Anti-human tigit monoclonal antibody tigit-m003 or functional fragment thereof and application thereof

Abstract

The application belongs to the technical field of biomedical and antibody engineering, and more particularly relates to an anti-human TIGIT monoclonal antibody TIGIT-M003 or a functional fragment thereof, and application of the anti-human TIGIT monoclonal antibody TIGIT-M003 or the functional fragment thereof as an immunoconjugate, a chimeric antigen receptor and / or a composition, and specifically discloses a monoclonal antibody or an antigen binding fragment thereof capable of combining with human TIGIT protein. The TIGIT-specific monoclonal antibody screened by the application has high sensitivity, high specificity and high affinity.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and antibody engineering technology; more specifically, it relates to anti-human TIGIT antibodies or functional fragments thereof, and the use of anti-human TIGIT antibodies or functional fragments thereof as immunoconjugates, chimeric antigen receptors and / or compositions. Background Technology

[0002] Immunotherapy has provided a new avenue for cancer treatment. Antibody drugs, represented by immune checkpoint inhibitors such as programmed cell death receptor 1 (PD-1), programmed cell death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), and lymphocyte activation gene-3 (LAG-3), have benefited patients with various cancers. However, many patients show no initial response or only a partial response to the drugs, and those who do experience tumor progression. This may be related to the effects of other immunosuppressive molecules and incomplete activation of the immune system. Screening and combining new immunotherapeutic targets to optimize treatment strategies is a major trend in current cancer treatment.

[0003] TIGIT (T cell immunoglobulin and ITIM domains) is also known as V-set and transmembrane domain-containing protein 3 (VSTM3) and V-set and immunoglobulin domain-containing protein 9 (V... SIG9) and WUCAM (Washington University cell adhesion molecule) are among the novel immunosuppressive receptors that have attracted much attention in recent years. TIGIT consists of an extracellular IgV domain, a type I transmembrane protein region, and the cytoplasmic tail of an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoglobulin tail tyrosine-like motif (ITT). It is expressed on the surface of activated T cells, natural killer cells (NK), regulatory T cells (Treg), and CD4 helper T cells (Th). TIGIT has three ligands: CD155 (PVR), CD112 (PVRL2, Nectin-2), and CD113 (Nectin-3). TIGIT has the highest affinity for CD155, which has been identified in dendritic cells (DCs), macrophages, and many human cancer cells. Binding to TIGIT has been shown to downregulate T cell activation and cytokine secretion. Inhibition of the TIGIT / PVR interaction can mediate effective anti-tumor activity of immune cells. DNAM-1 (CD226) is an activation receptor found on NK cells, monocytes, and T cells. TIGIT and DNAM-1 compete for the shared ligands CD155 and CD112, expressed by tumor cells and antigen-producing cells. Binding of TIGIT to CD155 or CD112 leads to immunosuppression, while binding of DNAM-1 to the same ligand mediates immune activation. Therefore, TIGIT is also a potential target for tumor immunotherapy. Research on TIGIT antibodies for anti-tumor treatment is currently in clinical trials, with the most advanced being in Phase III clinical trials.

[0004] Most existing TIGIT monoclonal antibodies are prepared using mouse hybridoma technology. Advances in antibody engineering have facilitated antibody preparation and improved their detection performance. Phage antibody library technology can be used to screen for antibodies specific to any antigen. This study utilized an immunized mouse phage antibody library to screen for high-performance monoclonal antibodies against human TIGIT protein, achieving high-affinity antibodies compared to mouse hybridoma antibody screening technology. Summary of the Invention

[0005] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof capable of binding to human TIGIT protein, wherein the anti-human TIGIT monoclonal antibody comprises complementarity-determining region sequences of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region complementarity-determining region contain CDR1, CDR2 and CDR3, respectively.

[0006] The amino acid sequence of the heavy chain variable region CDR region is shown below:

[0007] The amino acid sequence of VHCDR1 is SEQ ID NO.1;

[0008] The VHCDR2 amino acid sequence is selected from one or more of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5;

[0009] The amino acid sequence of VHCDR3 is SEQ ID NO.6 and / or SEQ ID NO.7;

[0010] The amino acid sequence of the CDR region of the light chain variable region is shown below:

[0011] The VLCDR1 amino acid sequence is selected from one or more of SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11;

[0012] The amino acid sequence of VLCDR2 is SEQ ID NO.12 and / or SEQ ID NO.13;

[0013] The VLCDR3 amino acid sequence is selected from one or more of SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17.

