Activated anti-ox40 antibodies, methods of production, and uses

Abstract

The application provides an antibody or fragment thereof of an activation receptor (OX40) expressed on the surface of activated CD4+ T and CD8+ T cells, and use of the antibody or fragment thereof for preventing or treating diseases. The antibody of the application has high affinity for OX40, obvious effect of activating the OX40 signal pathway, relatively broad immune enhancement effect, and the ability of enhancing T cell response immune memory. In an animal model in vivo experiment, good tumor inhibition and killing effects are achieved, and the antibody has good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antibody engineering, specifically relating to an anti-OX40 antibody, its production method and use, and particularly to a humanized antibody against human OX40, its recombinant expression method and its use in the treatment of solid tumors. Background Technology

[0002] OX40, also known as CD134, ACT45, or TNFRSF4, is a member of the tumor necrosis factor receptor (TNFR) superfamily and is an activating receptor expressed on the surface of activated CD4+ T and CD8+ T cells. OX40 signaling can activate downstream NF-κB, PI3K, and PKB pathways. Sustained activation of these pathways ultimately prolongs T cell survival time, expands T cell memory, and promotes T cell cytotoxicity. Furthermore, OX40 can enhance effector T cell function by inhibiting the differentiation and activity of regulatory T cells (Tregs) and improving immunosuppression in the tumor microenvironment.

[0003] The OX40 gene is located on human chromosome 1 (mouse chromosome 4) and encodes a 50kD type I transmembrane glycoprotein. Its extracellular region contains 191 amino acids, including three complete and one shorter cysteine-rich domain (CRD). It is primarily expressed on activated effector T cells (Teffs) and regulatory T cells (Tregs), and also on NKT cells, NK cells, and neutrophils.

[0004] OX40 binds to its ligand OX40L (CD252, TNFSF4) to transmit co-stimulatory signals. The OX40L gene is located on chromosome 1 of both humans and mice, encoding a 34kD type II transmembrane glycoprotein. OX40L can be expressed on antigen-presenting cells (APCs), such as B cells, dendritic cells, and macrophages; it can also be induced to express in other cell types, such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and NK cells.

[0005] The binding of OX40 and OX40L is involved in various physiological responses between T cells, lymphocytes, and non-lymphocytes. The interaction between OX40 and OX40L recruits TNFR-related (TRAF) molecules within the intracellular region of OX40, forming a signaling complex containing IKKα and IKKβ, as well as PI3k and PKB (Akt). OX40 also synergizes with TCR signaling to enhance intracellular Ca2+ through an unknown mechanism, thereby enhancing NFAT nuclear translocation. OX40 can activate the classical NF-κB1 pathway or the non-classical NF-κB2 pathway, PI3k / PKB, and NFAT pathways, thereby regulating genes controlling T cell division and survival, and promoting the transcription of cytokine genes and the expression of cytokine receptors, which is crucial for cell survival. OX40 signaling induces the downregulation of CTLA-4 and Foxp3.

[0006] Anti-OX40 activating antibodies can activate antigen-dependent T effector cells (T cells) similar to OX40L and exert anti-tumor effects by eliminating the inhibitory function of Treg cells. Several anti-OX40 activating antibodies are currently in clinical trials, with three studies publishing partial experimental data: Pfizer's PF-04518600, BMS's BMS-986178, and AbbVie's ABBV-368. In China, two companies have entered clinical trials for OX40: Innovent Biologics' recombinant fully human anti-tumor necrosis factor receptor superfamily member 4 (OX40) monoclonal antibody and Livzon Pharmaceutical Group's recombinant fully human anti-OX40 monoclonal antibody injection. Most existing OX40 monoclonal antibodies are obtained through hybridoma technology. However, target antibodies can also be obtained through transgenic mouse technology, phage antibody library technology, and B cell sorting technology, with evaluation and analysis methods similar to or the same as those used in hybridoma antibody preparation.

[0007] Although several anti-OX40 activating antibodies are currently in clinical trials, there is still a need to develop more activating anti-OX40 antibodies with high affinity, high bioactivity, and especially better immune memory stimulation to meet the urgent needs of clinical treatment. This is due to reasons such as the inconsistency between early screening performance and final clinical treatment effects, unpredictable adverse reactions, and different effects on different indications. Summary of the Invention

[0008] To address the aforementioned problems, this invention utilizes hybridoma technology to obtain mouse antibodies. After identifying candidate activating anti-OX40 mouse antibodies through antibody binding activity, activation activity, and blocking activity analysis, the gene of the activating anti-OX40 mouse antibody is used for antibody sequence humanization design. The resulting humanized antibody is then subjected to further antibody activity analysis (binding activity, activation activity, and blocking activity) and in vivo pharmacological analysis in mice to ultimately determine the activating anti-OX40 humanized antibody. Specifically:

[0009] On one hand, the present invention provides an antibody or a fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein

[0010] The heavy chain variable region contains:

[0011] VH CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 40, 52, 64.

[0012] VH CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 41, 53, 65.

[0013] VH CDR3 is selected from the amino acid sequences shown in SEQ ID NO: 42, 54, 66;

[0014] The variable region of the light chain contains:

[0015] VL CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 46, 58, 70.

[0016] VL CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 47, 59, 71.

[0017] VL CDR3 is selected from the amino acid sequences shown in SEQ ID NO: 48, 60, and 72.

[0018] Furthermore, the antibody or fragment thereof described in this invention has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2, 6, 10, 14, 22, 24 or 30.

[0019] Furthermore, the antibody or fragment thereof described in this invention has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4, 8, 12, 16, 18, 20, 26, 28, 32.

[0020] Furthermore, the antibody or fragment thereof described in this invention is characterized in that:

[0021] (1) The heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 2, 14, 22, 30, and the light chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 4, 16, 18, 20;

[0022] (2) The heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 6 and 24, and the light chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 8, 26, 28 and 32;

[0023] (3) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 12.

[0024] Furthermore, the antibody or fragment thereof described in this invention is characterized in that:

[0025] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 4;

[0026] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 8;

[0027] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 12;

[0028] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16, 18, or 20.

[0029] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16, 18 or 20;

[0030] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 26 or 28.

[0031] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16;

[0032] or

[0033] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 32.

[0034] Furthermore, the antibody or fragment thereof described in this invention includes the heavy chain constant region shown in SEQ ID NO: 34 and / or the light chain constant region shown in SEQ ID NO: 36.

[0035] Furthermore, the antibody or its fragments described in this invention include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies; the antibody fragments include F(ab′)2, Fab′, Fv, scFv, Fd, nanobodies, etc.

[0036] In a second aspect, the present invention provides an immunoconjugate comprising:

[0037] (1) The antibody or fragment thereof described in the first aspect of the present invention.

[0038] (2) Coupled part.

[0039] Furthermore, in the immunoconjugate of the present invention, the conjugated portion includes detectable markers, cytotoxic molecules, bioactive molecules, etc.

[0040] Furthermore, the immunoconjugates of the present invention include detectable labels such as chemiluminescent labels, fluorescent labels, enzyme labels, radioactive labels, quantum dots, and nanoparticles.

[0041] Furthermore, the immunoconjugates of the present invention contain cytotoxic molecules including diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, and leukotoxin.

[0042] Furthermore, the immunoconjugates of the present invention contain bioactive molecules including cytokines, enzymes, chemotherapeutic agents, liposomes, viral particles, etc.

[0043] Thirdly, the present invention provides a multispecific antibody or a derivative thereof, characterized in that it includes at least one antigen-binding domain of the antibody or fragment thereof described in the first aspect of the present invention.

[0044] Furthermore, the multispecific antibody or its derivatives described in this invention further include an antigen-binding domain that binds to other targets.

[0045] Furthermore, the multispecific antibody or its derivatives described in this invention include other targets such as PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, CD47, 4-1BB, CD73, ROR1, HER2, HER3, EGFR, etc.

[0046] Fourthly, the present invention provides a heavy chain antibody, which is a dimer heavy chain antibody obtained based on the antibody or a fragment thereof described in the first aspect of the present invention.

[0047] Furthermore, the heavy chain antibody described in this invention does not have an Fc region.

[0048] Fifthly, the present invention provides a composition comprising:

[0049] (1) The antibody or fragment thereof described in the first aspect of the present invention, the antibody conjugate described in the second aspect of the present invention, the multispecific antibody or derivative thereof described in the third aspect of the present invention, or the heavy chain antibody described in the fourth aspect of the present invention;

[0050] (2) Pharmaceutically acceptable carrier.

[0051] Furthermore, the composition of the present invention further comprises other active ingredients for treating tumors.

[0052] In a sixth aspect, the present invention provides a kit comprising the antibody or a fragment thereof described in the first aspect of the present invention for qualitative or quantitative detection of OX40.

[0053] In a seventh aspect, the present invention provides a nucleic acid that encodes the antibody or a fragment thereof described in the first aspect of the present invention, or encodes the multispecific antibody or a derivative thereof described in the third aspect of the present invention.

[0054] Eighthly, the present invention provides a carrier comprising the nucleic acid described in the seventh aspect of the present invention.

[0055] In a ninth aspect, the present invention provides a recombinant host cell comprising the nucleic acid described in the seventh aspect of the present invention or comprising the vector described in the eighth aspect of the present invention.

[0056] In a tenth aspect, the present invention provides the use of the antibody or fragment thereof described in the first aspect of the present invention, the antibody-drug conjugate described in the second aspect of the present invention, the multispecific antibody or derivative thereof described in the third aspect of the present invention, the heavy chain antibody described in the fourth aspect of the present invention, and the composition described in the fifth aspect of the present invention in the preparation of drugs such as those binding to OX40, inhibiting the binding of OX40 and OX40L, activating OX40+ T cells, activating human immune responses, and tumor therapeutic vaccines. For example, it can be used as a molecular adjuvant to enhance specific immune responses, or in combination with adjuvants such as CpG for use in tumor vaccines and other applications.

[0057] In one aspect, the present invention provides the use of the antibody or fragment thereof described in the first aspect of the present invention, the antibody-drug conjugate described in the second aspect of the present invention, the multispecific antibody or derivative thereof described in the third aspect of the present invention, the heavy chain antibody described in the fourth aspect of the present invention, and the composition described in the fifth aspect of the present invention in the preparation of drugs that induce IL-8 production in OX40+ cells and initiate NFκB gene transcription.

[0058] In a twelfth aspect, the present invention provides the use of the antibody or fragment thereof described in the first aspect of the present invention, the antibody-drug conjugate described in the second aspect of the present invention, the multispecific antibody or derivative thereof described in the third aspect of the present invention, the heavy chain antibody described in the fourth aspect of the present invention, and the composition described in the fifth aspect of the present invention in the preparation of a drug that stimulates PBMCs to produce IL-2 and IFN-γ.

[0059] In a thirteenth aspect, the present invention provides the use of the antibody or fragment thereof described in the first aspect of the present invention, the antibody-drug conjugate described in the second aspect of the present invention, the multispecific antibody or derivative thereof described in the third aspect of the present invention, the heavy chain antibody described in the fourth aspect of the present invention, and the composition described in the fifth aspect of the present invention in the preparation of a drug for inhibiting the growth and metastasis of solid tumors.

[0060] Furthermore, the use described in this invention is characterized in that the solid tumor includes tumors of the digestive system, tumors of the respiratory system, and tumors of the urinary and reproductive systems.

[0061] Furthermore, the use described in this invention is characterized in that the digestive system tumors include liver cancer, pancreatic cancer, gastric cancer, duodenal cancer, colorectal cancer, and esophageal cancer.

[0062] Furthermore, the use described in this invention is characterized in that the respiratory system tumors include small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma, laryngeal carcinoma, mesothelioma, etc.

[0063] Furthermore, the use described in this invention is characterized in that the urogenital system tumors include breast cancer, ovarian cancer, etc.

[0064] In a fourteenth aspect, the present invention provides a method for producing antibodies, comprising:

[0065] (1) Culturing the host cells of the ninth aspect of the present invention

[0066] (2) Antibody recovery.

[0067] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0068] The term "OX40" refers to the fourth member of the tumor necrosis factor superfamily. This term includes variants, allotypes, homologs, orthologs, and parallel homologs. For example, antibodies specific to human OX40 may cross-react with OX40 proteins from another species, such as monkeys, under certain conditions. In other embodiments, antibodies specific to human OX40 proteins may be completely specific to human OX40 proteins without cross-reacting with proteins from other species or other types, or may cross-react with OX40 proteins from some other species but not all others.

[0069] The term "human OX40" refers to the OX40 protein having a human amino acid sequence, such as the amino acid sequence with GenBank accession number NP_003318. The terms "monkey OX40" and "mouse OX40" refer to the OX40 sequences of monkeys and mice, respectively, for example, sequences with GenBank accession numbers NP_001090870 and NP_035789, respectively.

