CN117285628A - anti-VISTA antibodies and uses thereof - Google Patents

Abstract

The invention discloses an anti-VISTA antibody and application thereof, relates to the field of antibodies, and provides an anti-VISTA antibody, wherein CDRs of the anti-VISTA antibody are selected from any one of (1) to (7), the antibody or antigen binding fragment has high affinity to VISTA, can specifically identify and bind to the VISTA, and is applied to construction of an antibody drug conjugate ADC, and through an in vitro killing experiment, the VISTA-MMAE ADC has remarkable anti-tumor effect, so that the anti-VISTA antibody can be widely applied to preparation of drugs for preventing, diagnosing and treating tumors and autoimmune diseases.

Description

Anti-VISTA antibodies and their applications

Technical Field

The present invention relates to the field of antibodies, and in particular, to anti-VISTA antibodies and applications thereof.

Background Art

As an innovative treatment method, tumor immunotherapy has become a hot topic in the field of tumor treatment research and is expected to bring new treatment options to patients. By blocking the PD-L1/PD-1 pathway or the CTLA-4 signaling pathway, the immunosuppressive state of T cells can be reversed, and the ability of the patient's own immune system to inhibit or kill tumors can be improved. However, currently, blockers of the PD-L1/PD-1 pathway or the CTLA-4 pathway are only effective for a small number of patients in clinical practice, and new immunotherapy targets or new immunotherapy strategies still need to be explored.

V-domain Ig suppressor of T cell activation (VISTA) belongs to the B7 family of molecules. Unlike other immune checkpoint molecules, VISTA is constitutively expressed in naive T cells ( In addition to T cells, VISTA is also expressed on the surface of antigen-presenting cells. VISTA inhibits T cell activation through mechanisms inside and outside T cells. In short, VISTA is a unique negative checkpoint regulator on naive T cells, which is essential for maintaining their stable state of rest and peripheral immune tolerance.

The human VISTA protein has a total of 279 amino acid residues, including an extracellular domain (ECD) of 162 amino acid residues, a transmembrane domain of 21 amino acid residues, and a cytoplasmic domain of 96 amino acid residues. VISTA is highly expressed in bone marrow-derived cells, such as CD11b+ monocytes, CD11c+ dendritic cells, and to a lesser extent CD4+ and CD8+ T cells. Overexpression of VISTA has been found in a variety of human cancers, including prostate cancer, renal clear cell carcinoma, non-small cell lung cancer, colorectal cancer, and pleural mesothelioma.

Therefore, the development of anti-VISTA antibodies with strong specificity and high affinity will provide possibilities for the treatment of various cancers and has huge application potential and market value.

In view of this, the present invention is proposed.

Summary of the invention

The object of the present invention is to provide anti-VISTA antibodies and applications thereof.

The present invention is achieved in that:

In a first aspect, an embodiment of the present invention provides an anti-VISTA antibody or antigen-binding fragment, comprising the CDRs shown in any one of items (1) to (7):

(1) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 2 to 4, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 6 to 8; (2) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 10 to 12, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 14 to 16; (3) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18 to 20, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 22 to 24; (4) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 26 to 28, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 30 to 32; (5) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: NO: 34-36, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 38-40, respectively; (6) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 42-44, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 46-48, respectively; (7) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 50-52, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 54-56, respectively.

In a second aspect, an embodiment of the present invention provides an antibody conjugate, which includes: the antibody or antigen-binding fragment thereof described in the above embodiment.

In a third aspect, embodiments of the present invention provide the use of the antibody or antigen-binding fragment thereof as described in the preceding embodiments in the preparation of a detection product for VISTA antigen.

In a fourth aspect, embodiments of the present invention provide the use of the antibody or antigen-binding fragment thereof as described in the preceding embodiments in the preparation of a product for targeting VISTA to diagnose, prevent or treat a disease.

In a fifth aspect, an embodiment of the present invention provides a reagent or a kit, which includes the antibody or antigen-binding fragment thereof as described in the above embodiments.

In a sixth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the preceding embodiments.

In a seventh aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

In an eighth aspect, an embodiment of the present invention provides a cell comprising the vector described in the preceding embodiment.

