CN116715769A

CN116715769A - anti-HVEM antibody, preparation method and application thereof

Info

Publication number: CN116715769A
Application number: CN202310557714.1A
Authority: CN
Inventors: 涂永燕; 谯凡; 杨金亮; 勾蓝图
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-05-26
Filing date: 2023-05-17
Publication date: 2023-09-08

Abstract

The invention relates to the technical field of antibodies, in particular to preparation of anti-HVEM antibodies or fragments thereof and application thereof. The technical problem to be solved by the invention is to provide a series of BTLA blocking type anti-HVEM antibodies or fragments thereof. The anti-HVEM antibody has good affinity, specificity and functional activity, can block the combination of BTLA and HVEM, promote the activation of T cells, and has application potential in the aspect of cancer treatment.

Description

anti-HVEM antibody, preparation method and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-HVEM antibody and fragments thereof, a preparation method and application thereof.

Background

Antibodies are biological macromolecules composed of heavy chains and light chains, are secreted by B lymphocytes, and play an important role in humoral immunity of organisms. The heavy chain or the light chain of the antibody molecule is respectively composed of a variable region and a constant region, wherein the variable region mainly plays a role in binding a target antigen, and the constant region mainly plays an immune regulation effect. Antibodies can be classified into IgG, igM, igE, igA, igD and the like according to spatial structure and amino acid sequence characteristics, and heavy and light chains can be further subdivided into a plurality of subtypes. For example, human IgG heavy chains can be classified as IgG1, igG2, igG3, and IgG4, and light chains can be classified as kappa and lambda. Since antibodies can specifically and efficiently bind to target molecules or target cells, regulate signaling pathways downstream of target molecules, or kill target cells by immune effects, antibodies can be developed as drugs for disease treatment. Antibodies have been developed currently as an important biotechnological drug, where monoclonal antibodies take up a substantial part, which in turn are predominantly of the IgG type, so that so-called monoclonal antibodies are often referred to as IgG type antibodies.

An IgG type antibody molecule is a tetramer consisting of 2 heavy chains and 2 light chains with a molecular weight of about 150kD via interchain disulfide bonds. Antibody molecules can be divided into variable and constant regions according to structural and functional characteristics, with the variable region acting primarily for antigen binding and the constant region acting primarily for immunological effects and transport. The variable region of an antibody can be further divided into complementarity determining region CDRs and framework regions FRs, wherein each of the heavy or light chain contains 3 CDR regions (heavy chain VH-CDR1, VH-CDR2, VH-CDR3, light chain VL-CDR1, VL-CDR2, VL-CDR 3.) and 4 FR regions flanking the CDR regions (FR 1, FR2, FR3, FR 4). The loop formed by the CDR region is the main part of the binding of the antibody molecule to the antigen, and the FR region forms the supporting structure of the CDR region by spatial folding. The specific recognition of antibodies against different antigen molecules is mainly achieved by amino acid polymorphisms of the 6 CDR regions (VH-CDR 1, 2, 3 and VL-CDR1, 2, 3) together with conformational polymorphisms of the loop. Because of the high structural similarity of the FR regions of different antibodies, when the CDR regions of one antibody are substituted for the CDR regions of other antibody molecules, if the FR regions of different antibody molecules are appropriately matched, the conformational change of the CDR regions before and after substitution is small, so that the novel variable region formed after substitution can still retain the antigen binding ability, which is the basis of CDR grafting (CDR grafting) technology. The CDR regions of the murine antibody may be recombined with the human FR regions into a humanized antibody by CDR grafting techniques to replace the CDRs of the human antibody (humanized antibody), and if the FR regions between the human-murine antibody are properly matched, the antigen binding capacity may still be maintained.

Hybridoma technology is currently an important technology in the antibody discovery process. After the antigen is used for immunizing the mouse, the B cells of the mouse develop into germinal centers in the lymphoid tissues, and the affinity of the antibody is gradually improved through somatic cell high frequency mutation (SHM). The mouse spleen cells are isolated and fused with mouse myeloma cells in vitro to form hybridomas, and hybridomas producing the target antibodies can be selected by measuring the antibody activity in the culture supernatants of the hybridoma cells. The hybridoma cells are gradually monoclonal after subcloning, mRNA of the subcloned cells is extracted for antibody variable region gene sequencing, and the amino acid sequence of the antibody variable region can be analyzed. Currently, most antibodies on the market are obtained by hybridoma technology screening. Since hybridoma technology derived antibody molecules complete the affinity maturation process in mice, those B cells that are cross-reactive with the mouse self-protein are cleared in the mouse bone marrow by "negative selection", and thus the antibody molecules are effective in reducing non-specific binding to the self-protein or its similar proteins.

Monoclonal antibodies can exert pharmacological effects through a variety of mechanisms. The antibody variable region can bind to extracellular soluble ligand, block the binding of ligand and receptor and cut off downstream signal transmission induced by ligand, so that the antibody medicine developed by taking immune cell factor as target can improve inflammatory diseases, for example, antibodies such as adalimumab, belimumab, siltuximab are already marketed in batches. The antibody constant region can exert immune regulation effects, including Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) and the like, so that antibody drugs developed by taking tumor cell surface molecules as targets can kill tumor cells, such as antibodies on the market such as rituximab, trastuzumab, cetuximab and the like, have been greatly successful. In addition, novel antibody technologies such as bispecific antibodies and Antibody Drug Conjugates (ADC) derived based on monoclonal antibodies have been developed rapidly in recent years, and have all achieved breakthrough of different degrees. Cancer and inflammatory diseases are currently the most used areas of disease for antibody drugs.

