WO2021143914A1 - Activated anti-ox40 antibody, production method therefor and application thereof - Google Patents

Abstract

Provided is an antibody or a fragment thereof to an activated receptor (OX40) expressed on the surfaces of CD4+T and CD8+T activated cells, and use of the antibody or the fragment thereof in preventing or treating diseases. The antibody has a high affinity for OX40 and has an obvious effect of activating the OX40 signaling pathway, and has a relatively broad spectrum of immune-enhancing effects, can enhance T cell response to immune memory, achieve better tumor inhibition and killing effects in in-vivo experiments on animal models, and thus has good clinical application prospect.

Description

An activated anti-OX40 antibody, production method and application

This patent application claims the priority rights of the Chinese invention patent application with the application number CN202010063141.3 filed on January 19, 2020, and the entire content of which is hereby incorporated by reference.

Technical field

The present invention belongs to the field of antibody engineering, and specifically relates to an anti-OX40 antibody, its production method and application, in particular to an anti-human OX40 humanized antibody, its recombinant expression method and its application in the treatment of solid tumors.

Background technique

OX40, also known as CD134, ACT45, and TNFRSF4, belongs to the tumor necrosis factor receptor (TNFR) superfamily, and is an activating receptor expressed on the surface of activated CD4+T and CD8+T cells. OX40 signal can activate the downstream NF-κB, PI3K and PKB pathways. The continuous activation of these pathways can ultimately prolong the survival time of T cells, expand T cell memory, and promote the cell killing ability of T cells. In addition, OX40 can also inhibit The differentiation and activity of regulatory T cells (Treg) improve the immunosuppressive effect in the tumor microenvironment and further enhance the function of effector T cells.

The OX40 gene is located on human chromosome 1 (mouse chromosome 4) and encodes a 50kD transmembrane glycoprotein. The extracellular region has 191 amino acids and contains three complete and one shorter cysteine-rich domains (CRDs). It is mainly expressed on activated effector T cells (Teffs) and regulatory T cells (Tregs), but also on NKT cells, NK cells and neutrophils.

OX40 combines with the ligand OX40L (CD252, TNFSF4) to deliver costimulatory signals. The OX40L gene is located on chromosome 1 of humans and mice, and encodes a 34kD type 2 transmembrane glycoprotein. OX40L can be expressed on antigen presenting cells (APC), such as B cells, dendritic cells, and macrophages; in addition, it can also be induced on other cell types such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and NK cells Express.

The combination of OX40 and OX40L participates in a variety of physiological reactions between T cells and lymphocytes and non-lymphocytes. The interaction of OX40 and OX40L can recruit TNFR-related (TRAFs) molecules in the intracellular region of OX40 to form a signaling complex containing IKKα and IKKβ as well as PI3k and PKB (Akt); OX40 also cooperates with TCR signaling through unknown The mechanism enhances intracellular Ca2+, thereby enhancing NFAT into the nucleus. OX40 can activate the classic NF-κB1 pathway or the non-canonical NF-κB2 pathway, PI3k/PKB and NFAT pathways, thereby regulating the genes that control T cell division and survival, and promoting the transcription of cytokine genes and the expression of cytokine receptors , Is essential for cell survival. OX40 signaling can cause down-regulation including CTLA-4 and Foxp3.

Anti-OX40 activating antibody can play a similar function to OX40L to activate antigen-dependent T effector cells, and can play an anti-tumor effect by eliminating the inhibitory function of Treg cells. There are several types of anti-OX40 activated antibodies in clinical research. Three of them have published some experimental data, namely Pfizer's PF-04518600, BMS's BMS-986178 and AbbVie's ABBV-368. There are currently two OX40 in the clinical stage in China, namely the recombinant fully human anti-tumor necrosis factor receptor superfamily member 4 (OX40) monoclonal antibody of Cinda Biopharmaceuticals and the recombinant fully human anti-OX40 monoclonal antibody of Livzon Pharmaceutical Group Clone antibody injection. Most of the OX40 monoclonal antibodies in the prior art obtain anti-OX40 antibodies through hybridoma technology. Of course, the target antibody can also be obtained through methods such as transgenic mouse technology, phage antibody library technology, and B cell sorting technology. The evaluation and analysis methods are the same as or similar to the hybridoma antibody preparation scheme.

Although there are currently a number of anti-OX40 activating antibodies in clinical research, due to the incomplete consistency between the early screening performance of anti-OX40 activating antibodies and the final clinical treatment effect, unpredictable adverse reactions and effects for different indications For other reasons, there is still a need to develop more activated anti-OX40 antibodies with high affinity, high biological activity, especially better stimulation of immune memory, to meet the urgent needs of clinical treatment.

Summary of the invention

In order to solve the above problems, the present invention obtains murine antibodies through hybridoma technology, obtains candidate activated anti-OX40 murine antibodies through analysis of antibody binding activity, activation activity, and blocking activity; After designing, the obtained humanized antibody is analyzed again through antibody activity analysis (binding activity, activation activity, blocking activity) and pharmacodynamic analysis in mice to finally determine the activated anti-OX40 humanized antibody. in particular:

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

The variable region of the heavy chain contains:

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

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

VH CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 42, 54, 66;

The light chain variable region contains:

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

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

VL CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 48, 60, 72.

Further, in the antibody or fragment thereof of the present invention, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 2, 6, 10, 14, 22, 24, or 30.

Furthermore, the heavy chain variable region of the antibody or fragment thereof of the present invention includes the amino acid sequence shown in SEQ ID NO: 4, 8, 12, 16, 18, 20, 26, 28, and 32.

Furthermore, the antibody or fragment thereof of the present invention is characterized in that:

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

(2) The heavy chain variable region is selected from the amino acid sequence shown in 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 the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 12.

Furthermore, the antibody or fragment thereof of the present invention is characterized in that:

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

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

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

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

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

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

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

or

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

Furthermore, the antibody or fragment thereof of the present invention includes the heavy chain constant region shown in SEQ ID NO: 34 and/or the light chain constant region shown in SEQ ID NO: 36.

Furthermore, the antibody or fragment thereof of the present invention, wherein the antibody includes a murine antibody, a chimeric antibody, a humanized antibody, and a human antibody; the antibody fragment includes F(ab′) ₂ , Fab′, Fv , ScFv, Fd, Nanobody, etc.

In the second aspect, the present invention provides an immunoconjugate comprising

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

(2) Coupling part.

Further, in the immunoconjugate of the present invention, the coupling part includes a detectable label, a cytotoxic molecule, a biologically active molecule, and the like.

Furthermore, in the immunoconjugate of the present invention, the detectable labels include chemiluminescence labels, fluorescent labels, enzyme labels, radioactive labels, quantum dots, nanoparticles and the like.

Furthermore, in the immunoconjugate of the present invention, wherein the cytotoxic molecules include diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, leuculin and the like.

Furthermore, in the immunoconjugate of the present invention, the biologically active molecules include cytokines, enzymes, chemotherapeutics, liposomes, viral particles, and the like.

In the third aspect, the present invention provides a multispecific antibody or derivative thereof, which is characterized by comprising at least one antigen binding domain of the antibody or fragment thereof according to the first aspect of the present invention.

Furthermore, the multispecific antibody or derivative thereof of the present invention further includes an antigen binding domain that binds to other targets.

Furthermore, the multispecific antibody or derivative thereof of the present invention, wherein the other targets include PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, CD47, 4-1BB, CD73, ROR1, HER2, HER3, EGFR, etc.

In the 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 in the first aspect of the present invention.

Furthermore, 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) The antibody or fragment thereof of the first aspect of the present invention, the antibody conjugate of the second aspect of the present invention, the multispecific antibody or its derivative of the third aspect of the present invention, or the fourth aspect of the present invention The heavy chain antibody;

(2) A pharmaceutically acceptable carrier.

Furthermore, the composition of the present invention further includes other active ingredients for treating tumors.

