CN112574312A - OX40 monoclonal antibodies and medical application thereof - Google Patents
OX40 monoclonal antibodies and medical application thereof Download PDFInfo
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Abstract
The invention belongs to the field of tumor therapy and molecular immunology; in particular to a group of monoclonal antibodies of OX40 receptor and medical application thereof. The invention obtains a group of anti-OX 40 monoclonal antibodies with excellent effect on activating human T lymphocytes by a hybridoma technology, and successfully carries out humanized transformation on the monoclonal antibodies. The antibody has wide application prospect in the preparation of related medicines for activating and regulating the action and level of OX40 and remarkably enhancing the immunity of the organism, in particular to medicines related to the treatment of cancers.
Description
Technical Field
The invention belongs to the field of tumor immunotherapy and molecular immunology, and relates to an OX40 antibody and application thereof. In particular, the invention relates to a plurality of OX40 monoclonal antibodies.
Technical Field
OX40 (also known as CD134, ACT35 or TNFRSF4) belongs to a member of the TNFR superfamily, and is a glycoprotein with a molecular weight of 50kD, consisting of a cytoplasmic tail, a transmembrane region and an extracellular region. OX40 was essentially absent in non-activated naive T cells, but was highly expressed in activated T cells (Paterson, et al., (1987), mol. Immunol.24: 1281-90; Mallett, et al., (1990), EMBO J.9: 1063-8; Calderhead, et al., (1993), J. Immunol.151: 5261-71). OX40 ligand (OX40L) is a 34kD type II membrane protein expressed as a trimer in antigen presenting cells (Stuber, et Al., (1995), Immunity 2: 507-21; Ohshima, et Al., (1997), J. Immunol.159: 3838-48; Al-Shamkani, et Al., (1997), J. biol.chem.272: 5275-. The CD4 and CD 8T cells of OX40 knockout mice were reduced in proliferation and defective immune memory responses after antigen stimulation, indicating that endogenous OX40 is important in T cell immune regulation (Kopf, et al., (1999), Immunity 11: 699-.
OX40 is essential for T cell proliferation and survival, and OX 40-depleted T cells failed to survive effectively both at and after division, mainly due to decreased expression levels of anti-apoptotic Bcl-2 family members (Rogers, et al., (2001), Immunity 15: 445-305455; Gramaglia, et al., (2000), J.Immunol 165: 3043-3050). Multiple signaling pathways, including the transcription factors Nuclear factor kappa-light-chain-enhancer of activated B (NF-kB) and activated T-Cell transcription factor (NFAT), can be activated by binding to the OX40 receptor on the surface of T cells using natural ligands (OX40L) or activating antibodies, thereby enhancing the expression of multiple protein molecules, such as survivin, cyclin A, cyclin-dependent kinase, Bcl-2 anti-apoptotic molecules, etc., and increasing the survival and Cell proliferation of various effector T Cell subsets following antigen stimulation (gerros, et al., (2001), Imnity.15: 445-. OX40L-OX40 signaling can regulate cytokine secretion by T cells, antigen presenting cells, NK cells and NKT cells, and regulate signal transduction of cytokine receptor transduction pathways (Gramaglia, et al., (1998), J.Immunol.161: 6510-17; Diana, et al., (2009), Immunity.30: 289-99; Ohshima, et al., (1997), J.Immunol.159: 3838-48).
The OX40 signaling pathway affects the generation and function of regulatory T cells (tregs). Tregs can suppress biological functions of effector T cells, such as transforming growth factor-beta (TGF- β) and interleukin 10(IL-10), by secreting immunosuppressive cytokines. OX40 signal can inhibit IL-10 production by type 1 regulatory T cells, and inhibit ICOS+IL-10+Treg cell proliferation, inhibition of TGF-beta and antigen-driven primary CD4+T transition to CD25+FOXP3+Regulatory T cells, while blocking the suppressive function of natural Tregs (Voo, et al., (2013), J.Immunol.191: 3641-50; So, et al., (2007), J.Immunol.179: 1427-. However, in some specific cases, such as the absence of gamma interferon (IFN-. gamma.) and interleukin 4(IL-4), OX40 can significantly stimulate Treg cell proliferation, suggesting that OX40 can bi-directionally regulate Treg cells, depending mainly on the inflammatory environment surrounding the cells (Ruby, et al., (2009), J. Immunol.183: 4853; Baeyens, et al., (2015), J. Immunol.194: 999-.
OX40 was expressed in tumor infiltrating T cells, including FOXP3+Treg cells, because OX40 can obviously enhance CD4+T and CD8+T cell biological activity and abrogation of Treg cell function, OX40 may be a potential immunomodulatory target for the treatment of cancer. Preclinical studies showTreatment of tumor-bearing animals with OX40 agonists (including anti-OX 40 monoclonal antibodies and OX40L-Fc fusion proteins) resulted in regression of the majority of the animal tumors (Redmond, et al., (2009), Crit. Rev. Immunol.29: 187-. The therapeutic efficacy of OX40 agonists may be affected by a number of factors, including tumor burden, tumor intrinsic immunogenicity, and the tissue site of tumor growth (Kjaergaard, et al., (2000), Cancer Res.60: 5514-. Although OX40 antibody therapy showed strong effects in mice bearing smaller volumes of tumor, it was less desirable for larger tumors or tumor metastases. Thus, various combination therapies were attempted to overcome the deficiencies of the OX40 antibody alone. It has been reported that treatment of OX40 antibodies in combination with arginase inhibitors induces more potent CD4+T and CD8+T cell responses lead to tumor regression (Gough, et al., (2012), Immunology 136: 437-447). Combination therapy with anti-OX 40 and anti-CTLA-4 antibodies can lead to a significant improvement in animal survival in prostate Cancer models with weakly immunogenic TRAMP-C1 antigen and in sarcoma models with highly immunogenic MCA-205 antigen (Redmond, et al, (2014), Cancer Immunol Res.2: 142-.
Various types of anti-OX 40 molecules are currently used in clinical trials for metastatic cancer. According to clinical data of the first OX40 antibody, although the biological activity of the molecule is strong, the clinical anti-tumor effect is not very significant. Therefore, there is still a need to develop new effective OX40 antibody drugs for tumor immunotherapy.
Disclosure of Invention
The invention obtains a group of anti-OX 40 monoclonal antibodies with excellent activation function by a hybridoma technology, and successfully carries out humanized transformation on the monoclonal antibodies. These antibodies can modulate the OX40 pathway and further activate body immune cells by cross-linking reactions, and some antibodies are able to block the interaction of OX40 ligand with OX40 receptor and show stronger anti-tumor activity in animal models than positive control antibodies. The antibody has great application prospect in the preparation of medicines for regulating OX40 level, medicines for activating T lymphocytes of organisms and medicines for treating cancers.
