CN113135994A

CN113135994A - Activated anti-OX 40 antibody, production method and application

Info

Publication number: CN113135994A
Application number: CN202010063141.3A
Authority: CN
Inventors: 王双; 曾大地; 王荣娟; 焦莎莎; 张畅
Original assignee: Maiwei Shanghai Biotechnology Co ltd
Current assignee: Maiwei Shanghai Biotechnology Co ltd
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2021-07-20
Also published as: WO2021143914A1

Abstract

The present invention provides an antibody or a fragment thereof expressing an activated receptor (OX40) on the surface of activated CD4+ T, CD8+ T cells, and the use of the antibody or the fragment thereof for preventing or treating diseases. The antibody of the invention has high affinity to OX40, and has obvious effect of activating OX40 signal path; has a relatively broad-spectrum immune enhancement effect and can enhance T cell response immunological memory; the in vivo experiment of the animal model obtains better tumor inhibition and killing effects, and has good clinical application prospect.

Description

Activated anti-OX 40 antibody, production method and application

Technical Field

The invention belongs to the field of antibody engineering, and particularly relates to an anti-OX 40 antibody, a production method and application thereof, in particular to a humanized antibody resisting human OX40, a recombinant expression method thereof and application thereof in treating solid tumors.

Background

OX40, also known as CD134, ACT45, 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, CD8+ T cells. OX40 signal can activate the downstream NF-kappa B, PI3K and PKB pathways, and the continuous activation of the pathways can finally prolong the survival time of T cells, expand T cell memory and promote the cell killing capacity of the T cells; in addition, OX40 can also improve immunosuppressive effects in the tumor microenvironment by inhibiting the differentiation and activity of regulatory T cells (tregs), further enhancing the function of effector T cells.

The OX40 gene is located on human chromosome 1 (mouse chromosome 4) and encodes a 50kD type of transmembrane glycoprotein. The extracellular domain is 191 amino acids and contains three intact and a slightly shorter cysteine-rich domains (CRDs). Expressed predominantly on activated effector T cells (Teffs) and regulatory T cells (Tregs), but also on NKT cells, NK cells and neutrophils.

OX40 bound to ligand OX40L (CD252, TNFSF4) to deliver a costimulatory signal. The OX40L gene is located on chromosome 1 in humans and mice and encodes the two-type transmembrane glycoprotein, 34 kD. OX40L may be present on Antigen Presenting Cells (APCs), such as: b cells, dendritic cells, macrophages; expression may also be induced in other cell types such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and NK cells.

The binding of OX40 and OX40L is involved in a variety of physiological responses between T cells and lymphocytes and non-lymphocytes. The interaction of OX40 and OX40L is capable of recruiting TNFR-associated (TRAFs) molecules within the intracellular region of OX40, forming a signaling complex comprising IKK α and IKK β and PI3k and pkb (akt); OX40 also synergizes with TCR signaling, enhancing intracellular Ca2+ by an unknown mechanism, thereby enhancing NFAT nuclear entry. OX40 activates the classical NF-. kappa.B 1 pathway or the non-classical NF-. kappa.B 2 pathway, PI3k/PKB and NFAT pathways, thereby regulating genes that control T cell division and survival, and promoting transcription of cytokine genes and expression of cytokine receptors, which are essential for cell survival. OX40 signaling causes down-regulation including CTLA-4 and Foxp 3.

anti-OX 40-activating antibodies can exert a similar function as OX40L to activate antigen-dependent T effector cells, and can exert an anti-tumor effect by abrogating the suppressive function of Treg cells. There are several clinically active varieties of anti-OX 40-activating antibodies, of which 3 experiments published partial experimental data, PF-04518600 for Pfizer, BMS-986178 for BMS and ABBV-368 for AbbVie, respectively. At present, two OX40 enter the clinical stage at home, namely recombinant human anti-tumor necrosis factor receptor superfamily member 4(OX40) monoclonal antibodies of the belief biopharmaceuticals and recombinant human anti-OX 40 monoclonal antibody injection of the Lizhu pharmaceutical group. Most of OX40 monoclonal antibodies in the prior art are obtained by hybridoma technology and are anti-OX 40 antibodies. Of course, the target antibody can also be obtained by transgenic mouse technology, phage antibody library technology, B cell sorting technology, etc., and the evaluation and analysis means are the same as or similar to the hybridoma antibody preparation scheme.

Although a plurality of anti-OX 40 activated antibodies are clinically researched, due to the reasons that the early screening expression of the anti-OX 40 activated antibody is not completely consistent with the final clinical treatment effect, adverse reactions are difficult to predict, the effect on different indications is obtained and the like, the development of more activated anti-OX 40 antibodies with high affinity, high biological activity and particularly better immunological memory arousing is still needed to meet the urgent needs of clinical treatment.

Disclosure of Invention

In order to solve the problems, the invention obtains a mouse antibody by a hybridoma technology, and obtains a candidate activated anti-OX 40 mouse antibody by analyzing the binding activity, the activation activity and the blocking activity of the antibody; the activated anti-OX 40 mouse antibody gene is subjected to antibody sequence humanization design, and the obtained humanized antibody is subjected to antibody activity analysis (binding activity, activation activity and blocking activity) and in-vivo drug effect analysis of a mouse, so that the activated anti-OX 40 humanized antibody is finally determined. Specifically, the method comprises the following steps:

in one aspect, the invention provides an antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises:

VH CDR1 is selected from SEQ ID NO: 40. 52, 64, or a pharmaceutically acceptable salt thereof,

VH CDR2 is selected from SEQ ID NO: 41. 53 and 65, respectively, or a pharmaceutically acceptable salt thereof,

VH CDR3 is selected from SEQ ID NO: 42. 54, 66;

the light chain variable region comprises:

VL CDR1 is selected from SEQ ID NO: 46. 58 and 70, or a pharmaceutically acceptable salt thereof,

VL CDR2 is selected from SEQ ID NO: 47. 59 and 71, or a pharmaceutically acceptable salt thereof,

VL CDR3 is selected from SEQ ID NO: 48. 60, 72, or a pharmaceutically acceptable salt thereof.

Further, the antibody or fragment thereof of the present invention has a heavy chain variable region comprising SEQ ID NO: 2. 6, 10, 14, 22, 24 or 30.

Still further, the antibody or fragment thereof of the present invention comprises a heavy chain variable region comprising SEQ ID NO: 4. 8, 12, 16, 18, 20, 26, 28 and 32.

Further, the antibody or the fragment thereof according to the present invention is characterized in that:

(1) the heavy chain variable region is selected from SEQ ID NO: 2. 14, 22 and 30, and the light chain variable region is selected from SEQ ID NO: 4. 16, 18, 20;

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

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

the heavy chain variable region is SEQ ID NO:2, and the light chain variable region is SEQ ID NO: 4;

the heavy chain variable region is SEQ ID NO:6 and the light chain variable region is SEQ ID NO: 8;

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

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

the heavy chain variable region is SEQ ID NO: 22 and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16. 18 or 20;

the heavy chain variable region is SEQ ID NO: 24, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 26 or 28;

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

or

The heavy chain variable region is SEQ ID NO: 24, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 32, or a pharmaceutically acceptable salt thereof.

Still further, the antibody or fragment thereof according to the present invention comprises SEQ ID NO: 34, and/or the heavy chain constant region of SEQ ID NO: 36, or a light chain constant region.

Still further, the antibody or fragment thereof of the present invention, wherein the antibody comprises a murine antibody, a chimeric antibody, a humanized antibody, a human antibody; the antibody fragment includes F (ab')₂Fab', Fv, scFv, Fd, nanobody, etc.

In a second aspect, the invention provides an immunoconjugate comprising

(1) The antibody or fragment thereof according to the first aspect of the invention,

(2) a coupling moiety.

Further, the immunoconjugate of the invention, wherein the conjugating moiety comprises a detectable label, a cytotoxic molecule, a biologically active molecule, and the like.

Still further, the immunoconjugate of the present invention, wherein the detectable label comprises a chemiluminescent label, a fluorescent label, an enzyme label, a radioactive label, a quantum dot, a nanoparticle, etc.

Still further, the immunoconjugate of the present invention, wherein the cytotoxic molecule comprises diphtheria toxin, pseudomonas exotoxin, ricin, leukotoxin, and the like.

Still further, the immunoconjugate of the invention, wherein the bioactive molecule comprises a cytokine, an enzyme, a chemotherapeutic agent, a liposome, a viral particle, and the like.

In a third aspect, the present invention provides a multispecific antibody or derivative thereof, characterised in that it comprises at least one antigen-binding domain of an antibody or fragment thereof according to the first aspect of the invention.

Further, the multispecific antibody or derivative thereof according to the present invention further comprises an antigen binding domain that binds to another target.

Still further, the multispecific antibody or derivative thereof of the present invention, wherein the other targets comprise PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, CD47, 4-1BB, CD73, ROR1, HER2, HER3, EGFR, and the like.

In a fourth aspect, the present invention provides a heavy chain antibody which is a dimeric heavy chain antibody obtained on the basis of the antibody or fragment thereof according to the first aspect of the present invention.

Further, the heavy chain antibody of the present invention does not have an Fc region.

In a fifth aspect, the present invention provides a composition comprising

(1) An antibody or fragment thereof according to the first aspect of the invention, an antibody conjugate according to the second aspect of the invention, a multispecific antibody or derivative thereof according to the third aspect of the invention, or a heavy chain antibody according to the fourth aspect of the invention;

(2) a pharmaceutically acceptable carrier.

Further, the composition of the present invention further comprises other active ingredients for treating tumors.

In a sixth aspect, the invention provides a kit comprising an antibody or fragment thereof according to the first aspect of the invention for use in the qualitative or quantitative detection of OX 40.