Cancer is a type of disease that is severely threatening to human health and life. With aging and changes in people's lifestyle, the incidence of cancer is increasing and also the trend of younger, so cancer treatment is always a research hotspot in the medical field. Cancer immunotherapy is a successful cancer treatment modality, and has gained widespread acceptance by the medical community. The immune system has the ability to recognize and kill tumor cells, however tumor cells can evade anti-tumor immunity through a variety of mechanisms. Immune checkpoint (checkpoint) molecules can regulate the degree of immune cell activation, playing an important role in the normal activation of the immune system of the body and in preventing autoimmunity. Overexpression of immune checkpoint molecules such as PD-1, PD-L1 and CTLA4 is one of important factors for tumor immune escape, and immune checkpoint inhibitors are hot spots of tumor immunotherapy. At present, although antibody drugs represented by PD-1, PD-L1, CTLA4 and other targets have achieved great success in clinic, the antibody drugs are effective only on a few tumors, but the curative effect on most tumors is still not ideal, so that development of antibody drugs aiming at other new targets is urgently needed to solve the unmet clinical demands.

The herpes virus invasion mediator (Herpes Virus Entry Mediator, HVEM) is one of the TNFR family members and 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, liver cancer, and the like. HVEM has a number of functionally diverse ligands, mainly including BTLA, LIGHT, LT- α, CD160, SALM5, and the like. BTLA is an important ligand of HVEM, mainly expressed in B cells, T cells and DC cells, whose cytoplasmic region contains ITIM domains, and can recruit SHP1, SHP2 to transmit inhibition signals upon binding to HVEM, inhibiting lymphocyte growth and cytokine release. Studies show that HVEM can be used as a target point of tumor immunotherapy, and BTLA/HVEM signal path inhibitor can inhibit the growth of tumors.

The invention screens a series of BTLA blocking-type anti-HVEM antibodies through hybridoma technology, has good affinity, specificity and functional activity, can block the combination of BTLA and HVEM, promotes the activation of T cells, and has application potential in the aspect of cancer treatment.

Disclosure of Invention

The invention aims to provide a series of BTLA blocking-type anti-HVEM antibodies or antibody fragments, which have good affinity, specificity and functional activity and can effectively promote the activation of T cells.

The present invention provides a series of anti-HVEM antibodies or fragments thereof.

An anti-HVEM antibody or fragment thereof, which comprises a heavy chain complementarity determining region in the heavy chain variable region VH, comprising a heavy chain complementarity determining region 1 (VH-CDR 1) having at least one of the amino acid sequences set forth in SEQ ID No.2, SEQ ID No.10, and SEQ ID No. 18.

An anti-HVEM antibody or fragment thereof, which comprises a heavy chain complementarity determining region in the heavy chain variable region VH, comprising a heavy chain complementarity determining region 2 (VH-CDR 2) having at least one of the amino acid sequences set forth in SEQ ID No.3, SEQ ID No.11, and SEQ ID No. 19.

An anti-HVEM antibody or fragment thereof, which comprises a heavy chain complementarity determining region in the heavy chain variable region VH, comprising a heavy chain complementarity determining region 3 (VH-CDR 3) having an amino acid sequence set forth in at least one of SEQ ID No.4, SEQ ID No.12, and SEQ ID No. 20.

Further, the heavy chain complementarity determining region of at least one of the above-mentioned heavy chain variable region VH is contained in the antibody or fragment thereof of HVEM.

Further, the amino acid sequence of the heavy chain variable region VH in the antibody or fragment thereof of HVEM described above is at least one of SEQ ID No.1, SEQ ID No.9 or SEQ ID No. 17.

The invention provides an anti-HVEM antibody or fragment thereof, wherein the light chain variable region contains a light chain complementarity determining region, and the amino acid sequence of the light chain complementarity determining region 1 (VL-CDR 1) is shown as at least one of SEQ ID NO.6, SEQ ID NO.14 and SEQ ID NO. 22.

An anti-HVEM antibody or fragment thereof, comprising a light chain complementarity determining region in the light chain variable region, comprising a light chain complementarity determining region 2 (VL-CDR 2) having an amino acid sequence set forth in at least one of SEQ ID No.7, SEQ ID No.15, and SEQ ID No. 23.

An anti-HVEM antibody or fragment thereof, comprising a light chain complementarity determining region in the light chain variable region VH, comprising a light chain complementarity determining region 3 (VL-CDR 3) having at least one of the amino acid sequences set forth in SEQ ID No.8, SEQ ID No.16, and SEQ ID No.24.

Further, the light chain complementarity determining region of at least one of claims 6, 7, and 8 is contained in the light chain variable region VL of the antibody or fragment thereof of HVEM.

Further, the amino acid sequence of the light chain variable region VL in the antibody or fragment thereof of the above HVEM is at least one of SEQ ID NO.5, SEQ ID NO.13 or SEQ ID NO. 21.