[0070] The term "antibody" in this document is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains. A full-length antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system.

[0071] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the OX40 protein is substantially free of antibodies that specifically bind to antigens other than the OX40 protein. However, an isolated antibody that specifically binds to the human OX40 protein may have cross-binding to other antigens, such as OX40 proteins from other species. Furthermore, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0072] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to antibody molecules that consist of a single molecule. A monoclonal antibody composition exhibits specific binding specificity and affinity for a particular epitope.

[0073] In this article, the term "antigen-binding fragment" (or simply antibody moiety) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., OX40 protein). It has been demonstrated that the antigen-binding function of an antibody can be exercised through fragments of a full-length antibody. Examples of binding fragments contained in the "antigen-binding moiety" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1; (ii) F(ab′)2 fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1; (iv) Fv fragments consisting of the antibody's single arm VL and VH; (v) dAb fragments consisting of VH (Ward et al., (1989) Nature 341: 544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) nanobodies, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via a synthetic linker that makes them single-chain proteins through recombination, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fc (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained using techniques commonly known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0074] The antigen-binding fragments of the present invention include those capable of specifically binding to OX40. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv) fragments, and single-domain fragments.

[0075] The Fab fragment contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it contains the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. The Fab' fragment is generated by cleaving the disulfide bond at the hinge cysteine ​​residue in the F(ab')2 pepsin digestion product. Further chemical conjugation of antibody fragments is known to those skilled in the art. The Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of the intact antibody, are cleared more rapidly from animal circulation, and may have less nonspecific tissue binding than the intact antibody (see, for example, Wahl et al., 1983, J. Nucl. Med. 24: 316).

[0076] As is generally understood in the art, the "Fc" region is a crystallizable constant region of an antibody fragment that does not contain an antigen-specific binding region. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody. The IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

[0077] The “Fv” fragment is the smallest fragment of an antibody containing a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of a heavy chain and a light chain variable domain bound together in a tight, non-covalent manner. In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of the Fv containing only the three CDRs for target specificity) can have the ability to recognize and bind to the target, although its affinity is lower than that of the entire binding site.

[0078] A "single-chain Fv" or "scFv" antibody-binding fragment contains the VH and VL domains of the antibody, which are located within a single polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure conducive to target binding.

[0079] A “single-domain fragment” consists of a single VH or VL domain that exhibits sufficient affinity for OX40. In one specific implementation, the single-domain fragment is camelized (see, for example, Riechmann, 1999, Journal of Immunological Methods 231: 25-38).

[0080] The anti-OX40 antibody of the present invention comprises a derivatized antibody. For example, derivatized antibodies are typically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. In addition, the derivative may contain one or more non-natural amino acids, for example, using the ambrx technology (see, for example, Wolfson, 2006, Chem. Biol. 13(10): 1011-2).

[0081] Anti-OX40 antibodies can be antibodies whose sequences have been modified to alter the function of at least one constant region-mediated biological effector. For example, in some embodiments, anti-OX40 antibodies can be modified to reduce the function of at least one constant region-mediated biological effector relative to unmodified antibodies, such as reduced binding to one or more Fc receptors (FcγRs) like FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB. FcγR binding can be reduced by mutating a segment of the antibody’s immunoglobulin constant region at a specific region necessary for FcγR interaction (see, for example, Canfield and Morrison, 1991, J. Exp. Med. 173: 1483-1491; and Lund et al., 1991, J. Immunol. 147: 2657-2662). A reduction in the FcγR binding capacity of an antibody can also reduce other effector functions dependent on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cytotoxicity (“ADCC”). In illustrative examples, variants with V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of the Fc region can exhibit reduced affinity for FcγRIIB compared to the corresponding wild-type constant region.

[0082] The anti-OX40 antibodies described herein include those modified to obtain or improve the biological effector function mediated by at least one constant region relative to unmodified antibodies, such as antibodies to enhance FcγR interactions (see, for example, U.S. Patent Application No. 2006 / 0134709). For example, the anti-OX40 antibodies of the present invention may have constant regions that bind FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB with greater affinity than the corresponding wild-type constant regions. In illustrative examples, variant CH2 domains substituted with V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y in the CH2 domain of the Fc region may exhibit greater affinity for FcγRIIIA compared to the corresponding wild-type constant regions.

[0083] Therefore, the anti-OX40 antibody of the present invention can have alterations in its biological activity that result in increased or decreased opsonization, phagocytosis, or ADCC. Such alterations are known in the art. For example, modifications in antibodies that reduce ADCC activity are described in U.S. Patent No. 5,834,597. An exemplary ADCC-reducing variant corresponds to "Mutant 3" (also known as "M3", U.S. Patent No. 5,834,597). Figure 4(As shown in the image), where residues 234 and 237 (using EU numbers) are replaced with alanine. The mutant 3 (also known as "M3") variant can be used in many antibody isotypes, such as human IaG2 M3.

[0084] Additional substitutions that can modify the FcγR binding and / or ADCC effector function of anti-OX40 antibodies include K322A substitution or L234A and L235A dual substitution in the Fc region, such as human IgG1 with L234A / L235A dual substitution. See, for example, Hezareh et al. J.Virol., 75(24): 12161-12168 (2001).

[0085] In some embodiments, anti-OX40 antibodies have low levels of fucose or are fucose-free. Fucose-free antibodies have been associated with enhanced ADCC activity, especially at low doses. See Shields et al., 2002, J. Biol. Chem. 277: 26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278: 3466-73. Methods for preparing fucose-free antibodies involve growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme essential for the fucosylation of peptides.

[0086] Anti-OX40 antibodies may comprise a modified (or variant) CH2 domain including amino acid substitutions, wherein the amino acid substitutions increase binding to FcγRIIB and / or technically bind to FcγRIIIA compared to the binding of the corresponding wild-type CH2 or Fc region. Variant CH2 or variant Fc domains have been described in U.S. Patent Application No. 2014 / 0377253. Variant CH2 or variant Fc domains typically include one or more substitutions at positions 263, 266, 273, and 305, wherein the residue numbering in the Fc domain is as in the EU index in Kabat. In some embodiments, relative to the wild-type CH2 domain, the anti-OX40 antibody comprises one or more substitutions selected from V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, and V305W. In a specific embodiment, one or more substitutions of the CH2 domain relative to the human IgG1 are selected from V263L, V273E, V273F, V273M, V273S, and V273Y. For example, one or more substitutions of the IgG1 CH2 domain may be V273E. In another specific embodiment, the anti-OX40 antibody of the present invention comprises a variant IaG1 CH2 domain containing an amino acid substitution of V263L.

[0087] Other examples of variant CH2 or variant Fc domains that can provide increased binding to FcγRIIB and / or reduced binding to FcγRIIIA compared to the corresponding wild-type CH2 or Fc domains include those found in Vonderheide et al., Clin. Cancer Res., 19(5), 1035-1043 (2013), such as S267E or S267E / L328F in human IgG1.

[0088] In some embodiments, the anti-OX40 antibody includes modifications that increase or decrease its binding affinity to the fetal Fc receptor FcRn, for example, by mutating a segment of the immunoglobulin constant region at a specific region involved in FcRn interaction (see, for example, WO 2005 / 123780). In a particular embodiment, the anti-OX40 antibody of the IgG class is mutated such that at least one of amino acid residues 250, 314, and 428 in the heavy chain constant region is substituted individually, or in any combination thereof, for example at positions 250 and 428, or at positions 250 and 314, or at positions 314 and 428, or at positions 250, 314, and 428, wherein positions 250 and 428 are a specific combination. For position 250, the substituted amino acid residue can be any amino acid residue except threonine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. For position 314, the substituted amino acid residue can be any amino acid residue except leucine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. For position 428, the substituted amino acid residue can be any amino acid residue other than methionine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. An exemplary substitution known to modify the function of the Fc effector is the Fc substitution M428L, which can combine with the Fc substitution T250Q. Other specific combinations of suitable amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797. Such mutations increase binding to FcRn, which protects the antibody from degradation and increases its half-life.

[0089] Anti-OX40 antibodies may have one or more amino acids inserted into one or more of their CDRs, as described, for example, in Jung and Plückthun, 1997, Protein Engineering 10:8, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5): 481-9. Epub 2004 Aug 17; and U.S. Patent Application No. 2007 / 0280931.

[0090] The term "mouse-derived antibody" refers to an antibody whose variable frame and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. The mouse-derived antibodies of this invention may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations. However, the term "mouse-derived antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse frame sequence.

[0091] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or, more broadly, a chimeric antibody is an antibody that combines genetic material from one species with genetic material from another species.

[0092] Non-human (e.g., mouse) antibodies in the term "humanized" form are chimeric immunoglobulins containing a minimum sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will contain at least one, and typically two, variable domains substantially all of which correspond entirely or substantially entirely to those of non-human immunoglobulins, and entirely or substantially entirely to those of human immunoglobulins. Humanized antibodies may also contain at least a portion of an immunoglobulin constant region (Fc), typically a common sequence to human immunoglobulins. Methods of antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. Patents to Queen et al.: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; PCT Publication WO 91 / 09967; U.S. Patent No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332.

[0093] "Human antibodies" include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from a human immunoglobulin library or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous immunoglobulins. Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO96 / 33735; and WO 91 / 10741. Human antibodies can also be generated using transgenic mice that do not express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT disclosures WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. Additionally, using techniques similar to those described above, companies such as LakePharma, Inc. (Belmont, CA) or Creative BioLabs (Shirley, NY) can engage in providing human antibodies against selected antigens. Fully human antibodies recognizing selected epitopes can be generated using a technique known as “guided selection.” In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope (see Jespers et al., 1988, Biotechnology 12: 899-903).

[0094] The terms “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” in this document.

[0095] In this paper, an antibody that "specifically binds to human OX40" refers to an antibody that binds to human OX40 (and possibly OX40 from other non-human species) but substantially does not bind to non-OX40 proteins. Preferably, the antibody binds to human OX40 protein with "high affinity," i.e., a KD value of 5.0 x 10⁻⁶. -8 For sizes below M, 1.0x10 is preferred. -8 M or less, preferably 5.0x10 -9 Below M.

[0096] The term "basically non-binding" protein or cell refers to proteins or cells that do not bind to each other, or do not bind to them with high affinity; that is, the KD of the bound protein or cell is 1.0 x 10⁻⁶. -6 M or higher, preferably 1.0x10 -5 M or higher, preferably 1.0x10 -4 M or more, 1.0x10 -3 M or higher, preferably 1.0x10 -2 M and above.

[0097] For IgG antibodies, the term "high affinity" refers to a KD of 1.0 x 10⁻⁶ for the antigen. -6 For sizes below M, 5.0x10 is preferred. -8 M or less, preferably 1.0x10 -s Below M, 5.0x10 -9 M or less, preferably 1.0x10 -9 Below M. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a KD of 10. -6 For sizes below M, 10 is preferred. -7 M or less, preferably 10 -8 Below M.

[0098] The terms "Kassoc" or "Ka" refer to the binding rate of a specific antibody-antigen interaction, while the terms "Kdis" or "Kd" refer to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, obtained from the ratio of Kd to Ka (Kd / Ka), and expressed as molar concentration (M). The KD value of an antibody can be determined by methods known in the art. A preferred method for determining the antibody KD is by measuring it using a surface plasmon resonance (SPR) instrument, preferably a biosensor system such as the Biacore™ system.

[0099] The term "EC50," also known as the half-maximal effect concentration, refers to the antibody concentration that produces a 50% maximum effect.

[0100] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0101] The term "agonist OX40 antibody" as used in this article refers to an OX40 antibody that can bind to OX40 and activate or trigger the OX40 signaling pathway, thereby promoting T cell proliferation and survival. The term "antagonist OX40 antibody" refers to an OX40 antibody that blocks the OX40 signaling pathway, thereby correcting overactive T cell pathology and can be used to treat conditions such as asthma, enteritis, and arthritis.

[0102] The term "therapeutic effective amount" refers to the amount of the antibody of the present invention sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer) and / or reduce the severity of the disease. Therapeutic effective amount is related to the disease being treated, and the actual effective amount can be readily determined by those skilled in the art.

[0103] Several aspects of the present invention are described in more detail below.

[0104] OX40 antibodies possess binding specificity to human OX40 and other beneficial functional characteristics.

[0105] The antibody of the present invention binds specifically to human OX40 with high affinity, for example, with a KD value of 1 x 10⁻⁶. -8 Below M. The antibody also exhibits cross-reactivity with monkey OX40 but does not bind to mouse OX40.