In a seventh aspect, the present invention provides a nucleic acid encoding an antibody or fragment thereof according to the first aspect of the present invention, or encoding a multispecific antibody or derivative thereof according to the third aspect of the present invention.

In an eighth aspect, the present invention provides a vector comprising a nucleic acid according to the seventh aspect of the present invention.

In a ninth aspect, the present invention provides a recombinant host cell comprising a nucleic acid according to the seventh aspect of the invention or comprising a vector according to the eighth aspect of the invention.

In a tenth aspect, the invention provides the use of an antibody or fragment thereof according to the first aspect of the invention, an antibody conjugate according to the second aspect of the invention, a multispecific antibody or derivative thereof according to the third aspect of the invention, a heavy chain antibody according to the fourth aspect of the invention, a composition according to the fifth aspect of the invention in the manufacture of a medicament which binds to OX40, inhibits the binding of OX40 to OX40L, activates OX40+ T cells, activates an immune response in humans, a vaccine for the treatment of tumors, and the like. For example, molecular adjuvants for enhancing specific immune response are used in combination with adjuvants such as CpG, etc. for tumor vaccines, etc.

In an eleventh aspect, the invention provides the use of an antibody or fragment thereof according to the first aspect of the invention, an antibody conjugate according to the second aspect of the invention, a multispecific antibody or derivative thereof according to the third aspect of the invention, a heavy chain antibody according to the fourth aspect of the invention, a composition according to the fifth aspect of the invention in the manufacture of a medicament for inducing OX40+ cells to produce IL-8 and for initiating transcription of NF κ B genes.

In a twelfth aspect, the invention provides the use of an antibody or fragment thereof according to the first aspect of the invention, an antibody conjugate according to the second aspect of the invention, a multispecific antibody or derivative thereof according to the third aspect of the invention, a heavy chain antibody according to the fourth aspect of the invention, or a composition according to the fifth aspect of the invention in the manufacture of a medicament for stimulating IL-2 and IFN- γ production by PBMCs.

In a thirteenth aspect, the present invention provides the use of an antibody or fragment thereof according to the first aspect of the present invention, an antibody conjugate according to the second aspect of the present invention, a multispecific antibody or derivative thereof according to the third aspect of the present invention, a heavy chain antibody according to the fourth aspect of the present invention, or a composition according to the fifth aspect of the present invention, in the manufacture of a medicament for inhibiting the growth and metastasis of a solid tumor.

Further, the use according to the present invention, wherein the solid tumor comprises a tumor of digestive system, a tumor of respiratory system, a tumor of urinary system and a tumor of reproductive system.

Further, the use of the present invention is characterized in that the tumor of the digestive system includes liver cancer, pancreatic cancer, gastric cancer, duodenal cancer, colorectal cancer, and esophageal cancer.

Further, the use of the present invention is characterized in that the tumor of the respiratory system includes small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma, laryngeal carcinoma, mesothelioma, and the like.

Further, the use according to the present invention is characterized in that the genitourinary tumor includes breast cancer, ovarian cancer, and the like.

In a fourteenth aspect, the present invention provides a method of producing an antibody comprising:

(1) culturing the host cell of the ninth aspect of the invention,

(2) recovering the antibody.

For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

The term "OX 40" refers to the fourth member of the tumor necrosis factor superfamily. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human OX40 may in some cases cross-react with an OX40 protein of another species, e.g., monkey. In other embodiments, an antibody specific to human OX40 protein may be completely specific to human OX40 protein without cross-reacting with other species or other types of proteins, or may cross-react with OX40 protein of some other species but not all other species.

The term "human OX 40" refers to an OX40 protein having a human amino acid sequence, e.g., the amino acid sequence of Genbank accession No. NP _ 003318. The terms "monkey OX 40" and "murine OX 40" refer to monkey and mouse OX40 sequences, respectively, e.g., having Genbank accession No. NP _001090870 and Genbank accession No. NP _035789, respectively.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed 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 is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigenic specificities. For example, an isolated antibody that specifically binds to an OX40 protein is substantially free of antibodies that specifically bind to antigens other than OX40 protein. However, isolated antibodies that specifically bind to human OX40 protein may have cross-binding to other antigens, such as OX40 protein of other species. Furthermore, the isolated antibody is substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., an OX40 protein). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments comprised in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) an Fd fragment consisting of VH and CH 1; (iv) an Fv fragment consisting of VL and VH antibody single arms; (v) dAb fragments consisting of VH (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined by recombinant methods via a synthetic linker that makes the two single protein chains, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fc (scFv); see, e.g., Bird et al., (1988) Science 242: 423-. These single chain antibodies are also intended to be included within the term meaning. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

Antigen-binding fragments of the invention include those capable of specifically binding to OX 40. Examples of antibody binding fragments include, for example, but are not limited to, Fab ', F (ab')₂Fv fragments, single chain Fv (scFv) fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F (ab') 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F (ab') 2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J.Nucl. Med.24: 316).

As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not comprise 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 of the two heavy chains of an antibody (CH2 and CH3 domains, respectively). The IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

The "Fv" fragment is the smallest fragment of an antibody that contains the entire target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain (VH-VL dimer) in tight non-covalent association. 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 on an antibody. However, in some cases, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) may have the ability to recognize and bind to a target, although at a lower affinity than the entire binding site.

"Single chain Fv" or "scFv" antibody binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a structure that facilitates target binding.

A "single domain fragment" consists of a single VH or VL domain which exhibits sufficient affinity for OX 40. In a particular embodiment, the single domain fragments are camelized (see, e.g., Riechmann, 1999, Journal of immunological Methods 231: 25-38).

anti-OX 40 antibodies of the invention include derivatized antibodies. For example, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins. Any of a number of chemical modifications can be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more unnatural amino acid, e.g., using ambrx technology (see, e.g., Wolfson, 2006, chem. biol.13 (10): 1011-2).

The anti-OX 40 antibody can be an antibody whose sequence has been modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, an anti-OX 40 antibody can be modified to reduce at least one constant region-mediated biological effector function, e.g., reduced binding to one or more Fc receptors (Fc γ R), such as Fc γ RI, Fc γ RIIA, Fc γ RIIB, Fc γ RIIIA, and/or Fc γ RIIIB, relative to an unmodified antibody. Fc γ R binding can be reduced by mutating the immunoglobulin constant region segment of the antibody at a specific region necessary for Fc γ R interaction (see, e.g., Canfield and Morrison, 1991, J.Exp.Med.173: 1483-. A reduction in the Fc γ R binding capacity of an antibody may also reduce other effector functions that are dependent on Fc γ R interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity ("ADCC"). In illustrative examples, variant CH2 domains having V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of an Fc region may exhibit reduced affinity for fcyriib compared to the corresponding wild-type constant region.

anti-OX 40 antibodies described herein include antibodies that have been modified to obtain or improve at least one constant region-mediated biological effector function relative to the unmodified antibody, e.g., to enhance Fc γ R interaction (see, e.g., U.S. patent application No. 2006/0134709). For example, an anti-OX 40 antibody of the invention can have a constant region that binds Fc γ RI, Fc γ RIIA, Fc γ RIIB, Fc γ RIIIA, and/or Fc γ RIIIB with greater affinity than the corresponding wild-type constant region. In illustrative examples, variant CH2 domains having V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of the Fc region may exhibit greater affinity for Fc γ RIIIA than the corresponding wild-type constant region.

Thus, the anti-OX 40 antibodies of the invention may have an alteration in biological activity that results in increased or decreased opsonization, phagocytosis, or ADCC. Such modifications 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", shown in figure 4 of U.S. patent No. 5, 834, 597) in which residues 234 and 237 (using EU numbering) are substituted with alanine. Mutant 3 (also referred to as "M3") variants can be used in many antibody isotypes, for example, human IaG 2M 3.

Additional substitutions that may modify fcyr binding and/or ADCC effector function of the anti-OX 40 antibody include K322A substitutions or double substitutions of L234A and L235A in the Fc region, such as human IgG1 with double substitutions of L234A/L235A. See, e.g., Hezareh et al j.virol, 75 (24): 12161-12168(2001).

In some embodiments, the anti-OX 40 antibody has a low level of fucose or lacks fucose. Antibodies lacking fucose have been associated with enhanced ADCC activity, particularly at low doses of the antibody. See Shields et al, 2002, j.biol.chem.277: 26733. about.26740; shinkawa et al, 2003, j.biol.chem.278: 3466-73. A method of making reduced fucose antibodies includes growth in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells expressed low levels of FUT8 mRNA encoding an alpha-1, 6-fucosyltransferase, an enzyme necessary for fucosylation of the polypeptide.

The anti-OX 40 antibody may comprise a modified (or variant) CH2 domain or the entire Fc domain including amino acid substitutions that increase binding to fcyriib and/or binding to fcyriiia by the technique as compared to binding to 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. The variant CH2 or variant Fc domain typically includes one or more substitutions at positions 263, 266, 273 and 305, wherein the numbering of the residues in the Fc domain is that of the EU index as in Kabat. In some embodiments, the anti-OX 40 antibody comprises one or more substitutions selected from the group consisting of V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, and V305W relative to the wild-type CH2 domain. In particular embodiments, the one or more substitutions of the CH2 domain relative to the CH2 domain of human IgG1 are selected from V263L, V273E, V273F, V273M, V273S, and V273Y. For example, one or more substitutions in the IgG1 CH2 domain may be V273E. In another specific embodiment, the anti-OX 40 antibody of the invention comprises a variant IaG1 CH2 domain comprising the amino acid substitution V263L.

Other examples of variant CH2 or variant Fc domains that may provide increased binding to fcyriib and/or decreased binding to fcyriiia compared to the binding of the corresponding wild-type CH2 or Fc region include those found in von dehheide et al clin. cancer res., 19(5), 1035-1043(2013), e.g., S267E or S267E/L328F in human IgG 1.