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

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

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

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

In the tenth aspect, the present invention provides the antibody or fragment thereof of the first aspect of the present invention, the antibody conjugate of the second aspect of the present invention, the multispecific antibody or derivative thereof of the third aspect of the present invention, and the first aspect of the present invention. Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in the preparation of drugs that bind OX40, inhibit the binding of OX40 and OX40L, activate OX40+ T cells, activate human immune response, tumor therapeutic vaccines, etc. . For example, molecular adjuvants used to enhance specific immune responses are used in combination with adjuvants such as CpG for tumor vaccines and other purposes.

In the eleventh aspect, the present invention provides the antibody or fragment thereof according to the first aspect of the present invention, the antibody conjugate according to the second aspect of the present invention, the multispecific antibody or derivative thereof according to the third aspect of the present invention, and Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in preparing drugs for inducing OX40+ cells to produce IL-8 and for initiating NFκB gene transcription.

In the twelfth aspect, the present invention provides the antibody or fragment thereof according to the first aspect of the present invention, the antibody conjugate according to the second aspect of the present invention, the multispecific antibody or derivative thereof according to the third aspect of the present invention, and Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in the preparation of drugs that stimulate PBMC to produce IL-2 and IFN-γ.

In the thirteenth aspect, the present invention provides the antibody or fragment thereof according to the first aspect of the present invention, the antibody conjugate according to the second aspect of the present invention, the multispecific antibody or derivative thereof according to the third aspect of the present invention, and the present invention Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in the preparation of drugs for inhibiting the growth and metastasis of solid tumors.

Further, the use of the present invention is characterized in that the solid tumors include digestive system tumors, respiratory system tumors, urinary and reproductive system tumors.

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

Further, the use of the present invention is characterized in that the urogenital system tumors include breast cancer, ovarian cancer and the like.

In a fourteenth aspect, the present invention provides a method for producing antibodies, including:

(1) Culturing the host cell of the ninth aspect of the present invention,

(2) Recover antibodies.

In the fifteenth aspect, the present invention provides a method for preventing and/or treating diseases mediated by OX40, the method comprising administering the antibody or fragments thereof, antibody conjugates, Multispecific antibodies or derivatives thereof, heavy chain antibodies, compositions, nucleic acids, recombinant vectors or recombinant host cells.

Furthermore, the method for preventing and/or treating diseases mediated by OX40 of the present invention, wherein the subject has a solid tumor and/or is at risk of growth and metastasis of the solid tumor.

Further, the method for preventing and/or treating a disease mediated by OX40 of the present invention, wherein the solid tumor that the subject suffers is a tumor of the digestive system, a tumor of the respiratory system, a tumor of the urinary and reproductive system.

In one embodiment, the method for preventing and/or treating diseases mediated by OX40 of the present invention, wherein the digestive system tumors that the subject suffers from include liver cancer, pancreatic cancer, gastric cancer, duodenal cancer, Colorectal cancer, esophageal cancer.

In another embodiment, the method for preventing and/or treating a disease mediated by OX40 of the present invention, wherein the respiratory tumor of the subject includes small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma , Laryngeal cancer, mesothelioma, etc.

In another embodiment, the method for preventing and/or treating diseases mediated by OX40 according to the present invention, wherein the urogenital system tumors suffered by the subject include breast cancer, ovarian cancer and the like.

To better understand the present invention, first define some terms. Other definitions are listed throughout the detailed implementation section.

The term "OX40" refers to the fourth member of the tumor necrosis factor superfamily. The term includes variants, allologs, homologs, orthologs and paralogs. For example, an antibody specific to human OX40 can cross-react with the OX40 protein of another species, such as monkey, under certain circumstances. In other embodiments, antibodies specific for human OX40 protein may be completely specific for human OX40 protein without cross-reacting with other species or other types of proteins, or may cross-react with OX40 proteins of some other species but not all other species .

The term "human OX40" refers to an OX40 protein having a human amino acid sequence, such as the amino acid sequence of Genbank Accession No. NP_003318. The terms "monkey OX40" and "mouse OX40" refer to the OX40 sequences of monkey and mouse respectively, for example, sequences with Genbank accession number NP_001090870 and Genbank accession number NP_035789, respectively.

The term "antibody" herein is meant to include full-length antibodies and any antigen-binding fragments (ie, antigen-binding portions) or single chains thereof. Full-length antibodies are glycoproteins containing at least two heavy (H) chains and two light (L) chains, the heavy and light chains are connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH for short) and a heavy chain constant region. The heavy chain constant region is composed of three domains, namely CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The constant region of the light chain consists of a domain CL. The VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDR), which are separated by more conservative framework regions (FR) regions. Each VH and VL is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, FR3, FR4 from the amino terminal to the carboxy terminal. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (for example, effector cells) and the first component (C1q) of the traditional complement system.

The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to OX40 protein does not substantially contain antibodies that specifically bind to antigens other than OX40 protein. However, isolated antibodies that specifically bind to human OX40 protein may have cross-binding properties to other antigens, such as OX40 proteins of other species. In addition, the isolated antibody contains substantially no other cellular materials and/or chemical substances.

The term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule product of single molecular composition. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

The term "homology" is used to refer to the degree of sequence similarity between two polypeptides or between two nucleic acids. When a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine), then the molecules are the same at that position. The "percent homology" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared × 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% homology. For example, the DNA sequences CTGACT and CAGGTT share 50% homology (3 out of 6 positions match). Generally, the comparison is made when two sequences are aligned to produce maximum homology. Such comparison can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol., 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) that have been integrated into the ALIGN program (version 2.0), and use the PAM120 weight residue table (weightresiduetable) A gap length penalty of 12 and a gap penalty of 4 are used to determine the percent homology between two amino acid sequences. In addition, the Needleman and Wunsch (J.MoI.Biol., 48:444-453, 1970) algorithms in the GAP program integrated into the GCG software package (available on www.gcg.com) can be used, and Blossum 62 Matrix or PAM250 matrix and gap weight of 16, 14, 12, 10, 8, 6 or 4 and length weight of 1, 2, 3, 4, 5 or 6 to determine the percentage difference between two amino acid sequences. Source.

As used herein, the term "antigen-binding fragment" (or antibody portion for short) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (for example, OX40 protein). It has been confirmed that the antigen-binding function of antibodies can be implemented by fragments of full-length antibodies. Examples of binding fragments included in the "antigen-binding portion" of the antibody include (i) Fab fragments, monovalent fragments composed of VL, VH, CL, and CH1; (ii) F(ab')2 fragments, including hinge region two A bivalent fragment of two Fab fragments connected by a sulfur bridge; (iii) an Fd fragment composed of VH and CH1; (iv) an Fv fragment composed of antibody single-arm VL and VH; (v) a dAb fragment composed of VH ( Ward et al., (1989) Nature 341: 544-546); (vi) Isolated complementarity determining regions (CDR); and (vii) Nanobodies, which contain a single variable domain and two constant domains Heavy chain variable region. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by recombination through a synthetic linker that makes the two into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called Single-chain Fc (scFv); see, for example, Bird et al., (1988) Science242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

The antigen-binding fragments of the present invention include those capable of specifically binding to OX40. Examples of antibody binding fragments include, for example, but not limited to, Fab, Fab', F(ab') ₂ , Fv 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 (CH1) of the heavy chain. The difference between Fab' fragments and Fab fragments is the addition of a few residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of an antibody. Fab' fragments are generated by cleaving 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 crystallizable (Fc) region of intact antibody fragments, are cleared more rapidly from the animal’s circulation, and may have less non-specific tissue binding than intact antibodies (see, for example, Wahl et al. Human, 1983, J. Nucl. Med. 24:316).

As is generally understood in the art, the "Fc" region is a crystallizable constant region of an antibody fragment that does not contain an antigen-specific binding region. In the IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody. 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 a complete target recognition and binding site. This region is composed of a dimer (VH-VL dimer) of one heavy chain and one light chain variable domain in tight non-covalent bonding. 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. Generally, the six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three CDRs specific for the target) may have the ability to recognize and bind to the target, although its affinity is lower than the entire binding site.

