Anti-VISTA fully human monoclonal antibody and application thereof

Abstract

The invention provides an anti-VISTA fully human monoclonal antibody, which consists of a heavy chain and a light chain, and is specifically combined with human VISTA protein. The invention also provides application of the antibody. The complete sequences of the antibody are all human sequences, and Western Blot binding analysis, ELISA / FACS epitope analysis, EC50 detection, SPR / BLI affinity analysis and other detection means prove that the human anti-VISTA recombinant monoclonal antibody is respectively bound with human VISTA protein with high affinity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to antibodies and their pharmaceutical applications, and in particular to an anti-VISTA antibody and its application. Background Art

[0002] VISTA refers to the V-type immunoglobulin domain-containing suppressor of T-cell activation, which is an important T-cell immune checkpoint. It also has other names such as B7-H5, PD-1H, Platelet receptor Gi24, Dies1 and SISP1. VISTA is highly regulated mainly on bone marrow antigen presenting cells (APCs) and T cells, and its extracellular domain contains only one IgV-like domain. Recombinant VISTA Ig fusion protein can inhibit CD4 + and CD8 + T cell proliferation, IL-2 and IFN-γ production, and promotion of naive T cell transformation to FoxP3 + Regulatory T cells. Overexpression of VISTA on bone marrow-derived dendritic cells reduced T cell proliferation and cytokine production.

[0003] VISTA is expressed in a variety of organisms, including humans, mice, and rhesus monkeys. The human VISTA protein has a total of 279 amino acid residues, including an extracellular region, a transmembrane region, and a cytoplasmic region. Its extracellular region has a conserved IgV-like folding structure and contains a unique C51 / C113 disulfide bond. VISTA protein binds to VISTA ligands (VSIG-3, PSGL-1, VISTA-unknown Ligand, etc.) to form a protective tumor environment that directly inhibits T cell function. As a co-inhibitory T cell receptor, VISTA has been shown to inhibit T cell function without relying on APC. VISTA-deficient mice aggregate spontaneously activated T cells, secrete high levels of proinflammatory cytokines, and develop more severe experimental autoimmune encephalomyelitis than control mice; at the same time, VISTA on T cells and APCs can lead to worsening of the disease. Studies have shown that both in vitro and in vivo treatment of CD4 with VISTA-specific agonistic antibodies + T cells, which can suppress CD4 + T cell activation, which proves that VISTA is a CD4 +T cell inhibitory receptors. VISTA interacts with itself or another receptor through T cell-T cell interactions. VISTA's ligands can also be expressed on tumor cells, which allows tumors to directly inhibit T cell activity through VISTA. Through the binding of VISTA therapeutic antibodies to VISTA ligands, the proliferation, infiltration and effector functions of tumor-specific T cells are enhanced, the number of myeloid-derived suppressor cells in the tumor microenvironment is reduced, and the development and activity of regulatory T cells and tumor growth are inhibited. The application of VISTA antibodies to treat a variety of cancers such as ovarian cancer, cervical squamous cell carcinoma, bladder cancer and melanoma has become a new anti-cancer method. In addition, VISTA antibodies can also inhibit T cell immunity in autoimmune diseases, allergies, infections and inflammatory conditions such as multiple sclerosis and joint conditions such as arthritis.

[0004] The difference between VISTA protein and other immune checkpoints (such as PD1 or CTLA4, which only bind to activated T-cells) is that it can bind to natural Anti-VISTA antibodies can mobilize the functions of all immune cells, especially cancer cells in solid tumors and pancreatic cancer cells, which have high VISTA expression and are important candidate targets for the treatment of advanced cancer.

[0005] To date, anti-VISTA antibodies Onvatilimab (such as CI-8993 monoclonal antibody from Curis), W0180 (manufacturer Pierre Fabre), HMBD-002 (manufacturer Hummingbird Bioscience), etc. have entered Phase I clinical trials, and SNS-101 (manufacturer Sensei Biotherapeutics) and others have begun Phase I / II clinical trials (SNS-101 has the potential to inhibit tumor growth as a monotherapy and can significantly enhance the anti-tumor effect of PD-1 blockers such as Cemiplimab (PD-1 antibody, trade name Libtayo, manufacturer Regeneron)).