In a ninth aspect, an embodiment of the present invention provides a drug or a pharmaceutical composition, the active ingredients of which include the antibody or antigen-binding fragment thereof as described in the preceding embodiments, the antibody conjugate as described in the preceding embodiments, the reagent or kit as shown in the preceding embodiments, the isolated nucleic acid as described in the preceding embodiments, and the vector as described in the preceding embodiments or at least one of the cells as described in the preceding embodiments.

The present invention has the following beneficial effects:

The present invention provides an anti-VISTA antibody, which has a high affinity for VISTA and can specifically recognize and bind to VISTA.

Based on the provided anti-VISTA antibody, the present invention constructed an antibody-drug conjugate (ADC). Through in vitro killing experiments, the anti-tumor effect of the VISTA-MMAE ADC provided by the present invention was verified. The VISTA-MMAE ADC had a significant killing effect on NCI-H2803-hVISTA-GFP tumor cells.

The anti-VISTA antibodies provided by the present invention can be widely used in the preparation of drugs for preventing, diagnosing and treating tumors and autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

Figure 1 is an immunofluorescence analysis of the binding results of recombinant mouse monoclonal antibodies 2D12, 16H8, 10A12, 1G2, JCY, F8, and KY to VISTA molecules overexpressed in cells;

FIG2 shows the in vitro tumor killing results of ADCs of 2D12, 16H8, 10A12, 1G2, JCY, F8, and KY coupled with MMAE, respectively;

FIG3 is an in vivo anti-tumor analysis result of ADCs of 2D12, 16H8, 10A12, 1G2, JCY, F8, and KY conjugated with MMAE, respectively, evaluated using the NCG mouse model;

FIG. 4 shows the immunohistochemical analysis of VISTA in clinical tissue samples of pleural mesothelioma.

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

In one aspect, an embodiment of the present invention provides an anti-VISTA antibody or antigen-binding fragment, comprising the CDRs shown in any one of items (1) to (7):

(1) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 2 to 4, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 6 to 8, respectively;

(2) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 14 to 16, respectively;

(3) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18 to 20, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 22 to 24;

(4) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 26 to 28, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 30 to 32;

(5) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 34 to 36, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 38 to 40;

(6) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 42 to 44, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 46 to 48, respectively;

(7) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 50 to 52, respectively. The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 54 to 56, respectively.

The present invention provides an anti-VISTA antibody, which has a high affinity for VISTA and can specifically recognize and bind to VISTA.

In the present invention, the term "antibody" covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, full-length antibodies, etc., as long as they exhibit the desired antigen-binding activity.

The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region gene must be cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody must be cloned as needed, and the mouse variable region gene and the human constant region gene must be connected into a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule must be expressed in a eukaryotic or prokaryotic industrial system.

In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the hypervariable region of the heavy and light chains of immunoglobulins, which is the region within the antibody variable domain that mainly contributes to specific binding to an antigen.

In the present invention, the heavy chain complementary determining region is represented by HCDR, and the three CDR regions contained in the heavy chain variable region are: HCDR1, HCDR2 and HCDR3, or VH-CDR1, VH-CDR2 and VH-CDR3; the light chain complementary determining region is represented by LCDR, and the three CDR regions contained in the light chain variable region are: LCDR1, LCDR2 and LCDR3, or VL-CDR1, VL-CDR2 and VL-CDR3.

In some embodiments, the antibody or antigen-binding fragment further comprises a framework region of a heavy chain variable region and a framework region of a light chain variable region.

In the present invention, "framework region" or "FR" region refers to the region other than CDR in the heavy chain variable region and light chain variable region of an antibody; the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR4), wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by LCDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (arranged from the amino terminal to the carboxyl terminal): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (arranged from the amino terminal to the carboxyl terminal): LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In some embodiments, the heavy chain variable region and the light chain variable region are as shown in any one of (a) to (g):

(a) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 1 and 5, respectively;

(b) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 9 and 13, respectively;

(c) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 17 and 21, respectively;

(d) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 25 and 29, respectively;

(e) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 33 and 37, respectively;

(f) the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 41 and 45, respectively;

(g) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 49 and 53, respectively.