In some embodiments, the anti-OX 40 antibody comprises a modification that increases or decreases its binding affinity for the fetal Fc receptor FcRn, e.g., by mutating an immunoglobulin constant region segment at a specific region involved in FcRn interaction (see, e.g., WO 2005/123780). In particular embodiments, an anti-OX 40 antibody of the IgG class is mutated such that at least one of amino acid residues 250, 314, and 428 of the heavy chain constant region is substituted alone, 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 other than 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 other than 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 Fc effector function is Fc substitution M428L, which may occur in combination with Fc substitution T250Q. Additional 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.

anti-OX 40 antibodies may have one or more amino acids inserted into one or more of its CDRs, e.g., as exemplified by Jung and plu ü ckthun, 1997, Protein Engineering 10: 8,959-966; yazaki et al, 2004, proteineng. des sel.17 (5): 481-9.Epub 2004 Aug 17; and U.S. patent application No. 2007/0280931.

The term "mouse-derived antibody" refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the invention may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "murine antibody" does not include antibodies having CDR sequences from other mammalian species inserted into the mouse framework sequences.

The term "chimeric antibody" refers to an antibody obtained by combining genetic material of non-human origin with genetic material of human origin. Or more generally, a chimeric antibody refers to an antibody that combines genetic material of one species with genetic material of another species.

The term "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin containing minimal sequences derived from a non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically one of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al, 1988, Nature 332: 323-7; U.S. patent numbers to Queen et al: 5, 530, 101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; PCT publications WO 91/09967; U.S. Pat. nos. 5,225,539; EP 592106; EP 519596; padlan, 1991, mol. 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. Pat. No. 5,565,332.

"human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins. Human antibodies can be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. nos. 4,444,887 and 4,716,111; and PCT publication WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741. Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but can express human immunoglobulin genes. See, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. patent nos. 5,413,923; 5,625,126, respectively; 5,633,425, respectively; 5,569,825; 5,661,016, respectively; 5,545,806; 5,814, 318; 5,885,793, respectively; 5,916,771, respectively; and 5,939,598. Alternatively, using techniques similar to those described above, companies such as LakePharma, Inc (Belmont, CA) or Creative BioLabs (Shirley, NY) may be engaged in providing human antibodies to selected antigens. Fully human antibodies that recognize selected epitopes can be generated using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (see Jespers et al, 1988, Biotechnology 12: 899-903).

The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used herein interchangeably with the term "antibody specifically binding to an antigen".

Herein, an "antibody that specifically binds to human OX 40" refers to an antibody that binds to human OX40 (and possibly also other non-human species of OX40) but does not substantially bind to non-OX 40 protein. Preferably, the antibody binds human OX40 protein with "high affinity", i.e. with a KD value of 5.0x10^-8M or less, preferably 1.0x10^-8M or less, more preferably 5.0x10^-9M is less than or equal to M.

The term "does not substantially bind" to a protein or cell means that it does not bind to a protein or cell, or does not bind to it with high affinity, i.e., binds to a protein or cell with a KD of 1.0x10^-6M or more, more preferably 1.0x10^-5M or more, more preferably 1.0x10^-41.0x10 above M^-3M or more, more preferably 1.0x10^-2M is more than M.

The term "high affinity" for IgG antibodies means a KD for the antigen of 1.0x10^-6M or less, preferably 5.0x10^-8M or less, more preferably 1.0x10^-sM below, 5.0x10^-9M or less, more preferably 1.0x10^-9M is less than or equal to M. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of an IgM subtype means a KD of 10^-6M is less, preferably 10^-7M is less, more preferably 10^-8M is less than or equal to M.

The term "Kassoc" or "Ka" refers to the association rate of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, derived from the KD to Ka ratio (KD/Ka), and expressed in molar concentration (M). The KD value of an antibody can be determined by methods known in the art. A preferred way of determining the KD of an antibody is by measurement using a Surface Plasmon Resonance (SPR), preferably a biosensing system such as the Biacore (TM) system.

The term "EC 50," also called half maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximal effect.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

The term "agonistic OX40 antibody" as used herein refers to an OX40 antibody that is capable of binding to OX40 and activating or priming the OX40 signaling pathway to promote T cell proliferation and survival. The term "antagonistic OX40 antibody" refers to an OX40 antibody that blocks the OX40 signaling pathway, thereby correcting hyperactive T cell pathologies, and may be used to treat, for example, asthma, inflammatory bowel disease, and arthritis.

The term "therapeutically effective amount" refers to an amount of an antibody of the invention sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer) and/or to reduce the severity of the disease. The therapeutically effective amount is related to the disease to be treated, wherein the actual effective amount can be readily determined by one skilled in the art.

Aspects of the invention are described in more detail below.

OX40 antibodies have binding specificity for human OX40 as well as other beneficial functional characteristics

The antibodies of the invention specifically bind human OX40 with high affinity, e.g., a KD value of 1x10^-8M is less than or equal to M. The antibody also has cross-reactivity with monkey OX40 and does not bind to mouse OX 40.

The antibodies of the invention are agonistic OX40 antibodies that activate or elicit the OX40 signaling pathway and participate in T cell co-stimulation, promoting IL-2 secretion and CD8+ T cell proliferation.

The antibody of the invention has good in vivo anti-tumor effect. After cessation of antibody administration, the tumor will not grow, or even be completely eliminated, and immunological memory can be developed.

The antibodies of the invention may be polyclonal, monoclonal, genetically engineered, and/or otherwise substantially modified, including but not limited to chimeric, humanized, and human antibodies. In some embodiments, the constant region is an isoform selected from the group consisting of: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), and IgM. In particular embodiments, the anti-OX 40 antibodies described herein comprise IgG 1. In other embodiments, the anti-OX 40 antibody comprises IgG2 or IgG 4. As used herein, the "constant region" of an antibody includes a native constant region, allotype or native variant, for example D356E and L358M in human IgG1, or a 431G. See, e.g., Jefferis and Lefranc, MAbs, 1 (4): 332-338 (months 7 to 8 in 2009). Preferred antibodies of the invention are monoclonal antibodies. Furthermore, the antibody may be, for example, a murine, chimeric or humanized monoclonal antibody.

The light chain constant region of the anti-OX 40 antibody can be a C kappa (kappa) region or a C lambda (lambda) region. The lambda region may be any of the known subtypes, such as lambda 1, lambda 2, lambda 3 or lambda 4. In some embodiments, the anti-OX 40 antibody comprises a C kappa (kappa) region.

An anti-OX 40 antibody with high affinity for human OX40(SEQ ID NO: 1) may be desirable for therapeutic and diagnostic uses. Accordingly, antibodies having high binding affinity to human OX40 are contemplated by the invention. In particular embodiments, the anti-OX 40 antibody binds to human OX40 with an affinity of at least about 100nM, but can exhibit a higher affinity, e.g., at least about 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 25nM, 20nM, 15nM, 10nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.1nM, 0.01nM, or even higher. In some embodiments, the antibody binds human OX40 with an affinity in the range of about 1pM to about 100nM, or an affinity in the range between any of the foregoing values, such as, but not limited to, about 0.001 to 10nM, 0.001 to 5nM, 0.01 to 100nM, 0.01 to 50nM, 0.01 to 10nM, 0.01 to 5nM, or 0.01 to 1 nM.

The affinity of anti-OX 40 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 assays.

anti-OX 40 antibodies generally include a heavy chain comprising a variable region (VH) having three complementarity determining regions ("CDRs"), referred to herein (in N → C order) as VH CDR #1, VH CDR #2 and VH CDR #3, and a light chain comprising a variable region (VL) having three complementarity determining regions, referred to herein (in N → C order) as VL CDR #1, VL CDR #2 and VL CDR # 3. Provided herein are amino acid sequences of exemplary CDRs, as well as amino acid sequences of VH and VL regions of heavy and light chains of exemplary anti-OX 40. Specific embodiments of anti-OX 40 antibodies include these exemplary CDR and/or VH and/or VL sequences, as well as antibodies that compete with such antibodies for binding to human OX 40.

Compared with the prior art, the technical scheme of the invention has the following advantages:

first, the present invention provides a novel humanized anti-OX 40 activated antibody with a definite amino acid sequence structure, high affinity for OX40, a definite effect of activating OX40 signaling pathway, low heterogeneity and high clinical application potential.

Secondly, the humanized anti-OX 40 activated antibody provided by the invention has good biological activity, can not only induce OX40+ cells to generate IL-8 and NF kappa B, but also stimulate PBMC to generate IL-2 and IFN-gamma, has relatively broad-spectrum immune enhancement effect, and can enhance T cell response immune memory. The results of in vivo immunological memory studies of treated mice treated with the MC38 model of OX40 humanized mouse colorectal cancer by hz25A7m8 and hz27G12H1L2 show that the treated mice are not tumorigenic after being inoculated with MC38 cells again, but the growth of tumors is obviously inhibited after being inoculated with Hepal-6 cells, and the inhibition effect of hz25A7m8 and hz27G12H1L2 is more obvious than that of PC2 (variable region sequences are from BMS patent: WO2016196228A 1; heavy chain: SEQ ID: 124, light chain: SEQ ID: 116; also abbreviated as BMS in the invention; the same below).

Thirdly, the humanized anti-OX 40 activated antibody provided by the invention achieves better tumor inhibition and killing effects in-vivo experiments of animal models, even if tumors are completely regressed in partial experimental individuals, the clinical use effect of the humanized anti-OX 40 activated antibody on OX40 related cancers such as colorectal cancer is more obvious. Results of in vivo pharmacodynamic studies of hz25A7m8, hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 model show that candidate antibodies hz25A7m8 and hz27G12H1L2 can obviously inhibit tumor growth and enable most tumors to regress in OX40 transgenic mice. Wherein, hz25A7m8 can completely regress the tumor, and the activity is better than that of a control antibody such as PC2 and PC3 (the variable region sequence is from a belief patent: WO2018177220A 1; heavy chain: SEQ ID: 111; light chain: SEQ ID: 130; also abbreviated as XD in the invention; the same below).