[0106] The antibody of the present invention is an agonist OX40 antibody that activates or triggers the OX40 signaling pathway and participates in T cell co-stimulation, promoting IL-2 secretion and CD8+ T cell proliferation.

[0107] The antibody of this invention exhibits a good in vivo anti-tumor effect. After the antibody administration is stopped, the tumor does not grow, and may even be completely eliminated, and immune memory can be generated.

[0108] The antibodies of the present invention may be polyclonal, monoclonal, genetically modified, and / or otherwise substantially modified, including but not limited to chimeric antibodies, humanized antibodies, and human antibodies. In some embodiments, the constant region is selected from the following isotypes: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In a specific embodiment, the anti-OX40 antibody described herein comprises IgG1. In other embodiments, the anti-OX40 antibody comprises IgG2 or IgG4. As used herein, the “constant region” of an antibody includes the natural constant region, allotype, or natural variant, such as D356E and L358M, or A431G, in human IgG1. See, for example, Jefferis and Lefranc, MAbs, 1(4): 332-338 (July-August 2009). Preferred antibodies of the present invention are monoclonal antibodies. Furthermore, the antibody may be, for example, a murine, chimeric, or humanized monoclonal antibody.

[0109] The light chain constant region of the anti-OX40 antibody can be a C kappa(κ) region or a C lambda(λ) region. The λ region can be any of the known isotypes, such as λ1, λ2, λ3, or λ4. In some embodiments, the anti-OX40 antibody contains a C kappa(κ) region.

[0110] Anti-OX40 antibodies with high affinity for human OX40 (SEQ ID NO: 1) are likely desirable for therapeutic and diagnostic uses. Accordingly, the present invention contemplates antibodies with high binding affinity for human OX40. In specific embodiments, the anti-OX40 antibody binds to human OX40 with an affinity of at least about 100 nM, but may exhibit higher affinity, such as at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even higher. In some embodiments, the antibody binds to human OX40 with an affinity in the range of about 1 pM to about 100 nM, or an affinity in the range of any of the aforementioned values, such as, but not limited to, about 0.001 to 10 nM, 0.001 to 5 nM, 0.01 to 100 nM, 0.01 to 50 nM, 0.01 to 10 nM, 0.01 to 5 nM, or 0.01 to 1 nM.

[0111] The affinity of anti-OX40 antibodies for human OX40 can be determined using techniques well known in the art or described herein, such as, but not limited to, ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance, or fluorescence polarization assay.

[0112] Anti-OX40 antibodies generally comprise a heavy chain containing a variable region (VH) having three complementarity-determining regions (“CDRs”), referred herein as VH CDR#1, VH CDR#2, and VH CDR#3 (in N→C order), and a light chain containing a variable region (VL) having three complementarity-determining regions, referred herein as VL CDR#1, VL CDR#2, and VL CDR#3 (in N→C order). Exemplary CDR amino acid sequences are provided herein, as well as the amino acid sequences of the VH and VL regions of exemplary anti-OX40 heavy and light chains. Specific embodiments of anti-OX40 antibodies include these exemplary CDR and / or VH and / or VL sequences, and antibodies that competitively bind to human OX40 with such antibodies.

[0113] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0114] First, this invention provides a novel humanized anti-OX40 activating antibody with a well-defined amino acid sequence structure, high affinity for OX40, and a definite effect in activating the OX40 signaling pathway, exhibiting low heterology and high clinical application potential.

[0115] Secondly, the humanized anti-OX40 activating antibody provided by this invention has good biological activity. It can not only induce OX40+ cells to produce IL-8 and NFκB, but also stimulate PBMCs to produce IL-2 and IFN-γ, exhibiting a relatively broad-spectrum immune-enhancing effect and enhancing T cell response immune memory. In vivo immune memory studies of hz25A7m8 and hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 models showed that mice in all treatment groups no longer developed tumors after re-inoculation with MC38 cells. Although inoculation with Hepal-6 cells could induce tumor formation, tumor growth was significantly inhibited. The inhibitory effect of hz25A7m8 and hz27G12H1L2 was more significant compared to PC2 (the variable region sequence is derived from BMS patent: WO2016196228A1; heavy chain: SEQ NOID: 124, light chain: SEQ NO ID: 116; also referred to as BMS in this invention; the same below).

[0116] Third, the humanized anti-OX40 activating antibody provided by this invention has achieved good tumor inhibition and killing effects in in vivo experiments in animal models, and even complete tumor regression in some experimental individuals, showing a clear clinical efficacy for OX40-related cancers such as colorectal cancer. In vivo pharmacodynamic studies of hz25A7m8 and hz27G12H1L2 in an OX40 humanized mouse colorectal cancer MC38 model showed that in OX40 transgenic mice, both candidate antibodies hz25A7m8 and hz27G12H1L2 significantly inhibited tumor growth and caused most tumors to regress. Among them, hz25A7m8 caused complete tumor regression and its activity was superior to control antibodies such as PC2 and PC3 (the variable region sequence is derived from Innovent Biologics patent: WO2018177220A1; heavy chain: SEQ NO ID: 111, light chain: SEQ NO ID: 130; also referred to as XD in this invention; the same below). Attached Figure Description

[0117] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0118] Figure 1 Figure: ELISA results of the binding activity of mouse monoclonal antibody to OX40.

[0119] Figure 2 Image showing the results of IL8 expression detection in HT1080-hOX40 cells activated by anti-OX40 murine monoclonal antibody. The antibody concentration of each antibody gradually decreases from left to right.

[0120] Figure 3 Figure: The binding results of anti-OX40 murine monoclonal antibody to HT1080-hOX40 cells.

[0121] Figure 4 Figure 1: Affinity assay results for anti-OX40 chimeric antibodies ch25A7, ch27G12, and ch11F7 by Fortebio.

[0122] Figure 5A Figure: Results of activating activity of OX40 chimeric antibody identified by IL-8 secretion method of HT1080-huOX40.

[0123] Figure 5B The results of NFκB pathway activation-fluorescein assay for identifying the activation activity of OX40 chimeric antibodies are shown in the figure. The antibody concentration of each antibody gradually decreases from left to right.

[0124] Figure 6Chimeric antibodies ch25A7 and ch27G12 blocked the activity of OX40L binding to HT1080-huOX40 cells.

[0125] Figure 7A .25A7 and 27G12 activation activity detection results before and after humanization; Figure 7B .27G12 humanized molecular mutant activation activity detection results; Figure 7C .25A7 humanized molecular mutant activation activity detection results (partial); Figure 7D Partial image of activation activity detection results for the .25A7 humanized molecular mutant.

[0126] Figure 8 Image showing the species-specific identification results of anti-OX40 antibody hz25A7m8.

[0127] Figure 9 Image showing the species-specific identification results of anti-OX40 antibody hz27G12H1L2.

[0128] Figure 10A Immunological functions (IFN-γ) of OX40 antibodies hz25A7 and hz27G12: Von is PC1, BMS is PC2, and XD is PC3.

[0129] Figure 10B In vitro testing of the immunological function (IL-2) of OX40 antibodies hz25A7 and hz27G12, Von as PC1, BMS as PC2, and XD as PC3.

[0130] Figure 10C In vitro testing of the immunological function (cell proliferation) of OX40 antibodies hz25A7 and hz27G12, Von as PC1, BMS as PC2, and XD as PC3.

[0131] Figure 11A hz25A7m8 and hz27G12H1L2 effectively inhibited tumor growth in the OX40 humanized mouse colorectal cancer MC38 model (individual data). BMS was PC2 and XD was PC3.

[0132] Figure 11B hz25A7m8 and hz27G12H1L2 effectively inhibited tumor growth in the OX40 humanized mouse colorectal cancer MC38 model (average data), BMS was PC2 and XD was PC3.

[0133] Figure 12AThe results of the subcutaneous tumor formation experiment in mice after treatment with anti-OX40 antibody were analyzed (MC38). BMS was PC2. The treatment regimens for hz25A7m8, hz27G12H1L2, BMS, and the isotype control group were 0h, 10mg / kg, ip, biw×4.

[0134] Figure 12B The results of the subcutaneous tumor formation experiment in mice after treatment with anti-OX40 antibody were analyzed (Hepal-6). BMS was PC2. The treatment regimen for hz25A7m8, hz27G12H1L2, BMS and isotype groups was 0h, 10mg / kg, ip, biw×4.

[0135] Figure 13 Image showing the results of FACS assay for immune memory T cell populations, with BMS representing PC2. Detailed Implementation

[0136] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0137] Specifically, the present invention provides an antibody or a fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein

[0138] The heavy chain variable region contains:

[0139] VH CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 40, 52, 64.

[0140] VH CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 41, 53, 65.

[0141] VH CDR3 is selected from the amino acid sequences shown in SEQ ID NO: 42, 54, 66;

[0142] The variable region of the light chain contains:

[0143] VL CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 46, 58, 70.

[0144] VL CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 47, 59, 71.

[0145] VL CDR3 is selected from the amino acid sequences shown in SEQ ID NO: 48, 60, and 72.

[0146] The antibody or fragment thereof described in this invention is characterized in that:

[0147] (1) The variable region of the heavy chain has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequences shown in SEQ ID NO: 2, 14, 22, 30, and the variable region of the light chain has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequences shown in SEQ ID NO: 4, 16, 18, 20;

[0148] (2) The heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 6 and 24 and has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology; the light chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 8, 26, 28, and 32 and has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology.

[0149] (3) The heavy chain variable region has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 12.

[0150] The present invention also provides an immunoconjugate comprising:

[0151] (1) The antibody or fragment thereof described in this invention.

[0152] (2) Coupled part.

[0153] The present invention also provides a multispecific antibody or a derivative thereof, comprising at least one antigen-binding domain of an antibody or fragment thereof as described in the present invention.

[0154] The present invention also provides a heavy chain antibody, which is a dimer heavy chain antibody obtained based on the antibody described in the present invention.

[0155] The present invention also provides a composition comprising:

[0156] (1) The antibody or its fragment described in this invention, the antibody conjugate described in this invention, the multispecific antibody or its derivative described in this invention, or the heavy chain antibody described in this invention.

[0157] (2) Pharmaceutically acceptable carrier.

[0158] The present invention also provides a nucleic acid encoding the antibody or fragment thereof described in the present invention, the multispecific antibody or derivative thereof described in the present invention, or the heavy chain antibody described in the present invention.

[0159] The present invention also provides a recombinant vector or recombinant host cell comprising the nucleic acid encoded by the present invention.

[0160] The present invention also provides the use of the antibody or a fragment thereof, the antibody-drug conjugate, the multispecific antibody or a derivative thereof, the heavy chain antibody, the composition, the nucleic acid, the recombinant vector or recombinant host cell, comprising...

[0161] It is used to prepare drugs that bind to OX40, inhibit the binding of OX40 and OX40L, activate OX40+ T cells, activate human immune response, and are used in tumor therapeutic vaccines, etc.

[0162] Molecular adjuvants used to prepare specific immune responses are preferably used in combination with adjuvants such as CpG for the preparation of tumor vaccines.

[0163] Used to prepare drugs that induce IL-8 production in OX40+ cells and / or initiate NFκB gene transcription;

[0164] Used to prepare drugs that stimulate PBMCs to produce IL-2 and IFN-γ;

[0165] Used to prepare drugs that inhibit the growth and metastasis of solid tumors;

[0166] Kits for the qualitative or quantitative detection of OX40.

[0167] The present invention also provides a method for producing antibodies, comprising:

[0168] (1) Culturing the recombinant host cells described in this invention

[0169] (2) Antibody recovery.

[0170] Example 1: Preparation of anti-human OX40 antibody hybridoma cells

[0171] Immunization: Balb / c mice were immunized with recombinant human OX40 / mFc protein (sequence number: P43489-1, 29aa-216aa). Serum titers were detected by ELISA using 96-well microplates coated with recombinant human OX40-his protein. Mice with serum titers meeting the fusion requirements were used for the next step of cell fusion.

[0172] Cell fusion and hybridoma preparation: On day 67 post-primary immunization, mice with the required cell titer were selected, and their spleens were aseptically harvested. A suspension of B lymphocytes was prepared and mixed with FO myeloma cells at a ratio of 5:1. The two cell types were then fused using PEG4000. The fused cells were resuspended in HAT medium and aliquoted into 96-well cell culture plates. The plates were incubated at 37°C in a 5% CO2 incubator.

