Anti-hvem antibodies

Abstract

This invention relates to the field of antibody technology, specifically to anti-HVEM antibodies or fragments thereof and their uses. The technical problem this invention aims to solve is to provide a series of BTLA-blocking anti-HVEM antibodies or fragments thereof. The anti-HVEM antibodies of this invention possess good affinity, specificity, and functional activity, can block the binding of BTLA to HVEM, promote T cell activation, and have potential applications in cancer treatment.

Description

[0001] The present application is a divisional application of the Chinese invention patent application with the application number “202310557714.1”, the title of “Anti-HVEM antibody, preparation method and uses thereof”, and the filing date of May 17, 2023. The Chinese invention patent application “202310557714.1” claims the priority of the Chinese invention patent application with the application number “202210582451.5” and the filing date of May 26, 2022. The present application also claims the priority of the Chinese invention patent application “202210582451.5”. TECHNICAL FIELD

[0002] The present application relates to the field of antibody technology, in particular to anti-HVEM antibodies and uses thereof. BACKGROUND

[0003] Antibodies are biological macromolecules composed of heavy and light chains, secreted by B lymphocytes, and play an important role in the humoral immunity of the body. The heavy or light chain of an antibody molecule is composed of a variable region and a constant region. The variable region mainly plays a role in target antigen binding, and the constant region mainly plays an immune regulation effect. According to the spatial structure and amino acid sequence characteristics, antibodies can be divided into IgG, IgM, IgE, IgA, IgD, etc., and the heavy and light chains can be further divided into multiple subtypes. For example, human IgG heavy chains can be divided into IgG1, IgG2, IgG3 and IgG4, and light chains can be divided into kappa and lambda. Because antibodies can specifically and efficiently bind to target molecules or target cells, regulate the downstream signaling pathway of target molecules, or kill target cells through immune effects, antibodies can be developed as drugs for disease treatment. Currently, antibodies have developed into an important biotechnology drug, among which monoclonal antibodies account for the majority, and IgG type antibodies are the main ones. Therefore, the commonly referred to monoclonal antibodies usually refer to IgG type antibodies.

[0004] IgG type antibody molecules are tetramer composed of 2 heavy chains and 2 light chains by interchain disulfide bond, with a molecular weight of about 150 kD. According to structural and functional characteristics, antibody molecules can be divided into variable region and constant region, in which the variable region mainly plays the role of antigen binding, while the constant region mainly plays the functions of immunological effect and transport, etc. The variable region of antibody can be further divided into complementarity determining region CDR and framework region FR, in which each heavy chain or light chain contains 3 CDR regions (heavy chain VH-CDR1, VH-CDR2, VH-CDR3, light chain VL-CDR1, VL-CDR2, VL-CDR3) and 4 FR regions (FR1, FR2, FR3, FR4) on both sides of the CDR region. The loop formed by the CDR region is the main part of the antibody molecule binding to the antigen, and the FR region forms the supporting structure of the CDR region through spatial folding. The specific recognition of different antigen molecules by antibodies is mainly realized through the amino acid polymorphism of the 6 CDR regions (VH-CDR1, 2, 3 and VL-CDR1, 2, 3) and the conformational polymorphism of the loop. Since the structure of different antibody FR regions is highly similar, when the CDR region of a certain antibody is replaced by the CDR region of another antibody molecule, if the FR regions of different antibody molecules match well, the conformation of the CDR region changes little before and after replacement, so the new variable region formed after replacement can still retain the antigen binding ability, which is the basis of CDR grafting technology. The CDR region of murine antibody can be replaced by CDR grafting technology to replace the CDR of human antibody, so as to recombine with human FR region to form humanized antibody. If the FR regions of human-mouse antibodies match well, the antigen binding ability can still be retained.

[0005] Hybridoma technology is an important technology in the current antibody discovery process. After immunization of mice with antigens, mouse B cells develop into germinal centers in lymphoid tissues, and the affinity of antibodies is gradually improved through somatic hypermutation (SHM). Mouse spleen cells are isolated and fused with mouse myeloma cells in vitro to form hybridomas. By determining the antibody activity in the culture supernatant of hybridoma cells, hybridomas producing target antibodies can be screened. Hybridoma cells are gradually monoclonalized after subcloning, and the mRNA of subcloned cells is extracted for antibody variable region gene sequencing to analyze the amino acid sequence of the antibody variable region. At present, most of the antibodies on the market are obtained by hybridoma technology screening. Since the antibody molecules derived from hybridoma technology complete the affinity maturation process in mice, those B cells with cross-reactivity to mouse proteins are eliminated by "negative selection" in mouse bone marrow, so the antibody molecules can effectively reduce non-specific binding to self proteins or similar proteins.

[0006] Monoclonal antibodies can exert pharmacological effects through various mechanisms. Antibody variable regions can bind to extracellular soluble ligands, block ligand binding to receptors, and cut off ligand-induced downstream signaling, so antibody drugs targeting immune cytokines can improve inflammatory diseases, such as adalimumab, belimumab, siltuximab, and other approved antibodies. Antibody constant regions can exert immune regulation, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and the like, so antibody drugs targeting tumor cell surface molecules can kill tumor cells, such as rituximab, trastuzumab, cetuximab, and other approved antibodies have achieved great success. In addition, new antibody technologies such as bispecific antibodies, antibody drug conjugates (ADC) derived from monoclonal antibodies have also developed rapidly in recent years, and have achieved varying degrees of breakthrough. So far, cancer and inflammatory diseases are the most common diseases for antibody drugs.