[0006] The application of antibodies as drugs is affected by their immunogenicity, affinity, stability, effector function, half-life, tissue permeability and distribution. The earliest mouse monoclonal antibodies used played a major role in promoting the study of human diseases, but the immune response of these mouse proteins limited their application. With the development of antibody humanization technology, a variety of humanized antibodies have become possible. Humanized antibodies refer to antibodies in which the variable region (i.e., VH and VL regions) or the entire antibody sequence is encoded by human antibody genes. Transgenic mice are the main method for producing humanized antibodies. The humanization of anti-VISTA antibodies has been studied. However, the humanization process of antibodies is complicated and expensive, and the technical difficulty is relatively high. The humanization process also weakens the efficacy of antibodies to a certain extent and produces certain side effects in clinical use, such as the production of antibodies against anti-antibody drugs.

[0007] Chinese patent application document CN111549072A discloses a method for constructing and applying a VISTA gene humanized animal cell and animal model, which utilizes CRISPR / Cas9 gene editing technology to replace the mouse VISTA gene with the human VISTA gene (specifically, the second and third exons of the mouse Vista gene extracellular region are replaced with the second and third exons of the human VISTA gene, while the intracellular region retains the complete mouse sequence), and constructs a mouse model that can interact with anti-human VISTA antibodies. Compared with ordinary mice, this model realizes the humanization of key target molecules and can be used to screen and evaluate drugs targeting the human VISTA gene. It is an ideal preclinical drug testing model.

[0008] However, this technology belongs to the humanization of antigens, not the humanization of antibodies, and the antibodies secreted are still mouse antibodies.

[0009] Fully humanized antibodies refer to the transfer of all human antibody-encoding genes to genetically engineered antibody gene-deficient animals through transgenic or transchromosome technology, so that the animals can express human antibodies and achieve the purpose of fully humanized antibodies. In the global monoclonal antibody market, fully humanized monoclonal antibodies are the future development direction, and fully humanized anti-VISTA antibodies have not yet been obtained. Summary of the invention

[0010] In order to solve the above-mentioned technical problems, the present invention provides an anti-VISTA fully human monoclonal antibody composed of a heavy chain and a light chain, which specifically binds to the human VISTA protein.

[0011] Preferably, the variable region of the heavy chain includes three CDR regions, wherein the amino acid sequence of the first CDR region of the heavy chain is SEQ ID NO: 7 or 13, the amino acid sequence of the second CDR region of the heavy chain is SEQ ID NO: 8, 14 or 19, and the amino acid sequence of the third CDR region of the heavy chain is SEQ ID NO: 9, 15 or 20.

[0012] Preferably, the three CDR regions of the heavy chain variable region of the antibody are selected from one of the following combinations: 1) SEQ ID NOs: 7, 8 and 9; 2) SEQ ID NOs: 13, 14 and 15; 3) SEQ ID NOs: 7, 19 and 20.

[0013] Further preferably, the variable region of the light chain includes three CDR regions, wherein the amino acid sequence of the first CDR region of the light chain is SEQ ID NO: 10, 16 or 21, the amino acid sequence of the second CDR region of the light chain is SEQ ID NO: 11, 17 or 22, and the amino acid sequence of the third CDR region of the light chain is SEQ ID NO: 12, 18 or 23.

[0014] Preferably, the three CDR regions of the light chain variable region of the antibody are selected from one of the following combinations: 1) SEQ ID NOs: 10, 11 and 12; 2) SEQ ID NOs: 16, 17 and 18; 3) SEQ ID NOs: 21, 22 and 23.

[0015] Preferably, the full-length amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1, 3 or 5; the full-length amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 2, 4 or 6.

[0016] The present invention also provides a nucleic acid molecule encoding the antibody of the present invention.

[0017] The present invention also provides an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule of the present invention.

[0018] The present invention also provides a pharmaceutical composition, comprising the antibody, nucleic acid molecule or expression cassette, recombinant vector or recombinant microorganism of the present invention, and a pharmaceutically acceptable excipient, diluent or carrier.

[0019] The present invention also provides the use of the antibody, nucleic acid molecule, expression cassette, recombinant vector or recombinant microorganism, or pharmaceutical composition of the present invention in the preparation of an agent or drug for diagnosing, preventing and / or treating tumors, cancers, autoimmune diseases, allergies, infections or inflammatory disorders.

[0020] The present invention has the following beneficial effects:

[0021] The anti-VISTA recombinant monoclonal antibody prepared by the present invention is derived from hybridoma cell screening, and then sequenced and analyzed in the present invention. Recombinant expression is carried out in mice, and the entire sequence of recombinant expression is a fully human sequence, which greatly avoids any safety risks such as allergies or viral reinfection, and is suitable for large-scale production of high-purity fully human anti-VISTA recombinant monoclonal antibodies.