Wherein the amino acid sequence of the heavy chain variable region VH of the antibody or the fragment thereof is shown as SEQ ID NO.1, and the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.2, SEQ ID NO.3 and 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 and VL-CDR3 are shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively. This antibody was designated antibody 52B7.

Wherein the amino acid sequence of the heavy chain variable region VH of the antibody or the fragment thereof is shown as SEQ ID NO.9, and the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.10, SEQ ID NO.11 and 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 and VL-CDR3 are shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 respectively. This antibody was designated antibody 22G5.

Wherein the amino acid sequence of the heavy chain variable region VH of the antibody or the fragment thereof is shown as SEQ ID NO.17, and the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.18, SEQ ID NO.19 and 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 and VL-CDR3 are shown as SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24 respectively. This antibody was designated antibody 26C3.

Furthermore, the corresponding CDR regions of the above antibodies or fragments thereof are spliced with FRs of the framework regions of a humanized antibody to form a humanized antibody.

Further, the heavy chain constant region of the antibody or the fragment thereof may be derived from the constant region of the heavy chain of human immunoglobulins IgG1, igG2, igG3, igG4, igM, igE, igA, igD, and the light chain constant region may be derived from the constant region of the light chain of human immunoglobulins kappa and lambda.

Further, the above antibody fragment may be Fab (antigen binding fragment) or scFv (single-chain fragment variable).

The invention also provides nucleic acid molecules encoding the above anti-HVEM antibodies or fragments thereof.

The present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the above anti-HVEM antibody or fragment thereof, which may be a plasmid or a viral vector.

The invention also provides a cell comprising the recombinant vector, wherein the cell can be a eukaryotic cell or a prokaryotic cell.

The invention also provides the use of the above anti-HVEM antibodies or fragments thereof in promoting T cell activation.

The invention also provides application of the anti-HVEM antibody or the fragment thereof in preparing medicaments for treating or preventing tumors.

Further, the tumor in the above uses is colon cancer or prostate cancer.

The antibody of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional pharmaceutical techniques. Liquid injections and freeze-dried injections are more preferred.

The above-described antibodies of the invention may form pharmaceutical compositions with other drugs that may be used in the treatment of diseases with other therapeutic methods including chemotherapy, radiation therapy, biological therapy, and the like.

The invention has the beneficial effects that:

the invention provides a series of BTLA blocking-type anti-HVEM antibodies, which have good affinity, specificity and functional activity, can block the combination of BTLA and HVEM, promote the activation of T cells, and have application potential in the aspect of cancer treatment.

Drawings

FIG. 1 competitive screening of hybridoma antibodies.

FIG. 2 cell binding activity screening of hybridoma antibodies. The left curve is the mIgG control and the right curve is the hybridoma antibody.

FIG. 3 cell function Activity screening of hybridoma antibodies.

FIG. 4 human, monkey, murine HVEM binding activity screening for hybridoma antibodies.

Fig. 5 recombinant anti-HVEM antibodies block BTLA binding to HVEM.

FIG. 6. Recombinant anti-HVEM antibody releases HVEM inhibitory effect on Jurkat/BTLA/NFAT-luc cells.

FIG. 7 binding activity of recombinant anti-HVEM antibodies to human, monkey, murine HVEM.

Detailed Description

The anti-HVEM antibody is obtained by screening by adopting a hybridoma technology. The human HVEM protein is mixed with an adjuvant and immunized into a mouse, after the serum titer is qualified, spleen cells of the mouse are separated, and after the mouse is fused with myeloma cells of the mouse in vitro, the mouse is cultured, and cell culture supernatant containing antibodies is obtained. First, hybridoma antibodies having HVEM binding activity were screened using affinity ELISA, and hybridoma antibodies capable of blocking BTLA binding to HVEM were further screened using competitive ELISA. Then, hybridoma antibodies having good binding activity to CHO-K1/HVEM cells were screened by flow cytometry, and hybridoma antibodies having good binding kinetics were screened by SPR. The invention further screens out hybridoma antibodies capable of relieving the inhibition effect of HVEM on Jurkat/BTLA/NFAT-luc cells through cell function activity, which shows that the anti-HVEM antibodies have the function of promoting T cell activation. The invention also detects the cross-reactivity of the antibody to human, monkey and mouse HVEM proteins, and discovers that the antibody 52B7 can better identify the monkey HVEM, and all the antibodies do not show binding signals with the mouse HVEM. Specific screening showed that the anti-HVEM antibodies of the invention did not have non-specific binding signals in both cell lysate ELISA and flow cytometry.

In order to obtain the amino acid sequence of the hybridoma antibody, the invention sequences the antibody mRNA of the hybridoma to obtain the variable region sequence of the hybridoma antibody, prepares a recombinant monoclonal antibody and performs functional activity verification. The antibody variable region mRNA gene is amplified by PCR through an upstream signal peptide primer and a downstream constant region primer, and further sequenced to obtain the antibody variable region gene. The expression vector is constructed by splicing and fusing the murine antibody variable region and the antibody constant region. The HEK293 cells are transfected by expression plasmids containing antibody genes for transient expression, and the purity and the content of the antibodies are confirmed by protein G affinity purification and SDS-PAGE and spectrophotometry for further verification.