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1: results of ELISA identification of binding activity of murine monoclonal antibodies to OX 40.

FIG. 2: the anti-OX 40 murine monoclonal antibody activates HT1080-hOX40 cells to express IL8 detection result graph. Each antibody was reduced in antibody concentration stepwise from left to right.

FIG. 3: graph of results of binding of murine monoclonal antibody against OX40 to HT1080-hOX40 cells.

FIG. 4: affinity assay results of anti-OX 40 chimeric antibodies ch25A7, ch27G12 and ch11F7 Fortebio.

FIG. 5A: HT1080-huOX40 IL-8 secretion method results in the identification of OX40 chimeric antibody activating activity.

FIG. 5B: NF κ B pathway activation-fluorescein assay a graph of the results of the activation activity of OX40 chimeric antibodies was identified, with each antibody having a stepwise decreasing antibody concentration from left to right.

FIG. 6: the chimeric antibodies ch25a7, ch27G12 blocked the activity of OX40L binding to HT1080-huOX40 cells.

FIG. 7 is a graph showing the results of detection of activation activity before and after humanization of 25A7 and 27G 12; FIG. 7B.27G12 humanized molecule mutant activation activity detection result chart; FIG. 7C.25A7 humanized molecule mutant activation activity assay results (partial); FIG. 7D.25A7 humanized molecule mutant activation activity assay results (section).

FIG. 8: species-specific identification results of anti-OX 40 antibody hz25A7m 8.

FIG. 9: species-specific identification results of anti-OX 40 antibody hz27G12H1L 2.

FIG. 10A: OX40 antibodies hz25A7, hz27G12 had immunological functions (IFN-. gamma.), Von was PC1, BMS was PC2, XD was PC 3.

FIG. 10B: in vitro OX40 antibodies hz25A7, hz27G12 were tested for immunological function (IL-2), Von being PC1, BMS being PC2, XD being PC 3.

FIG. 10C: in vitro OX40 antibodies hz25A7, hz27G12 were tested for immunological function (cell proliferation), Von being PC1, BMS being PC2 and XD being PC 3.

FIG. 11A: hz25A7m8 and hz27G12H1L2 effectively inhibit the growth of OX40 humanized mouse colorectal cancer MC38 model tumor (individual data), BMS is PC2, and XD is PC 3.

FIG. 11B: hz25A7m8 and hz27G12H1L2 effectively inhibit the growth of OX40 humanized mouse colorectal cancer MC38 model tumor (average data), BMS is PC2, and XD is PC 3.

FIG. 12A: an immune memory result chart (MC38) analyzed by a mouse subcutaneous tumor formation experiment after anti-OX 40 antibody treatment, wherein BMS is PC 2; the treatment regimens for hz25A7m8, hz27G12H1L2, BMS, and isotype control were all 0H, 10mg/kg ip, biw × 4.

FIG. 12B: an immune memory result chart (Hepal-6) analyzed by an anti-OX 40 antibody treatment sequential mouse subcutaneous tumor formation experiment, wherein BMS is PC 2; the treatment regimens for hz25A7m8, hz27G12H1L2, BMS, and isotype groups were all 0H, 10mg/kg ip, biw × 4.

FIG. 13: the result of FACS method detection of immune memory T cell clustering is shown in BMS PC 2.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below 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 embodied 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.

In particular, the invention provides antibodies or fragments thereof comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises:

VH CDR3 is selected from SEQ ID NO: 42. 54, 66;

the light chain variable region comprises:

The antibody or the fragment thereof according to the present invention is characterized in that:

(1) the heavy chain variable region is substantially identical to a sequence selected from SEQ ID NO: 2. 14, 22, 30, and a light chain variable region that is more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4. 16, 18, 20, or more than 99%, or 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous thereto;

(2) the heavy chain variable region is selected from SEQ ID NO: 6. 24, and the light chain variable region is selected from SEQ ID NOs: 8. 26, 28, 32, or more than 99%, or 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous thereto;

(3) the heavy chain variable region is SEQ ID NO:10 has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology, and the light chain variable region is SEQ ID NO:12 has more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology.

The invention also provides an immunoconjugate comprising

(1) The antibody or the fragment thereof according to the present invention,

(2) a coupling moiety.

The invention also provides a multispecific antibody or derivative thereof comprising at least one antigen-binding domain of an antibody or fragment thereof according to the invention.

The invention also provides a heavy chain antibody, and a dimer heavy chain antibody obtained on the basis of the antibody.

The invention also provides a composition comprising

(1) The antibody or fragment thereof of the present invention, the antibody conjugate of the present invention, the multispecific antibody or derivative thereof of the present invention, or the heavy chain antibody of the present invention

(2) A pharmaceutically acceptable carrier.

The invention also provides a nucleic acid encoding an antibody or fragment thereof according to the invention, a multispecific antibody or derivative thereof according to the invention, or a heavy chain antibody according to the invention.

The invention also provides a recombinant vector or a recombinant host cell comprising the coding nucleic acid of the invention.

The invention also provides the use of said antibody or fragment thereof, said antibody conjugate, said multispecific antibody or derivative thereof, said heavy chain antibody, said composition, said nucleic acid, said recombinant vector or recombinant host cell, comprising

Used for preparing medicines for binding OX40, inhibiting OX40 from binding OX40L, activating OX40+ T cells, activating human immune response, and treating tumor vaccine;

the molecular adjuvant is used for preparing a molecular adjuvant for enhancing specific immune response, and is preferably combined with adjuvants such as CpG and the like for preparing a tumor vaccine;

used for preparing drugs for inducing OX40+ cells to produce IL-8 and/or starting transcription of NF kappa B genes;

for the preparation of a medicament for stimulating IL-2 and IFN-gamma production by PBMC;

for preparing a medicament for inhibiting the growth and metastasis of solid tumors;

for the preparation of a kit for the qualitative or quantitative detection of OX 40.

The present invention also provides a method of producing an antibody comprising:

(1) culturing the recombinant host cell of the present invention,

(2) recovering the antibody.

Example 1: anti-human OX40 antibody hybridoma cell preparation

Immunization: adopting human OX40/mFc recombinant protein (sequence number: P43489-1, 29aa-216aa) to immunize Balb/c mice, and using a 96-hole enzyme label plate coated with recombinant human OX40-his protein to detect the serum titer by an ELISA method; mice with serum titers meeting the fusion requirements were used for the next cell fusion.

Cell fusion and hybridoma preparation: on the 67 th day after the primary immunization, a mouse with the required titer was selected, the spleen of the mouse was aseptically taken, a B lymphocyte suspension was prepared, and the B lymphocyte suspension was mixed with FO myeloma cells at a ratio of 5: 1, and the two cells were fused under the action of PEG 4000. The fused cells were resuspended in HAT medium and then plated in 96-well cell culture plates. The mixture was cultured at 37 ℃ in a 5% CO2 incubator.

Example 2: screening of anti-human OX40 antibody positive hybridoma cell strain

Scheme 1 Positive hybridoma ELISA binding screening

10-14 days after fusion, the ELISA plate was coated with human OX40-His recombinant protein (SEQ ID NO: P43489-1, 29aa-216aa) (10ug/ml, pH9.6, 0.1M NaHCO3), and left overnight at 4 ℃; sealing with 4% skimmed milk powder-PBS at 37 deg.C for 2 hr; washing with PBST (0.05% Tween20-PBS) for three times, adding culture supernatant of hybridoma clone, and heating at 37 deg.C for 1 hr. Let the following controls: (1) positive Control (PC): post-immunization mouse serum (diluted 1: 1000 with PBS); (2) negative Control (NC): pre-immune mouse sera (diluted 1: 1000 with PBS). Washing with PBST (0.05% Tween20-PBS) for three times, adding HRP-goat anti-mouse IgG (Fc γ), diluting at 1: 20000, and diluting at 37 deg.C for 1 hr; washing with PBST (0.05% Tween20-PBS) for five times, adding OPD color developing solution, developing in dark for 10-15min, adding 2M H₂SO₄Terminating the reaction; the microplate reader reads the A492 value. The A492 value of the detection well is more than 2.1 times of the A492 value of the negative control well, and the positive result is judged. Is composed ofAnd determining the reliability of the positive clone, and performing a second round of screening every other day after the first screening and liquid changing. Through detection and identification (figure 1), 14 antibody secretion positive cell strains (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 and 30812-5) are obtained in total, and the 14 antibody strains are further screened.

Scheme 2. Activity screening of anti-OX 40 murine monoclonal antibody to activate IL8 expression in HT1080-hOX40 cells

HT1080-hOX40 cells (Congyuan Boxua: Cat.No: KC-0140) were digested one day before the experiment, the cells were resuspended in complete medium (1640 medium + 10% FBS + O.5. mu.g/mL), and the cell density was adjusted to 10⁵Per mL, 200. mu.L of cell suspension was added per well in a 96-well cell culture plate. The cell culture plates were then incubated overnight at 37 ℃ in a 5% CO2 incubator. The next day, the test antibody was diluted to the appropriate concentration and added to the cell culture plate, and incubated for 6 hours at 37 ℃ in a 5% CO2 incubator. Meanwhile, a positive control PC1(PC1, namely Vonlleralizumab, variable region sequence from WHO Drug information, Vol.31, NO.3, 2017, also referred to as Von in the present invention, the same below) was set. Finally, the cell culture supernatant was aspirated, and the IL-8 content in the supernatant was measured by an IL-8ELISA quantitative kit (cat: lot) according to the instructions. As shown in FIG. 2, most clones activated HT1080-hOX40 cells well, resulting in increased IL8 expression level.