[0022] Western Blot binding analysis, ELISA / FACS antigen epitope analysis, EC 50 Detection and ForteBio Affinity analysis and other detection methods have demonstrated that the fully human anti-VISTA recombinant monoclonal antibodies of the present invention bind to human VISTA protein with high affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 VISTA antigen immunization was shown Serum titer test results after mice were immunized with human VISTA antigen After the mice were isolated, serum was collected and then used to detect VISTA-positive transgenic cells (VISTA + -293F) were used for FACS analysis. In the figure, the sera of the four mice had obvious antibodies binding to VISTA protein, and the immune titer was high.

[0025] Figure 2 The anti-human VISTA antibodies of the present invention and control antibodies were shown to be positive for human VISTA transgenic cells (VISTA + The figure shows the FACS analysis results of the anti-human VISTA antibodies CAM030703, CAM031004, CAM033002 and Onvatilimab (control) of the present invention and VISTA positive transgenic cells (VISTA + -293F) had significant binding.

[0026] Figure 3 The results show that different concentrations of anti-human VISTA antibodies and control antibodies of the present invention interact with human VISTA positive transgenic cells (VISTA + -293F) FACS analysis (EC 50 ) Results. Anti-human VISTA antibodies CAM030703, CAM031004, CAM031704, CAM032011, CAM033002 and Onvatilimab (control) of the present invention were compared with human VISTA positive transgenic cells (VISTA + -293F) ELISA analysis results are shown in the figure, and the EC values ​​of the antibodies were calculated based on this. 50 value.

[0027] Figure 4 The ELISA analysis results of different concentrations of anti-human VISTA antibodies and control antibodies of the present invention binding to human VISTA protein under different pH conditions are shown. Under the conditions of pH = 6.0 and pH = 7.4, the ELISA analysis of the anti-human VISTA antibodies CAM030703, CAM031004, CAM031704, CAM032011, CAM033002 and Onvatilimab (control) of the present invention and VISTA protein is shown in the figure, proving that the anti-human VISTA antibodies of the present invention bind to human VISTA protein to the same extent under acidic conditions. DETAILED DESCRIPTION

[0028] The following is further described in detail through specific implementation methods. However, it should be pointed out that the following embodiments of the present invention are only for better illustrating the content of the present invention, but do not mean that the content of the present invention is limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still belongs to the protection scope of the present invention and is subject to the protection scope of the attached claims.

[0029] It is well known to those skilled in the art that VISTA refers to the T cell activation inhibitor immunoglobulin variable region domain, which is an important T-cell immune checkpoint. VISTA is highly regulated mainly on bone marrow antigen presenting cells and T cells, and its extracellular domain contains only one IgV-like domain. The human VISTA sequence used in the present invention can be obtained according to UNIPROT (Q9H7M9) of Uniprot.

[0030] It is well known to those skilled in the art that Onvatilimab, W0180, HMBD-002 and SNS-101 are known anti-VISTA monoclonal antibodies and can be used as positive controls of the present invention.

[0031] The present invention uses the fully human monoclonal antibody platform obtained from the previous research results of the inventor's team Mice are used to prepare fully human monoclonal antibodies of the present invention. The mouse is a transgenic mouse strain containing human heavy and light chains, and inactivated mouse endogenous antibody genes. The mouse strain has normal B-cell development, high immune titer, and can produce chimeric human / mouse IgM and human IgG; in the case of antigen immunization, a fully human IgG antibody with therapeutic potential can be obtained at one time. The construction method of the transgenic mouse strain and the specific contents of the immune production of hybridomas and monoclonal antibodies can be found in the Chinese patent documents CN105316313A and CN108486126A, the previous research results of the inventor's team, and the contents of these patents are all incorporated into the present invention as part of the content of the present invention specification.

[0032] It is well known to those skilled in the art that the polyclonal antibody described in the present invention is a mixture of antibodies produced by different B cells in the body against the same antigen, and they can recognize and bind to different antigenic epitopes on the single antigen. Monoclonal antibodies are produced by the same B cell and can only recognize a specific single antigenic epitope.