[0007] Cancer immunotherapy is a successful cancer treatment method that has been widely recognized by the medical community. The immune system has the ability to recognize and kill tumor cells, but tumor cells can evade anti-tumor immunity through various mechanisms. Immune checkpoint molecules can regulate the degree of activation of immune cells and play an important role in the normal activation of the body's immune system and the prevention of autoimmune diseases. Overexpression of immune checkpoint molecules such as PD-1, PD-L1, and CTLA4 is one of the important factors leading to tumor immune escape, and immune checkpoint inhibitors are also a hot topic in tumor immunotherapy. Currently, although antibody drugs targeting PD-1, PD-L1, and CTLA4 have achieved great success in clinical trials, they are only effective for a small number of tumors, and the efficacy for most tumors is still not ideal, so there is an urgent need to develop antibody drugs targeting other new targets to address unmet clinical needs.

[0008] Herpes Virus Entry Mediator (HVEM) is one of the members of TNFR family, which is expressed in immune cells such as B cells, T cells, myeloid cells, NK cells and dendritic cells. In addition, HVEM is also highly expressed on the surface of tumor cells such as gastric cancer, colon cancer, melanoma and liver cancer. HVEM has multiple functionally different ligands, mainly including BTLA, LIGHT, LT-alpha, CD160 and SALM5. BTLA is an important ligand of HVEM, mainly expressed in B cells, T cells and DC cells, and its cytoplasmic region contains an ITIM domain, which can recruit SHP1 and SHP2 to transmit inhibitory signals after binding to HVEM, thereby inhibiting the growth of lymphocytes and the release of cytokines. Studies have shown that HVEM can be used as a target for tumor immunotherapy, and BTLA / HVEM signaling pathway inhibitors can inhibit tumor growth.

[0009] The present application screens a series of BTLA blocking anti-HVEM antibodies by hybridoma technology, which have good affinity, specificity and functional activity, can block the binding of BTLA and HVEM, promote the activation of T cells, and have application potential in cancer treatment. SUMMARY

[0010] The technical problem to be solved by the present application is to provide a series of BTLA blocking anti-HVEM antibodies or antibody fragments, which have good affinity, specificity and functional activity, and can effectively promote the activation of T cells.

[0011] The present application provides a series of anti-HVEM antibodies or fragments thereof.

[0012] The anti-HVEM antibody or fragment thereof contains a heavy chain complementarity determining region in the heavy chain variable region VH, and the amino acid sequence of the heavy chain complementarity determining region 1 (VH-CDR1) contained therein is at least one of SEQ ID NO. 2, SEQ ID NO. 10, and SEQ ID NO. 18.

[0013] The anti-HVEM antibody or fragment thereof contains a heavy chain complementarity determining region in the heavy chain variable region VH, and the amino acid sequence of the heavy chain complementarity determining region 2 (VH-CDR2) contained therein is at least one of SEQ ID NO. 3, SEQ ID NO. 11, and SEQ ID NO. 19.

[0014] The anti-HVEM antibody or fragment thereof contains a heavy chain complementarity determining region in the heavy chain variable region VH, and the amino acid sequence of the heavy chain complementarity determining region 3 (VH-CDR3) contained therein is at least one of SEQ ID NO. 4, SEQ ID NO. 12, and SEQ ID NO. 20.

[0015] Further, the antibody or fragment thereof against HVEM contains at least one of the heavy chain complementarity determining regions in the heavy chain variable region VH.

[0016] Further, the antibody or fragment thereof against HVEM contains the amino acid sequence of the heavy chain variable region VH as shown in at least one of SEQ ID NO. 1, SEQ ID NO. 9 or SEQ ID NO. 17.

[0017] The present application provides an antibody or fragment thereof against HVEM, which contains a light chain complementarity determining region in the light chain variable region, and the amino acid sequence of the light chain complementarity determining region 1 (VL-CDR1) contained therein is at least one of SEQ ID NO. 6, SEQ ID NO. 14, SEQ ID NO. 22.

[0018] The antibody or fragment thereof against HVEM, which contains a light chain complementarity determining region in the light chain variable region, and the amino acid sequence of the light chain complementarity determining region 2 (VL-CDR2) contained therein is at least one of SEQ ID NO. 7, SEQ ID NO. 15, SEQ ID NO. 23.

[0019] The antibody or fragment thereof against HVEM, which contains a light chain complementarity determining region in the light chain variable region, and the amino acid sequence of the light chain complementarity determining region 3 (VL-CDR3) contained therein is at least one of SEQ ID NO. 8, SEQ ID NO. 16, SEQ ID NO. 24.

[0020] Further, the antibody or fragment thereof against HVEM contains at least one of the heavy chain complementarity determining regions in the heavy chain variable region VH.

[0021] Further, the antibody or fragment thereof against HVEM contains the amino acid sequence of the heavy chain variable region VH as shown in at least one of SEQ ID NO. 1, SEQ ID NO. 9 or SEQ ID NO. 17.

[0022] The amino acid sequence of the heavy chain variable region VH of the antibody or fragment thereof is shown as SEQ ID NO. 1, wherein the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, VH-CDR3 are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 respectively; the amino acid sequence of the light chain variable region VL is shown as SEQ ID NO. 5, wherein the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2, VL-CDR3 are SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 respectively. The antibody is named as antibody 52B7.

[0023] The amino acid sequence of the heavy chain variable region VH of the antibody or fragment thereof is shown as SEQ ID NO. 9, wherein the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, VH-CDR3 are SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 respectively; the amino acid sequence of the light chain variable region VL is shown as SEQ ID NO. 13, wherein the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2, VL-CDR3 are SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 respectively. The antibody is named as antibody 22G5.