The invention provides murine or humanized OX40 antibodies or functional fragments comprising heavy chain sequences and light chain sequences. The amino acid sequence information of the heavy chain variable region and the light chain variable region of the anti-human OX40 murine antibody is as follows: the amino acid sequences of the 3D9 heavy chain variable region and the light chain variable region are SEQ ID NOs: 1. 2; the amino acid sequences of the 3C9 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 3. 4; 1G4 the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 5. 6; the amino acid sequences of the 15A7 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 7. 8; the amino acid sequences of the 17C1 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 9. 10; 1A2 the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 11. 12; the amino acid sequences of the 3F5 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 13. 14; the amino acid sequences of the 9G7 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 15. 16. The amino acid sequence information of the CDR1, CDR2 and CDR3 of the heavy chain and the light chain of the antibody is as follows: the amino acid sequences of the 3D9 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 17. 18 and 19, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 20. 21, 22; the amino acid sequences of the 3C9 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 23. 24, 25, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 26. 27, 28; 1G4 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 29. 30, 31, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 32. 33, 34; 15A7 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 35. 36 and 37, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 38. 39, 40; the amino acid sequences of the 17C1 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 41. 42, 43, having the light chain CDR1, CDR2, CDR3 amino acid sequences of SEQ ID NOs: 44. 45, 46; 1A2 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 47. 48 and 49, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 50. 51, 52; the amino acid sequences of the 3F5 heavy chain CDR1, CDR2 and CDR3 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; the amino acid sequences of the 9G7 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 59. 60 and 61, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 62. 63, 64.
Further, the anti-human OX40 antibody or fragment is engineered to be a humanized antibody.
The amino acid sequence of the heavy chain variable region of the anti-human OX40 humanized antibody 3C9 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 3F5 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 15A7 is SEQ ID NO: 69; the amino acid sequence of the light chain variable region 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.
an expression vector comprising a nucleic acid molecule of the above antibody.
A pharmaceutical composition comprising the above antibody or a functional fragment thereof, and a pharmaceutically acceptable carrier.
Use of the above antibody or functional fragment thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition for the preparation of a medicament for OX40 immune function.
The method adopts a mammalian cell expression system to prepare recombinant OX40 receptor protein as antigen, and after immunizing a mouse, spleen cells of the mouse are fused with myeloma cells to obtain hybridoma cells. A plurality of monoclonal hybridoma cell strains are obtained by cloning and screening a large number of hybridoma cells for a plurality of times. The hybridoma cell lines can secrete monoclonal antibodies (shown in figures 1 and 3) which are specifically combined with OX40 receptors, and the agonist activities of the monoclonal antibodies mainly depend on Fc gamma RII (CD32) mediated cross-linking reaction, so that a nuclear factor NF-kB signal channel (shown in figure 2) is activated, and a cytokine IL-2 secreted by Jurkat cells induced by a CD3 antibody (OKT3) is enhanced (shown in figure 2)5) Promoting human CD4+T cells secrete cytokines IL-2 and IFN-gamma (FIG. 6), with some monoclonal antibodies being effective in blocking OX40L binding to OX40 receptors (FIG. 4), suggesting that some antibodies may have concurrent antagonist function and may be used for future OX40 antagonist studies. Further, genes encoding light and heavy Chain variable regions of the antibody were cloned by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and a complementary-determinant grafting method (CDR-grafting) was used to construct a humanized antibody. In vitro functional experiments showed that humanized OX40 antibodies specifically bind to OX40 receptor protein (fig. 7, fig. 9). These antibodies can activate nuclear factor NF- κ B signaling pathway (FIG. 8), enhance cytokine IL-2 secretion from Jurkat cells induced by CD3 antibody (OKT3) (FIG. 10), and promote human CD4 through Fc γ RIIB (CD32B) -mediated crosslinking+T cells secrete cytokines (fig. 11). Animal test results showed that the OX40 humanized antibody in this patent has stronger anti-tumor activity than the positive control antibody (fig. 12), the sequence of the positive control antibody BMS-986178 is from patent US20170306035 of BMS, the sequence of the positive control antibody von lerolizumab is from patent US20160355597 of Genentech, and the sequence of the positive control antibody Hu3738 is from patent US20180346593a1 of AbbVie.
Drawings
FIG. 1: EC50 of OX40 hybridoma antibody binding to OX40-hFc protein was determined by the ELSIA method;
FIG. 2: measuring EC50 of OX40 hybridoma antibody cross-linked with CD32A for activating NF- κ B signal of Jurkat-OX40 cells by a Reporter Gene (Gene Reporter) method;
FIG. 3: EC50 of the OX40 hybridoma antibodies binding to Jurkat-OX40 cells was determined by FACS method;
FIG. 4: determining the IC50 of OX40 hybridoma antibodies that block binding of OX40L to Jurkat-OX40 cells using FACS methods;
FIG. 5: measuring by ELISA the secretion of IL-2 by Jurkat-OX40 cells stimulated by OX40 hybridoma antibodies cross-linked to CD 32A;
FIGS. 6A-6B: measuring the stimulation of IL-2 and IFN-gamma secretion by Fc-crosslinked OX40 hybridoma antibodies on human CD4+ T cells by ELISA;
FIG. 7: EC50 of humanized OX40 antibody binding to OX40-mFc protein was determined by ELSIA method;
FIG. 8: determining the EC50 of humanized OX40 antibody cross-linked with CD32B activated NF- κ B signal of Jurkat-OX40 cells by a reporter gene method;
FIG. 9: determining EC50 binding of the humanized OX40 antibody to Jurkat-OX40 cells using FACS methods;
FIG. 10: humanized OX40 antibodies cross-linked to CD32B stimulated IL-2 secretion by Jurkat-OX40 cells as determined by the ELSIA method;
FIG. 11: fc-crosslinked humanized OX40 antibodies stimulated IL-2 secretion by human CD4+ T cells as determined by the ELSIA method;
FIG. 12: the OX40 monoclonal antibody significantly inhibited MC38 tumor growth in the humanized OX40 mouse model.
Detailed Description
Example 1
Generation of anti-OX 40 monoclonal antibodies
After fully emulsifying the extracellular region of human OX40 and mouse Fc fusion protein (OX40-ECD-mFc) as antigen with equal volume of complete Freund's adjuvant (Sigma, Cat. No.: F5581), Balb/c mice (purchased from Showa Kagaku pharmaceutical research center, Ltd.) with 6-8 weeks of age were immunized subcutaneously with 50. mu.g/mouse of antigen. Mice were then immunized subcutaneously three times every 2 weeks after full emulsification with the same dose of antigen with incomplete freund's adjuvant (Sigma, cat. No.: F5506). After three immunizations, the serum titer of the mice was determined, and a booster immunization was performed by intraperitoneal injection 3 days before the fusion. Using PEG Hybri-Max (Sigma, Cat. No.: 7181) as a fusion agent, mouse spleen cells and SP2/0 cells were mixed at a ratio of 4:1, and the fused cells were added to a 96-well plate (1X 10)5One/well) containing 0.1mL of 1 XHAT (Invitrogen, Cat. No.:21060-017) medium per well. 0.1mL of HT (Invitrogen, Cat. No.: 11067-one 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. Supernatants were harvested on day 9 for ELISA and Reporter Gene (Gene Reporter) assay.