The obtained recombinant monoclonal antibody is respectively verified in aspects of competitiveness, cell function activity, species cross reactivity, binding kinetics and the like according to a detection method of hybridoma screening, so that the activity of each aspect of the recombinant monoclonal antibody is ensured to accord with expectations.

The CDR regions of the anti-HVEM antibody of the invention can be spliced with the FRs of the framework regions of the humanized antibody to form the humanized antibody.

The anti-HVEM antibody of the invention can be modified into antibody fragments such as Fab (antigen binding fragment), scFv (single-chain fragment variable) and the like by conventional gene recombination technology. Antibody fragments such as Fab, scFv and the like have smaller volume and strong tissue permeability, and have unique advantages in some application fields. Fab is a heterodimer consisting of a heavy chain variable region-constant region 1 (VH-CH 1) and a light chain variable region-constant region (VL-CL) with a molecular size of 1/3 of an IgG molecule. Because of the absence of the Fc segment, the Fab-induced immune effect is significantly reduced compared to IgG and cytokine release is weaker. Antibody drugs such as abciximab, ranibizumab, etc. having Fab as structures are currently approved for the market. The scFv is formed by fusing VH and VL and a connecting peptide linker between the VH and the VL, the molecular size is only 1/6 of that of the IgG, and the scFv has the characteristics of strong tissue permeability, short half-life and the like, has unique advantages in the fields of imaging diagnosis and some treatments, and a bispecific antibody blinatumomab based on the scFv is also approved to be marketed. The antibody fragments may further be fused to other proteins, or conjugated to other small molecules, for diagnosis and treatment of diseases by targeted delivery.

The anti-HVEM antibodies of the invention may be used to further increase affinity by engineering amino acid mutations in the CDR regions. The antibody CDR regions play a critical role in the binding of antibodies to antigens, wherein the amino acids may interact with the amino acids of the antigen via hydrogen bonding, ionic bonding, van der waals forces, and the like. By mutating amino acids in the CDR regions of an antibody, the interaction of the CDRs with the antigen can be further enhanced, thereby increasing the affinity of the antibody. The application of the antibody library technology in the aspect of antibody affinity evolution is mature, an antibody mutation library can be established through strategies such as alanine hot spot mutation, error-prone PCR and the like, high-throughput screening of mutant antibodies is carried out, and the affinity evolution of the antibodies is realized in vitro.

The antibodies of the invention can be expressed using stable cell lines for large-scale production of large amounts of proteins. The gene encoding the antibody amino acid can be obtained by conventional gene recombination technology, and can be inserted into an expression vector after DNA sequence optimization, synthesis and PCR amplification. The vectors used may be plasmids, viruses or gene fragments which are customary for molecular biology. A protein secretion signal peptide gene is added at the front end of a DNA sequence for encoding the antibody so as to ensure that the antibody can be secreted outside cells. The vector sequence contains a promoter for gene expression, a protein translation initiation and termination signal, polyadenylation (polyA) and other elements. The vector contains antibiotic resistance genes and replication elements to facilitate replication of the vector in a host cell, such as a bacterium, for vector preparation. In addition, a selectable gene may be included in the vector to facilitate selection of stably transfected host cells for construction of stably expressed cell lines.

After construction of the vector containing the DNA sequence encoding the antibody, the vector may be used to transfect or transform a host cell for expression of the corresponding protein. There are various expression systems that can be used to express antibodies, which can be eukaryotic cells, or prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, and the like. Mammalian cells are the preferred system for expressing the protein because of the ease of inclusion bodies when prokaryotic cells express intact antibodies. 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., all of which are included among the cells that can be used in the present invention. Recombinant vectors containing genes encoding antibodies can be transfected into host cells by a variety of methods including electroporation, lipofection, and calcium phosphate transfection.

A preferred method of protein expression is by stably transfected host cell expression comprising a selectable gene. For example, after stably transfecting a host cell lacking Neomycin resistance with a recombinant vector containing a Neomycin (Neomycin) resistance gene, the concentration of Neomycin may be increased in the cell culture broth to select a stable cell strain with high expression; for example, after stably transfecting a host cell lacking DHFR with a recombinant vector containing the dihydrofolate reductase (DHFR) gene, the concentration of Methotrexate (MTX) may be increased in the cell culture medium to select for stable cell lines with high expression.

Other expression systems besides mammalian cells, such as insect cells, yeast, bacteria, etc., may also be used to express the antibodies or fragments thereof of the present invention, and they are also encompassed by the host cells that can be used in accordance with the present invention. The protein expression level of these expression systems is in some cases higher than that of mammalian cells, but inclusion bodies are easily formed, and thus further protein renaturation is required.

Antibodies of the invention may also be carried and expressed using viral vectors including, but not limited to, adenovirus vectors (adenoviral vectors), adeno-associated virus vectors (adeno-associated viral vectors), retrovirus vectors (retroviral vectors), herpes simplex virus vectors (herpes simplex virus-based vectors), lentivirus vectors (lentiviral vectors), and the like.

The anti-HVEM antibodies of the invention can be used for detection of HVEM, including ELISA and flow cytometry. The anti-HVEM antibody of the present invention showed no binding signal with HVEM negative cell components of various tissue sources, which indicates that the antibody has good specificity, as analyzed by ELISA and flow cytometry. The anti-HVEM antibody 52B7 of the invention also recognizes monkey HVEM proteins, which facilitates pharmacokinetic studies and safety evaluation with primates such as cynomolgus monkeys.