It should be noted that the CDRs shown in (1) to (7) correspond one-to-one to the heavy chain variable regions and light chain variable regions shown in (a) to (g).

In some embodiments, the antibody or antigen-binding fragment further comprises a constant region. Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region. The light chain of the full-length antibody comprises a light chain variable region domain VL and a constant region domain CL, VL is at the amino terminus of the light chain, the CL domain is at the carboxyl terminus, and the light chain comprises a κ chain and a λ chain; the full-length antibody heavy chain comprises a heavy variable region domain VH and a constant region CH, VH is at the amino terminus of the heavy chain, and the CH domain is at the carboxyl terminus.

In some embodiments, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

In some embodiments, the species of origin of the constant region is cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

In some embodiments, the antibody is selected from any one of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a murine antibody, a chimeric antibody, and a full-length antibody.

The "antigen binding fragment" herein is a portion of a complete antibody that specifically binds to an antigen to which the complete antibody binds. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the antigen binding fragment can be prepared by methods known in the art, for example, by enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds, and can also be synthesized by recombinant genetics technology or by an automatic peptide synthesizer (such as the automatic peptide synthesizer of Applied BioSystems).

In some embodiments, the antigen-binding fragment is selected from any one of F(ab') ₂ , Fab', Fab, Fv and scFv of an antibody.

On the other hand, an embodiment of the present invention provides an antibody conjugate, which includes: the antibody or antigen-binding fragment thereof described in any of the above embodiments.

In some embodiments, the antibody conjugate further comprises a drug molecule conjugated to the antibody or antigen-binding fragment.

In some embodiments, the drug molecule comprises: monomethyl auristatin E.

In some embodiments, the antibody conjugate further comprises a solid support coupled to the antibody or antigen-binding fragment thereof.

In some embodiments, the antibody conjugate further comprises a detectable label coupled to the antibody or antigen-binding fragment thereof.

In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

In some embodiments, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle label.

On the other hand, an embodiment of the present invention provides the use of an antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments in the preparation of a detection product for a VISTA antigen.

In some embodiments, the product includes any one of a test strip, a reagent, and a kit;

In some embodiments, the detection method is selected from any one of ELISA, immunofluorescence, chemiluminescent immunoassay, Western blot, immunochromatography, electrochemical immunoassay and magnetic bead method.

On the other hand, an embodiment of the present invention provides a use of an antibody or an antigen-binding fragment thereof as described in any of the preceding embodiments in the preparation of a product for targeting VISTA to diagnose, prevent or treat a disease.

In some embodiments, the disease includes any one of a tumor and an autoimmune disease.

In some embodiments, the tumor comprises at least one of prostate cancer, renal clear cell carcinoma, non-small cell lung cancer, colorectal cancer, and pleural mesothelioma.

In some embodiments, the product is selected from any one of a reagent, a kit, and a medicine.

On the other hand, an embodiment of the present invention provides a reagent or a kit, which includes the antibody or antigen-binding fragment thereof as described in any of the above embodiments.

In another aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in any of the preceding embodiments.

In another aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in any of the preceding embodiments.

In some embodiments, the vector includes an expression vector. The "expression vector" described in the present invention refers to any recombinant polynucleotide construct, which can directly or indirectly (such as packaged into a virus) introduce the target DNA fragment into a host cell by transformation, transfection or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can connect the target DNA fragment to the plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, the target gene can be expressed.

A person skilled in the art can also transcribe the nucleic acid sequence of the present invention into RNA by in vitro transcription, and further transfect, transduce or transform the RNA into a host cell to express the antibody of the present invention or its functional fragment to exert the biological efficacy of the present invention.

In another aspect, an embodiment of the present invention provides a cell comprising the vector described in any of the above embodiments.

In some embodiments, the cell is a host cell, and the host cell includes a prokaryotic host cell, a eukaryotic host cell, and a bacteriophage. The prokaryotic host cell may be Escherichia coli, Streptomyces, or Bacillus subtilis, etc. The eukaryotic host cell may be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6, Saccharomyces cerevisiae, Pichia pastoris, Hansen yeast, Candida, some insect cells, and plant cells. 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

On the other hand, an embodiment of the present invention provides a method for producing the antibody or antigen-binding fragment thereof as described in any of the above embodiments, which comprises culturing cells capable of expressing the antibody or antigen-binding fragment thereof.