[0173] Example 2: Screening of anti-human OX40 antibody-positive hybridoma cell lines

[0174] Option 1. Screening using ELISA combined with positive hybridoma

[0175] 10-14 days after fusion, the microplates were coated with human OX40-His recombinant protein (sequence number: P43489-1, 29aa-216aa) (10ug / ml, pH 9.6, 0.1M NaHCO3) and incubated overnight at 4°C; blocked with 4% skim milk powder-PBS and incubated at 37°C for 2 hours; washed three times with PBST (0.05% Tween 20-PBS), and the hybridoma clone culture supernatant was added and incubated at 37°C for 1 hour. The following controls were set up: (1) Positive control (PC): serum of mice after immunization (diluted with PBS 1:1000); (2) Negative control (NC): serum of mice before immunization (diluted with PBS 1:1000). Wash three times with PBST (0.05% Tween 20-PBS), add HRP-goat anti-mouse IgG (Fcγ) diluted 1:20000, incubate at 37℃ for 1 hour; then wash five times with PBST (0.05% Tween 20-PBS), add OPD chromogenic solution, incubate in the dark for 10-15 minutes, and terminate the reaction with 2M H2SO4; read the A492 value using a microplate reader. A positive result is defined as an A492 value in the detection well that is more than 2.1 times greater than that in the negative control well. To confirm the reliability of positive clones, a second round of screening is performed one day after the first screening medium change. The clones are then identified by testing. Figure 1 A total of 14 antibody-secreting positive cell lines were obtained (11F7-1, 13D3-3, 17B9-5, 18D6-4, 18G11-1, 22H1-1, 24H1-11, 25A7-11, 27H7-1, 27G12-7, 28C2-4, 29G2-4, 30B8-8, 30812-5), and these 14 antibodies were further screened.

[0176] Option 2. Screening for IL8 expression activity in HT1080-hOX40 cells activated by anti-OX40 murine monoclonal antibody.

[0177] The day before the experiment, HT1080-hOX40 cells (Kangyuan Bochuang: Cat. No: KC-0140) were digested, and the cells were resuspended in complete culture medium (1640 medium + 10% FBS + 0.5 μg / mL) to adjust the cell density to 102. 5 Cells / mL were added to each well of a 96-well cell culture plate, with 200 μL of cell suspension. The cell culture plate was then incubated overnight at 37°C with 5% CO2. The next day, the antibody to be tested was diluted to an appropriate concentration and added to the cell culture plate, which was then incubated at 37°C with 5% CO2 for 6 hours. A positive control PC1 (PC1, i.e., Vonderolizumab, the variable region sequence of which is derived from WHO Drug Information, Vol. 31, NO. 3, 2017, also referred to as Vond in this invention) was set up. Finally, the cell culture supernatant was aspirated, and the IL-8 content in the supernatant was detected using the IL-8 ELISA quantitative kit (catalog number: batch number) according to the instructions. The results are as follows. Figure 2 As shown, most clones can effectively activate HT1080-hOX40 cells, increasing their IL8 expression levels.

[0178] Option 3. Screening by binding of positive hybridomas to OX40 on the surface of HT-1080 cells.

[0179] Based on the above activity results, the hybridoma supernatant of 10 clones (11F7-1, 18D6-4, 18G11-1, 22H1-1, 24H1-11, 25A7-11, 27H7-1, 27G12-7, 29G2-4, 3088-8) was purified by affinity. The obtained mouse antibody (5ug / ml) was incubated with a suspension of 293 cells (293 / OX40) expressing recombinant human OX40 at 37°C for 30 min. The following controls were set: (1) Positive control PC1, in this example, the Fc of the antibody molecule was replaced with mouse IgG1, 5μg / ml; (2) Negative control NC: irrelevant mouse IgG1 5μg / ml. After washing the cells 3 times with PBS, goat anti-mouse IgG-FITC (Cat: F9006, Sigma) diluted 1:64 was added and incubated for 30 min. After washing cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMANCOULTER) to verify whether the antibody secreted by the hybridoma could bind to OX40 on the surface of 293 cells. Figure 3 As shown, most clones bind well to OX40 on the surface of 293 cells.

[0180] Example 3: Sequencing of mouse-derived anti-human OX40 antibody

[0181] Hybridoma cells m25A7, m27G12, and m11F7, which secrete anti-human OX40 antibodies, were expanded and cultured. Subtype detection was performed using MouseMonoclonaI Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and MouseMonoclonaI Antibody Light / Heavy Chain Test Card (Cat.: A12401, VicNovo) according to the reagent operation procedures. The subtype identification results showed that the heavy chain of m25A7, m27G12, and m11F7 were all IgG1, and the light chain was all Kappa chain, providing a basis for antibody gene cloning.

[0182] Total RNA was extracted from m25A7, m27G12, and m11F7 hybridoma cells according to the TRIzol kit (Cat: 15596026, Invitrogen) instructions. The total RNA was reverse transcribed into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB). The antibody light chain variable region IgVL(κ) and heavy chain variable region V were amplified using degenerate primers and the Phusion kit (Cat: E0553L, NEB). H Sequences were obtained; PCR amplification products were purified using a gel extraction kit (Cat: AP-GX-250, Axygen); the amplified PCR products were ligated into a T vector and transformed into competent *E. coli* cells according to the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Biotechnology). After amplification and plasmid extraction, DNA sequencing was performed to obtain the variable region sequence of the monoclonal antibody. The sequencing results showed that the nucleotide sequence of the mouse antibody m25A7 heavy chain variable region DNA is shown in Sequence 1, and the amino acid sequence of the mouse antibody m25A7 heavy chain variable region deduced from this DNA sequence is shown in Sequence 2. The nucleotide sequence of the mouse antibody m25A7 light chain variable region DNA is shown in Sequence 3, and the amino acid sequence of the mouse antibody m25A7 light chain variable region deduced from this DNA sequence is shown in Sequence 4; the nucleotide sequence of the mouse antibody m27G12 heavy chain variable region DNA is shown in Sequence 5, and the amino acid sequence of the mouse antibody m27G12 heavy chain variable region deduced from this DNA sequence is shown in Sequence 6. The nucleotide sequence of the light chain variable region DNA of mouse antibody m27G12 is shown in Sequence 7, and the amino acid sequence of the light chain variable region of mouse antibody m27G12 deduced from this DNA sequence is shown in Sequence 8. The nucleotide sequence of the heavy chain variable region DNA of mouse antibody m11F7 is shown in Sequence 9, and the amino acid sequence of the heavy chain variable region of mouse antibody m11F7 deduced from this DNA sequence is shown in Sequence 10. The nucleotide sequence of the light chain variable region DNA of mouse antibody m11F7 is shown in Sequence 11, and the amino acid sequence of the light chain variable region of mouse antibody m11F7 deduced from this DNA sequence is shown in Sequence 12.

[0183] Example 4: Preparation of anti-human OX40 chimeric antibody

[0184] The cloned mouse antibody light chain variable region and heavy chain variable region genes were introduced into restriction enzyme sites via PCR and cloned into eukaryotic expression vectors containing the coding genes for the human-kappa light chain constant region and the human IgG1 heavy chain constant region, respectively. This yielded expression plasmids for human-mouse chimeric light chain (pKN019-Ch25A7L) and human-mouse chimeric heavy chain (pKN041-Ch25A7H) of m25A7, human-mouse chimeric light chain (pKN019-Ch27G12L) and human-mouse chimeric heavy chain (pKN041-Ch27G12H) of m27G12, and human-mouse chimeric light chain (pKN019-Ch11F7L) and human-mouse chimeric heavy chain (pKN041-Ch11F7H) of m11F7. A large number of plasmids containing the light and heavy chains of human-mouse chimeric antibodies were isolated and amplified by transfection into *E. coli*. Following the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the light and heavy chain plasmids of the chimeric antibodies Ch 25A7, Ch 27G12, and Ch11F7 were paired and transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the m25A7, m27G12, and m11F7 chimeric antibodies.

[0185] Example 5: Activity analysis of anti-human OX40 chimeric antibody

[0186] Option 1. Affinity analysis of anti-human OX40 chimeric antibody

[0187] Antibody affinity was determined using the Fortebio Octet QKe system with an AHC (antibody capture antibody) bioprobe to capture the Fc fragment of the anti-human antibody. During the assay, anti-human OX40 chimeric antibodies ch25A7, ch27G12, ch11F7 and control antibody PC1 were diluted to 4 μg / mL with PBS buffer and flowed through the AHC probe (Cat: 18-0015, PALL) for 120 s. Human OX40-His recombinant protein (serial number: P43489-1, 29aa-216aa) was used as the mobile phase at a concentration of 100 nM. The binding time was 300 s, and the dissociation time was 300 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0188] The reaction curves of anti-human OX40 chimeric antibodies ch25A7, ch27G12, and ch11F7 with recombinant human OX40 protein are shown below. Figure 4 (Von represents the positive control PC1). The fitted curves and affinity calculations showed that the affinity (KD, Table 1) of the chimeric antibodies ch25A7, ch27G12, and ch11F7 were 1.36E-08, 4.34E-08, and 6.61E-09, respectively. These results indicate that the chimeric antibodies ch25A7, ch27G12, and ch11F7 have high affinity for recombinant human OX40 protein, but lower affinity than the control antibody PC1.

[0189] Table 1. Affinity fitting results of anti-OX40 chimeric antibodies ch25A7, ch27G12, and ch11F7 as determined by Fortebio.

[0190]

[0191] Scheme 2. Activation activity analysis of anti-human OX40 chimeric antibody

[0192] Following the method in Scheme 2 of Example 2, the activation activity of chimeric antibodies ch25A7, ch27G12, and ch11F7 was analyzed. Furthermore, the activity of chimeric antibody x was analyzed based on an NFκB signaling pathway activation assay using fluorescein detection. Specifically, HT1080-hOX40 cells were digested one day prior to the experiment, resuspended in complete culture medium (1640 medium + 10% FBS + 0.5 μg / mL), and the cell density was adjusted to 102. 5Cells were cultured at a density of 100 cells / mL, with 200 μL of cell suspension added to each well of a 96-well cell culture plate. The cell culture plate was then incubated overnight at 37°C with 5% CO2. The next day, the NFκB reporter gene plasmid (pGL4.32[Iuc2P / NF-κB-RE / Hygro]) was transfected into HT1080-hOX40 cells using liposomes. The culture medium was changed 24 h after transfection, and the cells were cultured for another 24 h to allow for a 48-hour recovery period after transfection. The antibody was then diluted to an appropriate concentration and added to the cell culture plate, which was incubated at 37°C with 5% CO2 for 18 h. The cell treatment medium was removed, and the cells were thoroughly washed with PBS and then allowed to dry completely. 25 μL of lysis buffer was added to each well (approximately 50 μL can be added if sufficient lysis buffer is available to prevent air bubbles in the next step), ensuring the cell layer was covered, and the cells were lysed at room temperature with shaking for 20 min. Add 20 μL of cell lysis buffer to the corresponding well of the assay plate, prepare the luciferase reaction substrate (100 μL / sample), and prepare for assay. Plot an antibody concentration-RLU bar chart or curve based on the luciferase levels (RLU) read by the machine. If using a dual-luciferase reporter system to simultaneously read the experimental reaction luciferase levels (RLU1) and the internal control luciferase levels (RLU2), calculate the ratio between the two and plot an antibody concentration-RLU1 / RLU2 bar chart or curve. Results are as follows... Figure 5A (Von represents PC1) Figure 5B As shown, 25A7 and 27G12 exhibit good activation activity, which is no less than that of the control antibody PC1.

[0193] Example 6: ELISA detection of the inhibitory effect of anti-human OX40 chimeric antibody on the binding of human OX40 / OX40L.

[0194] Human OX40 was diluted to 1 μg / mL, coated overnight at 4°C, and blocked with 5% BSA in a 37°C incubator for 60 min. ch25A7, ch27G12, and control antibody PC1, as well as isotype control NC-hlgG1 (starting concentration 20 μg / mL, 3-fold serial dilution, 8 gradients), were incubated at 37°C for 60 min. Then, 10 μg / mL of OX40L-mFc (OX40L sequence number: NP_003317, 51-Leu 183, mFc Tag) was added and co-incubated with the antibody in a 37°C incubator for 60 min. The plate was washed four times with PBST; then HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research) diluted 1:5000 was added, and the reaction was allowed to proceed for 45 min. TMB (Cat: ME142, Beijing Taitianhe Biotechnology) substrate was added for color development for 15 min, and the reaction was terminated with 2M HCl. The absorbance values ​​(A450nm-630nm) of the wells were read and recorded at a wavelength of 450nm using 630nm as the reference wavelength. The results showed that ch25A7 effectively blocked the binding of recombinant human OX40 to its receptor OX40L, with a half-maximal inhibitory concentration (IC50) of 0.92 μg / mL, comparable to the control antibody PC1 (0.91 μg / mL). Figure 6 ) ch27G12 showed no significant inhibitory effect.