[0033] It is well known to those skilled in the art that the chain structure and region of the antibody molecule described in the present invention have the following meanings: antibody molecules are generally composed of two heavy chains (i.e., H chains) and two light chains (i.e., L chains), and each chain includes a variable region (i.e., V region), a constant region (i.e., C region) and a hinge region. The composition and arrangement of the residues near the amino terminus of the heavy chain and the light chain vary greatly, and this region is called the variable region, while the composition and arrangement order of the residues near the carboxyl terminus are relatively stable, so it is called the constant region. In the variable region of the heavy chain and the light chain, the amino acid composition and arrangement order in each of the three specific segments are highly variable, called the hypervariable region (i.e., HVR), which together constitute the antigen binding site of the antibody molecule, which can complementarily bind to the corresponding antigen epitope, so the hypervariable region is also called the complementary determining region (i.e., CDR), which is represented by CDR1, CDR2 and CDR3 respectively. The amino acid composition and sequence of the CDR of different antibodies are different, which determines the specificity of the antibody binding to the corresponding antigen epitope, and is responsible for recognizing and binding to the antigen, thereby exerting an immune effect. The amino acid composition and arrangement sequence of the variable region of the antibody molecule other than the hypervariable region or CDR region change relatively little, and are called the framework region (ie, FR region).

[0034] From the above common sense, we know that the basic immune function of antibodies depends on the three CDR regions on the heavy chain and light chain, which are all located in the variable regions of the heavy chain and light chain of the antibody. Other amino acid sequences on the antibody molecule are usually conservative, and even if there are slight changes, they usually do not affect the basic immune function of the antibody molecule. Therefore, expressing the sequence of all CDR regions can express an antibody or evaluate whether they belong to the same antibody.

[0035] The principle of ELISA (enzyme-linked immunosorbent assay) used in the present invention to detect antigen-antibody binding is mainly based on the principle of antigen-antibody specific binding and enzyme-catalyzed substrate color development. Among them, the direct method is to directly bind the enzyme-labeled antibody to the antigen to be tested for detection. The indirect method is to first bind the antigen to be tested with the antibody on the solid phase carrier, and then add the enzyme-labeled secondary antibody for detection. This indirect method is adopted in the present invention. The double antibody sandwich rule is suitable for detecting large molecular antigens, using two antibodies targeting different epitopes of the same antigen, one of which is coated with a solid phase carrier and the other is used as an enzyme-labeled antibody. The competition rule is used to detect small molecule antigens or haptens, and the antigen to be tested and a certain amount of enzyme-labeled antigen compete for binding with the solid phase antibody.

[0036] The basic principle of FACS (fluorescence activated cell sorting) used in the present invention is to utilize the specific antibody of fluorescein labeling to combine with the corresponding antigen on the cell surface, so that the cell is labeled with fluorescein. Under the action of the excitation light of a specific wavelength, these fluorescein-labeled cells will produce fluorescence, which is then detected by flow cytometry. Flow cytometry can analyze cells one by one, and measure the physical characteristics such as the fluorescence intensity and size of each cell, thereby realizing the quantitative analysis of antigen-antibody binding. It can be used to screen antibodies for specific antigens. By comparing the binding ability of different antibodies to the same antigen, antibodies with high affinity and specificity can be screened out.

[0037] The basic principle of SPR (Surface Plasmon Resonance) used in the present invention is that when the incident light is incident at a critical angle to the interface of two media with different refractive indices, the resonance of the free electrons of the metal can be caused. Due to the resonance, the electrons absorb the light energy, thereby greatly weakening the reflected light within a certain angle, and the incident angle at which the reflected light completely disappears within a certain angle is called the SPR angle. SPR changes with the change of the surface refractive index, and the change of the refractive index is proportional to the mass of the biomolecules bound to the metal surface. Therefore, the specific signal of the interaction between biomolecules can be obtained by obtaining the dynamic change of the SPR angle during the biological reaction. In the antibody competitive binding analysis, the SPR technology can monitor the binding and dissociation process of antibodies with antigens or other molecules in real time, and the competitive relationship between antibodies can be judged by comparing the binding ability of different antibodies with the same antigen.

[0038] The BLI (Bio-Layer Interferometry) used in the present invention adopts a probe-type biosensor to directly detect the sample without any fluorescence or isotope labeling of the test sample. The instrument emits white light to the sensor surface and collects the reflected light. The reflection spectra of different frequencies are affected by the thickness of the optical film layer of the biosensor and form interference. When the molecule binds to the ligand fixed on the surface of the biosensor, the thickness of the optical layer will increase, and the path length of the reflected light will also increase, causing the interference spectrum curve to change and shift to the right. When the molecule and the ligand dissociate, the molecule will dissociate from the surface of the biosensor into the solution, causing the interference spectrum curve to move to the left. The relationship between the offset distance (in nm) of this interference spectrum curve and the reaction time is called a sensorgram. Through the sensorgram, the binding constant (k a or k on ) and dissociation constant (k d or k off ), and the initial binding rate, and the affinity (K D ) and concentration information. In antibody competitive binding analysis, BLI technology can monitor the binding process of antibodies and antigens in real time. By comparing the binding curves and affinity constants of different antibodies with the same antigen, the competitive relationship between antibodies can be determined. System detection SPR / BLI detection.