[0024] The amino acid sequence of the heavy chain variable region VH of the antibody or fragment thereof is shown as SEQ ID NO. 17, wherein the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, VH-CDR3 are SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 respectively; the amino acid sequence of the light chain variable region VL is shown as SEQ ID NO. 21, wherein the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2, VL-CDR3 are SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24 respectively. The antibody is named as antibody 26C3.

[0025] Further, the CDR regions of the antibody or fragment thereof are grafted with human antibody framework regions FR to form a humanized antibody.

[0026] Further, the heavy chain constant region of the antibody or fragment thereof can be from the constant region of the heavy chain of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, IgD, and the light chain constant region can be from the constant region of the light chain of human immunoglobulin κ, λ.

[0027] Further, the above-mentioned antibody fragment can be Fab (antigen binding fragment) or scFv (single-chain fragment variable).

[0028] The present application also provides a nucleic acid molecule encoding the above-mentioned anti-HVEM antibody or fragment thereof.

[0029] The present application also provides a recombinant vector comprising the above-mentioned nucleic acid molecule encoding the anti-HVEM antibody or fragment thereof, which can be a plasmid or a viral vector.

[0030] The present application also provides a cell comprising the above-mentioned recombinant vector, which can be a eukaryotic cell or a prokaryotic cell.

[0031] The present application also provides the use of the above-mentioned anti-HVEM antibody or fragment thereof in promoting T cell activation.

[0032] The present application also provides the use of the above-mentioned anti-HVEM antibody or fragment thereof in the preparation of a medicament for treating or preventing a tumor.

[0033] Further, the tumor in the above-mentioned use is colon cancer or prostate cancer.

[0034] The above-mentioned antibody of the present application can be prepared into various forms of pharmaceutical preparations according to the conventional techniques of pharmacy. Preferably, the liquid injection and the freeze-dried injection are used.

[0035] The above-mentioned antibody of the present application can form a pharmaceutical composition with other drugs, which can be used to treat diseases together with other treatment methods, including chemotherapy, radiotherapy, biotherapy, etc.

[0036] The present application has the following advantages:

[0037] The present application provides a series of BTLA blocking anti-HVEM antibodies with good affinity, specificity and functional activity, which can block the binding of BTLA and HVEM, promote T cell activation, and have application potential in cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 . Competitive screening of hybridoma antibodies.

[0039] Figure 2 . Screening of cell binding activity of hybridoma antibodies. The left curve is the mlgG control, and the right curve is the hybridoma antibody.

[0040] Figure 3 . Screening of cell functional activity of hybridoma antibodies.

[0041] Figure 4 Screening of human, monkey, mouse HVEM binding activity of hybridoma antibodies.

[0042] Figure 5 Recombinant anti-HVEM antibodies block the binding of BTLA to HVEM.

[0043] Figure 6 Recombinant anti-HVEM antibodies relieve the inhibition of Jurkat / BTLA / NFAT-luc cells by HVEM.

[0044] Figure 7 Recombinant anti-HVEM antibodies binding activity to human, monkey, mouse HVEM. DETAILED DESCRIPTION

[0045] The present application provides a series of BTLA blocking anti-HVEM antibodies with good affinity, specificity and functional activity, which can block the binding of BTLA to HVEM, promote the activation of T cells, and have application potential in cancer treatment.

[0046] The anti-HVEM antibodies of the present application are obtained by screening using hybridoma technology. Human HVEM protein is mixed with adjuvant and immunized mice, and after the serum titer is qualified, the mouse spleen cells are separated, cultured in vitro after fusion with mouse myeloma cells, and the cell culture supernatant containing antibodies is obtained. First, affinity ELISA is used to screen hybridoma antibodies with HVEM binding activity, and further competitive ELISA is used to screen hybridoma antibodies that can block the binding of BTLA to HVEM. Then, flow cytometry is used to screen hybridoma antibodies with good binding activity to CHO-K1 / HVEM cells, and SPR is used to screen hybridoma antibodies with good binding kinetics. The present application further screens hybridoma antibodies that can relieve the inhibition of Jurkat / BTLA / NFAT-luc cells by HVEM through cell functional activity, indicating that the anti-HVEM antibodies have the function of promoting the activation of T cells. The present application also detects the cross-reactivity of the antibodies to human, monkey, mouse HVEM protein, and finds that antibody 52B7 can better recognize monkey HVEM, while all antibodies do not show binding signals with mouse HVEM. Specificity screening shows that the anti-HVEM antibodies of the present application do not have non-specific binding signals in cell lysate ELISA and flow cytometry.

[0047] In order to obtain the amino acid sequence of the hybridoma antibody, the antibody mRNA of the hybridoma is sequenced to obtain the variable region sequence of the hybridoma antibody, and a recombinant monoclonal antibody is prepared and verified for functional activity. The antibody variable region mRNA gene is amplified by PCR using an upstream signal peptide primer and a downstream constant region primer, and the antibody variable region gene is obtained by further sequencing. The mouse antibody variable region is spliced and fused with the antibody constant region to construct an expression vector. The expression plasmid containing the antibody gene is transfected into HEK293 cells for transient expression, and the antibody is purified by protein G affinity purification, and the purity and content of the antibody are confirmed by SDS-PAGE and spectrophotometry for further verification.