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

A "single domain fragment" consists of a single VH or VL domain that shows sufficient affinity for OX40. In a specific embodiment, the single domain fragments are camelized (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

The anti-OX40 antibodies of the present invention include derivatized antibodies. For example, derivatized antibodies are usually glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, and linked to cell ligands or other proteins. To modify. Any of numerous chemical modifications can be performed 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 acids, for example, using ambrx technology (see, for example, Wolfson, 2006, Chem. Biol. 13(10): 1011-2).

The anti-OX40 antibody may be an antibody whose sequence has been modified to change at least one constant region-mediated biological effector function. For example, in some embodiments, an anti-OX40 antibody can be modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody, for example, with one or more Fc receptors (FcγR) such as The binding of FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA and/or FcyRIIIB is reduced. FcγR binding can be reduced by mutating the immunoglobulin constant region segment of the antibody at specific regions necessary for FcγR interaction (see, for example, Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol. 147: 2657-2662). The reduction in the FcγR binding capacity of an antibody can also reduce other effector functions that depend on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cytotoxicity ("ADCC"). In an illustrative example, compared to the corresponding wild-type constant region, a variant CH2 domain with V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of the Fc region can show The affinity for FcγRIIB is reduced.

The anti-OX40 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, for example to enhance FcγR interaction (see, for example, U.S. Patent Application No. 2006/0134709). For example, the anti-OX40 antibody of the present invention may have a constant region that binds FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and/or FcyRIIIB with greater affinity than the corresponding wild-type constant region. In an illustrative example, compared to the corresponding wild-type constant region, a variant CH2 domain with V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of the Fc region can show Greater affinity for FcγRIIIA.

Therefore, the anti-OX40 antibodies of the present invention may have alterations in biological activity that result in increased or decreased opsonization, phagocytosis, or ADCC. Such changes are known in the art. For example, US Patent No. 5,834,597 describes modifications in antibodies that reduce ADCC activity. An exemplary ADCC lowering variant corresponds to "Mutant 3" (also referred to as "M3", shown in Figure 4 of US Patent No. 5,834,597), in which residues 234 and 237 (using EU numbering) are substituted with alanine. Mutant 3 (also called "M3") variants can be used in many antibody isotypes, such as human IgG2M3.

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

In some embodiments, the anti-OX40 antibody has low levels of fucose or lacks fucose. Antibodies lacking fucose have been associated with enhanced ADCC activity, especially at low doses of antibody. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. The method of making antibodies with less fucose involves growth in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme necessary for the fucosylation of polypeptides.

The anti-OX40 antibody may comprise a modified (or variant) CH2 domain or the entire Fc domain that includes amino acid substitutions that increase the binding to FcγRIIB and/or the binding of the corresponding wild-type CH2 or Fc region. Or the combination of technology and FcγRIIIA. The variant CH2 or variant Fc domain has been described in US Patent Application No. 2014/0377253. The variant CH2 or variant Fc domain usually includes one or more substitutions at position 263, position 266, position 273, and position 305, where the number of residues in the Fc domain is the same as the EU index in Kabat . In some embodiments, the anti-OX40 antibody comprises one or more substitutions selected from V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, and V305W relative to the wild-type CH2 domain. In a specific embodiment, with respect to the CH2 domain of human IgG1, one or more substitutions of the CH2 domain are selected from V263L, V273E, V273F, V273M, V273S, and V273Y. For example, one or more substitutions of the IgG1 CH2 domain can be V273E. In another specific embodiment, the anti-OX40 antibody of the invention comprises a variant IgG1 CH2 domain, which comprises the amino acid substitution V263L.

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

In some embodiments, the anti-OX40 antibody includes modifications that increase or decrease its binding affinity to the fetal Fc receptor FcRn, for example, by mutating immunoglobulin constant region segments at specific regions involved in FcRn interaction (see, e.g., WO 2005/123780). In a specific embodiment, the anti-OX40 antibody of the IgG class is mutated so that at least one of the 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, where positions 250 and 428 are a specific combination. For position 250, the substituted amino acid residue can be any amino acid residue except threonine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, Histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine acid. For position 314, the substituted amino acid residue can be any amino acid residue except leucine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, Histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine acid. For position 428, the substituted amino acid residue can be any amino acid residue except 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 acid. An exemplary substitution known to modify Fc effector function is the Fc substitution M428L, which can occur in combination with the Fc substitution T250Q. Additional specific combinations of suitable amino acid substitutions are identified in Table 1 of US Patent No. 7,217,797. Such mutations increase binding to FcRn, which protect the antibody from degradation and increase its half-life.

The anti-OX40 antibody may have one or more amino acids inserted into one or more of its CDRs, such as Jung and Plückthun, 1997, Protein Engineering 10:8, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17. (5): 481-9.Epub 2004Aug 17; and described in US Patent Application No. 2007/0280931.

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

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

The term "humanized" form of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequences derived from non-human immunoglobulins. Generally speaking, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all of the FR regions Or substantially all of those of human immunoglobulin sequences. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101 to Queen et al.; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; PCT Publication WO 91/09967; U.S. Patent Nos. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91: 969 -973; and US Patent No. 5,565,332.

"Human antibodies" include antibodies having the amino acid sequence of human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or animals that are transgenic for one or more human immunoglobulins and are not Express endogenous immunoglobulins. Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741. It is also possible to use transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes to produce human antibodies. 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; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, using technologies similar to the above, companies such as LakePharma, Inc. (Belmont, CA) or Creative BioLabs (Shirley, NY) can be engaged in providing human antibodies against selected antigens. A technique called "guided selection" can be used to generate fully human antibodies that recognize selected epitopes. In this method, selected non-human monoclonal antibodies, such as mouse antibodies, are 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 that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen" herein.

As used herein, an antibody that "specifically binds to human OX40" refers to an antibody that binds to human OX40 (or OX40 of other non-human species) but does not substantially bind to non-OX40 proteins. Preferably, the antibody "high affinity" binding human OX40 protein, i.e., a KD value of 5.0x10 ^-8 M or less, preferably 1.0x10 ^-8 M or less, more preferably 5.0x10 ^-9 M or less.

The term "substantially not binding" protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, that is, the KD of the binding protein or cell is 1.0x10 ^-6 M or more, more preferably 1.0x10 ^-5 M or more , More preferably 1.0x10 ^-4 M or more, 1.0x10 ^-3 M or more, and more preferably 1.0x10 ^-2 M or more.

The term "high affinity" for an IgG antibody means that the KD for the antigen is 1.0x 10 ^-6 M or less, preferably 5.0x 10 ^-8 M or less, more preferably 1.0x 10 ^-8 M or less, 5.0x 10 ^-9 M or less, more preferably 1.0 x 10 ^-9 M or less. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM subtypes means that the KD is 10 ^-6 M or less, preferably 10 ^-7 M or less, and more preferably 10 ^-8 M or less.

The term "Kassoc" or "Ka" refers to the rate of association of a specific antibody-antigen interaction, and the term "Kdis" or "Kd" refers to the rate of dissociation of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (Kd/Ka) and is expressed in molar concentration (M). The KD value of the antibody can be determined by a method known in the art. A preferred way to determine the KD of an antibody is to use a surface plasmon resonance (SPR) measurement, preferably to use a biosensing system such as the BiacoreTM system.

The term "EC50", also called half-maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximum effect.

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

The term "agonistic OX40 antibody" as used herein refers to an OX40 antibody that can bind to OX40 and activate or trigger the OX40 signaling pathway to promote the proliferation and survival of T cells. The term "antagonistic OX40 antibody" refers to the OX40 antibody that blocks the OX40 signaling pathway, thereby correcting the pathology of overactive T cells, and can be used to treat, for example, asthma, enteritis, and arthritis.

The term "therapeutically effective amount" refers to an amount of the antibody of the present invention that is sufficient to prevent or alleviate the symptoms associated with a disease or condition (e.g., cancer) and/or reduce the severity of the disease. The therapeutically effective amount is related to the disease to be treated, and those skilled in the art can easily distinguish the actual effective amount.

Various aspects of the present invention are described in more detail below.

OX40 antibody has binding specificity to human OX40 and other beneficial functional characteristics.