Scheme 3 Positive hybridomas screening for binding to HT-1080 cell surface OX40

According to the activity results, 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 selected, and the obtained murine antibody (5ug/ml) and 293 cell (293/OX40) suspension recombinantly expressing human OX40 were incubated at 37 ℃ for 30min, and the following controls were set: (1) positive control PC1, the antibody molecule Fc in this example was replaced with murine IgG1, 5 μ g/ml; (2) negative control NC: irrelevant mouse IgG 15. mu.g/ml. After washing the cells 3 times with PBS, 1: goat anti-mouse IgG-FITC (Cat: F9006, Sigma) at 64 dilutions was incubated for 30 min. After washing the cells 3 times with PBS, the Mean Fluorescence Intensity (MFI) of the cells was examined by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibodies secreted by the hybridomas bind to OX40 on the surface of 293 cells, as shown in fig. 3, most clones bind well to OX40 on the surface of 293 cells.

Example 3: sequencing of murine anti-human OX40 antibodies

After expanded culture of hybridoma cells m25A7, m27G12 and m11F7 secreting anti-human OX40 Antibody, subtype detection was performed using Mouse monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and Mouse monoclonal Antibody Light/Heavy Chain Test Card (Cat.: A12401, VicNovo) according to the reagent protocol, and the results of subtype identification showed that the Heavy chains of m25A7, m27G12 and m11F7 were IgG1, and the Light chains were Kappa chains, which provided basis for cloning of Antibody genes.

Extracting total RNA from m25A7, m27G12 and m11F7 hybridoma cells according to the procedures of a TRIzol kit (Cat: 15596026, Invitrogen); reverse transcribing hybridoma cell total RNA to cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB); amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V using degenerate primers and Phusion kit (Cat: E0553L, NEB)_HA sequence; purifying PCR amplification products by using a gel recovery kit (Cat: AP-GX-250, Axygen); connecting the amplified PCR product to a T vector according to the specification of a T vector cloning kit (Cat: ZC205, a franchise organism), transforming escherichia coli competent cells, amplifying strains, extracting plasmids, and then performing DNA sequencing to obtain the variable region sequence of the monoclonal antibody. The sequencing result shows 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 the 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 the 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 the DNA sequence is shown in sequence 6. The nucleotide sequence of the mouse antibody m27G12 light chain variable region DNA is shown in sequence 7, and the amino acid sequence of the mouse antibody m27G12 light chain variable region deduced from the DNA sequence is shown in sequence 8. Mouse antibodyThe nucleotide sequence of the m11F7 heavy chain variable region DNA is shown in sequence 9, and the amino acid sequence of the mouse antibody m11F7 heavy chain variable region deduced from the DNA sequence is shown in sequence 10. The nucleotide sequence of the mouse antibody m11F7 light chain variable region DNA is shown in sequence 11, and the amino acid sequence of the mouse antibody m11F7 light chain variable region deduced from the DNA sequence is shown in sequence 12.

Example 4: preparation of chimeric antibodies against human OX40

The cloned murine antibody light chain variable region and heavy chain variable region genes are introduced into enzyme cutting sites through PCR, and are respectively cloned into eukaryotic expression vectors containing human-kappa light chain constant region and human IgG1 heavy chain constant region coding genes upstream, so as to obtain human-murine chimeric light chain (pKN019-Ch25A7L) and human-murine chimeric heavy chain (pKN041-Ch25A7H) expression plasmids of m25A7, human-murine chimeric light chain (pKN019-Ch27G12L) and human-murine heavy chain chimeric (pKN041-Ch27G12H) expression plasmids of m27G12, and human-murine chimeric light chain (pKN019-Ch11F7L) and human-murine chimeric heavy chain (pKN041-Ch11F7H) expression plasmids of m11F 7. Transferring into colibacillus to amplify, separating to obtain great amount of plasmid containing human-mouse chimeric antibody light chain and heavy chain. According to the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the light and heavy chain plasmids of the chimeric antibodies Ch25A7, Ch27G12 and Ch11F7 were respectively paired and transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, culture supernatant was taken and purified by a ProA affinity chromatography column to obtain m25A7, m27G12, m11F7 chimeric antibodies.

Example 5: activity assay of chimeric antibodies against human OX40

Scheme 1 affinity assay for anti-human OX40 chimeric antibodies

The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. In the assay, anti-human OX40 chimeric antibodies ch25A7, ch27G12, ch11F7 and control antibody PC1 were diluted to 4ug/mL with PBS buffer and passed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 s. Human OX40-His recombinant protein (SEQ ID NO: P43489-1, 29aa-216aa) was used as a mobile phase, and the concentration of OX40-His recombinant protein was 100 nM. The binding time was 300s and the dissociation time was 300 s. After the experiment, blank control response values are deducted, and 1: 1 Langmuir binding pattern fitting is carried out by software, and the kinetic constant of antigen-antibody binding is calculated.

As shown in fig. 4 (Von is positive control PC1), the reaction curves of anti-human OX40 chimeric antibodies ch25a7, ch27G12, and ch11F7 and human OX40 recombinant protein were fitted and the affinities were calculated, and the results showed that the affinities (KD, table 1) of the chimeric antibodies ch25a7, ch27G12, and ch11F7 were: 1.36E-08, 4.34E-08, 6.61E-09. The results showed that the chimeric antibodies ch25a7, ch27G12, ch11F7 had high affinity with human OX40 recombinant protein, which was lower than the control antibody PC 1.

TABLE 1 affinity fitting results of anti-OX 40 chimeric antibodies ch25A7, ch27G12, ch11F7 Fortebio assay

Scheme 2. anti-human OX40 chimeric antibody activation Activity assay

The activation activity analysis was performed on the chimeric antibodies ch25a7, ch27G12, ch11F7 according to the method of example 2, scheme 2. In addition, the activity of the chimeric antibody x was analyzed based on the NF-. kappa.B signaling pathway activation assay by fluorescein detection. Specifically, HT1080-hOX40 cells were digested the day before the experiment, the cells were resuspended in complete medium (1640 medium + 10% FBS + 0.5. mu.g/mL), and the cell density was adjusted to 10⁵Per mL, 200. mu.L of cell suspension was added per well in a 96-well cell culture plate. The cell culture plates were then incubated overnight at 37 ℃ in a 5% CO2 incubator. The next day, the NF-. kappa.B reporter plasmid (pGL4.32[ Iuc 2P/NF-. kappa.B-RE/Hygro) was transfected by liposome method]) Into HT1080-hOX40 cells. And (3) replacing the culture medium 24h after transfection, and continuously culturing the cells for 24h to ensure that the cells recover 48h after transfection. The test antibody was then diluted to the appropriate concentration and added to the cell culture plate and incubated at 37 ℃ in a 5% CO2 incubator for 18 hours. The cell treatment medium was removed, the cells were washed thoroughly with PBS and finally dried thoroughly. Adding 25ul of lysis solution into each well (about 50ul if the lysis solution is sufficient to ensure that no bubbles are generated in the next operation), covering the cell layer, and performing room temperature oscillation to lyseAnd 20 min. 20ul of cell lysate is added to the corresponding well of the detection plate, and luciferase reaction substrate (100ul/sample) is prepared for detection on the computer. Antibody concentration-RLU histograms or graphs are plotted against machine-read fluorescein values (RLU). If a dual-luciferase reporter system is used to read the experimental response luciferin value (RLU1) and the internal reference luciferin value (RLU2) simultaneously, the ratio of the two is calculated and a bar graph or graph of antibody concentration versus RLU1/RLU2 is generated. As a result, as shown in FIG. 5A (Von stands for PC1) and FIG. 5B, 25A7 and 27G12 had better activation activity, which was not weaker than that of the control antibody PC 1.

Example 6: ELISA detection of inhibition of human OX40 chimeric antibody on human OX40/OX40L binding

Human OX40 was diluted to 1. mu.g/mL, coated overnight at 4 ℃ and blocked with 5% BSA in a 37 ℃ incubator for 60 min. After reacting ch25A7, ch27G12 and the control antibody PC1, as well as the isotype control NC-hlgG1 (initial concentration of 20. mu.g/mL, 3-fold serial dilution, 8 gradients) in a 37 ℃ incubator for 60min, 10. mu.g/mL of OX40L-mFc (OX40L SEQ ID NO: NP-003317, 51-Leu 183, mFc Tag) was added and incubated with the antibody, and the reaction was carried out in a 37 ℃ incubator for 60 min. PBST plate washing 4 times; then HRP-anti-mouse Fc (Cat: 115-. The absorbance values A450nm-630nm were read and recorded for a well plate at a wavelength of 450nm, using 630nm as the reference wavelength. The results show that ch25A7 can effectively block the binding of recombinant human OX40 to its receptor OX40L, with half the effective inhibitory concentration (IC50) value of 0.92. mu.g/mL, respectively, comparable to control antibody PC1 (0.91. mu.g/mL) (FIG. 6). ch27G12 had no significant inhibitory effect.

