Anti-vista fully human monoclonal antibody and application thereof

Abstract

The application provides an anti-VISTA fully human monoclonal antibody, which is composed of a heavy chain and a light chain, and the antibody specifically binds to human VISTA protein. 50 The fully human anti-VISTA recombinant monoclonal antibody of the application is proved to be high in affinity to human VISTA protein by means of detection and SPR / BLI affinity analysis.

Description

TECHNICAL FIELD

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

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

[0003] VISTA is expressed in various organisms, including humans, mice, rhesus monkeys, etc. The human VISTA protein has a total of 279 amino acid residues, including an extracellular region, a transmembrane region and a cytoplasmic region, and the extracellular region has a conserved IgV-like folding structure and contains a unique C51 / C113 disulfide bond. VISTA protein binds to VISTA ligand (VSIG-3, PSGL-1, VISTA-unknown Ligand, etc.) to form a protective tumor microenvironment to directly inhibit 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 will gather spontaneously activated T cells, secrete high levels of pro-inflammatory cytokines, and have more severe experimental autoimmune encephalomyelitis than control mice; at the same time, VISTA on T cells and APC will cause the disease to worsen. Studies have shown that, whether in vitro or in vivo, CD4 + T cells treated with VISTA-specific agonistic antibodies can inhibit 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 function 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 various 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, making them an important candidate target for the treatment of advanced cancer.

[0005] To date, anti-VISTA antibodies such as Onvatilimab (e.g., CI-8993 monoclonal antibody from Curis), W0180 (manufactured by Pierre Fabre), and HMBD-002 (manufactured by Hummingbird Bioscience) have entered Phase I clinical trials, and SNS-101 (manufactured by Sensei Biotherapeutics) has 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 effects of PD-1 blockers such as Cemiplimab (PD-1 antibody, trade name Libtayo, manufactured by Regeneron)).

[0006] The use of antibodies as drugs is affected by their immunogenicity, affinity, stability, effector function, half-life, tissue penetration, and distribution. The earliest use of murine monoclonal antibodies played a significant role in the research of human diseases, but the immune response of these murine proteins limited their application. With the development of antibody humanization technology, various humanized antibodies have become possible. Humanized antibodies refer to antibodies whose variable region parts (i.e., VH and VL regions) or all antibody sequences are 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 complex and expensive, and the technical difficulty is high. The humanization process also weakens the drug efficacy of antibodies to some extent and produces certain side effects in clinical use, such as the production of anti-antibody drug antibodies.

[0007] Chinese patent application document CN111549072A discloses a method for constructing a VISTA gene humanized animal cell and animal model and its application. The CRISPR / Cas9 gene editing technology is used to replace the murine VISTA gene with the human VISTA gene (specifically, the 2nd and 3rd exons of the extracellular region of the mouse Vista gene are replaced with the 2nd and 3rd exons of the human VISTA gene, while the intracellular region retains the complete murine sequence), and a mouse model capable of interacting with anti-human VISTA antibodies is constructed. Compared with ordinary mice, the model realizes the humanization modification of the key target molecule and can be used for screening and evaluating drugs targeting human VISTA genes. It is an ideal preclinical drug testing model.

[0008] However, this technology belongs to the humanization modification of antigens rather than the humanization modification of antibodies, and the secreted antibody is still of murine origin.

[0009] A fully human antibody refers to the transfer of human antibody genes to genetically engineered antibody gene-deficient animals through transgenic or transchromosomal technology, so that the animals express human antibodies, achieving the purpose of fully humanizing antibodies. In the global monoclonal antibody market, fully human monoclonal antibodies are the future development direction, and there is currently no fully humanized anti-VISTA antibody. SUMMARY

[0010] The present application provides an anti-VISTA fully human monoclonal antibody, which is composed of a heavy chain and a light chain, and the antibody specifically binds to 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 antibodies, nucleic acid molecules, expression cassettes, recombinant vectors or recombinant microorganisms, or pharmaceutical compositions of the present invention in the preparation of reagents or drugs for diagnosing, preventing and / or treating tumors, cancers, autoimmune diseases, allergies, infections or inflammatory conditions.

[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 sequence analysis. The 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 the human VISTA protein with high affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 VISTA antigen immunity was shown Serum titer test results after mice were immunized with human VISTA antigen After the mice were isolated, serum was collected and then treated with VISTA-positive transgenic cells (VISTA + -293F) were analyzed by FACS. The sera of the four mice in the figure showed clear antibodies that bound to the VISTA protein, and the immune titers were high.