The antibody of the present invention specifically binds to human OX40 with high affinity, for example, a KD value of 1×10 ^-8 M or less. The antibody also has cross-reactivity with monkey OX40 and does not bind to mouse OX40.

The antibody of the present invention is an agonistic OX40 antibody that activates or triggers the OX40 signal pathway and participates in T cell co-stimulation, and promotes IL-2 secretion and CD8+ T cell proliferation.

The antibody of the present invention has a good anti-tumor effect in vivo. After the antibody administration is stopped, the tumor will not grow, and even the tumor will be completely eliminated, and immune memory can be produced.

The antibodies of the present invention may be polyclonal, monoclonal, genetically engineered, and/or substantially modified in other ways, including but not limited to chimeric antibodies, humanized antibodies, and human antibodies. In some embodiments, the constant region is an isotype selected from: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In a specific embodiment, the anti-OX40 antibodies described herein comprise IgG1. In other embodiments, the anti-OX40 antibody comprises IgG2 or IgG4. As used herein, the "constant region" of an antibody includes natural constant regions, allotypes or natural variants, such as D356E and L358M in human IgG1, or A431G. See, for example, Jefferis and Lefranc, MAbs, 1(4): 332-338 (July to August 2009). A preferred antibody of the invention is a monoclonal antibody. In addition, the antibody may be, for example, a murine, chimeric or humanized monoclonal antibody.

The light chain constant region of an anti-OX40 antibody can be a C kappa (κ) region or a C lambda (λ) region. The lambda region can be any of the known subtypes, such as lambda 1, lambda 2, lambda 3, or lambda 4. In some embodiments, the anti-OX40 antibody comprises a C kappa (κ) region.

The anti-OX40 antibody with high affinity for human OX40 (SEQ ID NO: 1) may be expected for therapeutic and diagnostic purposes. Accordingly, the present invention considers antibodies with high binding affinity to human OX40. In specific embodiments, the anti-OX40 antibody binds to human OX40 with an affinity of at least about 100 nM, but may show a higher affinity, such as at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM , 10nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.1nM, 0.01nM or even higher. In some embodiments, the antibody binds to human OX40 with an affinity ranging from about 1 pM to about 100 nM, or an affinity ranging between any of the foregoing values, such as but not limited to about 0.001 to 10 nM, 0.001 to 5 nM, 0.01 to 100 nM, 0.01 to 50 nM, 0.01 to 10 nM, 0.01 to 5 nM, or 0.01 to 1 nM.

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 measurement, can be used to determine the affinity of anti-OX40 antibodies for human OX40.

Anti-OX40 antibodies generally include a heavy chain comprising a variable region (VH) with three complementarity determining regions ("CDR"), which are referred to herein as (in N→C order) VH CDR# 1. VH CDR#2 and VH CDR#3, and a light chain comprising a variable region (VL) with three complementarity determining regions, which are referred to herein as (in N→C order) VL CDR#1, VL CDR#2 and VL CDR#3. Provided herein are the amino acid sequences of exemplary CDRs, as well as the amino acid sequences of the VH and VL regions of the heavy and light chains of the exemplary anti-OX40. Specific embodiments of anti-OX40 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 OX40.

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

First, the present invention provides a new humanized anti-OX40 activating antibody with a clear structure of amino acid sequence, high affinity to OX40, definite effect of activating the OX40 signaling pathway, low heterogeneity and high clinical Application potential.

Second, the humanized anti-OX40 activating antibody provided by the present invention has good biological activity. It can not only induce OX40+ cells to produce IL-8 and NFκB, but also stimulate PBMC to produce IL-2 and IFN-γ, and has a relatively broad spectrum of immunity. Enhance the effect, and can enhance T cell response to immune memory. The results of the in vivo immune memory study of hz25A7m8 and hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 model after treatment showed that the mice in all groups after the treatment were re-vaccinated with MC38 cells and no tumors would no longer form. Inoculation with Hepa1-6 cells can Tumor formation, but tumor growth is obviously inhibited. The inhibitory effect of hz25A7m8 and hz27G12H1L2 is similar to that of PC2 (variable region sequence is derived from BMS patent: WO2016196228A1; heavy chain: SEQ NO ID: 124, light chain: SEQ NO ID: 116; in the present invention Also referred to as: BMS; the same below) is more obvious.

Third, the humanized anti-OX40 activating antibody provided by the present invention has achieved good tumor suppression and killing effects in in vivo experiments in animal models, and even complete tumor regression in some experimental individuals, which is effective for colorectal cancer, etc. The clinical effect of OX40-related cancers is relatively clear. The in vivo pharmacodynamic study of hz25A7m8 and hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 model showed that in OX40 transgenic mice, the candidate antibodies hz25A7m8 and hz27G12H1L2 can significantly inhibit tumor growth and make most tumors regress. Among them, hz25A7m8 can make the tumor completely regress, and the activity is better than PC2 and PC3 (the variable region sequence comes from Xinda patent: WO2018177220A1; heavy chain: SEQ NO ID: 111, light chain: SEQ NO ID: 130; also abbreviated in the present invention : XD; the same below) and other control antibodies.

Description of the drawings

By reading the detailed description of the preferred embodiments below, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only used for the purpose of illustrating the preferred embodiments, and are not considered as a limitation to the present invention. Also, throughout the drawings, the same reference symbols are used to denote the same components. In the attached picture:

Figure 1: ELISA results of the binding activity of murine monoclonal antibodies to OX40.

Figure 2: Detection result of IL8 expression in HT1080-hOX40 cells activated by anti-OX40 murine monoclonal antibody. The antibody concentration of each antibody gradually decreases from left to right.

Figure 3: The result of binding of anti-OX40 murine monoclonal antibody to HT1080-hOX40 cells.

Figure 4: Anti-OX40 chimeric antibodies ch25A7, ch27G12, and ch11F7 Fortebio determined affinity results.

Figure 5A: HT1080-huOX40 secretion IL-8 method to identify the activation activity of the OX40 chimeric antibody.

Figure 5B: NFκB pathway activation-fluorescein detection method to identify the activation activity of the OX40 chimeric antibody. The antibody concentration of each antibody gradually decreases from left to right.

Figure 6: Chimeric antibodies ch25A7 and ch27G12 block the activity of OX40L in binding to HT1080-huOX40 cells.

Figure 7A. 25A7, 27G12 humanized before and after activation activity detection results; Figure 7B. 27G12 humanized molecular mutants activation activity detection results; Figure 7C. 25A7 humanized molecular mutants activation activity detection results (part) ; Figure 7D. 25A7 humanized molecular mutant activation activity test results (partial).

Figure 8: The result of the species-specific identification of the anti-OX40 antibody hz25A7m8.

Figure 9: The result of species-specific identification of the anti-OX40 antibody hz27G12H1L2.

Figure 10A: Immunological function (IFN-γ) of OX40 antibody hz25A7 and hz27G12, Von is PC1, BMS is PC2, and XD is PC3.

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

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

Figure 11A: hz25A7m8, hz27G12H1L2 effectively inhibited tumor growth in the MC38 model of OX40 humanized mouse colorectal cancer (individual data), BMS is PC2, XD is PC3.

Figure 11B: hz25A7m8, hz27G12H1L2 effectively inhibited tumor growth in the MC38 model of OX40 humanized mouse colorectal cancer (average data), BMS is PC2, XD is PC3.

Figure 12A: The results of immunological memory analysis of subcutaneous tumor formation in mice after anti-OX40 antibody treatment successively (MC38), BMS is PC2; hz25A7m8, hz27G12H1L2, BMS, and isotype control groups are treated with 0h, 10mg/kg, ip , Biw×4.

Figure 12B: The results of immunological memory analysis of subcutaneous tumor formation in mice after anti-OX40 antibody treatment successively (Hepa1-6), BMS is PC2; hz25A7m8, hz27G12H1L2, BMS, and isotype groups have treatment regimens of 0h, 10mg/kg, ip, biw×4.

Figure 13: FACS method to detect immune memory T cell grouping results, BMS is PC2.