The anti-HVEM antibody can relieve the inhibition of HVEM on Jurkat/BTLA/NFAT-luc cells, thereby promoting the activation of T cells and having application potential in the aspect of cancer treatment.

The antibody of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional techniques of pharmacy, and liquid injections and freeze-dried injections are more preferable.

The antibodies of the invention may be formed into pharmaceutical compositions with other drugs that may be used in the treatment of diseases along with other therapeutic methods including chemotherapy, radiation therapy, biological therapy, and the like.

The following examples illustrate the discovery, preparation, testing and use of antibodies of the invention. The content and use of the invention is not limited to the scope of the embodiments.

Example 1 mouse immunization

Female BALB/c mice, 6-8 weeks old and weighing about 20g, were used as immunization hosts and were subjected to antigen immunization after one week of adaptive feeding. Purified HVEM-hFc (human HVEM ectodomain C-terminal fusion human IgG1 Fc) was formulated to 1mg/mL with PBS (pH 7.2), and after filtration through a 0.22 μm filter, 50. Mu.L was thoroughly mixed with 50. Mu.L of an immunoadjuvant (QuickAntibody) and injected into the calf muscle of the hind leg of the mouse. The immunization was boosted 1 time on day 21 in the same manner, and serum antibody titer was measured by tail vein blood sampling on day 35. HVEM-HSA-His (human HVEM ectodomain C-terminal fusion human albumin and 6 xhis tag) proteins were coated onto an ELISA plate (50 ng/well) and mouse serum antibody titers were determined by ELISA. Mice with antibody titers greater than 32000 were given an antigen impact once and spleen cell fusion was performed after 3 days.

Example 2 spleen cell fusion

After the mice were euthanized, spleens were isolated under aseptic conditions, spleen cell suspensions were prepared using a 70 μm screen, and washed 2 times with basal medium for cell counting. SP2/0 was mixed with splenocytes in a 1:3 ratio, the supernatant was discarded after centrifugation, 1mL of PEG preheated at 37℃was added dropwise over 1min, the mixture was allowed to stand at 37℃for 90s, and then 20mL of basal medium preheated at 37℃was added over 6 min. Cells were collected by centrifugation (at room temperature, 800rpm,3 min), resuspended in 20mL HAT medium pre-warmed at 37℃and then grown according to 1X 10 ⁵ The density of individual spleen cells/holes is that the fused cells are added into a 96-hole cell culture plate and placed into a carbon dioxide cell culture box for culture, when the cell reaches more than 70% confluence, the culture supernatant is taken for ELISA detection.

Example 3 affinity ELISA screening

HVEM-HSA-His solution at 1. Mu.g/mL was prepared with PBS, and an ELISA plate (50. Mu.L/well) was added thereto, and coated overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, hybridoma cell culture supernatants (50. Mu.L/well) were added, and incubated at 37℃for 1h. PBST plates were washed 3 times, added with HRP-goat anti-mouse solution (50. Mu.L/well) diluted 1:5000, and incubated for 1h at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 5-10min in the dark. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. OD value of control well of mouse IgG (2. Mu.g/mL)<0.3, a total of 210 positive original clones (OD value>1.3)。

Example 4 competitive ELISA screening

HVEM-HSA-His coated ELISA plate (200 ng)Well) at 4 ℃ overnight. PBST plates were washed 3 times, 5% BSA blocking solution (300. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, hybridoma cell culture supernatants (50. Mu.L/well), isotype control mIgG (2. Mu.g/ml, 50. Mu.L/well), competing antibody 4CB (2. Mu.g/ml, 50. Mu.L/well) were added and incubated for 1h at 37 ℃. PBST plates were washed 3 times, and a solution of BTLA-hFc (50. Mu.L/well) in PBS was added at a concentration of 20. Mu.g/mL and incubated for 1h at 37 ℃. PBST plates were washed 3 times, diluted 5000-fold with HRP-donkey anti-human lgG (H+L) (50. Mu.L/well) added with 5% BSA, and incubated for 1H at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 5-10min in the dark. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. A total of 17 positive original clones (competitive inhibition rate)>40％)。

Example 5 hybridoma subcloning

The hybridoma cells were subcloned by limiting dilution. Hybridoma cells secreting neutralizing antibodies were collected and counted, diluted with complete medium, added to 96-well cell culture plates at a cell density of 0.5 cells/well for continued culture, and the remaining cells were expanded for seed retention. After 10 days of subcloning culture, the culture supernatants of the monoclonal wells were subjected to affinity ELISA and competitive ELISA assays, respectively. And taking positive clones, continuing to perform secondary subcloning, and continuing to verify the secondary subcloning according to the screening mode of the primary subcloning. 10 hybridoma subclones with competition inhibition rate higher than 70% were obtained in total, and the next screening was performed (see FIG. 1).