[0048] The recombinant monoclonal antibody obtained is verified in terms of competition, cell functional activity, species cross-reactivity, binding kinetics, etc. according to the detection method of the hybridoma screening, so as to ensure that the activity of the recombinant monoclonal antibody meets the expectation.

[0049] The CDR region of the anti-HVEM antibody of the present application can be spliced with a human antibody framework region FR to form a humanized antibody.

[0050] The anti-HVEM antibody of the present application can be modified into an antibody fragment such as Fab (antigen binding fragment) and scFv (single-chain fragment variable) by conventional genetic recombination technology. The antibody fragment such as Fab and scFv has a small volume and strong tissue permeability, and has unique advantages in some application fields. Fab is a heterodimer composed of heavy chain variable region-constant region 1 (VH-CH1) and light chain variable region-constant region (VL-CL), and the molecular size is 1 / 3 of IgG. Since there is no Fc segment, the immune effect induced by Fab is significantly reduced compared with IgG, and the cytokine release effect is weak. At present, antibody drugs with Fab structure such as abciximab and ranibizumab have been approved for marketing. scFv is formed by fusion of VH and VL and the linker between them, and the molecular size is only 1 / 6 of IgG, which has the characteristics of strong tissue permeability and short half-life, and has unique advantages in imaging diagnosis and some treatment fields. The bispecific antibody blinatumomab based on scFv has also been approved for marketing. Antibody fragments can be further fused with other proteins or coupled with other small molecules for targeted delivery for diagnosis and treatment of diseases.

[0051] The anti-HVEM antibody of the present application can be further improved in affinity by mutating the amino acids in the CDR region through genetic engineering techniques. The CDR region of the antibody plays a key role in the binding of the antibody to the antigen, and the amino acids therein can interact with the amino acids of the antigen through hydrogen bonds, ionic bonds, van der Waals forces, etc. By mutating the amino acids in the CDR region of the antibody, the interaction between the CDR and the antigen can be further enhanced, thereby improving the affinity of the antibody. The application of antibody library technology in the evolution of antibody affinity is already quite mature, and the mutation library of the antibody can be established through strategies such as alanine hot spot mutation and error-prone PCR, and high-throughput screening of the mutated antibody can be performed to realize the evolution of the affinity of the antibody in vitro.

[0052] The antibody of the present application can be expressed by a stable cell line for large-scale production of the protein. The gene encoding the amino acid of the antibody can be obtained through conventional genetic recombination techniques, and after DNA sequence optimization, synthesis, and PCR amplification, it can be inserted into an expression vector. The vector used can be a plasmid, virus, or gene fragment commonly used in molecular biology. A protein secretion signal peptide gene is added at the front end of the DNA sequence encoding the antibody to ensure that the antibody can be secreted outside the cell. The vector sequence contains elements for gene expression, such as promoter, protein translation initiation and termination signal, and polyadenylate (PolyA). The vector contains an antibiotic resistance gene and a replication element to facilitate the replication of the vector in host cells such as bacteria for vector preparation. In addition, the vector can also contain a selection gene to facilitate the selection of stably transfected host cells for the construction of a stable expression cell line.

[0053] After the construction of the vector containing the DNA sequence encoding the antibody is completed, the vector can be used to transfect or transform host cells to express the corresponding protein. There are various expression systems that can be used to express the antibody, which can be eukaryotic cells or prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, etc. Since prokaryotic cells tend to form inclusion bodies when expressing complete antibodies, mammalian cells are the preferred system for expressing the protein. There are various mammalian cells that can be used for large-scale expression of antibodies, such as CHO cells, HEK293 cells, NS0 cells, COS cells, etc., which are all included in the list of cells that can be used in the present application. The recombinant vector containing the antibody gene can be transfected into the host cell, and there are various methods for transfecting the cell, including electroporation, liposome transfection, and calcium phosphate transfection, etc.

[0054] A preferred method for expressing the protein is to use a host cell stably transfected with a recombinant vector containing a selective gene. For example, after stably transfecting a host cell lacking neomycin resistance with a recombinant vector containing a neomycin resistance gene, the concentration of neomycin in the cell culture medium can be increased to select a stable cell line with high expression; for another example, after stably transfecting a host cell lacking dihydrofolate reductase (DHFR) with a recombinant vector containing a DHFR gene, the concentration of methotrexate (MTX) in the cell culture medium can be increased to select a stable cell line with high expression.

[0055] Other expression systems, such as insect cells, yeast, bacteria, etc., other than mammalian cells can also be used to express the antibody or fragment thereof of the present application, which are also included in the host cells usable in the present application. The protein expression amount of these expression systems is higher than that of mammalian cells in some cases, but inclusion bodies are easily formed, thus further protein renaturation is required.

[0056] The antibody of the present application can also be carried and expressed by viral vectors, including but not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, herpes simplex virus-based vectors, lentiviral vectors, etc.

[0057] The anti-HVEM antibody of the present application can be used for the detection of HVEM, including ELISA and flow cytometry. The anti-HVEM antibody of the present application shows no binding signal with various different tissue-derived HVEM-negative cell components by ELISA and flow cytometry analysis, indicating that the antibody has good specificity. The anti-HVEM antibody 52B7 of the present application can also recognize monkey HVEM protein, which facilitates the pharmacokinetic study and safety evaluation by means of cynomolgus monkeys and other primates.

[0058] The anti-HVEM antibody of the present application can relieve the inhibition of Jurkat / BTLA / NFAT-luc cells by HVEM, thereby promoting the activation of T cells, and has application potential in cancer treatment.