[0014] Specifically, when the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.2, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.12, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.14, the monoclonal antibody that can bind human TIGIT protein is TIGIT-M001. The VH and VL amino acid sequences of the monoclonal antibody that can bind human TIGIT protein, TIGIT-M001, are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively; and the VH and VL nucleotide sequences of TIGIT-M001 are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively.

[0015] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.3, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.7; the amino acid sequence of VLCDR1 is selected from SEQ ID NO.9, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.13, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.15, the monoclonal antibody that can bind human TIGIT protein is TIGIT-M003; the VH and VL amino acid sequences of the monoclonal antibody that can bind human TIGIT protein TIGIT-M003 are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively; the VH and VL nucleotide sequences of TIGIT-M003 are shown in SEQ ID NO.34 and SEQ ID NO.35, respectively.

[0016] The amino acid sequences of VHCDR1 are selected from SEQ ID NO.1, VHCDR2 from SEQ ID NO.2, and VHCDR3 from SEQ ID NO.6; the amino acid sequences of VLCDR1 from SEQ ID NO.10, VLCDR2 from SEQ ID NO.12, and VLCDR3 from SEQ ID NO.16; the monoclonal antibody that can bind human TIGIT protein is TIGIT-M006; the VH and VL amino acid sequences of the monoclonal antibody that can bind human TIGIT protein, TIGIT-M006, are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively; the VH and VL nucleotide sequences of TIGIT-M006 are shown in SEQ ID NO.36 and SEQ ID NO.37, respectively.

[0017] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.4, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.11, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.13, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.15, the monoclonal antibody that can bind human TIGIT protein is TIGIT-M007; the VH and VL amino acid sequences of the monoclonal antibody that can bind human TIGIT protein TIGIT-M007 are shown in SEQ ID NO.24 and SEQ ID NO.25, respectively; and the VH and VL nucleotide sequences of TIGIT-M007 are shown in SEQ ID NO.38 and SEQ ID NO.39, respectively.

[0018] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.10, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.13, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.17, the monoclonal antibody that can bind human TIGIT protein is TIGIT-M011; the VH and VL amino acid sequences of the monoclonal antibody that can bind human TIGIT protein TIGIT-M011 are shown in SEQ ID NO.26 and SEQ ID NO.27, respectively; and the VH and VL nucleotide sequences of TIGIT-M011 are shown in SEQ ID NO.40 and SEQ ID NO.41, respectively.

[0019] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.2, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.10, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.13, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.17, the monoclonal antibody that can bind human TIGIT protein is TIGIT-M013; the VH and VL amino acid sequences of the monoclonal antibody that can bind human TIGIT protein TIGIT-M013 are shown in SEQ ID NO.28 and SEQ ID NO.29, respectively; and the VH and VL nucleotide sequences of TIGIT-M013 are shown in SEQ ID NO.42 and SEQ ID NO.43, respectively.

[0020] Furthermore, the sequences of the complementarity-determining regions of the heavy chain variable region (VH) and light chain variable region (VL) of the monoclonal antibody capable of binding human TIGIT protein also include their mutant sequences, the mutant sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity.

[0021] Furthermore, the monoclonal antibody or its antigen-binding fragment capable of binding to human TIGIT protein includes an antibody constant region Fc, which comprises a heavy chain constant region and a light chain constant region.

[0022] Furthermore, the light chain constant region of the monoclonal antibody or its antigen-binding fragment capable of binding to human TIGIT protein includes a κ-type or λ-type light chain constant region.

[0023] Furthermore, the heavy chain constant region of the monoclonal antibody or antigen-binding fragment capable of binding to human TIGIT protein is selected from the heavy chain constant region of any one of the antibodies: IgD, IgE, IgM, IgA, and IgG.

[0024] Furthermore, the heavy chain constant region IgG includes IgG1, IgG2a, IgG2b, IgG3, and IgG4.