[0039] Other antibody-related technologies and instruments, equipment, materials, etc. used are well known to those skilled in the art, such as immunizing animals (such as mice) with antigens, performing hybridoma fusion and screening after immunization, expressing and purifying antibodies, analyzing the structure and performance of antibodies (binding ability, affinity, neutralization), etc. If the technologies, instruments, equipment, and materials used are not specifically specified in the present invention, any other technologies, instruments, equipment, and materials that are well known to those skilled in the art and can achieve the same purpose can be used; even if the technologies, instruments, equipment, and materials used are specifically specified in the present invention, it does not mean that the present invention can only use these technologies, instruments, equipment, and materials, but only represents the preferred embodiment of the present invention, and those skilled in the art can still use any other technologies, instruments, equipment, and materials that are well known to those skilled in the art and can achieve the same purpose.

[0040] Example 1: Antibody preparation and sequencing

[0041] The fully human monoclonal antibody platform obtained using the previous research results of the inventor's team (see Chinese patent documents CN105316313A and CN108486126A) Mice are used to prepare fully human monoclonal antibodies of the present invention. The mouse is a transgenic mouse strain containing human heavy and light chains, and inactivated mouse endogenous antibody genes. This mouse strain has normal B-cell development, high immune titer, and can produce chimeric human / mouse IgM and human IgG; in the case of antigen immunization, fully human IgG antibodies with therapeutic potential can be obtained at one time.

[0042] 1. Preparation of human VISTA antigen:

[0043] According to the sequence of human VISTA sequence UNIPROT (Q9H7M9) of Uniprot (FKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFYKTWYRSSRGEVQTCSERRPIRNL TFQDLHLHHGGHQAANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGLYCCLVVEIRH HHSEHRVHGAMELQVQTGKDAPSNCVVYPSSSQDSENITAAAHHHHHHHH), its C-terminus was connected to a His tag, and 293F cells were transfected with it for transient expression, and human VISTA antigen was obtained after purification according to conventional protein purification methods in the art.

[0044] 2. Immunization of mice:

[0045] The human VISTA antigen purified above was used to Mice were immunized by subcutaneous injection (antigen + adjuvant) or intramuscular electroporation (VISTA expression vector) and then blood was collected to prepare serum. The serum titer of mice was analyzed by ELISA and FACS, and mice with a titer higher than 1:51200 were selected to collect lymphocytes.

[0046] ELISA analysis of mouse serum titer uses the principle of specific binding between antigen and antibody, and determines the titer by detecting the maximum dilution of the antibody in the serum. The optical density value (OD value) of each well is measured at a specific wavelength (such as 450nm) by an ELISA reader, and the data is recorded. The critical value of the serum titer is determined based on the OD values ​​of the negative control and the positive control. If the OD value of the serum to be tested is greater than the critical value, the serum is considered positive; if the OD value is less than the critical value, the serum is considered negative. At the same time, the serum titer is calculated based on the maximum dilution of the serum to be tested.

[0047] Similarly, a FACS instrument can be used to detect the fluorescence intensity of antigen-antibody binding to cells, and the activity or titer of antibodies in serum can be evaluated based on changes in fluorescence intensity.

[0048] Figure 1Immunization with human VISTA antigen was shown Serum titer test results after mice were immunized with human VISTA antigen After the mice were isolated, serum was collected and then used to detect VISTA-positive transgenic cells (VISTA + -293F) were used for FACS analysis. The results in the figure show that there are obvious antibodies binding to human VISTA protein in the serum of 4 mice, and the immune titer is high.