[0059] The antibody of the present application can be prepared into various forms of pharmaceutical preparations according to the conventional pharmaceutical technology, and the liquid injection and freeze-dried injection are more preferred.

[0060] The antibody of the present application can form a pharmaceutical composition with other drugs, which can treat diseases together with other treatment methods, including chemotherapy, radiotherapy, biotherapy, etc.

[0061] The following examples illustrate the discovery, preparation, testing and use of the antibodies involved in the present application. However, the scope of the present application is not limited to the examples.

[0062] Example 1, Mouse Immunization

[0063] 6-8 weeks old, 20 g weight, female BALB / c mice were used as immunization hosts, and were adaptively fed for one week before antigen immunization. The purified HVEM-hFc (human HVEM extracellular domain C terminal fusion human IgG1 Fc) was prepared into 1 mg / mL with PBS (pH 7.2), and filtered with a 0.22 μm filter membrane. 50 μL was mixed with 50 μL of immunoadjuvant (QuickAntibody) and injected into the small leg muscles of the mouse. On the 21st day, the same method was used for one-time booster immunization, and serum antibody titer was measured by tail vein blood collection on the 35th day. The HVEM-HSA-His (human HVEM extracellular domain C terminal fusion human albumin and 6xHis tag) protein was coated on an enzyme-labeled plate (50 ng / well), and ELISA was used to determine the mouse serum antibody titer. The mice with antibody titers greater than 32000 were given one-time antigen shock, and the spleen cell fusion was performed 3 days later.

[0064] Example 2, Spleen Cell Fusion

[0065] After the mouse was euthanized, the spleen was isolated under sterile conditions, a 70 μm screen was used to prepare a spleen cell suspension, and the cell suspension was washed twice with a basic culture medium and counted. SP2 / 0 and spleen cells were mixed at a ratio of 1:3, the supernatant was discarded after centrifugation, 1 mL of PEG preheated at 37°C was added dropwise within 1 min, and the mixture was placed in a 37°C environment for 90 s. Then 20 mL of 37°C preheated basic culture medium was added within 6 min. The cells were collected by centrifugation (room temperature, 800 rpm, 3 min), and 20 mL of 37°C preheated HAT culture medium was added to resuspend the cells. The fused cells were added to a 96-well cell culture plate at a density of 1x10 5 The cells were cultured in a carbon dioxide cell culture incubator, and when the cell confluence was more than 70%, the culture supernatant was taken for ELISA detection.

[0066] Example 3, Affinity ELISA Screening

[0067] Coat ELISA plate with HVEM-HSA-His (200 ng / well) at 4°C overnight. Wash plate 3 times with PBST, add 5% BSA blocking solution (300 μL / well), incubate at 37°C for 2 h. Wash plate 3 times with PBST, add hybridoma cell culture supernatant (50 μL / well), isotype control mlgG (2 μg / ml, 50 μL / well), competitive antibody 4CB (2 μg / ml, 50 μL / well), incubate at 37°C for 1 h. Wash plate 3 times with PBST, add BTLA-hFc solution (50 μL / well) at a concentration of 20 μg / mL prepared in PBS, incubate at 37°C for 1 h. Wash plate 3 times with PBST, add 5% BSA diluted 5000 times with HRP-donkey anti-human IgG (H+L) (50 μL / well), incubate at 37°C for 1 h. Wash plate 3 times with PBST, add ready-to-use TMB developing solution (100 μL / well), incubate at 37°C in the dark for 5-10 min. Add 2M H2SO4 to stop color development (100 μL / well), detect OD value at 450 nm. A total of 17 positive original clones were obtained (competitive inhibition rate > 40%).

[0068] Example 4, competitive ELISA screening

[0069] Coat ELISA plate with HVEM-HSA-His (200 ng / well) at 4°C overnight. Wash plate 3 times with PBST, add 5% BSA blocking solution (300 μL / well), incubate at 37°C for 2 h. Wash plate 3 times with PBST, add hybridoma cell culture supernatant (50 μL / well), isotype control mlgG (2 μg / ml, 50 μL / well), competitive antibody 4CB (2 μg / ml, 50 μL / well), incubate at 37°C for 1 h. Wash plate 3 times with PBST, add BTLA-hFc solution (50 μL / well) at a concentration of 20 μg / mL prepared in PBS, incubate at 37°C for 1 h. Wash plate 3 times with PBST, add 5% BSA diluted 5000 times with HRP-donkey anti-human IgG (H+L) (50 μL / well), incubate at 37°C for 1 h. Wash plate 3 times with PBST, add ready-to-use TMB developing solution (100 μL / well), incubate at 37°C in the dark for 5-10 min. Add 2M H2SO4 to stop color development (100 μL / well), detect OD value at 450 nm. A total of 17 positive original clones were obtained (competitive inhibition rate > 40%).

[0070] Example 5, hybridoma subcloning

[0071] The hybridoma cells were subcloned by limiting dilution method. The hybridoma cells secreting neutralizing antibodies were collected and counted, and diluted with complete medium at a cell density of 0.5 cells per well in a 96-well cell culture plate for further culture. The remaining cells were expanded and preserved. After 10 days of subculture, the culture supernatant of the single clone well was taken for affinity ELISA and competitive ELISA verification. The positive clones were taken for the second subculture, and the second subculture was verified according to the screening method of the first subculture. A total of 10 hybridoma subclones with a competitive inhibition rate of more than 70% were obtained for further screening (see Table 1). Figure 1 ).