[0025] Furthermore, the antigen-binding fragment includes a functional fragment for antigen binding.

[0026] Furthermore, the functional fragment is selected from one or more of Fab, Fab', F(ab')2, Fv or scFv.

[0027] Furthermore, the heavy chain constant region is preferably IgG1, with the amino acid sequence shown in SEQ ID NO.30.

[0028] Furthermore, the light chain constant region is preferably κ-type, and its amino acid sequence is shown in SEQ ID NO.31.

[0029] In one embodiment, when the heavy chain constant regions of the monoclonal antibody or antigen-binding fragment that can bind to human TIGIT protein are all identical, the nucleotide sequence of its heavy chain constant region is as shown in SEQ ID NO.44.

[0030] In another embodiment, when the light chain constant regions of the monoclonal antibody or antigen-binding fragment that can bind to human TIGIT protein are all identical, the nucleotide sequence of its light chain constant region is as shown in SEQ ID NO.45.

[0031] In a second aspect, the present invention provides a nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment that can bind to the human TIGIT protein as described in the first aspect of the present invention.

[0032] Thirdly, the present invention provides a vector comprising a nucleic acid molecule that edits a monoclonal antibody or antigen-binding fragment of the human TIGIT protein as described in the first aspect of the present invention.

[0033] Fourthly, the present invention provides a host cell containing a nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment that can bind to the human TIGIT protein as described in the first aspect of the present invention.

[0034] Fifthly, the present invention provides an immunoconjugate comprising the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0035] In a sixth aspect, the present invention provides a chimeric antigen receptor comprising the monoclonal antibody or its antigen-binding fragment described in the first aspect of the present invention.

[0036] In a seventh aspect, the present invention provides a composition comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule, a vector or host cell or an immunoconjugate.

[0037] Eighthly, the use of the monoclonal antibody or antigen-binding fragment thereof, nucleic acid molecule, vector or host cell, immunoconjugate, chimeric antigen receptor or its encoding nucleic acid molecule, construct or vector, transformed immune cells and / or the composition described in the fifth aspect of the invention in the preparation of a medicament for positively regulating immune cell activity and / or enhancing immune response is provided.

[0038] Furthermore, the application may include its use in the preparation of medicaments for the prevention and / or treatment of tumors, infections, or infectious diseases.

[0039] In a ninth aspect, the present invention provides a kit for detecting TIGIT protein, the kit comprising instructions and detection reagents, wherein the detection reagents are reagents of the monoclonal antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors or host cells, immunoconjugates, chimeric antigen receptors or their encoding nucleic acid molecules, constructs or vectors, transformed immune cells and / or the compositions described in the fifth aspect of the present invention.

[0040] Beneficial effects

[0041] The TIGIT-specific monoclonal antibody finally screened in this invention has high sensitivity, high specificity, and high affinity, and can bind to CD8. + The TIGIT antibody binds to the TIGIT protein on the surface of T cells and can specifically recognize the TIGIT protein on the cell membrane surface. Compared with the commercially available TIGIT monoclonal antibody (A15153G), the TIGIT antibody in this invention has significantly higher affinity. Attached Figure Description

[0042] Figure 1 SDS-PAGE assay for TIGIT antibody purity. (A: Detection results under non-reducing conditions; B: Detection results under reducing conditions; M: Marker; 1: TIGIT-M001; 2: TIGIT-M003; 3: TIGIT-M006; 4: TIGIT-M007; 5: TIGIT-M011; 6: TIGIT-M013).

[0043] Figure 2 ELISA identification of TIGIT protein specificity for TIGIT antibody detection.

[0044] Figure 3 Western blotting was used to identify TIGIT protein specificity for TIGIT antibody detection. (M: Marker; 1: Recombinant TIGIT protein under non-reducing conditions; 2: Recombinant TIGIT protein under reducing conditions.)

[0045] Figure 4 Flow cytometry identification of TIGIT protein expressed on the surface of 293FT cells for recognition by TIGIT antibody.

[0046] Figure 5 ELISA identification of the sensitivity of TIGIT antibody to TIGIT protein.