[0024] Figure 2 The results show that the anti-human VISTA antibodies of the present invention and the control antibodies are sensitive to human VISTA positive transgenic cells (VISTA + -293F) FACS analysis results. The figure shows the anti-human VISTA antibodies CAM030703, CAM031004, CAM033002 and Onvatilimab (control) of the present invention and VISTA positive transgenic cells (VISTA + -293F) showed significant binding.

[0025] Figure 3 The results show that different concentrations of anti-human VISTA antibodies and control antibodies of the present invention are related to 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 of the antibody is calculated based on this. 50 value.

[0026] Figure 4 The ELISA analysis results of the binding of different concentrations of the anti-human VISTA antibodies of the present invention and control antibodies to the human VISTA protein under different pH conditions are shown. ELISA analysis of the anti-human VISTA antibodies CAM030703, CAM031004, CAM031704, CAM032011, CAM033002 and Onvatilimab (control) of the present invention with VISTA protein under pH = 6.0 and pH = 7.4 is shown in the figure, demonstrating that the anti-human VISTA antibodies of the present invention bind to the human VISTA protein to the same extent under acidic conditions. DETAILED DESCRIPTION

[0027] The following is further described in detail through specific embodiments. However, it should be noted that the following embodiments of the present invention are merely for the purpose of better illustrating the content of the present invention, and do not mean that the content of the present invention is limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the embodiments based on the above invention, which still fall within the scope of protection of the present invention and are subject to the scope of protection of the appended claims.

[0028] 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.

[0029] 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 in the present invention.

[0030] 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 mice are 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 titers, and can produce chimeric human / mouse IgM and human IgG. Upon antigen immunization, fully human IgG antibodies with therapeutic potential can be obtained in a single step. Details of the construction method of this transgenic mouse strain and the production of hybridomas and monoclonal antibodies by immunization are described in the previous research results of the inventor's team, Chinese patent documents CN105316313A and CN108486126A, which are fully incorporated into this invention as part of the present specification.

[0031] As is well known to those skilled in the art, the polyclonal antibodies described herein are mixtures of antibodies produced by different B cells in the body against the same antigen, capable of recognizing and binding to different epitopes on that single antigen. Monoclonal antibodies, on the other hand, are produced by the same B cell and can only recognize a specific single epitope.

[0032] It is well known to those skilled in the art that the chain structure and regions of the antibody molecule described herein 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), each of which includes a variable region (i.e., V region), a constant region (i.e., C region), and a hinge region. The composition and arrangement of residues near the amino termini of the heavy and light chains vary greatly, and this region is called the variable region. The composition and arrangement of residues near the carboxyl termini are relatively stable, and are therefore called the constant region. Within the variable regions of the heavy and light chains, there are three specific segments with highly variable amino acid composition and arrangement, known as hypervariable regions (HVRs). Together, these regions comprise the antigen-binding site of the antibody molecule, which is capable of complementary binding to the corresponding antigen epitope. These hypervariable regions are also known as complementarity-determining regions (CDRs), and are designated CDR1, CDR2, and CDR3, respectively. The amino acid composition and sequence of the CDRs vary between antibodies, determining the specificity of the antibody's binding to the corresponding antigen epitope and responsible for antigen recognition and binding, thereby exerting the immune response. 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).

[0033] As we know from the above common sense, the basic immune function of antibodies depends on the three CDR regions on each heavy and light chain, which are all located in the variable regions of the antibody heavy and light chains. The other amino acid sequences on the antibody molecule are generally relatively conserved, and even if there are minor 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 are identical antibodies.

[0034] 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 to 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 method is suitable for detecting large molecular antigens. Two antibodies targeting different epitopes of the same antigen are used, one of which is coated with a solid phase carrier and the other is used as an enzyme-labeled antibody. The competition method is used to detect small molecule antigens or haptens. The antigen to be tested and a certain amount of enzyme-labeled antigen compete for binding to the solid phase antibody.