[0072] Example 6, Screening of Cell Binding Activity

[0073] The CHO-K1 cell strain stably expressing HVEM (CHO-K1 / HVEM) was collected, and the number of cells in each group was 1 x 10 6 The cells were washed once with PBS and centrifuged at 3500 rpm for 3 min. The cell pellets were resuspended with 100 μL of hybridoma cell culture supernatant, PBS, isotype antibody mlgG (2 μg / mL), and anti-HVEM positive antibody (2 μg / mL) respectively, and incubated on ice for 60 min. After incubation, the cells were collected by centrifugation and washed with 500 μL of PBS twice. 100 μL of APC-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added to the cell pellets, resuspended and mixed well, and incubated on ice for 60 min in the dark. After incubation, the cells were collected by centrifugation and washed with 500 μL of PBS twice. Finally, the cells were resuspended with 300 μL of PBS for flow cytometry analysis. The results showed that the 10 hybridoma antibodies had good cell binding activity (see Table 1). Figure 2 ).

[0074] Example 7, SPR Screening

[0075] The appropriate coupling amount was calculated according to the formula RL = (Rmax x MWligand) / (Sm x MWanalyte), and the anti-mouse antibody was coupled to the CM5 chip using the amine coupling kit. The hybridoma cell culture supernatant was captured on the chip, and the response value of HVEM-hFc flowing through the Biacore 8K detection channel was detected. Data fitting was performed by Evaluation Software to obtain the binding curve and kinetic parameters. Seven hybridoma antibodies with good binding kinetics were preferred for further screening (see Table 1).

[0076] Table 1. Detection of binding kinetics of hybridoma antibodies

[0077]

[0078] Example 8, screening of cell functional activity

[0079] CHO-K1 / HVEM / anti-CD3-scFv cells were added to 96-well plates (5 x 10 4 cells / well, 50 μL / well) and incubated overnight. Different anti-HVEM antibodies or isotype antibody (2 μg / mL) were added to the 96-well plates and incubated at 37°C for 60 min. Jurkat / BTLA / NFAT-luc cells were added to the 96-well plates (5 x 10 4 cells / well, 50 μL / well) and incubated at 37°C for 6 h. 100 μL luciferase detection reagent was added to each well and incubated at room temperature for 3 min. After sufficient reaction, the luminescence value was detected using a multifunctional microplate reader. Relative to the mlgG isotype control group, all 7 hybridoma antibodies can relieve the inhibition of Jurkat / BTLA / NFAT-luc cells by HVEM, and 4 antibodies (26C3, 22G5, 37H4, 52B7) are better (see Figure 3 ).

[0080] Example 9, ELISA detection of species cross-reactivity

[0081] Human HVEM, monkey HVEM, and mouse HVEM were coated onto an enzyme-labeled plate (50 ng / well) and incubated at 4°C overnight. The plate was washed 3 times with PBST, 5% BSA blocking solution (200 μL / well) was added and incubated at 37°C for 2 h. The plate was washed 3 times with PBST, hybridoma cell culture supernatant (50 μL / well) was added and incubated at 37°C for 1 h. The plate was washed 3 times with PBST, 1:5000 diluted HRP-goat anti-mouse solution (50 μL / well) was added and incubated at 37°C for 1 h. The plate was washed 3 times with PBST, ready-to-use TMB color developing solution (100 μL / well) was added and incubated at 37°C for 10 min in the dark. Color development was terminated by adding 2M H2SO4 (100 μL / well), and the OD value was detected at 450 nm. ELISA results showed that antibody 52B7 exhibited similar binding signals with human and monkey HVEM, while all 7 hybridoma antibodies did not exhibit binding signals with mouse HVEM (see Figure 4 ).

[0082] Example 10, specificity screening

[0083] The cells were cultured and collected, and the binding of the antibodies to the cell surface proteins was detected by flow cytometry (FCM) using different anti-HVEM antibodies as the primary antibodies and APC-labeled goat anti-mouse IgG (H+L) as the secondary antibody. The results showed that none of the 7 antibodies showed binding signals with BxPC3, MCF-7, and HCT116 cells, but showed obvious binding signals with CHO-K1 / HVEM cells. On the other hand, the cells were cultured and collected, and cell lysate samples were prepared using RIPA lysis buffer and an ultrasonic cell disrupter, coated onto an enzyme-labeled plate (500 ng / well), and coated with HVEM-hFc protein as a positive control. The binding of the antibodies to the cell lysate components was detected by ELISA using different anti-HVEM antibodies as the primary antibodies and HRP-goat anti-mouse antibodies as the secondary antibodies. The results showed that none of the 7 antibodies showed binding signals with SKOV3, HEPG2, ACHN, HCT116, MCF-7, and HEK293 cells, but showed obvious binding signals with coated HVEM-hFc.

[0084] Example 11, Obtaining the Variable Region Sequences of the Antibodies

[0085] The hybridoma cell subclones were collected, and the RNA was extracted by the Trizol method. The extracted RNA was used as a template for reverse transcription to obtain cDNA. The heavy and light chain variable regions of the antibodies were amplified by PCR using degenerate primers (Novagen Ig-Primer Sets), and the PCR amplification products were detected by agarose gel electrophoresis. The target DNA fragments were obtained using a gel recovery kit, followed by TA cloning to construct recombinant plasmids. The recombinant plasmids were transformed into competent cells by heat shock, and blue-white spot screening was performed by plating. White single colonies were picked into 0.5 mL of LB liquid medium, and incubated at 37°C, 220 rpm for 3 h. The bacterial solution was sent for sequencing. The variable region amino acid sequences of the anti-HVEM antibodies 52B7, 22G5, and 26C3 are shown in Table 2.