[0047] Figure 6The affinity of TIGIT antibody for TIGIT protein was determined (the lines from top to bottom represent TIGIT antibody concentrations of 250 nM, 125 nM, 62.5 nM and 31.3 nM, respectively).

[0048] Figure 7 Flow cytometry identification of TIGIT protein on the surface of T cells for recognition by TIGIT antibody. Detailed Implementation

[0049] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0051] As used in this article, the terms "VH" and "VL" refer to the variable regions of the antibody heavy chain and light chain, respectively. Variable regions consist of discrete, well-defined subregions called complementarity-determining regions (CDRs, also known as HVRs) and framework regions (FRs). A CDR is an amino acid within the antibody variable region that confers antigen specificity and / or binding affinity, separated by FRs. Each antibody light chain variable region contains three CDRs (VLCDR1, VLCDR2, and VLCDR3), and each antibody heavy chain variable region contains three CDRs (VHCDR1, VHCR2, and VHCDR3). The complementarity-determining regions (CDRs) of the VH and VL regions alternate with more conserved regions of the framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0052] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0053] Example 1: Preparation of mouse anti-human TIGIT monoclonal antibody

[0054] 1.1 Animal Immunization

[0055] BALB / c mice aged 6-8 weeks were selected and numbered after one week of normal feeding. Approximately 0.1 ml of blood was collected via the retro-orbital venous plexus. After standing at room temperature for 1 hour, the blood was incubated overnight at 4°C. The supernatant serum was collected after centrifugation at 1500g and stored at -20°C for later analysis. For primary immunization, 100 μg of TIGIT-His recombinant protein (Essential) was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites in the abdomen and back. Booster immunizations were performed 3 weeks later, with an equal volume of TIGIT recombinant protein emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites. Seven days after three booster immunizations, serum titers were measured via blood collection via the retro-orbital venous plexus. Mice with high titers were selected for intraperitoneal injection of 100 μg of TIGIT recombinant protein for pulse immunization. Mice were sacrificed on the 3rd day post-immunization, and their spleens were collected and rapidly transferred to liquid nitrogen for cryopreservation.

[0056] 1.2 Valence Testing Process

[0057] The TIGIT recombinant protein was diluted to 5 μg / ml with coating buffer and added to a 96-well ELISA plate (100 μl / well). The plate was vortexed, sealed tightly with plastic wrap, and incubated overnight at 4°C. The plate was washed three times, blotted dry, and 200 μl / well of blocking buffer was added to block non-specific binding sites. The plate was incubated at 37°C for 2 hours. After washing three times, serially diluted serum was added (100 μl / well), and the plate was incubated at 37°C for 2 hours. After washing five times, horseradish peroxidase-labeled goat anti-mouse IgG (H+L) diluted 1:5000 (100 μl / well) was added, and the plate was incubated at 37°C for 1 hour. After washing five times, chromogenic buffer was added (100 μl / well), and the plate was incubated at 37°C for 30 minutes. Finally, 50 μl of stop solution was added to each well. The OD was measured using an ELISA reader. 450 Value. Mouse serum dilution at 1:16000, antibody detection positive, OD value selected. 450 Mice with high values ​​were subjected to shock immunization.

[0058] 1.3 Construction of scFv phage antibody library and antibody screening

[0059] Total RNA was extracted from the spleen of immunized mice and reverse transcribed into cDNA. The antibody heavy and light chain variable regions (V) sequences were amplified by RT-PCR. Overlap extension splicing PCR was used to assemble the antibody-encoding heavy and light chain variable region sequences (VH and VL) into a nucleic acid sequence encoding scFv. The light and heavy chain variable regions were linked using a linker, and then ligated into a phage vector by restriction endonuclease digestion. Electroporation into X-Blue competent cells yielded an scFv phage antibody library with a volume of at least 10⁻⁶ cells. 8 .

[0060] The ELISA method was used to coat a phage library with TIGIT recombinant protein for screening, employing a "wash-amplification-enrichment" cycle, typically requiring two or more rounds to obtain positive clones. Positive clones detected by ELISA were then amplified by PCR, digested with enzymes for typing, and sequenced. Suitable variable region sequences were selected based on the sequencing results.