[0035] The basic principle of FACS (fluorescence-activated cell sorting) used in the present invention is to utilize fluorescein-labeled specific antibodies to bind to the corresponding antigen on the cell surface, so that the cells are fluorescein-labeled. Under the action of excitation light of a specific wavelength, these fluorescein-labeled cells will produce fluorescence and are subsequently detected by flow cytometry. Flow cytometry can analyze cells one by one and measure physical characteristics such as the fluorescence intensity and size of each cell, thereby achieving quantitative analysis of antigen-antibody binding. It can be used to screen for antibodies against specific antigens. By comparing the binding ability of different antibodies to the same antigen, antibodies with high affinity and specificity can be screened out.

[0036] The basic principle of SPR (Surface Plasmon Resonance) used in the present invention is that when incident light is incident at a critical angle to the interface of two media with different refractive indices, resonance of free electrons in 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. 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 directly proportional to the mass of the biomolecules bound to the metal surface. Therefore, by obtaining the dynamic changes of the SPR angle during the biological reaction, specific signals of the interaction between biomolecules can be obtained. In antibody competitive binding analysis, SPR technology can monitor the binding and dissociation processes of antibodies with antigens or other molecules in real time. By comparing the binding abilities of different antibodies with the same antigen, the competitive relationship between antibodies can be determined.

[0037] The BLI (Bio-Layer Interferometry) used in the present invention uses a probe-type biosensor to directly detect samples without any fluorescence or isotope labeling of the test samples. The instrument emits white light to the sensor surface and collects 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 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 and SPR / BLI detection.

[0038] Other antibody-related technologies and the instruments, equipment, and materials 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, neutralizing ability), etc. If the present invention does not specifically specify the technologies, instruments, equipment, and materials used therein, any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose can be used; even if the present invention specifically specifies the technologies, instruments, equipment, and materials used therein, it does not mean that the present invention can only use these technologies, instruments, equipment, and materials. They only represent the preferred embodiments of the present invention, and those skilled in the art can still use any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose.

[0039] Example 1: Antibody Preparation and Sequencing

[0040] The fully human monoclonal antibody platform was obtained using the previous research results of the inventors' team (see Chinese patent documents CN105316313A and CN108486126A). Mice are used to prepare fully human monoclonal antibodies of the present invention. The mice are 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.

[0041] 1. Preparation of human VISTA antigen:

[0042] According to the sequence of the 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. After purification according to conventional protein purification methods in the art, human VISTA antigen was obtained.

[0043] 2. Immunization of mice:

[0044] The human VISTA antigen purified above was used to Mice were immunized by subcutaneous injection (antigen + adjuvant) or intramuscular electroporation (VISTA expression vector). Blood was collected from the mice and serum was prepared. The serum titer of the mice was analyzed by ELISA and FACS. Lymphocytes were collected from mice with a titer higher than 1:51200.

[0045] ELISA analysis of mouse serum titer utilizes the principle of specific binding between antigen and antibody, and the titer is determined by detecting the maximum dilution of the antibody in the serum. The optical density (OD value) of each well is measured at a specific wavelength (e.g., 450 nm) using a microplate reader, and the data is recorded. The critical value of the serum titer is determined based on the OD values ​​of the negative and positive controls. 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.

[0046] Similarly, a FACS instrument can be used to detect the fluorescence intensity of antigen-antibody binding on cells, and the activity or titer of antibodies in serum can be evaluated based on changes in fluorescence intensity.

[0047] 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 treated with VISTA-positive transgenic cells (VISTA + -293F) were analyzed by FACS. The results in the figure show that the sera of four of the mice clearly contained antibodies that bound to the human VISTA protein, and the immune titers were high.

[0048] 3. Hybridoma and antibody preparation:

[0049] Mice with high immune titers were immunized intraperitoneally (antigen, without adjuvant). 3-4 days after immunization, the spleen and lymph nodes of the mice were taken to prepare lymphocytes. B-cells were fused with SP2 / 0 cells by electrical stimulation using a NEPA-21 electroporator and cultured in a semi-solid culture medium (ClonaCell TM -HY Medium E, STEMCELL Technologies, #03805) for screening and cultivation. Single clones were selected and cultured in 96-well plates, and their 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 from the supernatants to the human VISTA antigen protein were then analyzed by ELISA, FACS, and / or BLI (for specific methods, see Examples 2-4 below).

[0050] Select hybridoma clones with strong binding ability and high affinity to VISTA and prepare mRNA and cDNA. Use RT-PCR to obtain the sequence of hybridoma and seamlessly link to -Blunt Zero (full gold -Blunt Zero Cloning Kit CB501). Appropriate T7 primers were designed for sequence analysis.