[0086] Table 2. Variable region amino acid sequences of anti-HVEM antibodies

[0087]

[0088] Example 12, Construction and Preparation of Recombinant Monoclonal Antibodies

[0089] The heavy and light chain variable region gene fragments were spliced with signal peptide and mouse heavy chain (IgG2a) and light chain (K) constant region gene fragments, respectively, by overlap PCR, and sequencing identification was performed. The spliced antibody heavy chain and light chain genes were inserted into pTT5 plasmid, respectively, and the recombinant plasmid was transfected into HEK293 cells by PEI method, and the antibody was transiently expressed by serum-free suspension culture. The supernatant of the cells cultured for 7 days was collected, filtered through a 0.22 μm filter membrane, and the antibody was purified by protein G affinity chromatography. The antibody was ultrafiltrated and replaced into PBS solution, and the purity and concentration of the antibody were identified by reducing SDS-PAGE and NanoDrop 2000, and the antibody was stored at -80°C after aliquotting.

[0090] Example 13, verification of blocking activity of recombinant monoclonal antibody

[0091] The HVEM-HSA-His coated enzyme-labeled plate (200 ng / well) was incubated at 4°C overnight. The plate was washed with PBST for 3 times, 5% BSA blocking solution (300 μL / well) was added, and incubated at 37°C for 2 h. The plate was washed with PBST for 3 times, the concentration of recombinant antibodies 52B7, 22G5 and 26C3 was set as 20 μg / mL-0.02 μg / mL, and incubated at 37°C for 1 h. The plate was washed with PBST for 3 times, BTLA-hFc solution (50 μL / well) with a concentration of 20 μg / mL prepared in PBS was added, and incubated at 37°C for 1 h. The plate was washed with PBST for 3 times, HRP-donkey anti-human IgG (H+L) diluted 5000 times with 5% BSA (50 μL / well) was added, and incubated at 37°C for 1 h. The plate was washed with PBST for 3 times, ready-to-use TMB color developing solution (100 μL / well) was added, and incubated at 37°C in the dark for 10 min. 2M H2SO4 was added to terminate the color development (100 μL / well), and the OD value was detected at 450 nm. The results showed that the recombinant monoclonal antibodies 52B7, 22G5 and 26C3 all showed good BTLA / HVEM blocking activity (see Figure 5 ).

[0092] Example 14, functional verification of recombinant monoclonal antibody

[0093] According to the detection method of hybridoma screening, the cell function activity of recombinant monoclonal antibodies 52B7, 22G5 and 26C3 was verified. The results showed that the antibodies 52B7, 22G5 and 26C3 could all relieve the inhibition of HVEM on Jurkat / BTLA / NFAT-luc cells (see Figure 6 ).

[0094] Example 15, detection of species cross-reactivity

[0095] Human, monkey, and mouse HVEM were coated onto ELISA plates (50 ng / well) at 4°C overnight. Plates were washed 3 times with PBST, and 5% BSA blocking solution (200 μL / well) was added and incubated at 37°C for 2 hours. Plates were washed 3 times with PBST, and PBS control (50 μL PBS), mouse IgG isotype control (2 ug / mL, 50 μL / well) were added, and recombinant monoclonal antibodies 52B7, 22G5, 26C3 (2 ug / mL, 50 μL / well) were added, and incubated at 37°C for 1 hour. Plates were washed 3 times with PBST, and 1:5000 diluted HRP-goat anti-mouse solution (50 μL / well) was added, and incubated at 37°C for 1 hour. Plates were washed 3 times with PBST, and ready-to-use TMB color developing solution (100 μL / well) was added, and incubated at 37°C for 5-10 minutes in the dark. Color development was stopped by adding 2M H2SO4 (100 μL / well), and OD values were detected at 450 nm. ELISA results showed that antibody 52B7 exhibited similar binding signals to human and monkey HVEM, while all 3 antibodies did not exhibit binding signals to mouse HVEM (see Figure 2). Figure 7 Further Biacore detection showed that antibody 52B7 exhibited good binding kinetics to both human and monkey HVEM (see Table 3).

[0096] Table 3. Binding kinetics analysis of antibody 52B7 to human and monkey HVEM.

[0097] Ka (1 / Ms) Kd (1 / s) KD (M) Human HVEM 2.14E+05 1.51E-07 7.05E-13 Monkey HVEM 1.78E+05 5.56E-04 3.11E-09