[0049] 3. Hybridoma and antibody preparation:

[0050] Mice with high immune titers were immunized intraperitoneally (antigen, without adjuvant). 3-4 days after immunization, spleen and lymph nodes of mice were taken to prepare lymphocytes. B-cells and SP2 / 0 cells were fused by electrical stimulation using NEPA-21 electroporator and cultured in semi-solid culture medium (ClonaCell TM -HY Medium E, STEMCELL Technologies, #03805) for screening and cultivation. The monoclones were selected and cultivated in 96-well plates, and the supernatants were analyzed by ELISA. Positive hybridomas with OD values ​​> 3.5 were selected for clonal expansion. The binding and affinity of the antibodies secreted by the supernatants to the human VISTA antigen protein were then analyzed by ELISA, FACS and / or BLI (see Examples 2-4 below for specific methods).

[0051] Select hybridoma clones with strong binding ability and high affinity to VISTA, prepare mRNA and cDNA. Use RT-PCR to obtain the sequence of the hybridoma and seamlessly link it to -Blunt Zero (full gold -Blunt ZeroCloning Kit CB501). Design appropriate T7 primers for sequence analysis.

[0052] The results of sequence analysis revealed eight antibodies with strong binding ability and high affinity (CAM030703, CAM030804, CAM031004, CAM031704, CAM031912, CAM032011, CAM033012, and CAM033002), of which the full-length amino acid sequences of the heavy chain variable region and the light chain variable region of three antibodies (CAM030703, CAM031004, and CAM033002) are shown in the sequence listing as SEQ ID NOs: 1 and 2, 3 and 4, and 5 and 6, respectively, and their corresponding coding DNA sequences are shown in the sequence listing as SEQ ID NOs: 24 and 25, 26 and 27, and 28 and 29, respectively. The results of sequence analysis also revealed three CDR regions of each heavy chain variable region and light chain variable region, which constitute the functional active regions of the antibodies.

[0053] The heavy chain and light chain of the monoclonal antibody obtained by the present invention were synthesized by PCR and then transfected into 293F cells through the expression vector combination. After purifying the antibody, the binding ability and affinity of the obtained antibody to the VISTA antigen protein were repeated by ELISA, FACS and / or BLI analysis (see Examples 2-4 below for specific methods).

[0054] Example 2: Affinity analysis of the anti-human VISTA antibody of the present invention and human VISTA (FACS method)

[0055] The eight antibodies obtained in Example 1 (CAM030703, CAM030804, CAM031004, CAM031704, CAM031912, CAM032011, CAM033012, and CAM033002) were analyzed by ELISA and FACS to detect the binding ability and specificity between the antigen and the antibody.

[0056] 1. Preparation of human VISTA positive cells:

[0057] The secretory peptide of immunoglobulin was linked to human VISTA protein and then stably transfected into 293F cells to screen monoclonal positive cell lines.

[0058] 2. FACS analysis:

[0059] The 293F cells stably transfected with the human VISTA gene were cultured with KO-DMEM + 10% FCS, and single cells were collected, washed once with PBS + 2% FCS, and counted (the cell dilution concentration was 1×10 7 / ml); 100 μl of cell suspension (including positive and negative controls) was taken from each group, and antibodies of corresponding concentrations (hybridoma supernatant or purified human IgG monoclonal antibody) were added, cultured at 4°C for 30 minutes, washed once with PBS + 2% FCS, and then 100 μl of secondary antibody (FITC labeled, antibody dilution ratio: 1:200-1000) diluted in PBS-2% FCS was added, cultured at 4°C for 30 minutes, washed once with PBS + 2% FCS, and then 100 μl of PBS-2% FCS was added, the cells were mixed, and FACS analysis was performed.

[0060] Figure 2 The results show the relationship between anti-human VISTA antibody and positive control antibody (Onvatilimab) and VISTA positive transgenic cells (VISTA + -293F) FACS analysis results. The results showed that anti-human VISTA antibodies CAM030703, CAM031004, CAM033002 and Onvatilimab (control) were positive for VISTA transgenic cells (VISTA + -293F) had significant binding.

[0061] Figure 3 Different concentrations of anti-human VISTA antibody and control antibody are shown in Figure 5. + -293F) FACS analysis (EC 50 ) Results. Antibodies CAM030703, CAM031004, CAM031704, CAM032011, CAM033002 and Onvatilimab (control) of the present invention were used to detect VISTA-positive transgenic cells (VISTA + -293F) for FACS analysis. According to the recorded fluorescence intensity values, a curve of the relationship between antibody concentration and fluorescence intensity was drawn.

[0062] Then, by curve fitting, we find the antibody concentration when the fluorescence intensity reaches 50% of the maximum fluorescence intensity, which is the EC 50 EC value of the antibody 50 The results are shown in Table 1. The EC values ​​of CAM030703, CAM032011 and CAM033002 50 The EC values ​​of the control (ONVATILIMAB) 50 Equal or better value.