Example 2
For human beingsExtracellular region of source OX40 was screened for hybridomas with human Fc fusion protein (OX 40-hFc). Coating a 96-well ELISA plate (Corning, Cat. No.:9018) with 50. mu.L of OX40-hFc at a concentration of 2. mu.g/mL overnight at room temperature; after washing 3 times with washing buffer (PBS + 0.05% Tween20), blocking buffer (PBS + 2% BSA) was added and incubated for 1 hour at room temperature, and the ELISA plate was washed 3 times with washing buffer; adding hybridoma supernatant, incubating at room temperature for 1 hr, and washing for 3 times; 100 μ L of 10000-fold diluted HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.:31432) was added to each well, incubated at room temperature for 1 hour in the dark, and washed 3 times; adding 100 μ L of TMB (Cat. No.: ES-002) to each well, incubating at room temperature for color development for 2 min, and adding 100 μ L of 2N H to each well2SO4The color reaction was stopped and the OD450 of each well was read with a microplate reader (Tecan Spark).
A Reporter Gene (Gene Reporter) method is adopted to screen positive hybridoma antibodies which cause activation of cell transcription factor NF-kB by Fc gamma RIIA (CD32A) mediated cross-linking reaction. mu.L of hybridoma supernatant or purified hybridoma antibody positive in the above ELISA assay was combined with 25. mu.L of Jurkat-OX40 cells (4X 10)4One/well) were mixed, added to a 96-well half-pan flat plate, and 25. mu.L of 293T-CD32A cells (2.5X 10) were added4One/well), mixed well and incubated in an incubator at 37 ℃ for 4 hours. mu.L of pre-warmed Bright-Glo solution (Promega, Cat. No.: E2620) was added to each well, allowed to stand at room temperature in the dark for 3 minutes, and the Luminescence (Luminescence) signal value of each sample well was measured using a microplate reader (Tecan Spark).
In a 96-well U-bottom cell plate, 50. mu.L of hybridoma supernatant or purified hybridoma antibody positive in the above assay was added and mixed with 50. mu.L of Jurkat-OX40 cells (2X 10)5One/well), incubated at 4 ℃ for 1 hour, washed with FACS buffer (PBS + 3% FCS) and centrifuged twice, a 400-fold dilution of PE-labeled goat anti-mouse secondary antibody (Biolegend, cat. No.:405307) was added, incubated at 4 ℃ for 30 minutes in the dark, washed with FACS buffer and centrifuged twice, and the cell signal values on the PE channel were detected using BD Accuri C6 flow cytometer.
Competitive FACS was used to detect the activity of hybridoma antibodies to block the binding of OX40L to OX40 receptor. Hybridoma supernatants or purified antibodies were conjugated to Jurkat-OX40 cells (2X 10)5One/well) in 96-well U-bottom cell plates at 4 ℃ for 1 hour, adding a biotin-labeled OX40L to a final concentration of 1. mu.g/mL, mixing, incubating at 4 ℃ for 1 hour, centrifuging, washing twice with FACS buffer, adding a 400-fold dilution of a PE-labeled secondary avidin antibody (Biolegend, Cat. No.:409004), incubating at 4 ℃ for 30 minutes in the absence of light, washing twice with FACS buffer and centrifuging, and detecting the signal value of the cells on the PE channel by using a BD Accuri C6 flow cytometer.
Example 3
And carrying out subcloning on the positive hybridoma cells in the Reporter gene detection by adopting a limiting dilution method, and then repeatedly carrying out detection screening by adopting an ELISA (enzyme-Linked immuno sorbent assay) method and a Reporter method to obtain a positive hybridoma monoclonal. Positive monoclonal hybridoma cells 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 ultrafiltration using a Millipore centrifuge tube (Millipore, Cat. No.: ACS500024), the Protein concentration was measured by the BCA method, and the endotoxin content of the purified antibody samples was measured by limulus reagent (Tachypleus tridentatus, Mth.). The reporter gene method is used for detecting the activation capability of the transcription promoting factor NF-kB, ELISA and FACS are used for detecting the binding capability of a purified antibody sample and OX40, and FACS is used for detecting the blocking activity of the antibody on the combination of OX40L and OX40, and the specific method refers to example 2. Specific results are shown in tables 1-4 and FIGS. 1-4.
TABLE 1 detection of OX40 hybridoma antibodies binding to OX40-hFc protein by ELSIA
TABLE 2 reporter Gene assay for activation of Jurkat cells by OX40 hybridoma antibodies cross-linked to CD32A
TABLE 3 measurement of binding of OX40 hybridoma antibodies to human OX40 by FACS
TABLE 4 measurement of blocking of OX40L binding to OX40 by OX40 hybridoma antibodies by FACS
Example 4
Detection of IL-2 secretion by Jurkat-OX40 cells by OX40 hybridoma antibody crosslinked with CD32A
Purified OX40 antibody was diluted in a gradient, 25. mu.L of antibody dilution was added to a 96-well U-bottom cell plate, and 50. mu.L of Jurkat-OX40 cells (1X 10)5Cell/well), 25. mu.L of 293T-Fc γ RIIA (CD32A) cells treated with mitomycin (2.5X 10)4One/well) and 50. mu.L of 293T-OKT3 cells (2X 10)4One/well), mixed well and incubated in an incubator at 37 ℃ for 48 hours. Cell supernatants were collected and assayed using IL-2ELISA kit (R)&D Systems, Cat.No. DY202) assay the IL-2 concentration in cell supernatants. As shown in FIG. 5, crosslinking of OX40 antibody by CD32A significantly enhanced IL-2 secretion by OKT 3-induced Jurkat-OX40 cells.
Human CD4 after Fc cross-linking of OX40 hybridoma antibodies+Promoting effect of T cell for secreting IL-2 and IFN-gamma
A50 mL sterile centrifuge tube was charged with lymphocyte isolate Ficoll Paque PLUS (GE Healthcare, Cat. No.:17-1440-03) followed by an equal volume of a healthy donor blood diluent (V)Anticoagulation : VPBS1: 1) at room temperature 1500rpm for 30 minutes. 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, a red blood cell layer and a granulocyte layer from top to bottom. White cells in the middleThe layers were collected in a new centrifuge tube, washed by adding 10 volumes of wash buffer (PBS + 3% FBS) and mixing well, centrifuged at 1500rpm for 10 minutes at 4 ℃, washed 2 times, resuspended cells in wash buffer and counted.
