy chain CDR1 region amino acid sequence
<400> 23
Asn Tyr Gly Met Asn
1 5
<210> 24
<211> 17
<212> PRT
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<223> 8H8 heavy chain CDR2 region amino acid sequence
<400> 24
Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Glu Phe Lys
1 5 10 15
Gly
<210> 25
<211> 8
<212> PRT
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<223> 8H8 heavy chain CDR3 region amino acid sequence
<400> 25
Val Leu Thr Met Val Met Asp Tyr
1 5
<210> 26
<211> 15
<212> PRT
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<223> 8H8 light chain CDR1 region amino acid sequence
<400> 26
Arg Ala Ser Glu Ile Ile Asp Gly Ser Gly Asn Ser Phe Val His
1 5 10 15
<210> 27
<211> 7
<212> PRT
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<223> 8H8 light chain CDR2 region amino acid sequence
<400> 27
Arg Thr Ser Thr Leu Glu Ser
1 5
<210> 28
<211> 9
<212> PRT
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<223> 8H8 light chain CDR3 region amino acid sequence
<400> 28
Leu Gln Thr Val Glu Asp Pro Trp Thr
1 5
<210> 29
<211> 5
<212> PRT
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<223> 10B2 heavy chain CDR1 region amino acid sequence
<400> 29
Thr Phe Gly Leu His
1 5
<210> 30
<211> 17
<212> PRT
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<223> 10B2 heavy chain CDR2 region amino acid sequence
<400> 30
Tyr Ile Ser Ser Asp Ser Asn Thr Ile Tyr Tyr Ala Asp Thr Met Lys
1 5 10 15
Gly
<210> 31
<211> 18
<212> PRT
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<223> 10B2 heavy chain CDR3 region amino acid sequence
<400> 31
Gly Asp Pro Ala Tyr Tyr Gly Tyr Gly Gly Arg Phe Val Tyr Ala Leu
1 5 10 15
Asp Phe
<210> 32
<211> 16
<212> PRT
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<223> 10B2 light chain CDR1 region amino acid sequence
<400> 32
Arg Ser Ser Gln Ser Leu Thr Asn Ser Tyr Gly His Thr Tyr Leu Ser
1 5 10 15
<210> 33
<211> 7
<212> PRT
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<223> 10B2 light chain CDR2 region amino acid sequence
<400> 33
Gly Ile Ser Ile Arg Phe Ser
1 5
<210> 34
<211> 9
<212> PRT
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<223> 10B2 light chain CDR3 region amino acid sequence
<400> 34
Leu Gln Gly Thr His Gln Pro Trp Thr
1 5
<210> 35
<211> 5
<212> PRT
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<223> 3F10 heavy chain CDR1 region amino acid sequence
<400> 35
His His His Ile Asn
1 5
<210> 36
<211> 17
<212> PRT
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<223> 3F10 heavy chain CDR2 region amino acid sequence
<400> 36
Tyr Ile Asn Pro Tyr Asn Asp Tyr Ser Ile Tyr Asn Gln Lys Phe Glu
1 5 10 15
Gly
<210> 37
<211> 10
<212> PRT
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<223> 3F10 heavy chain CDR3 region amino acid sequence
<400> 37
Gly Gly Leu Gly Arg Gly Tyr Phe Asp Tyr
1 5 10
<210> 38
<211> 12
<212> PRT
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<223> 3F10 light chain CDR1 region amino acid sequence
<400> 38
Arg Ala Ser Ser Ser Val Thr Ser Ser Tyr Leu His
1 5 10
<210> 39
<211> 7
<212> PRT
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<223> 3F10 light chain CDR2 region amino acid sequence
<400> 39
Ser Thr Ser Asn Leu Ala Ser
1 5
<210> 40
<211> 9
<212> PRT
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<223> 3F10 light chain CDR3 region amino acid sequence
<400> 40
Gln Gln Tyr Ser Gly Asn Pro Tyr Thr
1 5
<210> 41
<211> 5
<212> PRT
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<223> 8E9 heavy chain CDR1 region amino acid sequence
<400> 41
Gly Tyr Trp Met His
1 5
<210> 42
<211> 17
<212> PRT
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<223> 8E9 heavy chain CDR2 region amino acid sequence
<400> 42
Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Leu Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 43
<211> 11
<212> PRT
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<223> 8E9 heavy chain CDR3 region amino acid sequence
<400> 43
Trp Arg Tyr Tyr Gly Tyr Asp Gly Ile Ala Tyr
1 5 10
<210> 44
<211> 17
<212> PRT
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<223> 8E9 light chain CDR1 region amino acid sequence
<400> 44
Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Arg Asn Tyr Leu
1 5 10 15
Thr
<210> 45
<211> 7
<212> PRT
<213> Artificial
<223> 8E9 light chain CDR2 region amino acid sequence
<400> 45
Trp Ala Ser Thr Arg Glu Ser
1 5
<210> 46
<211> 9
<212> PRT
<213> Artificial
<223> 8E9 light chain CDR3 region amino acid sequence
<400> 46
Gln Asn Asp Tyr Thr Tyr Pro Phe Thr
1 5
<210> 47
<211> 5
<212> PRT
<213> Artificial
<223> 8A8 heavy chain CDR1 region amino acid sequence
<400> 47
Asn Tyr Gly Val His
1 5
<210> 48
<211> 16
<212> PRT
<213> Artificial
<223> 8A8 heavy chain CDR2 region amino acid sequence
<400> 48
Ala Ile Trp Ser Gly Gly Asn Thr Asp Tyr Ser Ala Ala Phe Met Ser
1 5 10 15
<210> 49
<211> 10
<212> PRT
<213> Artificial
<223> 8A8 heavy chain CDR3 region amino acid sequence
<400> 49
Asn Pro Tyr Tyr Thr Asn Val Met Asp Tyr
1 5 10
<210> 50
<211> 11
<212> PRT
<213> Artificial
<223> 8A8 light chain CDR1 region amino acid sequence
<400> 50
Arg Ala Ser Gly Asn Ile Gly Asn Phe Leu Ala
1 5 10
<210> 51
<211> 7
<212> PRT
<213> Artificial
<223> 8A8 light chain CDR2 region amino acid sequence
<400> 51
Asn Gly Glu Thr Leu Ala Asp
1 5
<210> 52
<211> 9
<212> PRT
<213> Artificial
<223> 8A8 light chain CDR3 region amino acid sequence
<400> 52
Gln His Phe Trp Ser Thr Pro Trp Thr
1 5
<210> 53
<211> 5
<212> PRT
<213> Artificial
<223> 2F10 heavy chain CDR1 region amino acid sequence
<400> 53
Asp Thr Tyr Leu Asn
1 5
<210> 54
<211> 17
<212> PRT
<213> Artificial
<223> 2F10 heavy chain CDR2 region amino acid sequence
<400> 54
Lys Ile Tyr Pro Ala Asn Gly Asp Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 55
<211> 9
<212> PRT
<213> Artificial
<223> 2F10 heavy chain CDR3 region amino acid sequence
<400> 55
Gly Tyr Gly Ser Asn Phe Phe Asp Cys
1 5
<210> 56
<211> 15
<212> PRT
<213> Artificial
<223> 2F10 light chain CDR1 region amino acid sequence
<400> 56
Arg Ala Ser Glu Ser Val Asp Ser Tyr Asp Asn Ser Phe Met His
1 5 10 15
<210> 57
<211> 7
<212> PRT
<213> Artificial
<223> 2F10 light chain CDR2 region amino acid sequence
<400> 57
Arg Ala Ser Asn Leu Glu Ser
1 5
<210> 58
<211> 9
<212> PRT
<213> Artificial
<223> 2F10 light chain CDR3 region amino acid sequence
<400> 58
Gln Gln Ser Asn Glu Asp Pro Tyr Thr
1 5
<210> 59
<211> 5
<212> PRT
<213> Artificial
<223> 3A11 heavy chain CDR1 region amino acid sequence
<400> 59
Thr Asn Ala Met Asn
1 5
<210> 60
<211> 19
<212> PRT
<213> Artificial
<223> 3A11 heavy chain CDR2 region amino acid sequence
<400> 60
Arg Ile Arg Ser Lys Ile Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser
1 5 10 15
Val Lys Asp
<210> 61
<211> 4
<212> PRT
<213> Artificial
<223> 3A11 heavy chain CDR3 region amino acid sequence
<400> 61
Asp Glu Ala Tyr
1
<210> 62
<211> 11
<212> PRT
<213> Artificial
<223> 3A11 light chain CDR1 region amino acid sequence
<400> 62
Lys Ala Ser Asp His Ile Asn Asn Trp Leu Ala
1 5 10
<210> 63
<211> 7
<212> PRT
<213> Artificial
<223> 3A11 light chain CDR2 region amino acid sequence
<400> 63
Gly Ala Thr Ser Leu Glu Thr
1 5
<210> 64
<211> 9
<212> PRT
<213> Artificial
<223> 3A11 light chain CDR3 region amino acid sequence
<400> 64
Gln Gln Tyr Trp Ser Ile Pro Tyr Thr
1 5
<210> 65
<211> 117
<212> PRT
<213> Artificial
<223> humanized antibody 8H8 heavy chain variable region amino acid sequence
<400> 65
Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Glu Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Leu Thr Met Val Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 66
<211> 111
<212> PRT
<213> Artificial
<223> humanized antibody 8H8 light chain variable region amino acid sequence
<400> 66
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ile Ile Asp Gly Ser
20 25 30
Gly Asn Ser Phe Val His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Thr Ser Thr Leu Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gln Thr Val
85 90 95
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 67
<211> 127
<212> PRT
<213> Artificial
<223> humanized antibody 10B2 heavy chain variable region amino acid sequence
<400> 67
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe
20 25 30
Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Asp Ser Asn Thr Ile Tyr Tyr Ala Asp Thr Met
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Asp Pro Ala Tyr Tyr Gly Tyr Gly Gly Arg Phe Val Tyr
100 105 110
Ala Leu Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 68
<211> 112
<212> PRT
<213> Artificial
<223> humanized antibody 10B2 light chain variable region amino acid sequence
<400> 68
Asp Val Val Val Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Thr Asn Ser
20 25 30
Tyr Gly His Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gly Ile Ser Ile Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Gly
85 90 95
Thr His Gln Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 69
<211> 115
<212> PRT
<213> Artificial
<223> humanized antibody 3A11 heavy chain variable region amino acid sequence
<400> 69
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asn Thr Asn
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ala Arg Ile Arg Ser Lys Ile Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Asn Trp Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 70
<211> 107
<212> PRT
<213> Artificial
<223> humanized antibody 3A11 light chain variable region amino acid sequence
<400> 70
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Phe Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Ala Ser Gly Asn Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Ile Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 71
<211> 118
<212> PRT
<213> Artificial
<223> humanized antibody 1A2 heavy chain variable region amino acid sequence
<400> 71
Glu Ala Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Ala Pro Ala Asn Gly Asn Thr Lys Tyr Ala Pro Gln Phe
50 55 60
Gln Asp Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Tyr Gly Ser Asn Phe Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 72
<211> 111
<212> PRT
<213> Artificial
<223> humanized antibody 1A2 light chain variable region amino acid sequence
<400> 72
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Val Asp Asn Tyr
20 25 30
Asp Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Thr Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Val
85 90 95
Glu Asn Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 73
<211> 118
<212> PRT
<213> Artificial
<223> humanized antibody 2F10 heavy chain variable region amino acid sequence
<400> 73
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Glu Asp Thr
20 25 30
Tyr Leu Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Lys Ile Tyr Pro Ala Asn Gly Asp Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Gly Tyr Gly Ser Asn Phe Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 74
<211> 111
<212> PRT
<213> Artificial
<223> humanized antibody 2F0 light chain variable region amino acid sequence
<400> 74
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Asp Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45
Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
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