Detailed ways

Hereinafter, exemplary embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings. Although the drawings show exemplary embodiments of the present disclosure, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

Specifically, the present invention provides antibodies or fragments thereof, including heavy chain variable regions and light chain variable regions, wherein

The variable region of the heavy chain contains:

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

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

VH CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 42, 54, 66;

The light chain variable region contains:

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

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

VL CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 48, 60, 72.

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

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

(2) The heavy chain variable region is selected from SEQ ID NO: 6, the amino acid sequence shown in 24 has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% homology , The light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 8, 26, 28, 32 with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% identical Origin

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

The present invention also provides an immunoconjugate, which comprises

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

(2) Coupling part.

The present invention also provides a multispecific antibody or derivative thereof, which comprises at least one antigen binding domain of the antibody or fragment thereof according to the present invention.

The present invention also provides a heavy chain antibody, which is a dimeric heavy chain antibody obtained on the basis of the antibody of the present invention.

The present 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 present invention also provides a nucleic acid that encodes the antibody or fragment thereof of the present invention, the multispecific antibody or derivative thereof of the present invention, or the heavy chain antibody of the present invention.

The present invention also provides a recombinant vector or recombinant host cell, which includes the encoding nucleic acid of the present invention.

The present invention also provides the antibody or its fragment, the antibody conjugate, the multispecific antibody or its derivative, the heavy chain antibody, the composition, the nucleic acid, the recombinant vector or the recombinant Uses of host cells include:

It is used to prepare drugs that bind OX40, inhibit the combination of OX40 and OX40L, activate OX40+ T cells, activate human immune response, and tumor therapeutic vaccines;

Used to prepare molecular adjuvants that enhance specific immune responses, preferably in combination with adjuvants such as CpG for the preparation of tumor vaccines;

Used to prepare drugs for inducing OX40+ cells to produce IL-8 and/or for initiating NFκB gene transcription;

Used to prepare drugs that stimulate PBMC to produce IL-2 and IFN-γ;

Used to prepare drugs for inhibiting the growth and metastasis of solid tumors;

Used to prepare qualitative or quantitative detection kits for OX40.

The present invention also provides a method for producing antibodies, including:

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

(2) Recover antibodies.

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

Immunization: Balb/c mice were immunized with human OX40/mFc recombinant protein (serial number: P43489-1, 29aa-216aa), and serum titer was detected by ELISA with a 96-well ELISA plate coated with recombinant human OX40-his protein ; Mice whose serum titers meet the fusion requirements are used for the next cell fusion.

Cell fusion and hybridoma preparation: On the 67th day after the initial immunization, select mice with the required titer, take the mouse spleen aseptically, prepare a B lymphocyte suspension, and mix it with FO myeloma cells at a ratio of 5:1. The two cells are fused under the action of PEG4000. After the fused cells were resuspended in HAT medium, they were divided into 96-well cell culture plates. Place 37°C, 5% CO2 incubator for culture.

Example 2: Screening of anti-human OX40 antibody-positive hybridoma cell lines.

Scheme 1. ELISA binding screening of positive hybridomas.

10-14 days after fusion, coat the ELISA plate with human OX40-His recombinant protein (serial number: P43489-1, 29aa-216aa) (10ug/ml, pH9.6, 0.1M NaHCO3), at 4°C, overnight; Block with 4% skimmed milk powder-PBS, 37°C, 2hr; wash with PBST (0.05% Tween20-PBS) three times, add hybridoma clone culture supernatant, 37°C, 1hr. Set up the following controls: (1) positive control (PC): mouse serum after immunization (diluted with PBS 1:1000); (2) negative control (NC): mouse serum before immunization (diluted with PBS 1:1000). Wash three times with PBST (0.05% Tween20-PBS), add HRP-goat anti-mouse IgG (Fcγ), diluted 1:20000, 37°C, 1hr; then wash five times with PBST (0.05% Tween20-PBS), add OPD color developing solution, protect from light for 10-15 minutes, add 2M H ₂ SO _{4 to} stop the reaction; read the A492 value with the microplate reader. The test hole A492 value is greater than 2.1 times the negative control hole A492 value and it is judged as positive. In order to determine the reliability of positive clones, a second round of screening was carried out every other day after the first screening change. After testing and identification (Figure 1), a total of 14 antibody secretion-positive cell lines (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, 30B12-5), continue to further screen these 14 antibodies.

Scheme 2. Anti-OX40 murine monoclonal antibody activates HT1080-hOX40 cells to express IL8 activity screening.

Digest HT1080-hOX40 cells (Kangyuan Bochuang: Cat.No: KC-0140) the day before the experiment, resuspend the cells in complete medium (1640 medium + 10% FBS + 0.5 μg/mL), and adjust the cell density to 10 ⁵ cells/mL, add 200μL of cell suspension to each well of a 96-well cell culture plate. Then the cell culture plate was placed in a 37°C 5% CO2 incubator and cultured overnight. The next day, the antibody to be tested was diluted to an appropriate concentration, and added to the cell culture plate, and incubated in a 37°C 5% CO2 incubator for 6 hours. At the same time, a positive control PC1 (PC1, namely Vonlerolizumab, the variable region sequence is derived from WHO Drug information, Vol. 31, NO. 3, 2017, also referred to as Von in the present invention, the same below). Finally, the cell culture supernatant was aspirated, and the IL-8 ELISA quantitative kit (product number: batch number) was used to detect the content of IL-8 in the supernatant according to the instructions. The results are shown in Figure 2. Most of the clones can activate HT1080-hOX40 cells well and increase the level of IL8 expression.

Scheme 3. Screening of positive hybridomas combined with OX40 on the surface of HT-1080 cells.

According to the above activity results, 10 clones were selected (11F7-1, 18D6-4, 18G11-1, 22H1-1, 24H1-11, 25A7-11, 27H7-1, 27G12-7, 29G2-4, 30B8-8) The hybridoma supernatant was purified by affinity, and the mouse antibody (5ug/ml) obtained was incubated with a suspension of recombinant human OX40-expressing 293 cells (293/OX40) at 37°C for 30 minutes. The following controls were set: (1) Positive control PC1 In this example, the antibody molecule Fc was replaced with mouse IgG1, 5μg/ml; (2) Negative control NC: irrelevant mouse IgG1 5μg/ml. After washing the cells 3 times with PBS, goat anti-mouse IgG FITC (Cat: F9006, Sigma) diluted 1:64 was added and incubated for 30 min. After washing the cells with PBS three times, the average fluorescence intensity (MFI) of the cells was measured by a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma can bind to the OX40 on the surface of the 293 cell, as shown in Figure 3. It shows that most of the clones bind well to OX40 on the surface of 293 cells.

Example 3: Sequence determination of murine anti-human OX40 antibody.

After expanding the hybridoma cells m25A7, m27G12, and m11F7 that secrete anti-human OX40 antibodies, use Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and Mouse Monoclonal Antibody Light/Heavy Chain Test Card (Cat.:A12401) , VicNovo) carried out subtype detection in accordance with the reagent operating procedures. The subtype identification results showed that the heavy chains of m25A7, m27G12, and m11F7 are all IgG1, and the light chains are all Kappa chains, providing a basis for cloning of antibody genes.