After cell counting, human CD4 was used+T cell isolation magnetic bead kit (Miltenyi, Cat. No.: 130-096-533) for sorting and enriching human CD4+T cells. The isolated PBMCs were collected and pooled in washing buffer (40. mu.L/10)7Cells) resuspended cells, and a suitable volume of biotin-labeled antibody cocktail (10. mu.L/107Cells), mixed well and incubated at 4 ℃ for 10 minutes. Add washing buffer (30. mu.L/10)7Cells) and magnetic beads coupled with anti-biotin antibody (20. mu.L/10)7Cells), mixed well and incubated at 4 ℃ for 10 minutes. The LS separation column was placed in an immunomagnetic bead separator, and 3mL of washing solution was added to wash the LS separation column. The cell magnetic bead suspension was added to the upper end of the LS separation column, and 3mL of washing buffer was added for washing 3 times. Collecting the effluent to obtain CD4+T cells, centrifuged with the appropriate volume of wash buffer. Cell count, resuspension of human CD4 in complete medium (RPMI1640+ 10% FBS)+Experiments were performed immediately after T cells, or CD4 was used+The T cells were frozen to liquid nitrogen for use.
A96-well plate (Corning, Cat. No.:9018) was coated with 50. mu.L of Goat anti-mFc antibody (final concentration: 2. mu.g/mL), sterilized at room temperature overnight, washed 3 times with sterile PBS, and the liquid was aspirated off. Conditioning CD4 with complete Medium+T cell concentration, 100. mu.L of CD4 was added per well+T cells, 50. mu.L of OX40 antibody dilution (initial concentration 10. mu.g/mL, 10-fold gradient dilution) and 50. mu.L of CD3 antibody solution (final concentration: 80ng/mL) were mixed, and the 96-well cell plate was placed at 37 ℃ and 5% CO2The culture was carried out in an incubator for 48 hours and 72 hours, respectively. Cell culture supernatants were collected and assayed using IL-2ELISA kit (R)&D Systems, Cat.No. DY202) assayed the IL-2 concentration in the supernatant of cells cultured for 48 hours. Using IFN-gamma ELISA kit (R)&D Systems, Cat. No. DY285) measured IFN-. gamma.concentration in the supernatant of cells cultured for 72 hours. The results are shown in figure 6, OX40 antibody significantly promoted human CD4 after Fc cross-linking+T cells secrete IL-2 and IFN- γ.
Example 5
Cloning of OX40 antibody variable region genes
OX40 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 using cDNA as a template and degenerate primers (Kettleborough, et al, (1993), Eur.J.immunology.23: 206-211; Strebe, et al, (2010), Antibody Engineering 1: 3-14). 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 amino acid sequences of the variable regions and CDRs of the heavy and light chains of the antibodies are shown in Table 5-1.
TABLE 5-1 OX40 antibody SEQ ID Nos 1-64
Example 6
Construction and preparation of humanized OX40 antibodies 3C9, 3F5, 15A7, 1A2
Humanization of the OX40 antibody was performed using complementarity determining region grafting. First, the IMGT database was searched for human germline antibody (germline antibody) sequences with the highest homology to the light and heavy chain variable regions of the murine antibodies 3C9, 3F5, 15a7, and 1a2, respectively. The humanized selected germ line of 3C9 antibody light chain variable region is IGKV2-28 x 01, and the humanized selected heavy chain variable region is IGHV1-2 x 02. The humanized selected embryo line of 3F5 antibody light chain variable region is IGKV1-17 x 01, and the humanized selected heavy chain variable region is IGHV3-7 x 01. The light chain of the 15A7 antibody is humanized by IGKV1-5 × 01, and the heavy chain is humanized by IGHV4-59 × 01. The light chain of the 1A2 antibody is humanized and selected from IGKV3-11 x 01, and the heavy chain is humanized and selected from IGHV2-5 x 09. 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.
Partial amino acids of the humanized antibody framework regions of 3C9, 3F5, 15a7, 1a2 were back mutated to murine sequences according to structural modeling. The 48 th Met of the humanized heavy chain of the 3C9 antibody is back mutated into Ile, the 67 th Val is back mutated into Ala, the 69 th Met is back mutated into Leu, the 71 th Arg is back mutated into Ser, and the 73 th Thr is back mutated into Lys. The position 64 Gly of the humanized light chain of the 3C9 antibody is back-mutated into Ser. The 30 th Ser of the humanized heavy chain of the 3F5 antibody is back mutated into Gly, the 69 th Ile is back mutated into Thr, and the 93 th Ala is back mutated into Val. The 36 th Tyr of the humanized light chain of the 3F5 antibody is back mutated into Leu, the 66 th Gly is back mutated into Arg, the 69 th Thr is back mutated into Ser, and the 71 th Phe is back mutated into Tyr. The 27 th Gly of the humanized heavy chain of the 15A7 antibody is subjected to reverse mutation to Tyr, the 30 th Ser is subjected to reverse mutation to Thr, the 47 th Trp is subjected to reverse mutation to Tyr, the 48 th Ile is subjected to reverse mutation to Met, the 67 th Val is subjected to reverse mutation to Ile, the 71 th Val is subjected to reverse mutation to Arg, and the 78 th Phe is subjected to reverse mutation to Tyr. The 46 th Leu of the humanized light chain of the 1A2 antibody is back mutated into Pro, the 47 th Leu is back mutated into Trp, and the 71 th Phe is back mutated into Tyr. The 30 th Ser of the humanized heavy chain of the 1A2 antibody was back mutated to Ala.
The amino acid sequence numbers of the variable regions of the heavy chain and the light chain of the 3C9 humanized antibody are respectively SEQ ID NO: 65 and SEQ ID NO: 66. the amino acid sequence numbers of the variable regions of the heavy chain and the light chain of the 3F5 humanized antibody are respectively SEQ ID NO: 67 and SEQ ID NO: 68. 15a7 the amino acid sequence numbers of the variable regions of the heavy and light chains of the humanized antibody are SEQ ID NOs: 69 and SEQ ID NO: 70. 1A2 humanized antibody heavy and light chain variable region amino acid sequence numbers are SEQ ID NOs: 71 and SEQ ID NO: 72. the amino acid sequences of the variable regions of the heavy and light chains of the humanized antibodies are shown in tables 5-2.
TABLE 5-2 OX40 antibody SEQ ID Nos 65-72
Nucleic acid sequences encoding the light and heavy chains of the humanized antibodies 3C9, 3F5, 15a7, 1a2 were synthesized and inserted into the expression vector pcdna3.1. 200mL of 293 cells (cell density 1X 10) were co-transfected with 0.1mg of antibody light chain and 0.1mg of antibody heavy chain expression plasmid6/mL), shaking and culturing at 37 ℃ for 6 daysThe supernatant was collected by centrifugation, and the humanized antibody was purified using Protein A, and the activity of the purified humanized antibody was examined.