[0195] Example 7: Humanization and Recombinant Expression of Anti-human OX40 Monoclonal Antibodies 25A7 and 27G12

[0196] Scheme 1.25A7's humanized design and recombinant expression

[0197] First, a comprehensive analysis of the mouse antibody heavy chain sequence was performed to determine the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). VH3 (3-21) was selected as the basic template. Combining the full-sequence BLAST results, the frequency of amino acids at specific FR sites in the rearranged antibody was considered, along with the HCDR3 sequence. CDR transplantation was then performed, achieving a high degree of humanization of the m25A7 heavy chain variable region (VH) within the framework region. Based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). VK I (L5), VK II (O1), and VK VI (A26) were selected as the basic templates. Combined with the full-sequence BLAST results, considering the frequency of amino acids at specific FR sites of the rearranged antibody and the LCDR3 sequence, CDR grafting was performed, achieving full humanization of the m25A7 light chain Framework region. The nucleotide sequence of the humanized heavy chain variable region of the m25A7 antibody CDR-grafted (CDRGrafted) is shown in Sequence 13, and the amino acid sequence is shown in Sequence 14; the nucleotide sequence of the humanized light chain 1 variable region is shown in Sequence 15, and the amino acid sequence is shown in Sequence 16; the nucleotide sequence of the humanized light chain 2 variable region is shown in Sequence 17, and the amino acid sequence is shown in Sequence 18; the nucleotide sequence of the humanized light chain 3 variable region is shown in Sequence 19, and the amino acid sequence is shown in Sequence 20. Subsequently, based on the characteristics of the mouse 25A7 sequence, reverse mutations were designed for the humanized heavy chain variable region sequence of the CDR transplantation. The reverse mutation sites are shown in Table 2 below. The final selected sequences after the 25A7 humanized reverse mutation include the heavy chain variable region nucleotide sequence (Sequence 21), amino acid sequence (Sequence 22), light chain variable region nucleotide sequence (Sequence 15), and amino acid sequence (Sequence 16). The humanized light chain hz25A7_L1 and humanized heavy chain mutants (parental hz25A7_H1, mutants hz25A7_H1m1-hz25A7_H1m8) were then paired (Table 3) and transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain various humanized antibodies against hz25A7 mutants (referred to sequentially as hz25A7_H1m1-hz25A7_H1m9).

[0198] Table 2.25A7 Heavy Chain Humanization Reversion Mutation Sequence Design

[0199]

[0200] Note: R16E indicates that the 16th amino acid R has been mutated back to E according to the Kabat numbering system.

[0201] Table 3.25A7 Light and Heavy Chain Sequence Combinations

[0202]

[0203] Note: This table represents sequences obtained by various combinations of humanized 25A7 light and heavy chains. For example, 25A7-1 indicates that the antibody is composed of the humanized 25A7 light chain hz25A7_L1 and the humanized heavy chain hz25A7_H1, and so on.

[0204] Scheme 2.27G12 Humanized Design and Recombination Expression

[0205] First, a comprehensive analysis of the murine antibody heavy chain sequence was performed to determine the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). VH1 (1-46) was selected as the basic template. Combining the full-sequence BLAST results, considering the frequency of amino acids at specific FR sites in rearranged antibodies, PTM risk, and HCDR3 sequence information, CDR transplantation was performed, achieving full humanization of the m27G12 heavy chain variable region (VH) within the framework region. Based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). VK III (L2) and VK II (A2) were selected as the base templates. Combining the full-sequence BLAST results with the LCDR3 sequence, CDR grafting was performed, achieving full humanization of the m27G12 light chain framework region. The nucleotide sequence of the humanized heavy chain variable region of the m27G12 antibody CDR grafted (CDR Grafted) is shown in Sequence 23, and the amino acid sequence in Sequence 24; the nucleotide sequence of the variable region of humanized light chain 1 is shown in Sequence 25, and the amino acid sequence in Sequence 26; the nucleotide sequence of the variable region of humanized light chain 2 is shown in Sequence 27, and the amino acid sequence in Sequence 28. Subsequently, the humanized heavy chain hz27G12H1 and humanized light chains hz27G12L1 and hz27G12L2 of 27G12 were paired and transfected into HEK293 cells for recombinant expression. Five to six days after transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the mutant humanized antibodies hz27G12H1L1 and hz27G12H1L2 of hz27G12.

[0206] Example 8: Activity analysis of anti-human OX40 humanized antibody

[0207] Option 1. Fortebio assay for antibody affinity

[0208] Following the method described in Scheme 1 of Example 5, affinity analysis was performed on various mutants of hz25A7 and hz27G12. The results (Table 4) showed that the mutants hz25A7m8 and hz27G12H1L2 maintained affinity levels comparable to or slightly higher than those of the chimeric antibody, with affinity (KD) of 0.65E-08M and 1.32E-08M, respectively.

[0209] Table 4.25 Affinity Test Results of Humanized Mutant Ability of A.25A7

[0210]

[0211]

[0212] Following the method in Scheme 2 of Example 5, H1080 activation activity analysis was performed on hz25A7, hz27G12, and each mutant. The results are as follows... Figure 7A As shown in -D (Von represents PC1), the humanized 25A7 and 27G12 and their mutants all maintained good activity in activating H1080 expression of IL-8.

[0213] Example 9. Genera-species crossover of hz25A7-mut8 and hz27G12-H1L2

[0214] Recombinant extracellular proteins from human OX40 (serial number: P43489-1, 29aa-216aa), cynomolgus monkey OX40 (Cat: 90846-C08H, Beijing Yiqiao Shenzhou), and mouse OX40 (Cat: 50808-MCCH, Beijing Yiqiao Shenzhou) were diluted to 1 μg / mL and coated overnight at 4°C. After washing three times with PBS, 5% BSA PBS was added, and the plates were blocked at 37°C for 60 min, followed by three washes with PBST. Serially diluted h25A7mut8 and h27G12H1L2 were added to PBS, and the plates were incubated at 37°C for 60 min, followed by four washes with PBST. Finally, HRP-goat anti-human IgG antibody (Cat: 115-035-071, Jackson Immuno) diluted 1:5000 was added. Research), incubated at 37℃ for 30 min, washed 4 times with PBST; TMB substrate was added for color development, incubated at 37℃ for 10 min, and then 2M HCl was added to terminate the reaction; using 630 nm as the reference wavelength, the absorbance A450nm-630nm of the plate at a wavelength of 450 nm was read and recorded. Results ( Figure 8 , Figure 9 The results showed that h25A7mut8 and h27G12H1L2 bound to both human and monkey OX40, but not to mouse OX40. The binding activity of h25A7mut8 to human and monkey OX40 was comparable, while the binding activity of h27G12H1L2 to monkey OX40 was lower than that to human OX40.

[0215] Example 10: In vitro testing of the immunological function of OX40 antibodies hz25A7 and hz27G12

[0216] Peripheral anticoagulated blood was collected from healthy subjects, and peripheral blood mononuclear cells (PBMCs) were isolated. Serially diluted OX40 antibody or control antibody was co-incubated with PBMCs. The functional activity of the OX40 antibody was evaluated by detecting cell proliferation and cytokine (IL-2, IFN-γ) secretion levels. Specifically, anti-human CD3 antibody and anti-human CD28 antibody were coated alone or together on an ELISA plate as control wells; anti-human OX40 antibody was coated on an ELISA plate as the test wells. Peripheral blood was collected from healthy subjects and processed according to Dynabeads... TM FlowComp TM Following the instructions of the Human CD3 Kit, CD3+ T cells were isolated and obtained. After washing twice, the cells were adjusted to an appropriate concentration using culture medium and added to 96-well plates. Five days later, cell proliferation was assessed using the CTG method. The cell culture supernatant was harvested, and IL-2 and IFN-γ were detected using ELISA. Finally, the Anti-Human CD3 / CD28 group was used as a positive control, and the PBS group as a negative control to evaluate the biological activity of the OX40 antibody. The experimental results showed that, similar to the control antibody, hz25A7m8 (labeled KNAb-1 in this example) and hz27G12H1L2 (labeled KNAb-2 in this example) significantly increased the concentrations of cytokines IL-2 and IFN-γ in the culture medium in a dose-dependent manner. Figure 10A , Figure 10B This suggests that it can activate T lymphocytes and enhance their cytokine secretion function. Cell proliferation assay results showed ( Figure 10C Both hz25A7m8 and hz27G12H1L2 can effectively promote cell proliferation, and their ability to promote cell proliferation is comparable to that of the control antibodies PC2 and PC3.

[0217] Example 12: In vivo activity analysis of anti-OX40 antibodies hz25A7m8 and hz27G12H1L2

[0218] Algorithm 1. In vivo pharmacodynamic study of hz25A7m8 and hz27G12H1L2 in an OX40 humanized mouse model of colorectal cancer MC38.

[0219] MC38 mouse colorectal cancer cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged every 3-4 days after reaching confluence, and tumor cells in the logarithmic growth phase were harvested, resuspended in PBS, and subcutaneously injected into the right flank of human OX40 transgenic mice. When the average tumor volume reached approximately 60-100 mm³, the mice were divided into groups of six for drug administration. The mice received 10 mg / kg twice weekly for a total of four administrations. After grouping, tumor volume was measured three times weekly using calipers, measuring both the long and short diameters of the tumor. The volume was calculated using the formula: Volume = 0.5 × Long Diameter × Short Diameter 2 While measuring tumor volume, the mice were weighed. The relationship between changes in mouse weight and drug administration time was recorded. Simultaneously, the survival and health status of the mice, such as activity and feeding during drug administration, were observed. At the end of the experiment, the mice were euthanized, the tumors were removed and weighed, and the removed tumors from the control and test groups were neatly arranged and photographed. Results ( Figure 11A , Figure 11B The results showed that in OX40 transgenic mice, the candidate antibodies hz25A7m8 and hz27G12H1L2 could significantly inhibit tumor growth and cause most tumors to regress. Among them, hz25A7m8 could completely regress the tumor, and its activity was superior to control antibodies such as PC2 and PC3.

[0220] Algorithm 2. In vivo immune memory study of hz25A7m8 and hz27G12H1L2 in OX40 humanized mouse colorectal cancer MC38 model.

[0221] MC38 mouse colorectal cancer cells and Hepa1-6 mouse liver cancer cells were cultured in DMEM medium containing inactivated 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM glutamine in an incubator at 37°C and 5% CO2. After reaching confluence every 3-4 days, the cells were passaged into individual flasks, and tumor cells in the logarithmic growth phase were harvested. These cells were then inoculated into huOX40 mice (MC38 tumors completely regressed after treatment with hz25A7m8, hz27G12H1L2, and PC2 as described in Example 1) and C57BL / 6J mice that had not received tumor inoculation. The tumor cells, resuspended in PBS, were subcutaneously inoculated into the left and right flanks of the experimental animals. After inoculation, the tumor volume was measured 2-3 times per week using calipers, measuring the long and short diameters of the tumor. The volume was calculated using the formula: Volume = 0.5 × Long diameter × Short diameter². While measuring tumor volume, the mice were weighed. The relationship between changes in mouse weight and inoculation time was recorded. Simultaneously, the survival and health status of the mice were observed, such as their activity and feeding after inoculation. At the end of the experiment, the mice were euthanized, the tumors were removed and weighed, and the removed tumors from the control and test groups were neatly arranged and photographed. Results ( Figure 12A The results showed that mice in all treatment groups no longer developed tumors after re-inoculation with MC38 cells. Although inoculation with Hepa1-6 cells did induce tumor formation, tumor growth was significantly inhibited. The inhibitory effects of hz25A7m8 and hz27G12H1L2 were more pronounced compared to PC2. Figure 12B ).

[0222] Option 3. Detection of immune memory T cell populations using FACS method

[0223] Spleens from mice in Scheme 2 of this embodiment were used to prepare single-cell suspensions. Anti-mouse CD3 / CD4 / CD44 / CD62L was added and co-incubated with the spleen cells to analyze the immune memory T cell populations. Specifically, tumor-bearing mice were dissected under sterile conditions to obtain spleens. A 70 μM cell sieve was placed in a sterile petri dish, and the spleen was transferred into the sieve. The spleen was then ground into dispersed single cells using a grinding stick. The cell suspension was collected into a 50 mL centrifuge tube and centrifuged at 200 × g for 10 min. The supernatant was discarded, red blood cells were lysed, and the cells were washed twice. The cell suspension was then processed through a 40 μM cell sieve to obtain a spleen single-cell suspension. The spleen single-cell suspension was transferred to flow cytometry tubes, and the corresponding antibodies (anti-mouse CD3 / CD4 / CD44 / CD62L) were added according to the experimental design. The cells were incubated in the dark for 30 min. After washing once with PBS, the cells were resuspended and analyzed by flow cytometry. Results ( Figure 13The results showed that, compared with the control group, mice treated with hz25A7m8, hz27G12H1L2, and PC2 had a significant increase in helper T cells (CD3+CD4+). Further analysis revealed that the proportions of memory T cells (CD3+CD4+CD44hiCD62Lhi) and natural T cells (CD3+CD4+CD44loCD62Lhi) were both increased to varying degrees.