The m25A7, m27G12, and m11F7 hybridoma cells were extracted according to the TRIzol kit (Cat:15596026, Invitrogen) instructions to extract total cellular RNA; M-MuLV reverse transcriptase (Cat:M0253S, NEB) was used to reverse the total RNA of hybridoma cells Record into cDNA; use degenerate primers and Phusion kit (Cat: E0553L, NEB) to amplify antibody light chain variable region IgVL (κ) and heavy chain variable region V _H sequence; use gel recovery kit (Cat: AP -GX-250, Axygen) Purify the PCR amplification product; according to the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Biological), connect the amplified PCR product to the T vector and transform E. coli competent cells. The strain is amplified, After extracting the plasmid, DNA sequencing was performed to obtain the monoclonal antibody variable region sequence. Sequencing results showed that the nucleotide sequence of the murine antibody m25A7 heavy chain variable region DNA is shown in sequence 1, and the amino acid sequence of the murine antibody m25A7 heavy chain variable region inferred from the DNA sequence is shown in sequence 2. The nucleotide sequence of the light chain variable region DNA of the mouse antibody m25A7 is shown in sequence 3. From this DNA sequence, the amino acid sequence of the light chain variable region of the mouse antibody m25A7 is inferred in sequence 4; the nucleoside of the mouse antibody m27G12 heavy chain variable region DNA The acid sequence is shown in sequence 5, and the amino acid sequence of the variable region of the murine antibody m27G12 heavy chain is inferred from the DNA sequence in sequence 6. The nucleotide sequence of the light chain variable region DNA of the murine antibody m27G12 is shown in Sequence 7. From the DNA sequence, the amino acid sequence of the light chain variable region of the murine antibody m27G12 is shown in Sequence 8. The nucleotide sequence of the variable region DNA of the murine antibody m11F7 heavy chain is shown in Sequence 9. From the DNA sequence, the amino acid sequence of the variable region of the murine antibody m11F7 heavy chain is inferred in Sequence 10. The nucleotide sequence of the light chain variable region DNA of the mouse antibody m11F7 is shown in Sequence 11, and the amino acid sequence of the light chain variable region of the mouse antibody m11F7 is inferred from this DNA sequence.

Example 4: Preparation of anti-human OX40 chimeric antibody.

The cloned murine antibody light chain variable region and heavy chain variable region genes were introduced into restriction sites by PCR, and cloned to the upstream of the human-kappa light chain constant region and human IgG1 heavy chain constant region coding genes respectively. In the nuclear expression vector, the human-mouse chimeric light chain (pKN019-Ch25A7L) and human-mouse chimeric heavy chain (pKN041-Ch25A7H) expression plasmids of m25A7 were obtained, and the human-mouse chimeric light chain of m27G12 (pKN019-Ch27G12L) And human-mouse chimeric heavy chain (pKN041-Ch27G12H) expression plasmids, and m11F7 human-mouse chimeric light chain (pKN019-Ch11F7L) and human-mouse chimeric heavy chain (pKN041-Ch11F7H) expression plasmids. A large number of plasmids containing human-mouse chimeric antibody light chain and heavy chain were isolated and amplified by transferring into E. coli. According to the operating instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the light and heavy chain plasmids of the chimeric antibodies Ch 25A7, Ch 27G12 and Ch11F7 were paired and transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography column to obtain the m25A7, m27G12, and m11F7 chimeric antibodies.

Example 5: Activity analysis of anti-human OX40 chimeric antibody.

Scheme 1. Affinity analysis of anti-human OX40 chimeric antibody.

Using Fortebio's Octet QKe system instrument, the anti-human antibody Fc segment capture antibody (AHC) biological probe is used to capture the Fc segment of the antibody to determine the affinity of the antibody. During the determination, the anti-human OX40 chimeric antibodies ch25A7, ch27G12, ch11F7 and the control antibody PC1 were diluted to 4ug/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat:18-0015, PALL) for 120s. Human OX40-His recombinant protein (serial number: P43489-1, 29aa-216aa) was used as the mobile phase, and the concentration of OX40-His recombinant protein was 100nM. The binding time is 300s, and the dissociation time is 300s. After the experiment, the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.

The reaction curves of anti-human OX40 chimeric antibodies ch25A7, ch27G12, ch11F7 and human OX40 recombinant protein are shown in Figure 4 (Von is the positive control PC1). The curves are fitted and the affinity is calculated. The results show that the chimeric antibodies ch25A7, ch27G12, The affinities (KD, Table 1) of ch11F7 are: 1.36E-08, 4.34E-08, 6.61E-09. The results show that the chimeric antibodies ch25A7, ch27G12, and ch11F7 have high affinity with human OX40 recombinant protein, which is lower than the control antibody PC1.

Table 1. Anti-OX40 chimeric antibody ch25A7, ch27G12, ch11F7 Fortebio determination of affinity fitting results

Scheme 2. Analysis of activation activity of anti-human OX40 chimeric antibody.

According to the method of Example 2 Scheme 2, the chimeric antibodies ch25A7, ch27G12, and ch11F7 were analyzed for activation activity. In addition, the activity analysis of chimeric antibody x was carried out based on the NFκB signal pathway activation experiment detected by fluorescein. Specifically, digestion of HT1080-hOX40 cells one day before the experiment, with complete medium (1640 medium + 10% FBS + 0.5μg / mL ) resuspended cells, cell density was adjusted to ¹⁰⁵ / mL, and 96-well cell culture plate Add 200μL of cell suspension to each well. The cell culture plate was then placed in a 37°C 5% CO2 incubator and cultured overnight. On the second day, the NFκB reporter gene plasmid (pGL4.32[luc2P/NF-κB-RE/Hygro]) was transfected into HT1080-hOX40 cells by liposome method. Change the medium 24 hours after transfection and continue to culture the cells for 24 hours to ensure that the cells recover for 48 hours after transfection. The antibody to be tested is then diluted to an appropriate concentration and added to the cell culture plate, and incubated in a 37°C 5% CO2 incubator for 18 hours. Remove the cell treatment medium, wash the cells thoroughly with PBS, and finally dry the cells thoroughly. Add 25ul lysis solution to each well (if the lysis solution is sufficient, add about 50ul to ensure that no bubbles will be generated in the next operation) to ensure that the cell layer is covered, and lyse with shaking at room temperature for 20 minutes. Pipette 20ul of cell lysate into the corresponding wells of the detection plate, configure the luciferase reaction substrate (100ul/sample), and prepare for testing. Draw an antibody concentration-RLU histogram or curve chart based on the fluorescein value (RLU) read by the machine. If the dual luciferase reporter system is used to read the experimental reaction luciferin value (RLU1) and the internal control luciferin value (RLU2) at the same time, calculate the ratio of the two, and draw the antibody concentration-RLU1/RLU2 histogram or curve. The results are shown in Figure 5A (Von stands for PC1) and Figure 5B. 25A7 and 27G12 have good activation activity, and their activity is not weaker than that of the control antibody PC1.

Example 6: ELISA to detect the inhibitory effect of anti-human OX40 chimeric antibody on the binding of human OX40/OX40L.

The human OX40 was diluted to 1 μg/mL, coated overnight at 4°C, and sealed with 5% BSA in a constant temperature incubator at 37°C for 60 minutes. The ch25A7, ch27G12 and the control antibody PC1, and the isotype control NC-hIgG1 (initial concentration 20μg/mL, 3-fold serial dilution, 8 gradients) were reacted in a 37°C constant temperature incubator for 60 minutes, and then 10μg/mL OX40L- mFc (OX40L serial number: NP_003317, 51-Leu 183, mFc Tag) was incubated with the antibody, and reacted in a constant temperature incubator at 37°C for 60 minutes. Wash the plate 4 times with PBST; then add 1:5000 diluted HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research), react for 45 minutes, add TMB (Cat: ME142, Beijing Taitianhe Biological) substrate The color is developed for 15 minutes, and the plate is read after 2M HCl is stopped. With 630nm as the reference wavelength, read and record the absorbance value A450nm-630nm of the orifice plate at a wavelength of 450nm. The results show that ch25A7 can effectively block the binding of recombinant human OX40 to its receptor OX40L, and its half effective inhibitory concentration (IC50) value is 0.92μg/mL, which is equivalent to the control antibody PC1 (0.91μg/mL) (Figure 6). ch27G12 has no obvious inhibitory effect.

Example 7: Humanization and recombinant expression of anti-human OX40 monoclonal antibodies 25A7 and 27G12.

Scheme 1. Humanized design and recombinant expression of 25A7.