Example 7
The activation of NF- κ B signaling pathway after purified humanized OX40 antibody was cross-linked with CD32A or CD32B was determined by reporter gene method, and the binding activity of humanized OX40 antibody sample to OX40 receptor protein was examined by ELISA, FACS, as detailed in example 2. The results of the measurement of humanized antibodies hu3C9, hu3F5, hu15A7, hu1A2 are shown in tables 6 to 8 and FIGS. 7 to 9.
TABLE 6 detection of binding of humanized OX40 antibody to OX40-mFc protein by ELSIA
TABLE 7 reporter Gene assays for activation of Jurkat-OX40 by humanized OX40 antibody cross-linked to CD32B
TABLE 8 measurement of binding of humanized OX40 antibody to human OX40 by FACS
Example 8
Effect of humanized OX40 antibody on IL-2 secretion by Jurkat-OX40 cells after cross-linking CD 32B. Humanized OX40 antibody dilution, Jurkat-OX40 cells, mitomycin-treated 293T-CD32B and 293T-OKT3 were mixed well, placed in a cell culture chamber at 37 ℃ for 48 hours, cell supernatants were collected, and the concentration of IL-2 in the supernatants was measured, and the specific measurement method was as described in example 4. As a result, as shown in FIG. 10, the humanized OX40 antibody cross-linked with CD32B significantly promoted IL-2 secretion by Jurkat-OX40 cells.
Example 9
Fc crosslinking of humanized OX40 antibodies promotes human CD4+T cells secrete IL-2. Humanized OX40 antibody diluent, CD4+ T cells and CD3 antibody are mixed evenly, added into a 96-well plate coated with PBS or Goat anti-hFc and Goat anti-mFc, placed in a cell culture box at 37 ℃ for culturing for 72 hours, cell supernatant liquid is collected, and the IL-2 concentration in the supernatant liquid is detected, wherein the specific determination method refers to example 4. The results are shown in FIG. 11, and the humanized OX40 antibody significantly promoted human CD4 after Fc crosslinking+T cells secrete IL-2.
Example 10
Affinity assay for humanized OX40 antibody. The determination method mainly comprises the following steps: protein A was immobilized on a CM5 chip by amino coupling followed by capture of OX40 antibody at a flow rate of 10. mu.L/min. The flow rate was switched to 30. mu.L/min, different concentrations of histidine-tagged OX40 antigen (100nM, 50nM,25nM,12.5nM,6.25nM,3.125nM) were passed through the sample and reference channels in sequence, with a binding time of 3 min and a dissociation time of 10 min, and the chip was finally regenerated with glycine buffer pH 2.0. The results for the affinity of OX40 antibodies to OX40 antigen are shown in table 9. In addition, in order to improve the humanization degree of the antibody, partial amino acid positions in the CDR region of the 3F5 antibody were mutated, including Lys at position 24 of the 3F5 light chain to Arg, Ser at position 25 to Ala, Asp at position 55 to gin, and Val at position 56 to Ser, and the affinity of the mutants to the OX40 antigen was determined using Biacore (table 9).
TABLE 9 determination of binding of humanized OX40 antibody samples to OX40 using Biacore
Example 11
Inhibition of MC38 tumor growth in OX40 humanized mice by OX40 monoclonal antibody
The MC38 mouse tumor model and OX40 humanization were used for the experimentsC57BL/6 mice. 6 week old female OX40 humanized C57BL/6 mice were selected and inoculated subcutaneously at the lower right of their backs with 1X106MC38 tumor cells. 2 weeks after tumor cell inoculation, mice were divided into 6 different experimental groups of 6 mice each, with similar mean tumor volumes and tumor volumes of 70-100mm3In the meantime. Mice received 5mg/kg of anti-OX 40 antibody or isotype control antibody IgG1 (negative control). anti-OX 40 antibodies were humanized 3C9, 3F5, 15A7 (i.e., hu3C9-hIgG1, hu3F5-hIgG1, and hu15A7-hIgG1) and hybridoma antibody 1A2(mIgG 1). BMS-986178 and Vonllerolizumab were used as positive controls. Mice were dosed by intraperitoneal injection twice weekly at an antibody dose of 5mg/kg for a total treatment period of 2.5 weeks. During the experiment, the tumor volume is periodically measured by a vernier caliper, and when the tumor of the mouse is ulcerated or the tumor volume exceeds 2000mm3Mice were euthanized at time. The humanized anti-OX 40 antibodies hu3C9-hIgG1, hu3F5-hIgG1, hu15a7-hIgG1, and OX40 hybridoma antibody 1a2-mIgG1 all showed stronger inhibition of MC38 tumor growth compared to positive control antibodies for BMS and Roche (fig. 12).
The results of the above examples show that the monoclonal antibody or antigen binding fragment thereof of the present invention, or the conjugate comprising the monoclonal antibody or antigen binding fragment thereof of the present invention, has a good application prospect in the preparation of drugs for modulating the activity of OX40 or the expression level of OX40, drugs for promoting OX40 activation and enhancing immunity of the body, drugs for activating T lymphocytes, drugs for increasing the expression of IL-2 and INF-gamma by T lymphocytes, drugs for blocking the binding of OX40 ligand to OX40 receptor, and drugs for preventing and treating or adjunctively treating tumors.
The foregoing is only a preferred embodiment of this invention and it should be noted that modifications may be made by those skilled in the art without departing from the principles of the invention and these modifications should also be considered as within the scope of the invention.