Example 6 cell binding Activity screening

The CHO-K1 cell line (CHO-K1/HVEM) stably expressing HVEM was collected, and the number of cells in each group was 1X 10 ⁶ And each. Cells were washed 1 time with PBS and centrifuged at 3500rpm for 3min. The cell pellet was resuspended in 100. Mu.L of hybridoma cell culture supernatant, PBS, isotype antibody mIgG (2. Mu.g/mL), anti-HVEM positive antibody (2. Mu.g/mL), and incubated on ice for 60min. After the incubation was completed, the cells were collected by centrifugation, washed with 500 μl PBS, and repeated 2 times. To the cell pellet, 100. Mu.L of APC-labeled goat anti-mouse IgG (H+L) (1:200 dilution) was added, resuspended, and incubated on ice for 60min in the absence of light. After incubation, cells were collected by centrifugation usingWashing with 500. Mu.L PBS was repeated 2 times. Finally, the cells were resuspended in 300. Mu.L PBS and analyzed by flow cytometry. The results showed that 10 hybridoma antibodies all had good cell binding activity (see fig. 2).

EXAMPLE 7 SPR screening

The appropriate coupling amount was calculated according to the formula rl= (rmax×mwligand)/(sm×mwanalyte), and anti-mouse anti was coupled to CM5 chip using amine coupling kit. Hybridoma cell culture supernatants were captured on chip and the response values of the flow through HVEM-hFc in the channels were detected using Biacore 8K. Data fitting was performed by Evaluation Software to obtain binding curves and kinetic parameters. Preferably 7 hybridoma antibodies with good binding kinetics are subjected to further screening (see Table 1).

TABLE 1 detection of binding kinetics of hybridoma antibodies

Example 8 cell function Activity Screen

CHO-K1/HVEM/anti-CD3-scFv cells were added to 96-well plates (5X 10) ⁴ Individual/well, 50 μl/well), and incubated overnight. Different anti-HVEM antibodies or isotype antibodies (2. Mu.g/mL) were added to 96-well plates and incubated at 37℃for 60min. Jurkat/BTLA/NFAT-luc cells were added to 96-well plates (5X 10) ⁴ 50. Mu.L/well), incubation at 37℃for 6h. And adding 100 mu L of luciferase detection reagent into each hole, standing for 3min at room temperature, and detecting a luminescence value by using a multifunctional enzyme-labeled instrument after full reaction. All 7 hybridoma antibodies released the inhibition of Jurkat/BTLA/NFAT-luc cells by HVEM, with 4 antibodies (26C 3, 22G5, 37H4, 52B 7) being preferred over the mIgG isotype control (see FIG. 3).

Example 9 ELISA detection of species Cross-reactivity

Human HVEM, monkey HVEM, and mouse HVEM were coated on an ELISA plate (50 ng/well) at 4 DEG CIncubate overnight. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, hybridoma cell culture supernatants (50. Mu.L/well) were added, and incubated at 37℃for 1h. PBST plates were washed 3 times, added with HRP-goat anti-mouse solution (50. Mu.L/well) diluted 1:5000, and incubated for 1h at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 10min in the absence of light. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. ELISA results showed that antibody 52B7 showed similar binding signals to human, monkey HVEM, whereas none of the 7 hybridoma antibodies showed binding signals to mouse HVEM (see FIG. 4).

Example 10 specific screening

Multiple cell lines were cultured and collected, and binding of the antibodies to cell surface proteins was detected by Flow Cytometry (FCM) using different anti-HVEM antibodies as primary antibodies and APC-labeled goat anti-mouse IgG (h+l) as secondary antibodies. The results showed that none of the 7 antibodies showed binding signals to BxPC3, MCF-7, HCT116 cells, whereas a significant binding signal was shown to CHO-K1/HVEM cells. On the other hand, a variety of cell lines were cultured, collected, cell-lysed samples were prepared using RIPA lysate and an ultrasonic cytobreaker, coated onto an elisa plate (500 ng/well), and coated with HVEM-hFc protein as a positive control. Different anti-HVEM antibodies are used as primary antibodies, HRP-goat anti-mouse antibodies are used as secondary antibodies, and ELISA is used for detecting the binding condition of the antibodies and cell lysis components. The results showed that none of the 7 antibodies showed binding signals to SKOV3, HEPG2, ACHN, HCT116, MCF-7, HEK293 cells, whereas a significant binding signal was shown for coated HVEM-hFc.

Example 11 acquisition of antibody variable region sequences

Hybridoma subclones were collected and RNA was extracted using Trizol method. Reverse transcription is carried out by taking the extracted RNA as a template to obtain cDNA. The heavy and light chain variable regions of the antibodies were PCR amplified using degenerate primers (Novagen Ig-Primer Sets), respectively, and the PCR amplified products were detected by agarose gel electrophoresis. And obtaining a target DNA fragment by adopting a gel recovery kit, and then performing TA cloning to construct a recombinant plasmid. And (3) transforming the recombinant plasmid into competent cells by adopting a heat shock method, and plating to perform blue and white spot screening. White single colony is picked up to 0.5mL LB liquid culture medium, shake culture is carried out for 3h at 37 ℃ and 220rpm, and bacterial liquid is taken and sent for sequencing. The variable region amino acid sequences of anti-HVEM antibodies 52B7, 22G5, 26C3 are shown in Table 2.