[0063]

[0064] Example 3 Affinity analysis of the anti-human VISTA antibody of the present invention and human VISTA (ELISA analysis)

[0065] VISTA protein (antigen) was diluted with carbonate buffer (pH = 8.0) to a concentration of 0.5 μg / mL, 50 μl was added to each well, and incubated overnight at 4°C (embedding), washed 3 times with PBS-0.05% Tween20, and then 200 μl of 1% BSA was added to each well for 1 hour (blocking), washed 3 times with PBS-0.05% Tween20, and a certain concentration of antibody was added for 2 hours, washed 3 times with PBS-0.05% Tween20, and 50 μl of HRP-labeled secondary antibody (Mouse Anti-Human IgG Fab Antibody (12H3C4A6) [HRP], mAb, GenScript, A01855, 5000X) was added to each well, and incubated at 37°C for 2 hours; washed 3 times with PBST, 50 μl of TMB solution was added to each well, protected from light for 10 min, and then 50 μl of 2% oxalic acid solution was added to each well, and the OD was adjusted to 0.5. 450 The detection buffer used in normal ELISA is PBS + 2% FCS, and its pH is between 7.0 and 7.4; in ELISA tests under different pH conditions, PBS + 2% FCS buffer is used, and 1N NaOH or HCL is used to adjust pH = 6.0 and pH = 7.4.

[0066] The ELSIA analysis results of all antibodies tested in the present invention binding to VIST antigen are shown in Table 2.

[0067]

[0068] The above results prove that the antibody of the present invention specifically binds to the human VISTA protein, and does not bind to other human proteins tested or has weak binding ability.

[0069] Figure 4 The results of ELISA analysis (OD 450 The results show that under the conditions of pH = 6.0 and pH = 7.4, the anti-human VISTA antibodies CAM030703, CAM031004, CAM031704, CAM032011, CAM033002 and Onvatilimab (control) of the present invention within the test concentration range have similar or better binding abilities to human VISTA protein.

[0070] Example 4: Affinity analysis of the anti-human VISTA antibody of the present invention and human VISTA (SPR / BLI method)

[0071] Using Fortebio The system detects the affinity of antibody to antigen (K D ), which is the equilibrium dissociation constant between the antibody and its antigen. The specific steps are as follows:

[0072] 1) Pre-wetting the probe: Use 1% BSA-PBST (or PBST) solution to pre-wet the probe to ensure compatibility between the probe surface and the sample and to reduce nonspecific adsorption.

[0073] 2) Dilute antigen and antibody: Use PBST buffer to dilute antigen and antibody to appropriate concentration range such as 5-20 μg / ml.

[0074] 3) Set experimental parameters: According to Fortebio System requirements, set experimental parameters, including the order of loading samples, detection time, temperature, etc.

[0075] 4) Load samples: Load the diluted antigen and antibody samples into the Fortebio The corresponding position of the system.

[0076] 5) Start detection: Start Fortebio The system starts to detect the binding rate of antigen and antibody (k on ) and dissociation rate (k off ). The system will monitor the interaction between antigen and antibody in real time through biofilm interferometry (BLI).

[0077] 6) Data collection: During the testing process, Fortebio The system will automatically collect and record the binding and dissociation curves of antigen and antibody, as well as the related kinetic parameters.

[0078] K D It is a constant that describes the affinity between antibody and antigen, reflecting the ratio of the rate at which the antibody-antigen complex dissociates into free antibody and antigen to the rate at which they reassociate under equilibrium conditions. D The smaller the value, the stronger the affinity between the antibody and the antigen, that is, the tighter the binding between them and the less likely they are to dissociate; K D The larger the value, the weaker the affinity between the antibody and the antigen, that is, the binding between them is relatively loose and easy to dissociate.

[0079] K A It is a constant that describes the ability of antibodies to bind to antigens and is related to K D They are inversely proportional to each other. It reflects the ratio of the rate at which free antibodies and antigens combine to form antibody-antigen complexes to the rate at which the complexes dissociate into free antibodies and antigens in a state of equilibrium. AThe larger the value, the stronger the binding ability between the antibody and the antigen, that is, the easier it is for them to combine to form a complex; K A The smaller the value, the weaker the binding ability between the antibody and the antigen, that is, the less likely they are to bind.