SEQUENCE LISTING
<110> east China Biotechnology (Suzhou) Ltd
<120> a group of OX40 monoclonal antibodies and medical uses thereof
<130> 2020
<160> 72
<170> PatentIn version 3.5
<210> 1
<211> 116
<212> PRT
<213> Artificial
<223> 3D9 heavy chain variable region amino acid sequence
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr
20 25 30
Gly Ile Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Asp Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asn Asn Tyr Lys Ser Gln Val Phe Leu
65 70 75 80
Thr Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys Leu Leu Pro Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 2
<211> 107
<212> PRT
<213> Artificial
<223> 3D9 light chain variable region amino acid sequence
<400> 2
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asn Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 3
<211> 120
<212> PRT
<213> Artificial
<223> 3C9 heavy chain variable region amino acid sequence
<400> 3
Asp Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ile Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Ser Arg Tyr Asn Asp Lys Phe
50 55 60
Lys Val Lys Ala Thr Leu Ala Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Tyr Asp Asp Gly Ala Trp Phe Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 4
<211> 112
<212> PRT
<213> Artificial
<223> 3C9 light chain variable region amino acid sequence
<400> 4
Glu Ile Gln Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly
1 5 10 15
Glu Ser Val Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Ile Thr Tyr Leu Phe Trp Tyr Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn
85 90 95
Leu Glu Leu Pro Val Thr Phe Gly Ala Gly Thr Lys Val Glu Leu Lys
100 105 110
<210> 5
<211> 117
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<223> 1G4 heavy chain variable region amino acid sequence
<400> 5
Arg Val 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 Thr Asp Tyr
20 25 30
Ser Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Ser Glu Ile Ser Ala Thr Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Ser Leu Lys Asn Glu Asp Thr Ser Thr Tyr Phe Cys
85 90 95
Ala Trp Val Pro Pro Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr
100 105 110
Val Thr Val Ser Ser
115
<210> 6
<211> 107
<212> PRT
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<223> 1G4 light chain variable region amino acid sequence
<400> 6
Asp Ile Val Met Thr Gln Ser Thr Thr Ser Leu Ser Val Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Asn Cys Arg Thr Ser Gln Asp Ile Thr Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Met Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser His Leu Glu Gln
65 70 75 80
Asp Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Pro Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 7
<211> 123
<212> PRT
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<223> 15A7 heavy chain variable region amino acid sequence
<400> 7
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Ala Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Gly Gly Tyr Tyr Tyr Gly Ser Ser Tyr Pro Tyr Tyr Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 8
<211> 106
<212> PRT
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<223> 15A7 light chain variable region amino acid sequence
<400> 8
Asp Ile Val Met Thr Gln Ser Thr Lys Phe Met Ser Thr Thr Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Pro Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 9
<211> 115
<212> PRT
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<223> 17C1 heavy chain variable region amino acid sequence
<400> 9
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Thr Tyr Ser Gly Phe Thr Arg Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Phe Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Val Tyr Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
<210> 10
<211> 112
<212> PRT
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<223> 17C1 light chain variable region amino acid sequence
<400> 10
Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Val Gly
1 5 10 15
His Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asn Gly Glu Thr Tyr Leu Lys Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Gly
85 90 95
Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 11
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<223> 1A2 heavy chain variable region amino acid sequence
<400> 11
Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Ile Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Thr Ser
20 25 30
Gly Val Ala Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Val Lys Arg Tyr Asn Pro Ser
50 55 60
Leu Gln Gly Arg Leu Thr Ile Ser Lys Asp Thr Ala Ser Ser Gln Val
65 70 75 80
Phe Leu Lys Ile Ser Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Met Asp Trp Asp Gly Leu Ala Tyr Trp Gly Ala Gly Thr
100 105 110
Thr Val Thr Val Ser Ala
115
<210> 12
<211> 108
<212> PRT
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<223> 1A2 light chain variable region amino acid sequence
<400> 12
Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser
20 25 30
Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp
35 40 45
Ile Tyr Gly Thr Ser Asn Leu Ala Thr Gly Val Pro Val Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro
85 90 95
Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 13
<211> 117
<212> PRT
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<223> 3F5 heavy chain variable region amino acid sequence
<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Arg Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Ser Gly Gly Ile Tyr Thr Tyr Tyr Ala Asp Ile Val
50 55 60
Thr Gly Arg Phe Thr Thr Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Val Arg Asp Phe Asp Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 14
<211> 107
<212> PRT
<213> Artificial
<223> 3F5 light chain variable region amino acid sequence
<400> 14
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Lys Ser Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ala Trp Leu Gln Gln Lys Pro Asp Gly Thr Val Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Asp Val Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Phe Gln Tyr Ala Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 15
<211> 119
<212> PRT
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<223> 9G7 heavy chain variable region amino acid sequence
<400> 15
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Asn Gly Asp Ile Ser Thr Tyr Tyr Pro Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Asp Asp Gly Tyr Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 16
<211> 113
<212> PRT
<213> Artificial
<223> 9G7 light chain variable region amino acid sequence
<400> 16
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly
1 5 10 15
Gln Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ile 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 Asp Tyr Phe Cys Gln Gln
85 90 95
His Tyr Tyr Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 17
<211> 5
<212> PRT
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<223> 3D9 heavy chain CDR1 region amino acid sequence
<400> 17
Thr Tyr Gly Ile Thr
1 5
<210> 18
<211> 16
<212> PRT
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<223> 3D9 heavy chain CDR2 region amino acid sequence
<400> 18
Val Ile Trp Asp Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile Ser