Example 7: humanized and recombinant expression of anti-human OX40 monoclonal antibody 25A7, 27G12

Scheme 1.25 humanized design and recombinant expression of A7

First, the heavy chain sequence of the murine antibody is comprehensively analyzed to determine the antigen Complementarity Determining (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conserved three-dimensional conformation of the antibody. Then, according to the homology comparison result, the most similar human antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), VH3(3-21) is selected as a basic template, and combined with the full sequence blast result, the CDR transplantation is carried out in consideration of the occurrence frequency of the amino acid of the rearranged (rearranged) antibody at a specific FR region point and the HCDR3 sequence condition, so as to realize the high humanization of the m25A7 heavy chain variable region (VH) in the Framework region. According to the homology comparison result, the most similar human antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), VK I (L5), VK II (O1) and VK VI (A26) are respectively selected as basic templates, and CDR transplantation is carried out by combining the whole sequence blast result and considering the occurrence frequency of amino acids at a specific FR region of a rearranged (rearranged) antibody and the sequence condition of LCDR3, thereby realizing the whole humanization of the m25A7 light chain Framework region. The nucleotide sequence of the humanized heavy chain variable region of the m25A7 antibody CDR Grafted (CDR Grafted) is shown in a sequence 13, and the amino acid sequence is shown in a sequence 14; the nucleotide sequence of the humanized light chain 1 variable region is shown in a sequence 15, and the amino acid sequence is shown in a sequence 16; the nucleotide sequence of the humanized light chain 2 variable region is shown in a sequence 17, and the amino acid sequence is shown in a sequence 18; the nucleotide sequence of the humanized light chain 3 variable region is shown in a sequence 19, and the amino acid sequence is shown in a sequence 20. The CDR-grafted humanized heavy chain variable region sequence was then back-mutated according to the murine 25A7 sequence features, with the back-mutation sites shown in Table 2 below. After the humanized back mutation of 25A7, the nucleotide sequence of the heavy chain variable region of the selected sequence is shown as sequence 21, the nucleotide sequence of the amino acid sequence is shown as sequence 22, the nucleotide sequence of the light chain variable region is shown as sequence 15, and the nucleotide sequence of the amino acid sequence is shown as sequence 16. The humanized light chain hz25A7_ L1 of 25A7 and the humanized heavy chain mutant (parent hz25A7_ H1, mutant hz25A7_ H1m1-hz25A7_ H1m8) were then paired in combination (Table 3) respectively and transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, culture supernatants were collected and purified by ProA affinity chromatography to obtain various humanized antibodies against hz25A7 (designated as hz25A7_ H1m1-hz25A7_ H1m 9).

TABLE 2.25A7 heavy chain humanization back-mutation sequence design

Note: mutation of amino acid 16R back to E according to the Kabat numbering system as denoted by R16E

TABLE 3.25A7 light and heavy chain sequence combinations

Note: the table shows the sequences obtained for various combinations of humanized 25a7 light and heavy chains, as indicated by 25a7-1, the antibody consisting of 25a7 humanized light chain hz25a7_ L1 and humanized heavy chain hz25a7_ H1, and so on.

Scheme 2.27 humanization design and recombinant expression of G12

First, the heavy chain sequence of the murine antibody is comprehensively analyzed to determine the antigen Complementarity Determining (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conserved three-dimensional conformation of the antibody. Then, according to the homology comparison result, the most similar human antibody template is searched in a human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), VH1(1-46) is selected as a basic template, and the CDR transplantation is carried out by combining the result of full sequence blast and considering the occurrence frequency of amino acids at a specific FR region point of a rearranged (rearranged) antibody and the situation of the PTM risk and HCDR3 sequence, so that the full humanization of the m27G12 heavy chain variable region (VH) in the Framework region is realized. According to the results of homology comparison, the most similar human antibody template was searched for in the human antibody germline library (http:// www2.mrc-lmb. cam. ac. uk/vbase/alignment 2.php # VHEX), VK III (L2) and VK II (A2) were selected as basic templates, and the full sequence blast result was combined with the LCDR3 sequence to perform CDR grafting, thereby achieving the 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 is shown in sequence 24; the nucleotide sequence of the humanized light chain 1 variable region is shown in a sequence 25, and the amino acid sequence is shown in a sequence 26; the nucleotide sequence of the humanized light chain 2 variable region is shown in a sequence 27, and the amino acid sequence is shown in a sequence 28. The humanized heavy chain hz27G12H1 and the humanized light chains hz27G12L1 and hz27G12L2 of 27G12 were then combined and paired and transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, culture supernatants were taken and purified by ProA affinity chromatography to obtain the mutant humanized antibodies hz27G12H1L1 and hz27G12H1L2 of hz27G 12.

Example 8: activity assay of humanized antibody against human OX40

Scheme 1 Fortebio detection of antibody affinity

Affinity analysis was performed on each of the mutants of hz25A7 and hz27G12 by the method described in scheme 1 of example 5. The results show (Table 4) that the mutants hz25A7M8, hz27G12H1L2 maintained an affinity level comparable to or slightly higher than that of the chimeric antibody, with affinity (KD) of 0.65E-08M, 1.32E-08M, respectively.

TABLE 4.25A7 humanized mutant affinity assay results Table

H1080 activation activity analysis was performed on hz25a7, hz27G12 and each mutant according to the method of example 5, scheme 2. The results are shown in FIGS. 7A-D (Von represents PC1), and the humanized 25A7 and 27G12 and the mutant thereof all maintain good activity of activating the expression of IL-8 by H1080.

Example 9 genus crossover of hz25A7-mut8, hz27G12-H1L2

Human OX40(SEQ ID NO: P43489-1, 29aa-216aa), cynomolgus monkey OX40 (Cat: 90846-C08H, Beijing Yi Qiaoshengzhou), and mouse OX40 (Cat: 50808-MCCH, Beijing Yi Qiaoshengzhou) extracellular region recombinant proteins were diluted to 1 μ g/mL and coated overnight at 4 ℃; after washing the plate for 3 times with PBS, adding 5% BSA PBS, blocking for 60min at 37 ℃, and washing the plate for 3 times with PBST; adding PBS gradient diluted H25A7mut8 and H27G12H1L2, incubating at 37 ℃ for 60min, and washing the plate for 4 times by PBST; HRP-goat anti-human IgG antibody (Cat: 115-; adding TMB substrate for color development, incubating at 37 deg.C for 10min, and adding 2M HCI to terminate reaction; the absorbance A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded using 630nm as the reference wavelength. The results (FIGS. 8 and 9) show that H25A7mut8 and H27G12H1L2 all bound to human and monkey OX40 and no binding to murine OX 40. The binding activity of H25A7mut8 to human and monkey OX40 was comparable, and the binding activity of H27G12H1L2 to monkey OX40 was lower than that to human OX 40.

Example 10: in vitro testing of the immunological function of OX40 antibodies hz25A7, hz27G12

Peripheral anticoagulation of healthy subjects is carried out, mononuclear cells (PBMC) are obtained through separation, a gradient dilution of OX40 antibody or control antibody is incubated with the PBMC, and the functional activity of the OX40 antibody is evaluated through detecting the cell proliferation condition and the secretion level of cytokines (IL-2, IFN-gamma). Specifically, anti-human CD3 antibody and anti-human CD28 antibody were coated on the enzyme-linked plate individually or together as control wells; an anti-human OX40 antibody was coated on the enzyme-linked plate as a well to be detected. Taking peripheral blood of healthy person according to Dynabeads^TM FlowComp^TMHuman CD3 Kit specification, separating to obtain CD3+ T cells; after washing for 2 times, the cells were adjusted to an appropriate concentration using a culture medium and added to a 96-well plate; after 5 days, the cell proliferation condition is detected by adopting a CTG method, cell culture supernatant is harvested, and IL-2 and IFN-gamma are detected by adopting an ELISA method. Finally, the Anti-Human CD3/CD28 group was used as a positive control, and the PBS group was used as a negative control to evaluate the biological activity of the OX40 antibody. The experimental results show that, like the control antibody, hz25A7m8 (labeled as KNAb-1 in the present example) and hz27G12H1L2 (labeled as KNAb-2 in the present example) can significantly increase the concentrations of cytokines IL-2 and IFN-gamma in the culture medium in a dose-dependent manner (FIG. 10A and FIG. 10B), suggesting that the antibodies can activate T lymphocytes and enhance the function of secreting cytokines. The cell proliferation assay results (fig. 10C) showed that both hz25A7m8 and hz27G12H1L2 were effective in promoting cell proliferation, and the ability to promote cell proliferation was comparable to that of control antibodies PC2 and PC 3.

Example 12: in vivo Activity analysis of anti-OX 40 antibodies hz25A7m8, hz27G12H1L2

In vivo pharmacodynamic study of hz25A7m8, hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 model

MC38 mouse colorectal cancer cells, using DMEM medium containing 10% inactivated fetal calf serum, 100U/ml penicillin and 100 mug/ml streptomycin to culture in an incubator of 5% CO2 at 37 ℃, after the cells are overgrown every 3 to 4 days, bottle-dividing and passaging, harvesting tumor cells in logarithmic growth phase, after PBS heavy suspension, inoculating the cells under the right flank of human OX40 transgenic mice, when the average tumor volume reaches about 60-100mm3, group administration is carried out, 6 animals in each group are administrated twice a week according to the dose of 10mg/kg, and the total administration is 4 times. After grouping, the tumor volume is measured for 3 times every week by using a vernier caliper, the long diameter and the short diameter of the tumor are measured, and the volume calculation formula is as follows: volume is 0.5 x long diameter x short diameter². Mice were weighed while tumor volume measurements were performed. The change in body weight of the mice was recorded as a function of time of administration. The survival and health of the mice were also observed as a general state of the animals during administration, such as activity, food intake, etc. At the end of the experiment, mice were euthanized, tumors were stripped and weighed, and the stripped tumors of the control and test groups were placed in order for photography. The results (fig. 11A, fig. 11B) show that candidate antibodies hz25A7m8, hz27G12H1L2 can significantly inhibit tumor growth and regress most of tumors in OX40 transgenic mice. Wherein, hz25A7m8 can completely regress the tumor, and the activity is better than that of control antibodies such as PC2 and PC 3.