TABLE 2 variable region amino acid sequences of anti-HVEM antibodies

EXAMPLE 12 construction and preparation of recombinant monoclonal antibodies

And splicing the heavy chain variable region gene fragment and the light chain variable region gene fragment with the signal peptide, the mouse heavy chain (IgG 2 a) constant region gene fragment and the light chain (kappa) constant region gene fragment by adopting overlap PCR, and sequencing and identifying. And (3) inserting the correctly spliced heavy chain genes and light chain genes of the antibody into pTT5 plasmid respectively, transfecting the recombinant plasmid into HEK293 cells by adopting a PEI method, performing serum-free suspension culture, and transiently expressing the antibody. Cell supernatants cultured for 7 days were collected, filtered through a 0.22 μm filter, and the antibodies were purified by protein G affinity chromatography. The antibody is ultrafiltered and replaced to PBS solution, reduced SDS-PAGE and NanoDrop 2000 are adopted to identify the purity and concentration of the antibody, and the antibody is subpackaged and stored at-80 ℃ for standby.

EXAMPLE 13 verification of blocking Activity of recombinant monoclonal antibodies

HVEM-HSA-His coated ELISA plates (200 ng/well) were incubated overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (300. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, and the recombinant antibodies 52B7, 22G5, 26C3 concentration ranges were set as follows: 20. Mu.g/mL-0.02. Mu.g/mL, incubated at 37℃for 1h. PBST plates were washed 3 times, and a solution of BTLA-hFc (50. Mu.L/well) in PBS was added at a concentration of 20. Mu.g/mL and incubated for 1h at 37 ℃. PBST plates were washed 3 times, diluted 5000-fold with HRP-donkey anti-human lgG (H+L) (50. Mu.L/well) added with 5% BSA, and incubated for 1H at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 10min in the absence of light. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. The results show that the recombinant monoclonal antibodies 52B7, 22G5 and 26C3 all show good BTLA/HVEM blocking activitySex (see fig. 5).

Example 14 functional validation of recombinant monoclonal antibodies

The cell function activities of the recombinant monoclonal antibodies 52B7, 22G5, 26C3 were confirmed according to the detection method of hybridoma screening, respectively. The results showed that antibodies 52B7, 22G5, 26C3 all released the inhibitory effect of HVEM on Jurkat/BTLA/NFAT-luc cells (see FIG. 6).

Example 15 species Cross-reactivity detection

Human HVEM, monkey HVEM, murine HVEM were coated onto an elisa plate (50 ng/well) and incubated overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, PBS control (50. Mu.L PBS) was set, murine lgG isotype control (2 ug/mL, 50. Mu.L/well) added recombinant monoclonal antibodies 52B7, 22G5, 26C3 (2 ug/mL, 50. Mu.L/well) and incubated for 1h at 37 ℃. PBST plates were washed 3 times, added with HRP-goat anti-mouse solution (50. Mu.L/well) diluted 1:5000, and incubated for 1h at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 5-10min in the dark. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. ELISA results showed that antibody 52B7 showed similar binding signals to human, monkey HVEM, whereas all 3 antibodies did not show binding signals to murine HVEM (see FIG. 7). Further Biacore assays showed that antibody 52B7 showed good binding kinetics to both human and monkey HVEM (see table 3).

Table 3. Binding kinetics analysis of antibody 52B7 for human, monkey HVEM.

	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

Sequence listing

SEQ ID NO.1

Antibody 52B7 heavy chain variable region VH amino acid sequence

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTIYGVHWIRQSPGKGLEWLGVIWTGGNTDYNAAFMSRLSITKDNSKSQVFF

KMNSLQPDDTAIYYCAKRGYNGNFGFAYWGQGTLVTVSA

SEQ ID NO.2

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

GFSLTIYGVH

SEQ ID NO.3

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

VIWTGGNTDYNAAFMS

SEQ ID NO.4

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

RGYNGNFGFAY

SEQ ID NO.5

Antibody 52B7 light chain variable region VL amino acid sequence

EIVLTQSPALMAASPGEKVTITCSVSSNISSGNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSRSGTSYSLTISSME

AEDAATYYCQQWSRYPLTFGAGTILELK

SEQ ID NO.6

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

SVSSNISSGNLH

SEQ ID NO.7

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

GTSNLAS

SEQ ID NO.8

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

QQWSRYPLT

SEQ ID NO.9

Antibody 22G5 heavy chain variable region VH amino acid sequence

EVQLQESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAFISSGSSSIYYADTVKGRFTISRDNPRNTLI

LQMTSLRSEDTAMYYCVRGLYGNFAWFAYWGQGTLVTVSA

SEQ ID NO.10

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

GFTFSSFGMH

SEQ ID NO.11

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

FISSGSSSIYYADTVKG

SEQ ID NO.12

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

GLYGNFAWFAY

SEQ ID NO.13

Antibody 22G5 light chain variable region VL amino acid sequence

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYFHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI

IRVEAEDLGVYFCSQSSHAPFTFGSGTKLEIK

SEQ ID NO.14

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

RSSQSLVHSNGNTYFH

SEQ ID NO.15

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

KVSNRFS

SEQ ID NO.16

Antibody 22G5 light chain variable region VL-CDR3 amino acid sequence

SQSSHAPFT

SEQ ID NO.17

Antibody 26C3 heavy chain variable region VH amino acid sequence

QVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGDIYPGSDGTNYDEKFKSKATLTVDTSSSTAY