[0080] K dis Used to describe the rate at which the antibody-antigen complex dissociates into free antibody and antigen. dis The larger the value, the faster the dissociation rate of the antibody-antigen complex, that is, the easier it is for the complex to dissociate into free antibodies and antigens; K dis The smaller the value, the slower the dissociation rate of the antibody-antigen complex, that is, the more stable the complex is and the less likely it is to dissociate.

[0081] The specific test results are shown in Table 3. The data show that the antibodies CAM030703, CAM031004, and CAM033002 obtained in the present invention have similar or even better affinity to the human VISTA antigen than the positive control antibody Onvatilimab.

[0082]

[0083] On the same sensor chip or biosensor, a certain amount of antigen is first fixed, and then antibody 1 and antibody 2 are added (different concentration gradients can be set), and their binding is observed and recorded. By comparing the binding and dissociation curves of antibody 1 and antibody 2, as well as their K D value, to determine whether there is a competitive relationship between the two.

[0084] K D The value reflects the affinity between the antibody and the antigen, K D The smaller the value, the stronger the affinity between the antibody and the antigen. D The K values ​​can be used to determine their ability to bind to the antigen. If antibody 1 and antibody 2 show obvious differences in their binding curves when added to the antigen on the same sensor chip or biosensor (such as reduced binding degree, slower binding rate, etc.), it may indicate that there is a competitive relationship between the two. If antibody 1 and antibody 2 exist at the same time, their K D The value increased compared with that when it existed alone (i.e., the affinity was weakened), which further confirmed the competitive relationship between the two.

[0085] The antibodies CAM030703, CAM031004 and CAM033002 obtained in the present invention were subjected to competition binding tests with the positive control antibody Onvatilimab, and the results are shown in Table 4. The data prove that some antibodies have no direct competition, and the use of multiple antibodies at the same time helps to enhance their effects.

[0086]

[0087] The above is only an embodiment of the present invention. The commonly known technical common sense in the scheme is not described in detail here. The ordinary technicians in the relevant field are aware of all the common technical knowledge in the technical field to which the invention belongs before the application date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical commonly known technologies should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, they can also make some adjustments and improvements, which should also be regarded as the scope of protection of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection claimed by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. An anti-VISTA fully human monoclonal antibody, consisting of a heavy chain and a light chain, characterized in that: The antibody specifically binds to human VISTA protein.

2. The anti-VISTA fully human monoclonal antibody according to claim 1, characterized in that The variable region of the heavy chain includes three CDR regions, wherein the amino acid sequence of the first CDR region of the heavy chain is SEQ ID NO: 7 or 13, the amino acid sequence of the second CDR region of the heavy chain is SEQ ID NO: 8, 14 or 19, and the amino acid sequence of the third CDR region of the heavy chain is SEQ ID NO: 9, 15 or 20.

3. The anti-VISTA fully human monoclonal antibody according to claim 2, characterized in that The three CDR regions of the heavy chain variable region of the antibody are selected from one of the following combinations: 1) SEQ ID NOs: 7, 8 and 9; 2) SEQ ID NOs: 13, 14 and 15; 3) SEQ ID NOs: 7, 19 and 20.

4. The anti-VISTA fully human monoclonal antibody according to claim 1, characterized in that The variable region of the light chain includes three CDR regions, wherein the amino acid sequence of the first CDR region of the light chain is SEQ ID NO: 10, 16 or 21, the amino acid sequence of the second CDR region of the light chain is SEQ ID NO: 11, 17 or 22, and the amino acid sequence of the third CDR region of the light chain is SEQ ID NO: 12, 18 or 23.

5. The anti-VISTA fully human monoclonal antibody according to claim 4, characterized in that The three CDR regions of the light chain variable region of the antibody are selected from one of the following combinations: 1) SEQ ID NOs: 10, 11 and 12; 2) SEQ ID NOs: 16, 17 and 18; 3) SEQ ID NOs: 21, 22 and 23.

6. The anti-VISTA fully human monoclonal antibody according to claim 5, characterized in that The full-length amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1, 3 or 5; the full-length amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 2, 4 or 6.

7. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 6.

8. An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule according to claim 7.

9. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the expression cassette, recombinant vector or recombinant microorganism according to claim 8, and a pharmaceutically acceptable excipient, diluent or carrier.

10. Use of the antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression cassette, recombinant vector or recombinant microorganism according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of an agent or drug for diagnosing, preventing and / or treating tumors, cancers, autoimmune diseases, allergies, infections or inflammatory disorders.

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