In vivo Immunomagnetic memory study following treatment of the OX40 humanized mouse colorectal cancer MC38 model with hz25A7m8, hz27G12H1L2

MC38 mouse colorectal cancer tumor cells and Hepa1-6 mouse liver cancer tumor cells were cultured in DMEM medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin, 100. mu.g/ml streptomycin and 2mM glutamine at 37 ℃ in 5% CO₂The culture box of (3) was cultured, after the cells were overgrown every 3 to 4 days, the cells were passaged in flasks, and tumor cells in the logarithmic growth phase were harvested, and inoculated into huOX40 mice in which MC38 tumors completely regressed after treatment with hz25A7m8, hz27G12H1L2, and PC2 in the present example, scheme 1, and C57BL/6J mice which were not inoculated with tumors, and the tumor cells resuspended in PBS were inoculated subcutaneously into the left and right flanks of the experimental animals. After inoculation of tumor cells2-3 times of tumor volume measurement is carried out by using a vernier caliper every week, the long diameter and the short diameter of the tumor are measured, and the volume calculation formula is as follows: the volume is 0.5 × long diameter × short diameter 2. Mice were weighed while tumor volume measurements were performed. Changes in body weight of mice were recorded as a function of time of inoculation. The survival and health of the mice were also observed, as was the general status of the animals after vaccination, food intake, etc. At the end of the experiment, mice were euthanized, tumors were stripped and weighed, and the stripped tumors of the control and test groups were placed in order for photography. The results (fig. 12A) show that all groups of treated mice were not further inoculated with MC38 cells for tumor formation, and that inoculated with Hepa1-6 cells for tumor formation, but tumor growth was significantly inhibited, and that hz25A7m8 and hz27G12H1L2 had a more significant inhibitory effect than PC2 (fig. 12B).

Protocol 3 detection of Immunomagnetic memory T cell populations by FACS method

Spleen cells of mice of example 2 were collected, single cell suspensions were prepared, and anti-mouse CD3/CD4/CD44/CD62L was added to incubate with spleen cells, and immune memory T cell populations were analyzed. Specifically, dissecting a tumor-bearing mouse in a sterile environment to obtain a spleen; placing a 70 μ M cell screen in a sterile plate, transferring the spleen to the cell screen, and milling the spleen to disperse single cells using a milling rod; collecting the cell suspension into a 50mL centrifuge tube, centrifuging at 200 Xg for 10 min; discarding the supernatant, cracking red blood cells, washing for 2 times, and treating the cell suspension with a 40 mu M cell screen to obtain a spleen single cell suspension; transferring the spleen single cell suspension into a flow tube, adding a corresponding antibody (anti-mouse CD3/CD4/CD44/CD62L) according to the experimental design, and incubating for 30min in the dark; after washing 1 time with PBS, the cells were resuspended and flow-tested. The results (fig. 13) show a significant increase in helper T cells (CD3+ CD4+) in mice treated with hz25A7m8, hz27G12H1L2, PC2 compared to the control. Further analysis revealed that the ratio of memory T cells (CD3+ CD4+ CD44hicD62LIo) and natural T cells (CD3+ CD4+ CD44loCD62Lhi) increased to different extents.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

The sequences referred to in this application are as follows:

sequence 1: m25A7 heavy chain variable region DNA nucleotide sequence

Wherein, the coding region of CDRs is sequence 37-39

Sequence 2: m25A7 heavy chain variable region amino acid sequence

Wherein, the CDRs are shown as sequences 40-42

And (3) sequence: m25A7 light chain variable region DNA nucleotide sequence

Wherein the coding region of CDRs is sequence 43-45

And (3) sequence 4: m25A7 light chain variable region amino acid sequence

Wherein, the CDRs are shown as sequences 46-48

And (5) sequence: m27G12 heavy chain variable region DNA nucleotide sequence

Wherein the coding region of CDRs is shown as sequence 49-51

And (3) sequence 6: m27G12 heavy chain variable region amino acid sequence

Wherein, the CDRs are shown as sequences 52-54

And (3) sequence 7: m27G12 light chain variable region DNA nucleotide sequence

Wherein, the coding region of CDRs is sequence 55-57

And (2) sequence 8: m27G12 light chain variable region amino acid sequence

Wherein, the CDRs are shown as sequence 58-60

Sequence 9: m11F7 heavy chain variable region DNA nucleotide sequence

Wherein, the coding region of CDRs is sequence 61-63

Sequence 10: m11F7 heavy chain variable region amino acid sequence

Wherein the CDRs are shown as sequences 64-66

Sequence 11: m11F7 light chain variable region DNA nucleotide sequence

Wherein, the coding region of CDRs is sequence 67-69

Sequence 12: m11F7 light chain variable region amino acid sequence

Wherein, the CDRs region is the sequence 70-72

The nucleotide sequence of the humanized heavy chain variable region of m25A7 is shown in a sequence 13;

SEQ.ID NO.13：

wherein, the coding region of CDRs is sequence 73-75

25A7 humanized heavy chain variable region amino acid sequence shown in sequence 14;

SEQ.ID NO.14：

wherein the CDRs are shown as sequences 76-78

25A7 humanized light chain 1 variable region nucleotide sequence shown in sequence 15;

SEQ.ID NO.15：

wherein, the coding region of CDRs is sequence 79-81

The amino acid sequence of the 25A7 humanized light chain 1 variable region is shown in the sequence 16

SEQ.ID NO.16：

Wherein, the CDRs are shown as sequence 82-84

25A7 humanized light chain 2 variable region nucleotide sequence shown in sequence 17;

SEQ.ID NO.17：

wherein, the coding region of CDRs is sequence 85-87

25A7 humanized light chain 2 variable region amino acid sequence shown in sequence 18;

SEQ.ID NO.18：

wherein, the CDRs region is 88-90

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

SEQ.ID NO.19：

Wherein, the coding region of CDRs is sequence 91-93

25A7 humanized light chain 3 variable region amino acid sequence shown in sequence 20.

SEQ.ID NO.20：

Wherein the CDRs are shown as sequences 94-96

The nucleotide sequence of the heavy chain variable region after the 25A7 humanized backsmuttation is shown as a sequence 21

SEQ.ID NO.21：

Wherein, the coding region of CDRs is sequence 97-99

The amino acid sequence of the heavy chain variable region after humanization of 25A7 backsmutation is shown in a sequence 22

SEQ.ID NO.22∶

Wherein the CDRs region is sequence 100-102

27G12 humanized heavy chain variable region coding nucleotide sequence shown in sequence 23

SEQ.ID NO.23：

Wherein, the coding region of CDRs is sequence 103-105

The amino acid sequence of the humanized heavy chain variable region of 27G12 is shown in sequence 24

SEQ.ID NO.24：

Wherein the CDRs are shown as sequence 106-108

27G12 humanized light chain 1 variable region coding nucleotide sequence shown in sequence 25

SEQ.ID NO.25：

Wherein, the coding region of CDRs is sequence 109-111

27G12 humanized light chain 1 variable region amino acid sequence shown in sequence 26

SEQ.ID NO.26：

Wherein the CDRs region is sequence 112-

27G12 humanized light chain 2 variable region encoding nucleotide sequence 27

SEQ.ID NO.27：

Wherein, the coding region of CDRs is sequence 115-117

27G12 humanized light chain 2 variable region amino acid sequence shown in sequence 28

SEQ.ID NO.28：

Wherein the CDRs region is sequence 118-120

The nucleotide sequence of the humanized final heavy chain variable region of 25A7 is shown in sequence 29

SEQ.ID NO.29：

Wherein, the coding region of CDRs is sequence 121-123

The amino acid sequence of the humanized final heavy chain variable region of 25A7 is shown in SEQ ID No. 30

SEQ.ID NO.30：

Wherein the CDRs region is sequence 124-126

25A7 humanized Final light chain variable region encoding nucleotide sequence shown in SEQ ID No. 15

SEQ.ID NO.15：

Wherein, the coding region of CDRs is sequence 127-129

The amino acid sequence of the humanized final light chain variable region of 25A7 is shown in SEQ ID No. 16

SEQ.ID NO.16：

Wherein the CDRs region is sequence 130-132

27G12 humanized Final heavy chain variable region encoding nucleotide sequence shown in SEQ ID No. 23

SEQ.ID NO.23：

Wherein, the coding region of CDRs is sequence 133-135

The amino acid sequence of the humanized final heavy chain variable region of 27G12 is shown in SEQ ID No. 24

SEQ.ID NO.24：

Wherein the CDRs region is sequence 136-138

27G12 humanized Final light chain variable region encoding nucleotide sequence shown in SEQ ID No. 31

SEQ.ID NO.31：

Wherein, the coding region of CDRs is sequence 139-141

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

SEQ.ID NO.32：

Wherein the CDRs are represented by sequence 142-144

Heavy chain constant region nucleotide sequence 33

SEQ.ID NO.33：

Heavy chain constant region amino acid sequence 34

SEQ.ID NO.34：

Light chain constant region nucleotide sequence 35

SEQ.ID NO.35：

Light chain constant region amino acid sequence 36

SEQ.ID NO.36：

Sequence listing

<110> Applicant's name (Miwei)

<120> an activated anti-OX 40 antibody, production method and application

<130> do not

<160> 144

<170> SIPOSequenceListing 1.0

<210> 1

<211> 357

<212> DNA

<213> Artificial Sequence (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 (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 (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 (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 (Artificial Sequence)

<400> 5

aaggtccagc tgcagcagtc tggagctggg ctggtgaaac ccggggcatc agtgaagctg 60

tcctgcaagg cttctggcta caccttcact gaatatatta tacactgggt aaaacagagg 120

tctggacagg gtcttgagtg gattgggtgg ttttaccctg aaagtggtag tataaagtac 180

aacgagaaat tcaaggacaa ggccacattg actgcggaca aatcctccaa cacagtctat 240

atggagctta gtagattgac atctgaagac tctgcggtct atttctgtgc aagacacgaa 300

gatccgatta cctttgctta ctggggccaa gggactctgg tcactgtctc tgca 354

<210> 6

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Lys Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Ile Ile His Trp Val Lys Gln Arg Ser 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 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 (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 (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 (Artificial Sequence)