Based on the amino acid sequence of the antibody disclosed in the present invention, those skilled in the art can prepare the antibody by genetic engineering technology or other technologies (chemical synthesis, recombinant expression), for example, separating and purifying the antibody from the culture product of a recombinant cell capable of recombinantly expressing the antibody as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody of the present invention, it belongs to the protection scope of the present invention.

In addition, an embodiment of the present invention further provides a drug or a pharmaceutical composition, the active ingredient of which includes the antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, the antibody conjugate as described in any of the preceding embodiments, the reagent or kit as shown in any of the preceding embodiments, the isolated nucleic acid as described in any of the preceding embodiments, and the vector as described in any of the preceding embodiments or at least one of the cells as described in any of the preceding embodiments.

The "pharmaceutical composition" of the present invention refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and/or space, as long as they can work together to achieve the purpose of the present invention. Some pharmaceutical compositions are achieved by co-administering some pharmaceutically acceptable ingredients or compounds to enhance the biological efficacy of the present invention or reduce the side effects of the drug (for example, it can be used in combination with other anti-tumor drugs to enhance the anti-tumor effect). The purpose of other pharmaceutical compositions is to promote administration to an organism, facilitate the absorption of active ingredients, enhance stability or targeting, prolong the half-life, and thus better exert the biological efficacy of the present invention.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

(1) Preparation of VISTA recombinant protein: The nucleic acid sequence encoding human VISTA (NM_022153.2) was synthesized by Anhui General Biotechnology Co., Ltd. PCR amplification and subcloning into the pcDNA3.1 expression vector (Invitrogen). Then, the extracellular domain of VISTA was subcloned into the pcDNA3.1 expression vector carrying an Fc or His tag at the C-terminus. The Fc tag includes human Fc (hFc) and mouse Fc (mFc). By transient transfection of 293FT, FreeStyle ^TM serum-free medium (Life Technologies) was used for shake flask culture for 5-7 days, the supernatant was collected, subjected to centrifugal ultrafiltration, and then purified by Protein A/G or nickel column affinity chromatography and molecular sieve chromatography column. The recombinant VISTA protein carrying the Fc or His tag.

(2) Preparation of stable cell lines expressing human VISTA antigen: The full-length sequence encoding human VISTA was constructed into the pcDNA3.1 expression vector carrying IRES-EGFP. CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies); Hela cells were cultured in DMEM containing 10% fetal bovine serum. Cells were transfected with Lipofectamine LTX (Life Technologies) transfection reagent, flow sorted 48 hours later, cultured to 96-well plates, and monoclonal stable cell lines were screened and identified. CHO (CHO-VISTA-EGFP) and Hela (Hela-VISTA-EGFP) cells stably expressing VISTA were maintained.

Example 2

1. Preparation of anti-VISTA monoclonal antibodies:

(1) Animal immunization

5-6 week old Balb/c female mice were used as immunized animals, and the immunization dose was 100 μg/mouse. For the first immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant VISTA protein, and multiple subcutaneous injections were performed after full emulsification. Every 2 weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, and multiple subcutaneous injections were performed after full emulsification. The booster immunization was performed 4 times in total. On the 10th day after the last booster immunization, the mouse antibody titer was detected by cutting the tail and collecting blood. 3 days before cell fusion, 100 μg of recombinant protein was intraperitoneally impacted once.

(2) Cell fusion and hybridoma screening

Under sterile conditions, the spleen of mice was taken to prepare a suspension rich in B cells, and the cells were fused with SP2/0 according to the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium for culture. Fresh HAT medium was used for half-medium replacement culture on the 5th and 10th days after fusion. ELISA, immunofluorescence and flow cytometry were performed on the 11th to 15th day after fusion to screen positive clones.