[0224] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

[0225] The sequences involved in this application are as follows:

[0226] Sequence 1: m25A7 heavy chain variable region DNA nucleotide sequence

[0227]

[0228] The CDR coding region is sequence 37-39.

[0229] Sequence 2: Amino acid sequence of the m25A7 heavy chain variable region

[0230]

[0231] The CDRs region is sequence 40-42.

[0232] Sequence 3: m25A7 light chain variable region DNA nucleotide sequence

[0233]

[0234] The CDRs coding region is sequence 43-45.

[0235] Sequence 4: Amino acid sequence of the m25A7 light chain variable region

[0236]

[0237] The CDRs region is sequence 46-48.

[0238] Sequence 5: m27G12 heavy chain variable region DNA nucleotide sequence

[0239]

[0240] The CDRs coding region is sequence 49-51.

[0241] Sequence 6: Amino acid sequence of the m27G12 heavy chain variable region

[0242]

[0243] The CDRs region is sequence 52-54.

[0244] Sequence 7: m27G12 light chain variable region DNA nucleotide sequence

[0245]

[0246] The CDR coding region is sequence 55-57.

[0247] Sequence 8: Amino acid sequence of the m27G12 light chain variable region

[0248]

[0249] The CDRs region is sequence 58-60.

[0250] Sequence 9: DNA nucleotide sequence of the m11F7 heavy chain variable region

[0251]

[0252] The CDRs coding region is sequence 61-63.

[0253] Sequence 10: Amino acid sequence of the m11F7 heavy chain variable region

[0254]

[0255] The CDRs region is sequence 64-66.

[0256] Sequence 11: DNA nucleotide sequence of the m11F7 light chain variable region

[0257]

[0258] The CDR coding region is sequence 67-69.

[0259] Sequence 12: Amino acid sequence of the m11F7 light chain variable region

[0260]

[0261] The CDRs region is sequence 70-72.

[0262] The m25A7 humanized heavy chain variable region nucleotide sequence is shown in Sequence 13;

[0263] SEQ.ID NO.13:

[0264]

[0265] The CDRs coding region is sequence 73-75.

[0266] The amino acid sequence of the variable region of the 25A7 humanized heavy chain is shown in Sequence 14;

[0267] SEQ.ID NO.14:

[0268]

[0269] The CDRs region is sequence 76-78.

[0270] The nucleotide sequence of the variable region of the 25A7 humanized light chain 1 is shown in Sequence 15;

[0271] SEQ.ID NO.15:

[0272]

[0273]

[0274] The CDRs coding region is sequence 79-81.

[0275] The amino acid sequence of the variable region of the 25A7 humanized light chain 1 is shown in Sequence 16.

[0276] SEQ.ID NO.16:

[0277]

[0278] The CDRs region is sequence 82-84.

[0279] The nucleotide sequence of the variable region 25A7 humanized light chain is shown in Sequence 17;

[0280] SEQ.ID NO.17:

[0281]

[0282] The CDR coding region is sequence 85-87.

[0283] The amino acid sequence of the variable region 25A7 humanized light chain is shown in Sequence 18;

[0284] SEQ.ID NO.18:

[0285]

[0286] The CDRs region is sequence 88-90.

[0287] The nucleotide sequence of the variable region 3 of the humanized light chain 25A7 is shown in Sequence 19.

[0288] SEQ.ID NO.19:

[0289]

[0290]

[0291] The CDRs coding region is sequence 91-93.

[0292] The amino acid sequence of the variable region 3 of the humanized light chain 25A7 is shown in Sequence 20.

[0293] SEQ.ID NO.20:

[0294]

[0295] The CDRs region is sequence 94-96.

[0296] The nucleotide sequence of the heavy chain variable region after humanization backmutation of 25A7 is shown in Sequence 21.

[0297] SEQ.ID NO.21:

[0298]

[0299] The CDR coding region is sequence 97-99.

[0300] The amino acid sequence of the heavy chain variable region after humanization backmutation of 25A7 is shown in Sequence 22.

[0301] SEQ.ID NO.22∶

[0302]

[0303] The CDRs region is sequence 100-102.

[0304] The nucleotide sequence encoding the variable region of the 27G12 humanized heavy chain is shown in Sequence 23.

[0305] SEQ.ID NO.23:

[0306]

[0307] The CDRs coding region is sequence 103-105.

[0308] The amino acid sequence of the variable region of the 27G12 humanized heavy chain is shown in Sequence 24.

[0309] SEQ.ID NO.24:

[0310]

[0311] The CDRs region is sequence 106-108.

[0312] The nucleotide sequence encoding the variable region of the 27G12 humanized light chain 1 is shown in Sequence 25.

[0313] SEQ.ID NO.25:

[0314]

[0315] The CDRs coding region is sequence 109-111.

[0316] The amino acid sequence of the variable region 1 of the 27G12 humanized light chain is shown in Sequence 26.

[0317] SEQ.ID NO.26:

[0318]

[0319]

[0320] The CDRs region is sequence 112-114.

[0321] 27G12 humanized light chain 2 variable region coding nucleotide sequence 27

[0322] SEQ.ID NO.27:

[0323]

[0324] The CDRs coding region is sequence 115-117.

[0325] The amino acid sequence of the variable region 27G12 humanized light chain is shown in Sequence 28.

[0326] SEQ.ID NO.28:

[0327]

[0328] The CDRs region is sequence 118-120.

[0329] The nucleotide sequence of the variable region of the 25A7 humanized final heavy chain is shown in Sequence 29.

[0330] SEQ.ID NO.29:

[0331]

[0332] The CDRs coding region is sequence 121-123.

[0333] The amino acid sequence of the variable region of the 25A7 humanized final heavy chain is shown in Sequence 30.

[0334] SEQ.ID NO.30:

[0335]

[0336] The CDRs region is sequence 124-126.

[0337] The nucleotide sequence encoding the variable region of the 25A7 humanized final light chain is shown in Sequence 15.

[0338] SEQ.ID NO.15:

[0339]

[0340] The CDRs coding region is sequence 127-129.

[0341] The amino acid sequence of the final light chain variable region of the 25A7 humanized chain is shown in Sequence 16.

[0342] SEQ.ID NO.16:

[0343]

[0344] The CDRs region is sequence 130-132.

[0345] The nucleotide sequence encoding the variable region of the 27G12 humanized final heavy chain is shown in Sequence 23.

[0346] SEQ.ID NO.23:

[0347]

[0348]

[0349] The CDRs coding region is sequence 133-135.

[0350] The amino acid sequence of the variable region of the 27G12 humanized final heavy chain is shown in Sequence 24.

[0351] SEQ.ID NO.24:

[0352]

[0353] The CDRs region is sequence 136-138.

[0354] The nucleotide sequence encoding the variable region of the 27G12 humanized final light chain is shown in Sequence 31.

[0355] SEQ.ID NO.31:

[0356]

[0357] The CDRs coding region is sequence 139-141.

[0358] The amino acid sequence of the final light chain variable region of the 27G12 humanized chain is shown in sequence 32.

[0359] SEQ.ID NO.32:

[0360]

[0361] The CDRs region is sequence 142-144.

[0362] nucleotide sequence of heavy chain constant region 33

[0363] SEQ.ID NO.33:

[0364]

[0365]

[0366] Heavy chain constant region amino acid sequence 34

[0367] SEQ.ID NO.34:

[0368]

[0369] nucleotide sequence 35 of the light chain constant region

[0370] SEQ.ID NO.35:

[0371]

[0372]

[0373] 36 amino acid sequences in the light chain constant region

[0374] SEQ.ID NO.36:

[0375] sequence list <110> Applicant Name (Maiwei) <120> An activating anti-OX40 antibody, its production method and application <130> none <160> 144 <170> SIPOSequenceListing 1.0 <210> 1 <211> 357 <212> DNA <213> Artificial Sequence <400> 1 gaggttcagc tggtggagtc tgggggaggc ttagtgcagc ctggagagtc cctgaaactc 60 tcctgtgaat ccaatgaata cgaattccct tcccatgaca tgtcttgggt ccgcaagact 120 ccggagaaga ggctggagtt ggtcgcagcc attaatagtg atggtggtag aatctactat 180 ccagacacca tggagagacg attcatcatc tccagagaca ataccaagaa gaccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acagccttgt attactgtac aagacactat 300 gatggttacg cctggtttgc ttactggggc caagggactc tggtcactgt ctctgca 357 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His 20 25 30 Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asp Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met 50 55 60 Glu Arg Arg Phe Ile Ile Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Arg His Tyr Asp Gly Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 3 <211> 333 <212> DNA <213> Artificial Sequence <400> 3 gacatccagc tgactcagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcatgca gggccagcaa aagtgtcagt acatctggct ctagttatat acactggtac 120 caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga gcttccgctc 300 acgttcggtg ctgggaccaa gctggagctg aga 333 <210> 4 <211> 111 <212> PRT <213> Artificial Sequence <400> 4 Asp 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 Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Ser Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg 100 105 110 <210> 5 <211> 354 <212> DNA <213> Artificial Sequence <400> 5 aaggtccagc tgcagcagtc tggagctggg ctggtgaaac ccggggcatc agtgaagctg 60 aaggtccagc tgcagcagtc tggagctggg ctggtgaaac ccggggcatc agtgaagctg 60 tcctgcaagg cttctggcta caccttcact gaatatatta tacactgggt aaaacagagg 120 tcctgcaagg cttctggcta caccttcact gaatatatta tacactgggt aaaacagagg 120 tctggacagg gtcttgagtg gattgggtgg ttttaccctg aaagtggtag tataaagtac 180 tctggacagg gtcttgagtg gattgggtgg ttttaccctg aaagtggtag tataaagtac 180 aacgagaaat tcaaggacaa ggccacattg actgcggaca aatcctccaa cacagtctat 240 aacgagaaat tcaaggacaa ggccacattg actgcggaca aatcctccaa cacagtctat 240 atggagctta gtagattgac atctgaagac tctgcggtct atttctgtgc aagacacgaa 300 atggagctta gtagattgac atctgaagac tctgcggtct atttctgtgc aagacacgaa 300 <000,0947>gatccgatta cctttgctta ctggggccaa gggactctgg tcactgtctc tgca 354 gatccgatta cctttgctta ctggggccaa gggactctgg tcactgtctc tgca 354 <210> 6<UNK>210> 6 <211> 118<211> 118 <212> PRT<212> PRT <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 6<400> 6 Lys Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Val Lys Pro Gly Ala Lys Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Val Lys Pro Gly Ala 1 5 10 15 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 20 25 30 Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile Ile Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile 35 40 45 35 40 45 Gly Trp Phe Tyr Pro Glu Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe Gly Trp Phe Tyr Pro Glu Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe 50 55 60 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg His Glu Asp Pro Ile Thr Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 7 <211> 333 <212> DNA <213> Artificial Sequence <400> 7 gacattgtga tgacccagtc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatat gaactggtac 120 caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctagaatct 180 gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaatga ggatccttac 300 acgttcggag gggggaccaa gctggaaatt aaa 333 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala 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 Lys 100 105 110 <210> 9 <211> 360 <212> DNA <213> Artificial Sequence <400> 9 gaggtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagagtc cctgaaactc 60 tcctgtgaat ccaatgaata cgaattccct tcccatgaca tgtcttgggt ccgcaagact 120 tcctgtgaat ccaatgaata cgaattccct tcccatgaca tgtcttgggt ccgcaagact 120 ccggagaaga ggctggagtt ggtcgcaacc attaatagtg atggtgataa cacctactat 180 ccggagaaga ggctggagtt ggtcgcaacc attaatagtg atggtgataa cacctactat 180 ccagacacct tggagagacg attcatcatc tccagagaca ataccaagaa gaccctgtac 240 ccagacacct tggagagacg attcatcatc tccagagaca ataccaagaa gaccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acagcctttt attactgtac aagacactat 300 ctgcaaatga gcagtctgag gtctgaggac acagcctttt attactgtac aagacactat 300 gataattact acgcctggtt tgcttactgg ggccaaggga ctctagtcac tgtctctgca 360 gataattact acgcctggtt tgcttactgg ggccaaggga ctctagtcac tgtctctgca 360 <210> 10<210> 10 <211> 120<211> 120 <212> PRT<212> PRT <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 10 <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 1 5 10 15 1, 5, 10, 15 <0,001,017> Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His 20 25 30 20, 25, 30 Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 35, 40, 45 Ala Thr Ile Asn Ser Asp Gly Asp Asn Thr Tyr Tyr Pro Asp Thr Leu Ala Thr Ile Asn Ser Asp Gly Asp Asn Thr Tyr Tyr Pro Asp Thr Leu 50 55 60 50, 55, 60 Glu Arg Arg Phe Ile Ile Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr Glu Arg Arg Phe Ile Ile Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Thr Arg His Tyr Asp Asn Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Click Download to save Gly Thr Leu Val Thr Val Ser Ala mp3 youtube com 115 120 <210> 11 <211> 333 <212> DNA <213> Artificial Sequence <400> 11 60. gacattgtga tgacccagtc tcctgcttcc ttaactgtat ctctggggca gagggccacc atctcatgca gggccagcga aagtgtcagt acatctggct atagttatat gcactggtac 180. acccaaactc ctcatctatc ttgcatcca cctagaatct ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggagg tgcaacctat tactgtcagc acagtagg gcttccgctc acgttcggtg ctgggacca gctggagctg aaa <210> 12 <211> 111 <212> PRT <213> Artificial Sequence <400> 12 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 13 <211> 357 <212> DNA <213> Artificial Sequence <400> 13 gaggtgcagc tggtggagtc cggaggaggc ctggtgcagc ctggacggtc cctgcgactg 60 tcctgcgctg cctctggctt caccttctcc tcccacgaca tgtcctgggt gcggcaggct 120 cctggcaagg gactggagtg ggtggctgcc atcaactccg acggaggccg gatctactac 180 cctgacacca tggagcgacg gttcaccatc tctcgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg agccgaggac accgccgtgt actactgcac ccggcactac 300 gacggctacg cctggttcgc ctactggggc cagggcaccc tggtgaccgt gtcctcc 357 <210> 14 <211> 119 <212> PRT <213> Artificial Sequence <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val<O001082>35 40 45 Ala Ala Ile Asn Ser Asp Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met 50 55 60 Glu Arg 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 His Tyr Asp Gly Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 15 <211> 333 <212> DNA <213> Artificial Sequence <400> 15 gacatccaga tgacccagtc tccctcctcc ctgtctgcct ccgtgggcga ccgagtgacc 60 atcacctgcc gagcctccaa gtccgtgtcc acctctggct cctcctacat ccactggtac 120 cagcagaagc ctggcaaggc tcccaagctg ctgatctacc tggcctccaa cctggagtct 180 ggagtgccct ctcggttctc cggatctggc tccggcaccg acttcaccct gaccatctcc 240 tccctgcagc ccgaggactt cgccacctac tactgccagc actccaggga gctgcctctg 300 accttcggag gaggcaccaa ggtggagatc aag 333 <210> 16 <211> 111 <212> PRT <213> Artificial Sequence <400> 16 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 Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Ser Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 17 <211> 333 <212> DNA <213> Artificial Sequence <400> 17 gacatcgtga tgacccagac acctctgtcc ctgcccgtga cacctggcca gcctgcctcc 60 atctcctgcc gagcctccaa gtccgtgtcc acctccggct cctcctacat ccactggtac 120 ctgcagaagc ctggacagtc tcctcggctg ctgatctacc tggcctccaa cctggagtct 180 ggcgtgcccg accggttctc cggatctgga tctggcaccg acttcaccct gaagatctcc 240 agggtggagg ccgaggacgt gggcgtgtac tactgccagc actccaggga gctgcctctg 300 accttcggag gaggcaccaa ggtggagatc aag 333 <210> 18 <211> 111 <212> PRT <213> Artificial Sequence <400> 18 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser For positions 20 - 25 - 30 Gly Ser Ser Tyr Ile His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg Note: I added "For positions" in the translation of line 24 to make the meaning more clear as the original text seems a bit incomplete in that regard. If this is not allowed according to the strict rules, please let me know and I'll adjust accordingly.85 90 95 Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 19 <211> 333 <212> DNA <213> Artificial Sequence <400> 19 gagatcgtgc tgacccagtc tcctgccacc ctgtccctgt ctcctggcga gcgagccacc 60 ctgtcctgca gagcctccaa gtccgtgtcc acctctggct cctcctacat ccactggtac 120 cagcagaagc ctggacagtc tcccaagctg ctgatctacc tggcctccaa cctggagtct 180 ggagtgccct cccggttctc cggatctggc tctggcaccg acttcaccct gaccatcaac 240 tccctggagg ccgaggacgc tgccacctac tactgccagc actctcggga gctgcctctg 300 accttcggag gaggcaccaa ggtggagatc aag 333 <210> 20 <211> 111 <212> PRT <213> Artificial Sequence <400> 20 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 Lys Ser Val Ser Thr Ser 20 25 30 Gly Ser Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 21 <211> 357 <212> DNA <213> Artificial Sequence <400> 21 gaggtgcagc tggtggagtc cggaggaggc ctggtgcagc ctggacggtc cctgcgactg 60 tcctgcgctg cctctggctt caccttctcc tcccacgaca tgtcctgggt gcggcaggct 120 cctggcaagg gactggagct ggtggctgcc atcaactccg acggaggccg gatctactac 180 cctgacacca tggagcgacg gttcaccatc tctcgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg agccgaggac accgccgtgt actactgcgc tcggcactac 300 gacggctacg cctggttcgc ctactggggc cagggcaccc tggtgaccgt gtcctcc 357 <210> 22 <211> 119 <212> PRT <213> Artificial Sequence <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asp Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met 50 55 60 Glu Arg 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 Ala Arg His Tyr Asp Gly Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 23 <211> 354 <212> DNA <213> Artificial Sequence <400> 23 gaggtgcagc tggtgcagtc cggagctgag gtgaagaagc ctggagcctc cgtgaaggtg 60 tcctgcaagg cctccggcta caccttcacc gagtacatca tccactgggt gaggcaggct 120 cctggccagg gcctggagtg gatcggctgg ttctaccctg agtccggctc catcaagtac 180 aacgagaagt tcaaggaccg ggtgaccatc accagggaca cctccacctc caccgtgtac 240 atggagctgt cctccctgcg gtccgaggac accgctgtgt actactgcgc tcgacacgag 300 gaccccatca ccttcgccta ctggggccag ggcaccctgg tgaccgtgtc ctcc 354 <210> 24 <211> 118 <212> PRT <213> Artificial Sequence <400> 24 Glu 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 Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Glu Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Val 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 His Glu Asp Pro Ile Thr Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 25 <211> 333 <212> DNA <213> Artificial Sequence <400> 25 gagatcgtga tgacccagtc tcctgccacc ctgtccgtgt ctcctggcga gcgagccacc 60 ctgtcctgca aggcctccca gtccgtggac tacgacggcg actcctacat gaactggtac 120 cagcagaagc ctggccaggc tcccaagctg ctgatctacg ctgcctccaa cctggagtcc 180 ggcatccctg ctcggttctc cggatctggc tccggcaccg acttcaccct gaccatctcc 240 tccctggagc ccgaggactt cgccacctac tactgccagc agtccaacga ggacccctac 300 accttcggcg gaggcaccaa ggtggagatc aag 333 <210> 26 <211> 111 <212> PRT <213> Artificial Sequence <400> 26 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 3 "]] Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 27 <211> 333 <212> DNA <213> Artificial Sequence <400> 27 gacatcgtga tgacccagac acctctgtcc ctgtccgtga cacctggcca gcctgcctcc 60 atctcctgca aggcctccca gtccgtggac tacgacggcg actcctacat gaactggtac 120 ctgcagaagc ctggacagtc tcctcagctg ctgatctacg ctgcctccaa cctggagtct 180 ggcgtgcccg accggttctc cggatctgga tctggcaccg acttcaccct gaagatctcc 240 agggtggagg ccgaggacgt gggcgtgtac tactgccagc agtccaacga ggacccctac 300 accttcggcg gaggcaccaa ggtggagatc aag 333 <210> 28 <211> 111 <212> PRT <213> Artificial Sequence <400> 28 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 29 <211> 357 <212> DNA <213> Artificial Sequence <400> 29 gaggtgcagc tggtggagtc cggaggaggc ctggtgcagc ctggacggtc cctgcgactg 60 tcctgcgctg cctctggctt caccttctcc tcccacgaca tgtcctgggt gcggcaggct 120 cctggcaagg gactggagct ggtggctgcc atcaactccg agggaggccg gatctactac 180 { cctgacacca tggagcgacg gttcaccatc tctcgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg agccgaggac accgccgtgt actactgcgc tcggcactac 300 gacaactacg cctggttcgc ctactggggc cagggcaccc tggtgaccgt gtcctcc 357 <210> 30 <211> 119 <212> PRT <213> Artificial Sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Glu Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met 50 55 60 Glu Arg 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 Ala Arg His Tyr Asp Asn Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 31 <211> 333 <212> DNA <213> Artificial Sequence <400> 31 gagatcgtga tgacccagtc tcctgccacc ctgtccgtgt ctcctggcga gcgagccacc 60 gagatcgtga tgacccagtc tcctgccacc ctgtccgtgt ctcctggcga gcgagccacc 60 ctgtcctgca aggcctccca gtccgtggac tacgagggcg actcctacat gaactggtac 120 ctgtcctgca aggcctccca gtccgtggac tacgagggcg actcctacat gaactggtac 120 cagcagaagc ctggccaggc tcccaagctg ctgatctacg ctgcctccaa cctggagtcc 180 cagcagaagc ctggccaggc tcccaagctg ctgatctacg ctgcctccaa cctggagtcc 180 ggcatccctg ctcggttctc cggatctggc tccggcaccg acttcaccct gaccatctcc 240 ggcatccctg ctcggttctc cggatctggc tccggcaccg acttcaccct gaccatctcc 240 tccctggagc ccgaggactt cgccacctac tactgccagc agtccaacga ggacccctac 300 tccctggagc ccgaggactt cgccacctac tactgccagc agtccaacga ggacccctac 300 accttcggcg gaggcaccaa ggtggagatc aag 333 accttcggcg gaggcaccaa ggtggagatc aag 333 <210> 32<210> 32 <211> 111<211> 111 <212> PRT<212> PRT <213> Artificial Sequence<213> Artificial Sequence <400> 32<400> 32 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu 20 25 30 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 33 <211> 990 <212> DNA <213> Artificial Sequence <400> 33 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 34 <211> 330 <212> PRT <213> Artificial Sequence <400> 34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 35 <211> 321 <212> DNA <213> Artificial Sequence <400> 35 cggaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <400> 36 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 37 <211> 15 <212> DNA <213> Artificial Sequence <400> 37 tcccatgaca tgtct 15 <210> 38 <211> 51 <212> DNA <213> Artificial Sequence <400> 38 gccattaata gtgatggtgg tagaatctac tatccagaca ccatggagag a 51 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <400> 39 cactatgatg gttacgcctg gtttgcttac 30 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <400> 40 Ser His Asp Met Ser 1 5 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <400> 41 Ala Ile Asn Ser Asp Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met Glu 1 5 10 15 Arg <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <400> 42 His Tyr Asp Gly Tyr Ala Trp Phe Ala Tyr 1 5 10 <210> 43 <211> 45 <212> DNA <213> Artificial Sequence <400> 43 agggccagca aaagtgtcag tacatctggc tctagttata tacac 45 <210> 44 <211> twenty one <212> DNA <213> Artificial Sequence <400> 44 cttgcatcca acctagaatc t 21 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <400> 45 cagcacagta gggagcttcc gctcacg 27 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <400> 46 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Ser Ser Tyr Ile His 1 5 10 15 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <400> 47 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <400> 48 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 49 <211> 15 <212> DNA <213> Artificial Sequence <400> 49 gaatatatta tacac 15 <210> 50 <211> 51 <212> DNA <213> Artificial Sequence <400> 50 tggttttacc ctgaaagtgg tagtataaag tacaacgaga aattcaagga c 51 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <400> 51 cacgaagatc cgattacctt tgcttac 27 <210> 52 <211> 5 <212> PRT <213> Artificial Sequence <400> 52 Glu Tyr Ile Ile His 1 5 <210> 53 <211> 17 <212> PRT <213> Artificial Sequence <400> 53 Trp Phe Tyr Pro Glu Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <400> 54 His Glu Asp Pro Ile Thr Phe Ala Tyr 1 5 <210> 55 <211> 45 <212> DNA <213> Artificial Sequence <400> 55 aaggccagcc aaagtgttga ttatgatggt gatagttata tgaac 45 <210> 56 <211> 33 <212> DNA <213> Artificial Sequence <400> 56 gctgcatcca atctagaatc tgggatccca gcc 33 <210> 57 <211> 27 <212> DNA <213> Artificial Sequence <400> 57 cagcaaagta atgaggatcc ttacacg 27 <210> 58 <211> 15 <212> PRT <213> Artificial Sequence <400> 58 Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <400> 59 Ala Ala Ser Asn Leu Glu Ser 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <400> 60 Gln Gln Ser Asn Glu Asp Pro Tyr Thr 1 5 <210> 61 <211> 15 <212> DNA <213> Artificial Sequence <400> 61 tcccatgaca tgtct 15 <210> 62 <211> 51 <212> DNA <213> Artificial Sequence <400> 62 accattaata gtgatggtga taacacctac tatccagaca ccttggagag a 51 <210> 63 <211> 33 <212> DNA <213> Artificial Sequence <400> 63 cactatgata attactacgc ctggtttgct tac 33 <210> 64 <211> 5 <212> PRT <213> Artificial Sequence <400> 64 Ser His Asp Met Ser 1 5 <210> 65 <211> 17 <212> PRT <213> Artificial Sequence <400> 65 Thr Ile Asn Ser Asp Gly Asp Asn Thr Tyr Tyr Pro Asp Thr Leu Glu 1 5 10 15 Arg <210> 66 <211> 11 <212> PRT <213> Artificial Sequence <400> 66 His Tyr Asp Asn Tyr Tyr Ala Trp Phe Ala Tyr 1 5 10 <210> 67 <211> 45 <212> DNA <213> Artificial Sequence <400> 67 agggccagcg aaagtgtcag tacatctggc tatagttata tgcac 45 <210> 68 <211> 33 <212> DNA <213> Artificial Sequence <400> 68 cttgcatcca acctagaatc tggggtccct gcc 33 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <400> 69 cagcacagta gggagcttcc gctcacg 27 <210> 70 <211> 15 <212> PRT <213> Artificial Sequence <400> 70 Arg Ala Ser Glu Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 71 <211> 11 <212> PRT <213> Artificial Sequence <400> 71 Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 1 5 10 <210> 72 <211> 9 <212> PRT <213> Artificial Sequence <400> 72 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 73 <211> 15 <212> DNA <213> Artificial Sequence <400> 73 tcccacgaca tgtcc 15 <210> 74 <211> 51 <212> DNA <213> Artificial Sequence <400> 74 gccatcaact ccgacggagg ccggatctac taccctgaca ccatggagcg a 51 <210> 75 <211> 30 <212> DNA <213> Artificial Sequence <400> 75 cactacgacg gctacgcctg gttcgcctac 30 <210> 76 <211> 5 <212> PRT <213> Artificial Sequence <400> 76 Ser His Asp Met Ser 1 5 <210> 77 <211> 17 <212> PRT <213> Artificial Sequence <400> 77 Ala Ile Asn Ser Asp Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met Glu 1 5 10 15 Arg <210> 78 <211> 10 <212> PRT <213> Artificial Sequence <400> 78 His Tyr Asp Gly Tyr Ala Trp Phe Ala Tyr 1 5 10 <210> 79 <211> 45 <212> DNA <213> Artificial Sequence <400> 79 cgagcctcca agtccgtgtc cacctctggc tcctcctaca tccac 45 <210> 80 <211> twenty one <212> DNA <213> Artificial Sequence <400> 80 ctggcctcca acctggagtc t 21 <210> 81 <211> 27 <212> DNA <213> Artificial Sequence <400> 81 cagcactcca gggagctgcc tctgacc 27 <210> 82 <211> 15 <212> PRT <213> Artificial Sequence <400> 82 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Ser Ser Tyr Ile His 1 5 10 15 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <400> 83 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 84 <211> 9 <212> PRT <213> Artificial Sequence <400> 84 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 85 <211> 45 <212> DNA <213> Artificial Sequence <400> 85 cgagcctcca agtccgtgtc cacctccggc tcctcctaca tccac 45 <210> 86 <211> twenty one <212> DNA <213> Artificial Sequence <400> 86 ctggcctcca acctggagtc t 21 <210> 87 <211> 27 <212> DNA <213> Artificial Sequence <400> 87 cagcactcca gggagctgcc tctgacc 27 <210> 88 <211> 15 <212> PRT <213> Artificial Sequence <400> 88 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Ser Ser Tyr Ile His 1 5 10 15 <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <400> 89 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <400> 90 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 91 <211> 45 <212> DNA <213> Artificial Sequence <400> 91 agagcctcca agtccgtgtc cacctctggc tcctcctaca tccac 45 <210> 92 <211> twenty one <212> DNA <213> Artificial Sequence <400> 92 ctggcctcca acctggagtc t 21 <210> 93 <211> 27 <212> DNA <213> Artificial Sequence <400> 93 cagcactctc gggagctgcc tctgacc 27 <210> 94 <211> 15 <212> PRT <213> Artificial Sequence <400> 94 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Ser Ser Tyr Ile His 1 5 10 15 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <400> 95 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <400> 96 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 97 <211> 15 <212> DNA <213> Artificial Sequence <400> 97 tcccacgaca tgtcc 15 <210> 98 <211> 51 <212> DNA <213> Artificial Sequence <400> 98 gccatcaact ccgacggagg ccggatctac taccctgaca ccatggagcg a 51 <210> 99 <211> 30 <212> DNA <213> Artificial Sequence <400> 99 cactacgacg gctacgcctg gttcgcctac 30 <210> 100 <211> 5 <212> PRT <213> Artificial Sequence <400> 100 Ser His Asp Met Ser 1 5 <210> 101 <211> 17 <212> PRT <213> Artificial Sequence <400> 101 Ala Ile Asn Ser Asp Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met Glu 1 5 10 15 Arg <210> 102 <211> 10 <212> PRT <213> Artificial Sequence <400> 102 His Tyr Asp Gly Tyr Ala Trp Phe Ala Tyr 1 5 10 <210> 103 <211> 15 <212> DNA <213> Artificial Sequence <400> 103 gagtacatca tccac 15 <210> 104 <211> 51 <212> DNA <213> Artificial Sequence <400> 104 tggttctacc ctgagtccgg ctccatcaag tacaacgaga agttcaagga c 51 <210> 105 <211> 27 <212> DNA <213> Artificial Sequence <400> 105 cacgaggaccccatcacctt cgcctac 27 <210> 106 <211> 5 <212> PRT <213> Artificial Sequence <400> 106 Glu Tyr Ile Ile His 1 5 <210> 107 <211> 17 <212> PRT <213> Artificial Sequence <400> 107 Trp Phe Tyr Pro Glu Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 108 <211> 9 <212> PRT <213> Artificial Sequence <400> 108 His Glu Asp Pro Ile Thr Phe Ala Tyr 1 5 <210> 109 <211> 45 <212> DNA <213> Artificial Sequence <400> 109 aaggcctccc agtccgtgga ctacgacggc gactcctaca tgaac 45 <210> 110 <211> twenty one <212> DNA <213> Artificial Sequence <400> 110 gctgcctcca acctggagtc c 21 <210> 111 <211> 27 <212> DNA <213> Artificial Sequence <400> 111 cagcagtcca acgaggaccc ctacacc 27 <210> 112 <211> 15 <212> PRT <213> Artificial Sequence <400> 112 Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 113 <211> 7 <212> PRT <213> Artificial Sequence <400> 113 Ala Ala Ser Asn Leu Glu Ser 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <400> 114 Gln Gln Ser Asn Glu Asp Pro Tyr Thr 1 5 <210> 115 <211> 45 <212> DNA <213> Artificial Sequence <400> 115 aaggcctccc agtccgtgga ctacgacggc gactcctaca tgaac 45 <210> 116 <211> twenty one <212> DNA <213> Artificial Sequence <400> 116 gctgcctcca acctggagtc t 21 <210> 117 <211> 27 <212> DNA <213> Artificial Sequence <400> 117 cagcagtcca acgaggaccc ctacacc 27 <210> 118 <211> 15 <212> PRT <213> Artificial Sequence <400> 118 Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <400> 119 Ala Ala Ser Asn Leu Glu Ser 1 5 <210> 120 <211> 9 <212> PRT <213> Artificial Sequence <400> 120 Gln Gln Ser Asn Glu Asp Pro Tyr Thr 1 5 <210> 121 <211> 15 <212> DNA <213> Artificial Sequence <400> 121 tcccacgaca tgtcc 15 <210> 122 <211> 51 <212> DNA <213> Artificial Sequence <400> 122 gccatcaact ccgagggagg ccggatctac taccctgaca ccatggagcg a 51 <210> 123 <211> 30 <212> DNA <213> Artificial Sequence <400> 123 cactacgaca actacgcctg gttcgcctac 30 <210> 124 <211> 5 <212> PRT <213> Artificial Sequence <400> 124 Ser His Asp Met Ser 1 5 <210> 125 <211> 17 <212> PRT <213> Artificial Sequence <400> 125 Ala Ile Asn Ser Glu Gly Gly Arg Ile Tyr Tyr Pro Asp Thr Met Glu 1 5 10 15 Arg <210> 126 <211> 10 <212> PRT <213> Artificial Sequence <400> 126 His Tyr Asp Asn Tyr Ala Trp Phe Ala Tyr 1 5 10 <210> 127 <211> 45 <212> DNA <213> Artificial Sequence <400> 127 cgagcctcca agtccgtgtc cacctctggc tcctcctaca tccac 45 <210> 128 <211> twenty one <212> DNA <213> Artificial Sequence <400> 128 ctggcctcca acctggagtc t 21 <210> 129 <211> 27 <212> DNA <213> Artificial Sequence <400> 129 cagcactcca gggagctgcc tctgacc 27 <210> 130 <211> 15 <212> PRT <213> Artificial Sequence <400> 130 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Ser Ser Tyr Ile His 1 5 10 15 <210> 131 <211> 7 <212> PRT <213> Artificial Sequence <400> 131 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <400> 132 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 133 <211> 15 <212> DNA <213> Artificial Sequence <400> 133 gagtacatca tccac 15 <210> 134 <211> 51 <212> DNA <213> Artificial Sequence <400> 134 tggttctacc ctgagtccgg ctccatcaag tacaacgaga agttcaagga c 51 <210> 135 <211> 27 <212> DNA <213> Artificial Sequence <400> 135 cacgaggaccccatcacctt cgcctac 27 <210> 136 <211> 5 <212> PRT <213> Artificial Sequence <400> 136 Glu Tyr Ile Ile His 1 5 <210> 137 <211> 17 <212> PRT <213> Artificial Sequence <400> 137 Trp Phe Tyr Pro Glu Ser Gly Ser Ile Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 138 <211> 9 <212> PRT <213> Artificial Sequence <400> 138 His Glu Asp Pro Ile Thr Phe Ala Tyr 1 5 <210> 139 <211> 45 <212> DNA <213> Artificial Sequence <400> 139 aaggcctccc agtccgtgga ctacgagggc gactcctaca tgaac 45 <210> 140 <211> 33 <212> DNA <213> Artificial Sequence <400> 140 gctgcctcca acctggagtc cggcatccct gct 33 <210> 141 <211> 27 <212> DNA <213> Artificial Sequence <400> 141 cagcagtcca acgaggaccc ctacacc 27 <210> 142 <211> 15 <212> PRT <213> Artificial Sequence <400> 142 Lys Ala Ser Gln Ser Val Asp Tyr Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 143 <211> 7 <212> PRT <213> Artificial Sequence <400> 143 Ala Ala Ser Asn Leu Glu Ser 1 5 <210> 144 <211> 9 <212> PRT <213> Artificial Sequence <400> 144 Gln Gln Ser Asn Glu Asp Pro Tyr Thr 1 5