[0098] SEQUENCE LISTING

[0099] SEQ ID NO. 1

[0100] Antibody 52B7 heavy chain variable region VH amino acid sequence

[0101] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTIYGVHWIRQSPGKGLEWLGVIWTGGNTDYNAAFMSRLSITKDNSKSQVFF

[0102] KMNSLQPDDTAIYYCAKRGYNGNFGFAYWGQGTLVTVSA

[0103] SEQ ID NO. 2

[0104] Antibody 52B7 heavy chain variable region VH-CDR1 amino acid sequence

[0105] GFSLTIYGVH

[0106] SEQ ID NO. 3

[0107] Antibody 52B7 heavy chain variable region VH-CDR2 amino acid sequence

[0108] VIWTGGNTDYNAAFMS

[0109] SEQ ID NO. 4

[0110] Antibody 52B7 heavy chain variable region VH-CDR3 amino acid sequence

[0111] RGYNGNFGFAY

[0112] SEQ ID NO. 5

[0113] Antibody 52B7 light chain variable region VL amino acid sequence

[0114] EIVLTQSPALMAASPGEKVTITCSVSSNISSGNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSRSGTSYSLTISSME

[0115] AEDAATYYCQQWSRYPLTFGAGTILELK

[0116] SEQ ID NO. 6

[0117] Antibody 52B7 light chain variable region VL-CDR1 amino acid sequence

[0118] SVSSNISSGNLH

[0119] SEQ ID NO. 7

[0120] Antibody 52B7 light chain variable region VL-CDR2 amino acid sequence

[0121] GTSNLAS

[0122] SEQ ID NO. 8

[0123] Antibody 52B7 light chain variable region VL-CDR3 amino acid sequence

[0124] QQWSRYPLT

[0125] SEQ ID NO. 9

[0126] Antibody 22G5 heavy chain variable region VH amino acid sequence

[0127] EVQLQESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAFISSGSSSIYYADTVKGRFTISRDNPRNTLI

[0128] LQMTSLRSEDTAMYYCVRGLYGNFAWFAYWGQGTLVTVSA

[0129] SEQ ID NO. 10

[0130] Antibody 22G5 heavy chain variable region VH-CDR1 amino acid sequence

[0131] GFTFSSFGMH

[0132] SEQ ID NO. 11

[0133] Antibody 22G5 heavy chain variable region VH-CDR2 amino acid sequence

[0134] FISSGSSSIYYADTVKG

[0135] SEQ ID NO. 12

[0136] Antibody 22G5 heavy chain variable region VH-CDR3 amino acid sequence

[0137] GLYGNFAWFAY

[0138] SEQ ID NO. 13

[0139] Antibody 22G5 light chain variable region VL amino acid sequence

[0140] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYFHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI

[0141] IRVEAEDLGVYFCSQSSHAPFTFGSGTKLEIK

[0142] SEQ ID NO. 14

[0143] Antibody 22G5 light chain variable region VL-CDR1 amino acid sequence

[0144] RSSQSLVHSNGNTYFH

[0145] SEQ ID NO. 15

[0146] Antibody 22G5 light chain variable region VL-CDR2 amino acid sequence

[0147] KVSNRFS

[0148] SEQ ID NO. 16

[0149] Antibody 22G5 heavy chain variable region VH-CDR1 amino acid sequence

[0150] SQSSHAPFT

[0151] SEQ ID NO. 17

[0152] Antibody 26C3 heavy chain variable region VH amino acid sequence

[0153] QVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGDIYPGSDGTNYDEKFKSKATLTVDTSSSTAY

[0154] MQLSSLTSEDSAVYYCIREGNYVWFAYWGQGTLVTVSA

[0155] SEQ ID NO. 18

[0156] Antibody 26C3 heavy chain variable region VH-CDR1 amino acid sequence

[0157] GYTFTSYWMH

[0158] SEQ ID NO. 19

[0159] Antibody 26C3 heavy chain variable region VH-CDR2 amino acid sequence

[0160] DIYPGSDGTNYDEKFKS

[0161] SEQ ID NO. 20

[0162] Antibody 26C3 heavy chain variable region VH-CDR3 amino acid sequence

[0163] EGNYVWFAY

[0164] SEQ ID NO. 21

[0165] Antibody 26C3 light chain variable region VL amino acid sequence

[0166] DIQMTQTTSSLSASLGDRVTISCRASQDISNSLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ

[0167] EDIATYFCQQGNTLPYTFGGGTKLEIKR

[0168] SEQ ID NO. 22

[0169] Antibody 26C3 heavy chain variable region VH-CDR1 amino acid sequence

[0170] RASQDISNSLN

[0171] SEQ ID NO. 23

[0172] Antibody 26C3 heavy chain variable region VH-CDR2 amino acid sequence

[0173] YTSRLHS

[0174] SEQ ID NO. 24

[0175] Antibody 26C3 heavy chain variable region VH-CDR3 amino acid sequence QQGNTLPYT.

Claims

1. An antibody or antigen-binding fragment thereof against HVEM, characterized in that: The amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are represented by SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20, respectively; and the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 are represented by SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, respectively.

2. The antibody or antigen-binding fragment thereof against HVEM of claim 1, characterized in that At least one of the following is met: 1) the amino acid sequence of the heavy chain variable region VH is represented by SEQ ID NO. 17; 2) the amino acid sequence of the light chain variable region VL is represented by SEQ ID NO.

21.

3. The antibody or antigen-binding fragment thereof against HVEM of any one of claims 1 or 2, characterized in that: The variable region of the antibody is spliced from the complementarity determining regions CDR and the framework region FR of a human antibody.

4. The antibody or antigen-binding fragment thereof against HVEM of any one of claims 1 or 2, characterized in that At least one of the following is met: 1) the constant region of the heavy chain of the antibody is selected from the constant region of the heavy chain of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA or IgD; 2) the constant region of the light chain of the antibody is selected from the constant region of the light chain of human immunoglobulin κ or λ.

5. The antibody or antigen-binding fragment thereof against HVEM of any one of claims 1 or 2, characterized in that: The antigen binding fragment is Fab (antigen binding fragment) or scFv (single-chain fragment variable).

6. A nucleic acid molecule encoding the antibody or antigen binding fragment thereof against HVEM according to any one of claims 1 to 5.

7. A recombinant vector comprising the nucleic acid molecule according to claim 6.

8. A cell comprising the recombinant vector according to claim 7.

9. Use of the antibody or antigen binding fragment thereof against HVEM according to any one of claims 1 to 5 in the preparation of a medicament for treating a tumor; the tumor is colon cancer or prostate cancer.

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