First, a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then according to the homology comparison result, in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) to find the most similar human antibody template, select VH3( 3-21) as the basic template, combined with the full sequence blast results, considering the frequency of rearranged antibodies in specific FR region amino acids, and the HCDR3 sequence, CDR grafting, to achieve the m25A7 heavy chain variable region (VH) Highly humanized in the Framework area. According to the result of homology comparison, search for the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and select VK I respectively (L5), VK II (O1), VK VI (A26) as the basic template, combined with the full sequence blast results, consider the frequency of rearranged antibody amino acids at specific FR sites, and the LCDR3 sequence, and perform CDR grafting. Realize the full humanization of m25A7 light chain Framework area. The nucleotide sequence of the humanized heavy chain variable region of m25A7 antibody CDR Grafted is shown in Sequence 13, and the amino acid sequence is shown in Sequence 14. The nucleotide sequence of the variable region of the humanized light chain 1 is shown in Sequence 15 and the amino acid sequence. See sequence 16; the nucleotide sequence of the variable region of humanized light chain 2 is shown in sequence 17, and the amino acid sequence is shown in sequence 18; the nucleotide sequence of the variable region of humanized light chain 3 is shown in sequence 19, and the amino acid sequence is shown in sequence 20. Subsequently, according to the characteristics of the murine 25A7 sequence, the CDR-transplanted humanized heavy chain variable region sequence was designed for back mutation, and the back mutation site is shown in Table 2. After the 25A7 humanized back mutation, the final selection of the heavy chain variable sequence The region nucleotide sequence is shown in Sequence 21, the amino acid sequence is shown in Sequence 22, the light chain variable region nucleotide sequence is shown in Sequence 15, and the amino acid sequence is shown in Sequence 16. Subsequently, the 25A7 humanized light chain hz25A7_L1 and humanized heavy chain mutants (parental hz25A7_H1, mutant hz25A7_H1m1-hz25A7_H1m8) were paired (Table 3) and transferred into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain various mutant humanized antibodies of hz25A7 (denoted as hz25A7_H1m1-hz25A7_H1m9 in sequence).

Table 2. 25A7 heavy chain humanized back mutation sequence design

Note: If R16E is marked according to the Kabat numbering system, mutate amino acid R at position 16 back to E

Table 3. 25A7 light and heavy chain sequence combination

Note: This table shows the sequences obtained from various combinations of humanized 25A7 light and heavy chains. For example, 25A7-1, the antibody is composed of 25A7 humanized light chain hz25A7_L1 and humanized heavy chain hz25A7_H1, and others are analogized.

Scheme 2. Humanized design and recombinant expression of 27G12.

First, a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then according to the homology comparison result, in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) to find the most similar human antibody template, select VH1( 1‐46) as the basic template, combined with the full sequence blast results, considering the frequency of rearranged antibodies in the specific FR region, the PTM risk and the HCDR3 sequence situation, CDR grafting was performed to achieve the m27G12 heavy chain variable region ( VH) Full humanization in the Framework area. According to the result of homology comparison, search for the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and select VK III respectively (L2), VK II (A2) as the basic template, combined with the full sequence blast results, and LCDR3 sequence situation, CDR transplantation, to achieve the full humanization of the framework region of the m27G12 light chain. The nucleotide sequence of the humanized heavy chain variable region of m27G12 antibody 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 Sequence 25, and the amino acid sequence See sequence 26; the humanized light chain 2 variable region nucleotide sequence is shown in sequence 27, and the amino acid sequence is shown in sequence 28. Subsequently, the 27G12 humanized heavy chain hz27G12H1 and the humanized light chain hz27G12L1 and hz27G12L2 were paired and transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography column to obtain hz27G12 mutant humanized antibodies hz27G12H1L1 and hz27G12H1L2.

Example 8: Activity analysis of anti-human OX40 humanized antibody.

Scheme 1. Fortebio detects antibody affinity.

With reference to the method of Scheme 1 in Example 5, affinity analysis was performed on various mutants of hz25A7 and hz27G12. The results showed (Table 4) that the mutants hz25A7m8 and hz27G12H1L2 maintained the same or slightly higher affinity level than the chimeric antibody, with affinities (KD) of 0.65E-08M and 1.32E-08M, respectively.

Table 4. Affinity test results of 25A7 humanized mutant

Sample	Response	KD(M)	kon(1/Ms)	kdis(1/s)
ch25A7	0.2603	0.91E-08	1.82E+05	1.65E-03
hz25A7H1L1	0.1161	2.28E-08	3.14E+05	7.16E-03
hz25A7m1	0.1614	2.26E-08	3.02E+05	6.82E-03
hz25A7m2	0.1534	2.14E-08	3.01E+05	6.43E-03
hz25A7m3	0.1709	2.24E-08	2.97E+05	6.65E-03
hz25A7m4	0.1527	1.96E-08	2.52E+05	4.95E-03
hz25A7m5	0.243	1.19E-08	1.47E+05	1.75E-03
hz25A7m6	0.2039	1.63E-08	3.46E+05	5.63E-03
hz25A7m7	0.1519	1.48E-08	3.56E+05	5.26E-03
hz25A7m8	0.3062	0.65E-08	1.58E+05	1.02E-03
ch27G12	0.2562	1.71E-08	2.85E+05	4.88E-03
h27G12HL	0.2323	1.63E-08	3.35E+05	5.45E-03
27G12H1L1	0.208	1.43E-08	3.36E+05	4.82E-03
h27G12H1L2	0.1866	1.32E-08	3.15E+05	4.15E-03

With reference to the method of Scheme 2 in Example 5, hz25A7, hz27G12 and each mutant were analyzed for H1080 activation activity. The results are shown in Figure 7A-D (Von stands for PC1). After humanization, 25A7 and 27G12 and their mutants all maintained a good activity of activating H1080 to express IL-8.

Example 9. The species cross of hz25A7-mut8 and hz27G12-H1L2.

Human OX40 (serial number: P43489-1, 29aa-216aa), cynomolgus OX40 (Cat: 90846-C08H, Beijing Yiqiao Shenzhou), and mouse OX40 (Cat: 50808-MCCH, Beijing Yiqiao Shenzhou) cells The outer region recombinant protein was diluted to 1μg/mL and coated overnight at 4°C; after washing the plate 3 times with PBS, add 5% BSA PBS, block at 37°C for 60 min, wash the plate 3 times with PBST; add h25A7mut8, h27G12H1L2, 37 Incubate at ℃ for 60min, wash the plate with PBST 4 times; add 1:5000 diluted HRP-goat anti-human IgG antibody (Cat:115-035-071, Jackson ImmunoResearch), incubate at 37℃ for 30min, wash the plate with PBST 4 times; add TMB The substrate develops color. After incubating at 37°C for 10 minutes, add 2M HCl to terminate the reaction; using 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at the wavelength of 450nm. The results (Figure 8, Figure 9) showed that h25A7mut8 and h27G12H1L2 bound to human and monkey OX40, but did not bind to mouse OX40. The binding activity of h25A7mut8 to human and monkey OX40 is equivalent, and the binding activity of h27G12H1L2 to monkey OX40 is lower than that of human OX40.

Example 10: In vitro testing of the immunological functions of OX40 antibodies hz25A7 and hz27G12.

Take the peripheral blood mononuclear cell (PBMC) from healthy subjects to separate the peripheral blood mononuclear cell (PBMC), and incubate the serially diluted OX40 antibody or control antibody with PBMC to detect cell proliferation and cytokine (IL-2 , IFN-γ) secretion level to evaluate the functional activity of OX40 antibody. Specifically, the anti-human CD3 antibody and the anti-human CD28 antibody are individually or together coated on the enzyme-linked plate as a control well; the anti-human OX40 antibody is coated on the enzyme-linked plate as the well to be tested. Take the peripheral blood of healthy people ^{and separate and obtain CD3+ T cells according to the instructions of Dynabeads TM} FlowComp ^TM Human CD3Kit; after washing twice, adjust the cells to a suitable concentration with medium and add them to a 96-well plate; after 5 days, use CTG method to detect cells Proliferation, harvest the cell culture supernatant, and detect IL-2 and IFN-γ by ELISA. 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 KNAb-1 in this example), hz27G12H1L2 (labeled KNAb-2 in this example) can significantly increase the cytokine IL-2 and IL-2 in the culture medium in a dose-dependent manner. The concentration of IFN-γ (Figure 10A, Figure 10B) suggests that it can activate T lymphocytes and enhance their function of secreting cytokines. The cell proliferation test results showed (Figure 10C) that both hz25A7m8 and hz27G12H1L2 can effectively promote cell proliferation, and the ability to promote cell proliferation is equivalent to that of the control antibodies PC2 and PC3.