ELISA screening was performed using a 96-well plate. Briefly, VISTA recombinant protein was coated at 100 ng/well at 4 degrees overnight on the bottom of the well plate, and HRP-coupled anti-mouse IgG antibody and chemiluminescent reagent (Biyuntian Biotechnology) were used for color development, and the values were read on a microplate reader at a wavelength of 450 nm. Immunofluorescence staining used Hela cell lines that stably expressed VISTA. Briefly, the cell lines were cultured on 96-well plates, 50 μl of hybridoma supernatant was added as the primary antibody, incubated at 4 degrees for 2 hours, washed 3 times with PBS, and Cy3-labeled Goat Anti-Mouse IgG (Proteintech) was used as the secondary antibody, incubated at room temperature for 1 hour, washed 3 times with PBS, and images were collected using a fluorescence microscope. Flow cytometry analysis was performed using CHO cells stably expressing VISTA. Briefly, cells were collected by centrifugation and resuspended in PBS buffer containing 50 μl of hybridoma supernatant, incubated at 4°C for 2 h, washed three times with PBS, and CY3-labeled Goat Anti-Mouse IgG (Beyotime Biotech) was used as a secondary antibody. The cells were incubated at room temperature for 1 h, washed three times with PBS, and analyzed on a flow cytometer (Agilent-Novosampler Pro).

Based on the above ELISA analysis, immunofluorescence analysis and flow cytometry analysis results, seven optimal hybridoma clones (named 2D12, 16H8, 10A12, 1G2, JCY, F8, KY) were finally determined for subsequent experiments such as sequence cloning and affinity analysis.

Example 3

Hybridoma antibody variable region sequence cloning: Hybridoma cells in the logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen) and reverse transcribed (PrimeScriptTM Reverse Transcriptase, Takara). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen) and then sequenced, and finally the heavy chain and light chain variable region sequences of seven monoclonal antibodies were obtained. The variable region CDR sequences of the heavy chain and light chain are shown in Table 1.

Table 1. CDR sequences contained in the heavy and light chain variable regions of mouse monoclonal antibodies

Note: VH is the heavy chain variable region, and VL is the light chain variable region.

Example 4

Binding analysis of recombinant chimeric antibodies to HeLa cervical cancer cells overexpressing VISTA.

The human (IgG1) and mouse (IgG2a) heavy chain constant regions, as well as the human/mouse light chain constant regions, were cloned into the pcDNA3.1 (Invitrogen) plasmid vector, and then the VH and VL gene fragments of the hybridoma clones 2D12, 16H8, 10A12, 1G2, JCY, F8, and KY were constructed into the human IgG1 heavy chain constant region (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT, SEQ ID NO: 57) and the human IgGκ light chain constant region (TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO: 58). NO:58) on the gene recombination vector, the recombinant chimeric antibody heavy chain expression vector and light chain expression vector were obtained, and 293FT was transiently transfected, and FreeStyle ^TM serum-free medium (Life Technologies) was used to shake the flask for 5-7 days, and the supernatant was collected, and then centrifuged and ultrafiltered, and then purified by Protein A/G affinity chromatography and molecular sieve chromatography column to obtain the corresponding type of anti-VISTA recombinant monoclonal antibody. Hela-VISTA-EGFP cells were plated in a 24-well cell culture dish, and the recombinant chimeric antibodies 2D12, 16H8, 10A12, 1G2, JCY, F8, KY were used as primary antibodies on the second day, and CY3-labeled Goat Anti-Mouse IgG (Biyuntian Biotechnology Company) was used as a secondary antibody, and it was observed and photographed with fluorescence confocal.

As shown in Figure 1, the results in Figure 1 show that the recombinant chimeric antibodies 2D12, 16H8, 10A12, 1G2, JCY, F8, and KY can specifically bind to the cells Hela-VISTA-EGFP.

Example 5

Binding analysis of hybridoma antibodies to murine VISTA.

The cDNA clone of mouse VISTA was purchased from Sino Biological, and then the stable expression cell line CHO-mVISTA of mouse VISTA was constructed according to Example 2. Then the supernatants of hybridomas 2D12, 16H8, 10A12, 1G2, JCY, F8, and KY were taken and flow cytometry analysis was performed according to Example 2. The results showed that the monoclonal antibody could not bind to mouse VISTA.