1 5 10 15
<210> 19
<211> 8
<212> PRT
<213> Artificial
<223> 3D9 heavy chain CDR3 region amino acid sequence
<400> 19
Leu Leu Pro Tyr Tyr Phe Asp Ser
1 5
<210> 20
<211> 11
<212> PRT
<213> Artificial
<223> 3D9 light chain CDR1 region amino acid sequence
<400> 20
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 21
<211> 7
<212> PRT
<213> Artificial
<223> 3D9 light chain CDR2 region amino acid sequence
<400> 21
Tyr Thr Ser Arg Leu Leu Ser
1 5
<210> 22
<211> 9
<212> PRT
<213> Artificial
<223> 3D9 light chain CDR3 region amino acid sequence
<400> 22
Gln Gln Gly Tyr Thr Leu Pro Pro Thr
1 5
<210> 23
<211> 5
<212> PRT
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<223> 3C9 heavy chain CDR1 region amino acid sequence
<400> 23
Ser Tyr Ile Met His
1 5
<210> 24
<211> 17
<212> PRT
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<223> 3C9 heavy chain CDR2 region amino acid sequence
<400> 24
Tyr Ile Asn Pro Tyr Asn Gly Gly Ser Arg Tyr Asn Asp Lys Phe Lys
1 5 10 15
Val
<210> 25
<211> 11
<212> PRT
<213> Artificial
<223> 3C9 heavy chain CDR3 region amino acid sequence
<400> 25
Gly Asp Tyr Asp Asp Gly Ala Trp Phe Gly Tyr
1 5 10
<210> 26
<211> 16
<212> PRT
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<223> 3C9 light chain CDR1 region amino acid sequence
<400> 26
Lys Ser Ser Gln Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Phe
1 5 10 15
<210> 27
<211> 7
<212> PRT
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<223> 3C9 light chain CDR2 region amino acid sequence
<400> 27
Arg Met Ser Asn Arg Ala Ser
1 5
<210> 28
<211> 9
<212> PRT
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<223> 3C9 light chain CDR3 region amino acid sequence
<400> 28
Gly Gln Asn Leu Glu Leu Pro Val Thr
1 5
<210> 29
<211> 5
<212> PRT
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<223> 1G4 heavy chain CDR1 region amino acid sequence
<400> 29
Asp Tyr Ser Ile His
1 5
<210> 30
<211> 17
<212> PRT
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<223> 1G4 heavy chain CDR2 region amino acid sequence
<400> 30
Trp Ile Asn Thr Glu Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe Lys
1 5 10 15
Gly
<210> 31
<211> 8
<212> PRT
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<223> 1G4 heavy chain CDR3 region amino acid sequence
<400> 31
Val Pro Pro Trp Tyr Phe Asp Val
1 5
<210> 32
<211> 11
<212> PRT
<213> Artificial
<223> 1G4 light chain CDR1 region amino acid sequence
<400> 32
Arg Thr Ser Gln Asp Ile Thr Asn Tyr Leu Asn
1 5 10
<210> 33
<211> 7
<212> PRT
<213> Artificial
<223> 1G4 light chain CDR2 region amino acid sequence
<400> 33
Tyr Thr Ser Met Leu Lys Ser
1 5
<210> 34
<211> 9
<212> PRT
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<223> 1G4 light chain CDR3 region amino acid sequence
<400> 34
Gln Gln Gly Asn Thr Pro Pro Leu Thr
1 5
<210> 35
<211> 5
<212> PRT
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<223> 15A7 heavy chain CDR1 region amino acid sequence
<400> 35
Ser Asp Tyr Trp Asn
1 5
<210> 36
<211> 16
<212> PRT
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<223> 15A7 heavy chain CDR2 region amino acid sequence
<400> 36
Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 37
<211> 15
<212> PRT
<213> Artificial
<223> 15A7 heavy chain CDR3 region amino acid sequence
<400> 37
Gly Gly Tyr Tyr Tyr Gly Ser Ser Tyr Pro Tyr Tyr Phe Asp Tyr
1 5 10 15
<210> 38
<211> 11
<212> PRT
<213> Artificial
<223> 15A7 light chain CDR1 region amino acid sequence
<400> 38
Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala
1 5 10
<210> 39
<211> 7
<212> PRT
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<223> 15A7 light chain CDR2 region amino acid sequence
<400> 39
Ser Pro Ser Asn Arg Tyr Thr
1 5
<210> 40
<211> 8
<212> PRT
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<223> 15A7 light chain CDR3 region amino acid sequence
<400> 40
Gln Gln Tyr Ser Ser Tyr Arg Thr
1 5
<210> 41
<211> 6
<212> PRT
<213> Artificial
<223> 17C1 heavy chain CDR1 region amino acid sequence
<400> 41
Ser Asp Tyr Ala Trp Asn
1 5
<210> 42
<211> 16
<212> PRT
<213> Artificial
<223> 17C1 heavy chain CDR2 region amino acid sequence
<400> 42
Tyr Ile Thr Tyr Ser Gly Phe Thr Arg Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 43
<211> 6
<212> PRT
<213> Artificial
<223> 17C1 heavy chain CDR3 region amino acid sequence
<400> 43
Pro Ala Trp Phe Ala Tyr
1 5
<210> 44
<211> 16
<212> PRT
<213> Artificial
<223> 17C1 light chain CDR1 region amino acid sequence
<400> 44
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asn Gly Glu Thr Tyr Leu Lys
1 5 10 15
<210> 45
<211> 7
<212> PRT
<213> Artificial
<223> 17C1 light chain CDR2 region amino acid sequence
<400> 45
Leu Val Ser Lys Leu Asp Ser
1 5
<210> 46
<211> 9
<212> PRT
<213> Artificial
<223> 17C1 light chain CDR3 region amino acid sequence
<400> 46
Val Gln Gly Thr His Phe Pro Tyr Thr
1 5
<210> 47
<211> 7
<212> PRT
<213> Artificial
<223> 1A2 heavy chain CDR1 region amino acid sequence
<400> 47
Thr Ser Gly Val Ala Val Gly
1 5
<210> 48
<211> 16
<212> PRT
<213> Artificial
<223> 1A2 heavy chain CDR2 region amino acid sequence
<400> 48
His Ile Trp Trp Asp Asp Val Lys Arg Tyr Asn Pro Ser Leu Gln Gly
1 5 10 15
<210> 49
<211> 8
<212> PRT
<213> Artificial
<223> 1A2 heavy chain CDR3 region amino acid sequence
<400> 49
Met Asp Trp Asp Gly Leu Ala Tyr
1 5
<210> 50
<211> 12
<212> PRT
<213> Artificial
<223> 1A2 light chain CDR1 region amino acid sequence
<400> 50
Ser Val Ser Ser Ser Ile Ser Ser Ser Asn Leu His
1 5 10
<210> 51
<211> 7
<212> PRT
<213> Artificial
<223> 1A2 light chain CDR2 region amino acid sequence
<400> 51
Gly Thr Ser Asn Leu Ala Thr
1 5
<210> 52
<211> 9
<212> PRT
<213> Artificial
<223> 1A2 light chain CDR3 region amino acid sequence
<400> 52
Gln Gln Tyr Ser Ser Tyr Pro Phe Thr
1 5
<210> 53
<211> 5
<212> PRT
<213> Artificial
<223> 3F5 heavy chain CDR1 region amino acid sequence
<400> 53
Ser Tyr Ala Met Ser
1 5
<210> 54
<211> 17
<212> PRT
<213> Artificial
<223> 3F5 heavy chain CDR2 region amino acid sequence
<400> 54
Glu Ile Ser Ser Gly Gly Ile Tyr Thr Tyr Tyr Ala Asp Ile Val Thr
1 5 10 15
Gly
<210> 55
<211> 8
<212> PRT
<213> Artificial
<223> 3F5 heavy chain CDR3 region amino acid sequence
<400> 55
Asp Phe Asp Tyr Arg Phe Ala Tyr
1 5
<210> 56
<211> 11
<212> PRT
<213> Artificial
<223> 3F5 light chain CDR1 region amino acid sequence
<400> 56
Lys Ser Ser Gln Glu Ile Ser Gly Tyr Leu Ala
1 5 10
<210> 57
<211> 7
<212> PRT
<213> Artificial
<223> 3F5 light chain CDR2 region amino acid sequence
<400> 57
Ala Ala Ser Ser Leu Asp Val
1 5
<210> 58
<211> 9
<212> PRT
<213> Artificial
<223> 3F5 light chain CDR3 region amino acid sequence
<400> 58
Phe Gln Tyr Ala Ser Tyr Pro Tyr Thr
1 5
<210> 59
<211> 5
<212> PRT
<213> Artificial
<223> 9G7 heavy chain CDR1 region amino acid sequence
<400> 59
Asp Tyr Tyr Met Tyr
1 5
<210> 60
<211> 17
<212> PRT
<213> Artificial
<223> 9G7 heavy chain CDR2 region amino acid sequence
<400> 60
Tyr Ile Ser Asn Gly Asp Ile Ser Thr Tyr Tyr Pro Asp Thr Val Lys
1 5 10 15
Gly
<210> 61
<211> 10
<212> PRT
<213> Artificial
<223> 9G7 heavy chain CDR3 region amino acid sequence
<400> 61
Asp Asp Gly Tyr Ser Ala Trp Phe Ala Tyr
1 5 10
<210> 62
<211> 17
<212> PRT
<213> Artificial
<223> 9G7 light chain CDR1 region amino acid sequence
<400> 62
Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu
1 5 10 15
Ala
<210> 63
<211> 7
<212> PRT
<213> Artificial
<223> 9G7 light chain CDR2 region amino acid sequence
<400> 63
Phe Ala Ser Thr Arg Glu Ser
1 5
<210> 64
<211> 9
<212> PRT
<213> Artificial
<223> 9G7 light chain CDR3 region amino acid sequence
<400> 64
Gln Gln His Tyr Tyr Thr Pro Phe Thr
1 5
<210> 65
<211> 120
<212> PRT
<213> Artificial
<223> humanized antibody 3C9 heavy chain variable region amino acid sequence
<400> 65
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Ser Arg Tyr Asn Asp Lys Phe
50 55 60
Lys Val Arg Ala Thr Leu Thr Ser Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asp Tyr Asp Asp Gly Ala Trp Phe Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 66
<211> 112
<212> PRT
<213> Artificial
<223> humanized antibody 3C9 light chain variable region amino acid sequence
<400> 66
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Ile Thr Tyr Leu Phe Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Ser 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 Gly Gln Asn
85 90 95
Leu Glu Leu Pro Val Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 67
<211> 117
<212> PRT
<213> Artificial
<223> humanized antibody 3F5 heavy chain variable region amino acid sequence
<400> 67
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Ser Gly Gly Ile Tyr Thr Tyr Tyr Ala Asp Ile Val
50 55 60
Thr Gly Arg Phe Thr Thr Ser Arg Asp Asn Ala Lys Asn Ser 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
Val Arg Asp Phe Asp Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 68
<211> 107
<212> PRT
<213> Artificial
<223> humanized antibody 3F5 light chain variable region amino acid sequence
<400> 68
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 Ser Ser Gln Glu Ile Ser Gly Tyr
20 25 30
Leu Ala Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Asp Val Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Tyr Ala Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 69
<211> 123
<212> PRT
<213> Artificial
<223> humanized antibody 15A7 heavy chain variable region amino acid sequence
<400> 69
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Gly Tyr Tyr Tyr Gly Ser Ser Tyr Pro Tyr Tyr Phe Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 70
<211> 106
<212> PRT
<213> Artificial
<223> humanized antibody 15A7 light chain variable region amino acid sequence
<400> 70
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Pro Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val 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
Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Thr Ser
20 25 30
Gly Val Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Val Lys Arg Tyr Asn Pro Ser
50 55 60
Leu Gln Gly Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80
Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Met Asp Trp Asp Gly Leu Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 72
<211> 108
<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 Ser Val Ser Ser Ser Ile Ser Ser Ser
20 25 30
Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp
35 40 45
Ile Tyr Gly Thr Ser Asn Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro
85 90 95
Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
Claims (7)
1. A panel of OX40 monoclonal antibodies, or antigen-binding fragments thereof, comprising a heavy chain and a light chain, characterized in that the amino acid sequence of the CDR1 of said 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 OX40 monoclonal antibodies or antigen-binding fragments thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 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 OX40 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; the amino acid sequence of the humanized light chain variable region is selected from SEQ ID NO: 66. 68, 70, 72.
4. Use of a panel of OX40 monoclonal antibodies or antigen binding fragments thereof according to any one of claims 1-3 in the preparation of a medicament for: agents that modulate OX40 activity or OX40 levels, agents that promote OX40 activation to enhance immune function, agents that activate T lymphocytes or agents that increase IL-2, IFN- γ expression in T lymphocytes, agents that block OX40 ligand binding to OX40 receptors.
5. A monoclonal antibody conjugate comprising a monoclonal antibody that is the panel of OX40 monoclonal antibodies or antigen-binding fragments thereof of any one of claims 1-3 and a conjugate moiety that is one or more selected from the group consisting of a radionuclide, a drug, a toxin, a cytokine receptor fragment, an enzyme, fluorescein, and biotin.
6. Use of a monoclonal antibody conjugate as claimed in claim 5 for the preparation of a medicament comprising: agents that modulate OX40 activity or OX40 levels, agents that promote OX40 activation to enhance immune function, agents that activate T lymphocytes or agents that increase IL-2, IFN- γ expression in T lymphocytes, agents that block OX40 ligand binding to OX40 receptors.
7. Use of a monoclonal antibody conjugate as claimed in claim 6 in the manufacture of a medicament for the prophylaxis, treatment, or adjunctive treatment of a tumour.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114409781A (en) * | 2021-12-20 | 2022-04-29 | 安徽安科生物工程（集团）股份有限公司 | Fully human anti-human CD40 monoclonal antibody and application thereof |
| CN114478771A (en) * | 2021-12-14 | 2022-05-13 | 英诺湖医药(杭州)有限公司 | OX40 antibodies and medical uses thereof |
| CN114656564A (en) * | 2021-12-20 | 2022-06-24 | 安徽安科生物工程（集团）股份有限公司 | Nano antibody for resisting hu-OX40 antigen and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110078825A (en) * | 2019-01-23 | 2019-08-02 | 北京天广实生物技术股份有限公司 | In conjunction with the antibody and application thereof of OX40 |
| CN111196854A (en) * | 2018-11-20 | 2020-05-26 | 上海开拓者生物医药有限公司 | OX40 antibody and preparation method and application thereof |
| CN111363040A (en) * | 2018-12-25 | 2020-07-03 | 杭州翰思生物医药有限公司 | anti-OX 40 monoclonal antibodies and uses thereof |
-
2020
- 2020-12-30 CN CN202011610223.1A patent/CN112574312B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111196854A (en) * | 2018-11-20 | 2020-05-26 | 上海开拓者生物医药有限公司 | OX40 antibody and preparation method and application thereof |
| CN111363040A (en) * | 2018-12-25 | 2020-07-03 | 杭州翰思生物医药有限公司 | anti-OX 40 monoclonal antibodies and uses thereof |
| CN110078825A (en) * | 2019-01-23 | 2019-08-02 | 北京天广实生物技术股份有限公司 | In conjunction with the antibody and application thereof of OX40 |
Non-Patent Citations (1)
| Title |
|---|
| 毛辉等: "一株抗人OX40激发型单克隆抗体的研制及其生物学功能的初步研究", 《现代免疫学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114478771A (en) * | 2021-12-14 | 2022-05-13 | 英诺湖医药(杭州)有限公司 | OX40 antibodies and medical uses thereof |
| WO2023109976A3 (en) * | 2021-12-14 | 2023-08-03 | 英诺湖医药(杭州)有限公司 | Antibody against ox40 and medical use thereof |
| CN114409781A (en) * | 2021-12-20 | 2022-04-29 | 安徽安科生物工程（集团）股份有限公司 | Fully human anti-human CD40 monoclonal antibody and application thereof |
| CN114656564A (en) * | 2021-12-20 | 2022-06-24 | 安徽安科生物工程（集团）股份有限公司 | Nano antibody for resisting hu-OX40 antigen and application thereof |
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|---|---|
| CN112574312B (en) | 2022-08-16 |
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