MQLSSLTSEDSAVYYCIREGNYVWFAYWGQGTLVTVSA

SEQ ID NO.18

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

GYTFTSYWMH

SEQ ID NO.19

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

DIYPGSDGTNYDEKFKS

SEQ ID NO.20

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

EGNYVWFAY

SEQ ID NO.21

Antibody 26C3 light chain variable region VL amino acid sequence

DIQMTQTTSSLSASLGDRVTISCRASQDISNSLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ

EDIATYFCQQGNTLPYTFGGGTKLEIKR

SEQ ID NO.22

Antibody 26C3 light chain variable region VL-CDR1 amino acid sequence

RASQDISNSLN

SEQ ID NO.23

Antibody 26C3 light chain variable region VL-CDR2 amino acid sequence

YTSRLHS

SEQ ID NO.24

Antibody 26C3 light chain variable region VL-CDR3 amino acid sequence

QQGNTLPYT

Claims

1. An anti-HVEM antibody or fragment thereof, characterized in that: the heavy chain variable region VH contains a heavy chain complementarity determining region, and the amino acid sequence of the heavy chain complementarity determining region 1 (VH-CDR 1) is at least one of SEQ ID NO.2, SEQ ID NO.10 and SEQ ID NO. 18.

2. An anti-HVEM antibody or fragment thereof, characterized in that: the heavy chain variable region VH contains a heavy chain complementarity determining region, and the amino acid sequence of the heavy chain complementarity determining region 2 (VH-CDR 2) is at least one of SEQ ID NO.3, SEQ ID NO.11 and SEQ ID NO. 19.

3. An anti-HVEM antibody or fragment thereof, characterized in that: the heavy chain variable region VH contains a heavy chain complementarity determining region, and the amino acid sequence of the heavy chain complementarity determining region 3 (VH-CDR 3) is at least one of SEQ ID NO.4, SEQ ID NO.12 and SEQ ID NO. 20.

4. An anti-HVEM antibody or fragment thereof, characterized in that: a heavy chain variable region VH comprising a heavy chain complementarity determining region according to at least one of claims 1, 2 and 3.

5. The anti-HVEM antibody or fragment thereof according to claim 4, wherein: the amino acid sequence of the heavy chain variable region VH is shown as at least one of SEQ ID NO.1, SEQ ID NO.9 or SEQ ID NO. 17.

6. An anti-HVEM antibody or fragment thereof, characterized in that: the light chain variable region contains a light chain complementarity determining region, and the amino acid sequence of the light chain complementarity determining region 1 (VL-CDR 1) is shown as at least one of SEQ ID NO.6, SEQ ID NO.14 and SEQ ID NO. 22.

7. An anti-HVEM antibody or fragment thereof, characterized in that: the light chain variable region contains a light chain complementarity determining region, and the amino acid sequence of the light chain complementarity determining region 2 (VL-CDR 2) is shown as at least one of SEQ ID NO.7, SEQ ID NO.15 and SEQ ID NO. 23.

8. An anti-HVEM antibody or fragment thereof, characterized in that: the light chain variable region VH contains a light chain complementarity determining region, and the amino acid sequence of the light chain complementarity determining region 3 (VL-CDR 3) is at least one of SEQ ID NO.8, SEQ ID NO.16 and SEQ ID NO.24.

9. An anti-HVEM antibody or fragment thereof, characterized in that: light chain variable region VL comprising the light chain complementarity determining region according to at least one of claims 6, 7, 8.

10. An anti-HVEM antibody or fragment thereof, characterized in that: the amino acid sequence of the light chain variable region VL is shown as at least one of SEQ ID NO.5, SEQ ID NO.13 or SEQ ID NO. 21.

11. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 10, wherein: the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO.1, wherein the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.2, SEQ ID NO.3 and 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 and VL-CDR3 are shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.

12. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 10, wherein: the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO.9, wherein the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.10, SEQ ID NO.11 and 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 and VL-CDR3 are shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 respectively.

13. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 10, wherein: the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO.17, wherein the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.18, SEQ ID NO.19 and 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 and VL-CDR3 are shown as SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24 respectively.

14. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 9, wherein: the variable region of the antibody may be formed by splicing CDRs of the complementarity determining region of the corresponding antibody with FRs of the framework region of the human antibody.

15. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 14, wherein: the heavy chain constant region of the antibody may be derived from the constant region of a human immunoglobulin IgG1, igG2, igG3, igG4, igM, igE, igA, or IgD heavy chain.

16. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 15, wherein: the light chain constant region of the antibody may be derived from the constant region of a human immunoglobulin kappa or lambda light chain.

17. The anti-HVEM antibody or fragment thereof according to any one of claims 1 to 16, wherein: the antibody fragment is Fab (antigen binding fragment) or scFv (single-chain fragment variable).

18. A nucleic acid molecule encoding the anti-HVEM antibody or fragment thereof of any one of claims 1-17.

19. A recombinant vector comprising the nucleic acid molecule of claim 18.

20. A cell comprising the recombinant vector of claim 19.

21. Use of the anti-HVEM antibody or fragment thereof of any one of claims 1-17 to promote T cell activation.

22. Use of the anti-HVEM antibody or fragment thereof of any one of claims 1-17 in the manufacture of a medicament for treating or preventing a tumor.

23. Use according to claim 22, characterized in that: the tumor is colon cancer or prostate cancer.