Example 6

In vitro binding affinity and kinetics experiments.

This embodiment adopts the surface plasmon resonance (SPR) method to measure, and uses GE's Biacore 8K instrument for analysis. Using the kit provided by Biacore, the VISTA-His recombinant protein is covalently linked to the CM5 (GE) chip by the standard amino coupling method, and then the antibody to be tested (recombinant chimeric antibody 2D12, 16H8, 10A12, 1G2, JCY, F8, KY) is diluted in the same buffer according to different concentration gradients and injected. After injection, it is regenerated with the regeneration reagent in the kit. The analysis and collection of data are carried out using Biacore 8K supporting analysis software. The results are shown in Table 2 below.

Table 2. Antibody-antigen in vitro binding affinity and kinetic analysis

Antibody antigen Binding rate ka (1/M*s) Dissociation rate kd (1/s) Affinity KD(M) 2D12 VISTA-His 6.78E+05 2.33E-04 3.44E-10 16H8 VISTA-His 5.28E+05 2.31E-03 4.38E-09 10A12 VISTA-His 1.64E+06 2.87E-04 1.75E-10 1G2 VISTA-His 2.34E+05 3.55E-07 1.52E-12 JCY VISTA-His 4.32E+05 3.14E-05 7.28E-11 F8 VISTA-His 1.32E+06 6.02E-06 4.59E-12 KY VISTA-His 1.96E+06 3.82E-04 1.95E-10

Example 7

In vitro killing experiment of VISTA-MMAE ADC.

After three days of co-incubation with VISTA-MMAE and NCI-H2803-hVISTA-GFP cells, CCK8 reagent was used to detect cell viability. The drugs were divided into monoclonal antibody group and VISTA-MMAE group. The drug concentration was diluted three times from 1μM to the twelfth concentration. The control group was only added with cells and only added with culture medium. There were 3 replicates in each group. About 5000 NCI-H2803-hVISTA-GFP cells were plated in a 96-well plate. On the second day, each group of drugs was added to the cells and placed in an incubator at 37°C for 72h. Take out the 96-well plate, add 10μl CCK8 reagent to each well, place it in a 37°C incubator again, and incubate for 2h. Take out the well plate and detect the absorbance at 450nm. The results are shown in Figure 2.

As shown in the results of Figure 2, compared with the monoclonal antibody group, VISTA-MMAE ADC (recombinant chimeric antibodies 2D12, 16H8, 10A12, 1G2, JCY, F8, KY) has a significant killing effect on NCI-H2803-hVISTA-GFP tumor cells.

Example 8

Anti-tumor experiments in xenograft mouse models.

This example uses a xenograft mouse model to evaluate the in vivo anti-tumor activity of VISTA-targeted antibodies (recombinant chimeric antibodies 2D12, 16H8, 10A12, 1G2, JCY, F8, KY). An immunodeficient mouse model was used for evaluation.

NCG severe immunodeficiency mouse model: NCG severe immunodeficiency mice were purchased from the Model Animal Institute of Nanjing University, and 3×10 ⁶ logarithmic growth phase NCI-H2803-hVISTA-GFP cells were inoculated subcutaneously on the right back of NCG mice. After about 6 days when the tumor grew to 200mm ³ , mice with uniform tumor volume were randomly divided into groups, with 5 mice in each group. An ADC storage buffer equal to the volume of the drug was set as the control group. The ADC was administered by tail vein administration, 5, 10 or 15 mg/kg, once every 4 days, for a total of 3 times. The mice were weighed and the tumor size was measured every 4 days. The average volume of the transplanted tumor was calculated according to the formula V=1/2(L×W2), where L represents the length of the tumor and W represents the width of the tumor. When the mouse tumor volume reached 2000mm ³ or the tumor surface showed obvious ulceration, the mouse was killed and the animal experiment was terminated. The experimental results are shown in Figure 3.

As shown in the results of Figure 3, compared with the control group without medication, VISTA-MMAE ADC (2D12, 16H8, 10A12, 1G2, JCY, F8, KY) had a significant inhibitory effect on the growth of NCI-H2803-hVISTA-GFP tumor cells.