[0051] Sequence analysis revealed eight antibodies with strong binding ability and high affinity (CAM030703, CAM030804, CAM031004, CAM031704, CAM031912, CAM032011, CAM033012, and CAM033002). The full amino acid sequences of the heavy and light chain variable regions of three of these 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. Their corresponding encoding DNA sequences are shown in the sequence listing as SEQ ID NOs: 24 and 25, 26 and 27, and 28 and 29, respectively. Sequence analysis also revealed three CDR regions in each heavy and light chain variable region, which constitute the functionally active regions of the antibodies.

[0052] The heavy and light chains of the monoclonal antibodies obtained by PCR were synthesized and then transfected into 293F cells through expression vector combination. After purification of the antibodies, the binding ability and affinity of the obtained antibodies to the VISTA antigen protein were repeatedly analyzed by ELISA, FACS and / or BLI (for specific methods, see Examples 2-4 below).

[0053] Example 2: Affinity analysis of the anti-human VISTA antibody of the present invention and human VISTA (FACS method)

[0054] 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.

[0055] 1. Preparation of human VISTA positive cells:

[0056] The secretory peptide of immunoglobulin was linked to human VISTA protein and then stably transfected into 293F cells, and monoclonal positive cell lines were screened.

[0057] 2. FACS analysis:

[0058] 293F cells stably transfected with human VISTA gene were cultured with KO-DMEM + 10% FCS, single cells were collected, washed once with PBS + 2% FCS, and counted (cell dilution concentration was 1×10 7 / ml); 100 μl of cell suspension (including positive and negative controls) was added to each group, and the corresponding concentration of antibody (hybridoma supernatant or purified human IgG monoclonal antibody) was added. The cells were incubated 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. The cells were incubated 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 subjected to FACS analysis.

[0059] Figure 2 The results show the interaction 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 significantly associated with VISTA positive transgenic cells (VISTA + -293F) showed significant binding.

[0060] Figure 3 The results show that different concentrations of anti-human VISTA antibodies and control antibodies were detected in VISTA positive transgenic cells (VISTA + -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) were used for FACS analysis. Based on the recorded fluorescence intensity values, a curve of the relationship between antibody concentration and fluorescence intensity was drawn.

[0061] 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. Among them, the EC values ​​of CAM030703, CAM032011 and CAM033002 50 The EC values ​​of the control (ONVATILIMAB) 50 Equal or better value.

[0062]

[0063] Example 3 Affinity Analysis of the Anti-Human VISTA Antibody of the Present Invention and Human VISTA (ELISA Analysis)

[0064] 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 the cells were incubated at 4°C overnight (embedding), washed three times with PBS-0.05% Tween20, and then 200 μl of 1% BSA was added to each well for 1 hour (blocking), washed three times with PBS-0.05% Tween20, and a certain concentration of antibody was added for 2 hours, washed three 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 three times with PBST, 50 μl of TMB solution was added to each well, protected from light for 10 minutes, and then 50 μl of 2% oxalic acid solution was added to each well, and the OD was adjusted to 0. 450 The detection buffer used in normal ELISA is PBS + 2% FCS, with a pH between 7.0 and 7.4. In ELISA tests under different pH conditions, use PBS + 2% FCS buffer and adjust the pH to 6.0 and pH to 7.4 with 1N NaOH or HCl.

[0065] The ELSIA analysis results of the binding of all antibodies tested in the present invention to the VIST antigen are shown in Table 2.

[0066]

[0067] The above results demonstrate that the antibodies of the present invention specifically bind to the human VISTA protein and do not bind or have weak binding ability to other human proteins tested.

[0068] Figure 4 The results of ELISA analysis (OD 450 The binding ability of the anti-human VISTA antibodies of the present invention and the control antibody at different concentrations to the VISTA protein under different pH conditions (values) is reflected. 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 the human VISTA protein.

[0069] Example 4: Affinity Analysis of Anti-Human VISTA Antibodies of the Present Invention and Human VISTA (SPR / BLI Method)

[0070] 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:

[0071] 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 reduce nonspecific adsorption.

[0072] 2) Dilute antigen and antibody: Use PBST buffer to dilute the antigen and antibody to an appropriate concentration range, such as 5-20 μg / ml.

[0073] 3) Set experimental parameters: According to Fortebio According to the system requirements, set the experimental parameters, including the order of loading samples, detection time, temperature, etc.