<400> 9

gaggtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagagtc cctgaaactc 60

tcctgtgaat ccaatgaata cgaattccct tcccatgaca tgtcttgggt ccgcaagact 120

ccggagaaga ggctggagtt ggtcgcaacc attaatagtg atggtgataa cacctactat 180

ccagacacct tggagagacg attcatcatc tccagagaca ataccaagaa gaccctgtac 240

ctgcaaatga gcagtctgag gtctgaggac acagcctttt attactgtac aagacactat 300

gataattact acgcctggtt tgcttactgg ggccaaggga ctctagtcac tgtctctgca 360

<210> 10

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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 Thr Ile Asn Ser Asp Gly Asp Asn Thr Tyr Tyr Pro Asp Thr Leu

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 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

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 11

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gacattgtga tgacccagtc tcctgcttcc ttaactgtat ctctggggca gagggccacc 60

atctcatgca gggccagcga aagtgtcagt acatctggct atagttatat gcactggtac 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 aaa 333

<210> 12

<211> 111

<212> PRT

<213> Artificial Sequence (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 (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 (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

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 (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 (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 (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 (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

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

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 (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 (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 (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 (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 (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 (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 (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 (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 30

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 (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 (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 (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 (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 (Artificial Sequence)

<400> 31

gagatcgtga tgacccagtc tcctgccacc ctgtccgtgt ctcctggcga gcgagccacc 60

ctgtcctgca aggcctccca gtccgtggac tacgagggcg 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> 32

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

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 Glu

20 25 30

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> 33

<211> 990

<212> DNA

<213> Artificial Sequence (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 (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 (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 (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 (Artificial Sequence)

<400> 37

tcccatgaca tgtct 15

<210> 38

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gccattaata gtgatggtgg tagaatctac tatccagaca ccatggagag a 51

<210> 39

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cactatgatg gttacgcctg gtttgcttac 30

<210> 40

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Ser His Asp Met Ser

1 5

<210> 41

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 43

agggccagca aaagtgtcag tacatctggc tctagttata tacac 45

<210> 44

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

cttgcatcca acctagaatc t 21

<210> 45

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

cagcacagta gggagcttcc gctcacg 27

<210> 46

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 47

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 49

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

gaatatatta tacac 15

<210> 50

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

tggttttacc ctgaaagtgg tagtataaag tacaacgaga aattcaagga c 51

<210> 51

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

cacgaagatc cgattacctt tgcttac 27

<210> 52

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Glu Tyr Ile Ile His

1 5

<210> 53

<211> 17

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 54

His Glu Asp Pro Ile Thr Phe Ala Tyr

1 5

<210> 55

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

aaggccagcc aaagtgttga ttatgatggt gatagttata tgaac 45

<210> 56

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

gctgcatcca atctagaatc tgggatccca gcc 33

<210> 57

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

cagcaaagta atgaggatcc ttacacg 27

<210> 58

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 59

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 60

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

<210> 61

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

tcccatgaca tgtct 15

<210> 62

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

accattaata gtgatggtga taacacctac tatccagaca ccttggagag a 51

<210> 63

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

cactatgata attactacgc ctggtttgct tac 33

<210> 64

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Ser His Asp Met Ser

1 5

<210> 65

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 67

agggccagcg aaagtgtcag tacatctggc tatagttata tgcac 45

<210> 68

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

cttgcatcca acctagaatc tggggtccct gcc 33

<210> 69

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

cagcacagta gggagcttcc gctcacg 27

<210> 70

<211> 15

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 72

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 73

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

tcccacgaca tgtcc 15

<210> 74

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

gccatcaact ccgacggagg ccggatctac taccctgaca ccatggagcg a 51

<210> 75

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

cactacgacg gctacgcctg gttcgcctac 30

<210> 76

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Ser His Asp Met Ser

1 5

<210> 77

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 79

cgagcctcca agtccgtgtc cacctctggc tcctcctaca tccac 45

<210> 80

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

ctggcctcca acctggagtc t 21

<210> 81

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

cagcactcca gggagctgcc tctgacc 27

<210> 82

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 83

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 84

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 84

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 85

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

cgagcctcca agtccgtgtc cacctccggc tcctcctaca tccac 45

<210> 86

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

ctggcctcca acctggagtc t 21

<210> 87

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

cagcactcca gggagctgcc tctgacc 27

<210> 88

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 89

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 90

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 90

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 91

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

agagcctcca agtccgtgtc cacctctggc tcctcctaca tccac 45

<210> 92

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

ctggcctcca acctggagtc t 21

<210> 93

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

cagcactctc gggagctgcc tctgacc 27

<210> 94

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 95

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 96

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 96

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 97

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

tcccacgaca tgtcc 15

<210> 98

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

gccatcaact ccgacggagg ccggatctac taccctgaca ccatggagcg a 51

<210> 99

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

cactacgacg gctacgcctg gttcgcctac 30

<210> 100

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 100

Ser His Asp Met Ser

1 5

<210> 101

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 103

gagtacatca tccac 15

<210> 104

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

tggttctacc ctgagtccgg ctccatcaag tacaacgaga agttcaagga c 51

<210> 105

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

cacgaggacc ccatcacctt cgcctac 27

<210> 106

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 106

Glu Tyr Ile Ile His

1 5

<210> 107

<211> 17

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 108

His Glu Asp Pro Ile Thr Phe Ala Tyr

1 5

<210> 109

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

aaggcctccc agtccgtgga ctacgacggc gactcctaca tgaac 45

<210> 110

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

gctgcctcca acctggagtc c 21

<210> 111

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

cagcagtcca acgaggaccc ctacacc 27

<210> 112

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 113

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 114

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 114

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

<210> 115

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

aaggcctccc agtccgtgga ctacgacggc gactcctaca tgaac 45

<210> 116

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

gctgcctcca acctggagtc t 21

<210> 117

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

cagcagtcca acgaggaccc ctacacc 27

<210> 118

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 119

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 120

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 120

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

<210> 121

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

tcccacgaca tgtcc 15

<210> 122

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

gccatcaact ccgagggagg ccggatctac taccctgaca ccatggagcg a 51

<210> 123

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

cactacgaca actacgcctg gttcgcctac 30

<210> 124

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 124

Ser His Asp Met Ser

1 5

<210> 125

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 127

cgagcctcca agtccgtgtc cacctctggc tcctcctaca tccac 45

<210> 128

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

ctggcctcca acctggagtc t 21

<210> 129

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

cagcactcca gggagctgcc tctgacc 27

<210> 130

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 131

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 132

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 132

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 133

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

gagtacatca tccac 15

<210> 134

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

tggttctacc ctgagtccgg ctccatcaag tacaacgaga agttcaagga c 51

<210> 135

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

cacgaggacc ccatcacctt cgcctac 27

<210> 136

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 136

Glu Tyr Ile Ile His

1 5

<210> 137

<211> 17

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 138

His Glu Asp Pro Ile Thr Phe Ala Tyr

1 5

<210> 139

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

aaggcctccc agtccgtgga ctacgagggc gactcctaca tgaac 45

<210> 140

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

gctgcctcca acctggagtc cggcatccct gct 33

<210> 141

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

cagcagtcca acgaggaccc ctacacc 27

<210> 142

<211> 15

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 143

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 144

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 144

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

Claims

1. An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises:

VL CDR3 is selected from SEQ ID NO: 42. 54, 66;

the light chain variable region comprises:

2. The antibody or fragment thereof of claim 1, wherein:

(1) the heavy chain variable region has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence selected from SEQ ID NO 2, 14, 22, 30, and the light chain variable region has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence selected from SEQ ID NO 4, 16, 18, 20;

(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 more than 99% homology, and 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 more than 99% homology;

(3) the heavy chain variable region is the amino acid sequence shown in SEQ ID NO. 10 and has homology of 80%, 85%, 90%, 95%, 96%, 97%, 98% or more than 99%, and the light chain variable region is the amino acid sequence shown in SEQ ID NO. 12 and has homology of 80%, 85%, 90%, 95%, 96%, 97%, 98% or more than 99%.

3. An immunoconjugate comprising

(1) The antibody or fragment thereof according to any one of claims 1 to 2,

(2) a coupling moiety.

4. A multispecific antibody or derivative thereof, comprising at least one antigen-binding domain of an antibody or fragment thereof according to any one of claims 1 to 2.

5. A heavy chain antibody which is a dimeric heavy chain antibody obtained on the basis of the antibody of any one of claims 1-2.

6. A composition comprising

(1) The antibody or fragment thereof of any one of claims 1 to 2, the antibody conjugate of claim 3, the multispecific antibody or derivative thereof of claim 4, or the heavy chain antibody of claim 5

(2) A pharmaceutically acceptable carrier.

7. A nucleic acid encoding the antibody or fragment thereof of any one of claims 1-2, the multispecific antibody or derivative thereof of claim 4, or the heavy chain antibody of claim 5.

8. A recombinant vector or recombinant host cell comprising the nucleic acid of claim 7.

9. Use of the antibody or fragment thereof of any one of claims 1 to 2, the antibody conjugate of any one of claims 3, the multispecific antibody or derivative thereof of claim 4, the heavy chain antibody of claim 5, the composition of claim 6, the nucleic acid of claim 7, the recombinant vector or recombinant host cell of claim 8, wherein:

10. A method of producing an antibody comprising:

(1) culturing the recombinant host cell of claim 8,

(2) recovering the antibody.