Embodiment 9

Immunohistochemical analysis of VISTA in clinical tumor tissue specimens.

The tissue sections were incubated at 65°C for 1 h, blocked with PBS containing 10% goat serum for 30 minutes at room temperature, and then incubated with VISTA monoclonal antibodies (2D12 and 16H8 in Example 4) at 4°C overnight. The sections were incubated with goat anti-human secondary antibodies and stained with 3,3'-diaminobenzidine. The percentage of positively stained cells and the cell staining intensity of 5 randomly selected fields of view were simply counted.

Staining intensity score: 0 (negative); 1 (weakly positive); 2 (moderately positive); 3 (strongly positive). The total score was quantified as: histopathological score = (1×weakly positive staining%+2×positive staining%+3×strongly positive staining%)×100. The experimental results are shown in Figure 4.

As shown in Figure 4, VISTA is highly expressed in pleural mesothelioma.

The following is the sequence involved in the present invention:

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. An anti-VISTA antibody or antigen-binding fragment comprising the CDRs of any one of (1) to (7):

(1) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 2-4, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:6 to 8;

(2) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 10-12, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:14 to 16;

(3) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 18-20, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 22-24;

(4) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 26-28, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:30 to 32;

(5) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 34-36, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:38 to 40;

(6) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 42-44, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:46 to 48;

(7) The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 50-52, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:54 to 56.

2. The antibody or antigen-binding fragment of claim 1, further comprising a framework region of a heavy chain variable region and a framework region of a light chain variable region;

optionally, the heavy chain variable region and the light chain variable region are as set forth in any one of (a) to (g):

(a) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:1 and 5;

(b) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:9 and 13;

(c) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:17 and 21;

(d) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:25 and 29;

(e) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:33 and 37;

(f) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:41 and 45;

(g) The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:49 and 53;

optionally, the antibody or antigen binding fragment further comprises a constant region;

alternatively, the constant region is selected from the group consisting of a constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD;

optionally, the constant region is derived from bovine, equine, porcine, ovine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human;

alternatively, the antibody is selected from any one of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a murine antibody, a chimeric antibody, and a full-length antibody;

alternatively, the antigen binding fragment is selected from the group consisting of F (ab') ₂ Any of Fab', fab, fv and scFv.

3. An antibody conjugate, comprising: the antibody or antigen-binding fragment thereof of claim 1 or 2;

optionally, the antibody conjugate further comprises a drug molecule conjugated to the antibody or antigen binding fragment;

optionally, the drug molecule comprises: monomethyl auristatin E;

optionally, the antibody conjugate further comprises a solid support coupled to the antibody or antigen binding fragment thereof;

optionally, the antibody conjugate further comprises a detectable label conjugated to the antibody or antigen binding fragment thereof.

4. Use of the antibody or antigen binding fragment thereof of claim 1 or 2 in the preparation of a detection product for VISTA antigen;

optionally, the product comprises any one of a test paper, a reagent and a kit;

optionally, the method of detecting is selected from: ELISA, immunofluorescence, chemiluminescent immunoassay, western blot, immunochromatography, electrochemical immunoassay and magnetic bead method.

5. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2 in the manufacture of a product for targeting VISTA for diagnosis, prevention or treatment of a disease;

optionally, the disease comprises: any one of a tumor and an autoimmune disease;

optionally, the tumor comprises: at least one of prostate cancer, renal clear cell carcinoma, non-small cell lung cancer, colorectal cancer, and pleural mesothelioma;

optionally, the product is selected from any one of a reagent, a kit and a medicament.

6. A reagent or kit comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1 or 2.

8. A vector comprising the isolated nucleic acid of claim 7.

9. A cell comprising the vector of claim 8.

10. A medicament or pharmaceutical composition, characterized in that the active ingredients thereof comprise: at least one of the antibody or antigen binding fragment thereof of claim 1 or 2, the antibody conjugate of claim 3, the reagent or kit of claim 6, the isolated nucleic acid of claim 7 and the vector of claim 8 and the cell of claim 9.