Claims

1. Anti-human OX40 antibody or its antigen-binding fragment, including the heavy chain variable region and the light chain variable region, wherein... The amino acid sequences of VH CDR1, CDR2 and CDR3 in the heavy chain variable region are shown in SEQ ID NO:40-42, and the amino acid sequences of VL CDR1, CDR2 and CDR3 in the light chain variable region are shown in SEQ ID NO:46-48, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, 14 or 22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4, 16, 18 or 20, or The heavy chain variable region has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequences shown in SEQ ID NO:2, 14, and 22, and the light chain variable region has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequences shown in SEQ ID NO:4, 16, 18, and 20.

3. A composition comprising (1) The antibody or antigen-binding fragment thereof as described in any one of claims 1-2; (2) Pharmaceutically acceptable carrier.

4. A nucleic acid encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-2.

5. A recombinant vector or recombinant host cell comprising the nucleic acid of claim 4.

6. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2, the composition according to claim 3, the nucleic acid according to claim 4, the recombinant vector or recombinant host cell according to claim 5, characterized in that: This is used to prepare a drug for inhibiting the growth and metastasis of solid tumors, wherein the solid tumor is colorectal cancer or liver cancer.

7. A method for producing antibodies, comprising: (1) Culturing the recombinant host cells as described in claim 5, (2) Antibody recovery.

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