Example 11: In vivo activity analysis of anti-OX40 antibodies hz25A7m8 and hz27G12H1L2.

Scheme 1. In vivo pharmacodynamic study of hz25A7m8 and hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 model.

MC38 mouse colorectal cancer cells are cultured in DMEM medium containing 10% inactivated fetal bovine serum, 100U/ml penicillin and 100μg/ml streptomycin in a 37°C, 5% CO2 incubator, every 3 to After 4 days when the cells are overgrown, the cells are divided into flasks and passaged. The tumor cells in logarithmic growth phase are harvested, resuspended in PBS and inoculated subcutaneously on the right flank of human OX40 transgenic mice, and grouped when the average tumor volume reaches about 60-100mm3 Administration, 6 animals in each group, according to the dose of 10mg/kg, twice a week, a total of 4 administrations. After grouping, the tumor volume was measured with vernier calipers three times a week, and the long and short diameters of the tumor were measured. The volume calculation formula is: volume = 0.5 × long diameter × short diameter ² . While measuring the tumor volume, the mice were weighed. The relationship between the change of mouse body weight and the time of administration was recorded. At the same time, observe the survival and health of the mice, such as animal activity and eating during the administration period. At the end of the experiment, the mice were euthanized, the tumors were stripped and weighed, and the tumors stripped from the control group and the test group were placed neatly and photographed. The results (Figure 11A, Figure 11B) show that in OX40 transgenic mice, the candidate antibodies hz25A7m8 and hz27G12H1L2 can significantly inhibit tumor growth and make most tumors regress. Among them, hz25A7m8 can completely regress the tumor, and its activity is better than that of control antibodies such as PC2 and PC3.

Scheme 2. Study on in vivo immune memory after treatment of hz25A7m8 and hz27G12H1L2 on OX40 humanized mouse colorectal cancer MC38 model.

MC38 mouse colorectal cancer tumor cells and Hepa1-6 mouse liver cancer tumor cells use DMEM medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin, 100μg/ml streptomycin and 2mM glutamine Cultivate in a 37°C, 5% CO ₂ incubator, and sub-flask the cells every 3 to 4 days after the cells are fully grown, harvest the tumor cells in the logarithmic growth phase, and inoculate them in the scheme 1 of this embodiment through hz25A7m8, hz27G12H1L2 , HuOX40 mice whose MC38 tumors completely disappeared after PC2 treatment, and C57BL/6J mice that have not been inoculated with tumors, the tumor cells resuspended in PBS were inoculated subcutaneously on the left and right flanks of experimental animals. After the tumor cells are inoculated, the tumor volume is measured 2-3 times with a vernier caliper every week, and the long and short diameters of the tumor are measured. The volume calculation formula is: volume = 0.5 × long diameter × short diameter2. While measuring the tumor volume, the mice were weighed. The relationship between the change of mouse body weight and the time of vaccination was recorded. At the same time, observe the survival and health status of the mice, such as the general state of animal activity and eating after vaccination. At the end of the experiment, the mice were euthanized, the tumors were stripped and weighed, and the tumors stripped from the control group and the test group were placed neatly and photographed. The results (Figure 12A) showed that the mice in all treatment groups were no longer tumorigenic after being vaccinated with MC38 cells again. Although Hepa1-6 cells could form tumors, the tumor growth was significantly inhibited. The inhibitory effects of hz25A7m8 and hz27G12H1L2 were similar to those of PC2. The ratio is more obvious (Figure 12B).

Scheme 3. FACS method to detect immune memory T cell grouping.

Take the spleen of the mouse in Scheme 2 of this Example, prepare a single cell suspension, add anti-mouse CD3/CD4/CD44/CD62L to co-incubate with the spleen cells, and analyze the immune memory T cell population. Specifically, the tumor-bearing mice were dissected in a sterile environment to obtain the spleen; the 70μM cell screen was placed in a sterile dish, the spleen was transferred to the cell screen, and the spleen was ground into discrete single cells with a grinding rod; the cells were collected Transfer the suspension to a 50 mL centrifuge tube, centrifuge at 200×g for 10 min; discard the supernatant, lyse the red blood cells, wash twice, and process the cell suspension through a 40μM cell screen to obtain a spleen single cell suspension; suspend the spleen single cell Transfer the solution to a flow cytometer, add the corresponding antibody (anti-mouse CD3/CD4/CD44/CD62L) according to the experimental design, and incubate for 30 minutes in the dark; add PBS to wash once, resuspend the cells and perform flow cytometry. The results (Figure 13) showed that compared with the control group, the helper T cells (CD3+CD4+) of mice treated with hz25A7m8, hz27G12H1L2, and PC2 increased significantly. Further analysis showed that the ratio of memory T cells (CD3+CD4+CD44hiCD62Llo) and natural T cells (CD3+CD4+CD44loCD62Lhi) increased to varying degrees.

The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or changes within the technical scope disclosed by the present invention. All replacements shall be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (11)

1. Antibodies or fragments thereof, including heavy chain variable regions and light chain variable regions, wherein

   The variable region of the heavy chain contains:

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

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

   VH CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 42, 54, 66;

   The light chain variable region contains:

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

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

   VL CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 48, 60, 72.
2. The antibody or fragment thereof of claim 1, wherein:

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

   (2) The heavy chain variable region is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 6, 24 The light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 8, 26, 28, 32 with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% Homology

   (3) The variable region of the heavy chain has more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO: 10. The variable region has more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID NO: 12.
3. An immunoconjugate comprising

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

   (2) Coupling part.
4. A multispecific antibody or derivative thereof, characterized in that it comprises at least one antigen binding domain of the antibody or fragment thereof according to any one of claims 1-2.
5. A heavy chain antibody, which is a dimeric heavy chain antibody obtained on the basis of any one of the antibodies of claims 1-2; preferably a nanobody obtained by camel transformation.
6. A composition comprising

   (1) The antibody or fragment thereof according to any one of claims 1-2, the antibody conjugate according to claim 3, the multispecific antibody or derivative thereof according to claim 4, or the recombinant antibody according to claim 5. Chain antibody

   (2) A pharmaceutically acceptable carrier.
7. A nucleic acid encoding the antibody or fragment thereof according to any one of claims 1-2, the multispecific antibody or derivative thereof according to claim 4, or the heavy chain antibody according to claim 5.
8. A recombinant vector or recombinant host cell, which comprises the nucleic acid of claim 7.
9. The antibody or fragment thereof according to any one of claims 1-2, the antibody conjugate according to any one of claim 3, the multispecific antibody or derivative thereof according to claim 4, and the heavy chain according to claim 5 The use of the antibody, the composition according to claim 6, the nucleic acid according to claim 7, and the recombinant vector or recombinant host cell according to claim 8, characterized in that:

   It is used to prepare drugs that bind OX40, inhibit the combination of OX40 and OX40L, activate OX40+ T cells, activate human immune response, and tumor therapeutic vaccines;

   Used to prepare molecular adjuvants that enhance specific immune responses, preferably in combination with adjuvants such as CpG for the preparation of tumor vaccines;

   Used to prepare drugs for inducing OX40+ cells to produce IL-8 and/or for initiating NFκB gene transcription;

   Used to prepare drugs that stimulate PBMC to produce IL-2 and IFN-γ;

   Used to prepare drugs for inhibiting the growth and metastasis of solid tumors;

   Used to prepare qualitative or quantitative detection kits for OX40.
10. A method of producing antibodies, including:

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

    (2) Recover antibodies.
11. A method for preventing and/or treating a disease mediated by OX40, the method comprising administering the antibody or fragment thereof according to any one of claims 1 to 2, and the antibody according to claim 3 to a subject in need thereof Conjugate, 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 of claim 8, or Recombinant host cells, and optionally other drugs or means.

Images