[0074] 4) Loading samples: Load the diluted antigen and antibody samples into the Fortebio The corresponding position of the system.

[0075] 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).

[0076] 6) Data collection: During the testing process, Fortebio The system will automatically collect and record the binding and dissociation curves of the antigen and antibody, as well as the related kinetic parameters.

[0077] K D K is a constant that describes the affinity between antibodies and antigens, reflecting the ratio of the rate at which the antibody-antigen complex dissociates into free antibodies and antigens 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.

[0078] 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 equilibrium. AThe larger the value: the stronger the binding ability between the antibody and the antigen, that is, they are easier to bind to form a complex; K A The smaller the value: the weaker the binding ability between the antibody and the antigen, that is, they are less likely to bind.

[0079] K dis Used to describe the rate of dissociation of the antibody-antigen complex into free antibody and antigen. dis The larger the value, the faster the dissociation rate of the antibody-antigen complex, that is, the complex is easier to dissociate into free antibody and antigen; K dis The smaller the value, the slower the dissociation rate of the antibody-antigen complex, that is, the complex is more stable and less likely to dissociate.

[0080] The specific detection results are shown in Table 3. The data show that the antibodies CAM030703, CAM031004 and CAM033002 obtained by the application have an affinity for human VISTA antigen close to or even better than the positive control antibody Onvatilimab.

[0081]

[0082] On the same sensor chip or biosensor, a certain amount of antigen is first immobilized, and then antibody 1 and antibody 2 (different concentration gradients can be set) are added respectively, and their binding conditions are observed and recorded. By comparing the binding curves and dissociation curves of antibody 1 and antibody 2, and their K D values, it can be judged whether there is a competitive relationship between the two.

[0083] 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. By comparing the K D values of antibody 1 and antibody 2, it can be judged their binding ability to the antigen. If antibody 1 and antibody 2 have obvious differences in their binding curves when added to the same antigen on the 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 the K D value of antibody 1 and antibody 2 when they exist together is increased compared with when they exist alone (i.e., the affinity is weakened), it further confirms the competitive relationship between the two.

[0084] The antibodies CAM030703, CAM031004 and CAM033002 obtained by the application were subjected to competitive binding experiments with the positive control antibody Onvatilimab, and the results are shown in Table 4. The data prove that some antibodies do not directly compete, and the use of multiple antibodies at the same time helps to enhance their effect.

[0085]

[0086] The above is only an embodiment of the present application, and the technical knowledge in the art is not described in detail. Those skilled in the art know all the technical knowledge in the art before the filing date, can know all the prior art in the art, and have the ability to apply the conventional experimental means before the date. Those skilled in the art can improve and implement the present application based on their own ability under the guidance of the present application. Some typical known technologies should not be an obstacle for those skilled in the art to implement the present application. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, some adjustments and improvements can be made, which should be considered as the protection scope of the present application. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode in the specification can be used to explain 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 the human VISTA protein; The variable region of the heavy chain includes three CDR regions in order from 5' to 3' of the amino acid sequence, wherein the amino acid sequence of the first CDR region of the heavy chain is SEQ ID NO: 7, the amino acid sequence of the second CDR region of the heavy chain is SEQ ID NO: 8, and the amino acid sequence of the third CDR region of the heavy chain is SEQ ID NO: 9; The variable region of the light chain includes three CDR regions in order from 5' to 3' of the amino acid sequence, wherein the amino acid sequence of the first CDR region of the light chain is SEQ ID NO: 10, the amino acid sequence of the second CDR region of the light chain is SEQ ID NO: 11, and the amino acid sequence of the third CDR region of the light chain is SEQ ID NO:

12.

2. The anti-VISTA fully human monoclonal antibody according to claim 1, wherein The full-length amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1; the full-length amino acid sequence of the light chain variable region of the antibody is SEQ ID NO:

2.

3. A nucleic acid molecule encoding the antibody according to claim 1 or 2.

4. An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule according to claim 3.

5. A pharmaceutical composition comprising the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the expression cassette, recombinant vector or recombinant microorganism according to claim 4, and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, wherein The excipient is a diluent or a carrier.

7. Use of the antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression cassette, recombinant vector or recombinant microorganism according to claim 4, or the pharmaceutical composition according to claim 5 or 6 in the preparation of a detection reagent for detecting VISTA antigen.

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