[0061] 1.4 Construction of antibody expression vector

[0062] Using phage culture or plasmids as templates, PCR was used to amplify the variable region fragments of the light and heavy chains. These variable region gene fragments were then inserted into expression vectors containing light and heavy chain signal peptides and light and heavy chain constant regions, respectively, to obtain complete IgG heavy chain (pCMV3-H) and light chain (pCMV3-L) expression vectors. After amplification, the plasmids were transfected into HEK293 cells for culture, expression, and antibody purification.

[0063] Example 2: TIGIT antibody sequence analysis

[0064] Six TIGIT monoclonal antibodies were obtained through screening of the scFv phage antibody library and named TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, and TIGIT-M013. Positive clones were sequenced to obtain the light and heavy chain variable region sequences. Amino acid sequence analysis of the antibody variable regions was performed using IGBLAST software (https: / / www.ncbi.nlm.nih.gov / igblast / ), revealing that the scFv antibodies belong to the VH class and Vκ type. Further analysis of the three CDR regions (VH (heavy chain variable region) and VL (light chain variable region) of each antibody was conducted using ABodyBuilder software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ) in Kabat mode.

[0065] The amino acid sequences of the antibody heavy chain variable region CDR (VHCDR) and light chain variable region CDR (VLCDR) are as follows:

[0066] The amino acid sequences of TIGIT-M001-VHCDR1, TIGIT-M001-VHCDR2, and TIGIT-M001-VHCDR3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.6, respectively.

[0067] The amino acid sequences of TIGIT-M001-VLCDR1, TIGIT-M001-VLCDR2, and TIGIT-M001-VLCDR3 are shown in SEQ ID NO.8, SEQ ID NO.12, and SEQ ID NO.14, respectively.

[0068] The amino acid sequences of TIGIT-M003-VHCDR1, TIGIT-M003-VHCDR2, and TIGIT-M003-VHCDR3 are shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.7, respectively.

[0069] The amino acid sequences of TIGIT-M003-VLCDR1, TIGIT-M003-VLCDR2, and TIGIT-M003-VLCDR3 are shown in SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.15, respectively.

[0070] The amino acid sequences of TIGIT-M006-VHCDR1, TIGIT-M006-VHCDR2, and TIGIT-M006-VHCDR3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.6, respectively.

[0071] The amino acid sequences of TIGIT-M006-VLCDR1, TIGIT-M006-VLCDR2, and TIGIT-M006-VLCDR3 are shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.16, respectively.

[0072] The amino acid sequences of TIGIT-M007-VHCDR1, TIGIT-M007-VHCDR2, and TIGIT-M007-VHCDR3 are shown in SEQ ID NO.1, SEQ ID NO.4, and SEQ ID NO.6, respectively.

[0073] The amino acid sequences of TIGIT-M007-VLCDR1, TIGIT-M007-VLCDR2, and TIGIT-M007-VLCDR3 are shown in SEQ ID NO.11, SEQ ID NO.13, and SEQ ID NO.15, respectively.

[0074] The amino acid sequences of TIGIT-M011-VHCDR1, TIGIT-M011-VHCDR2, and TIGIT-M011-VHCDR3 are shown in SEQ ID NO.1, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0075] The amino acid sequences of TIGIT-M011-VLCDR1, TIGIT-M011-VLCDR2, and TIGIT-M011-VLCDR3 are shown in SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO.17, respectively.

[0076] The amino acid sequences of TIGIT-M013-VHCDR1, TIGIT-M013-VHCDR2, and TIGIT-M013-VHCDR3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.6, respectively.

[0077] The amino acid sequences of TIGIT-M013-VLCDR1-3 are shown in SEQ ID NO.10, SEQ ID NO.13 and SEQ ID NO.17, respectively.

[0078] The amino acid sequences of the variable regions of the heavy and light chains of the antibodies TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, and TIGIT-M013 are as follows:

[0079] The VH and VL amino acid sequences of TIGIT-M001 are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively.

[0080] The VH and VL amino acid sequences of TIGIT-M003 are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively.

[0081] The VH and VL amino acid sequences of TIGIT-M006 are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively.

[0082] The VH and VL amino acid sequences of TIGIT-M007 are shown in SEQ ID NO.24 and SEQ ID NO.25, respectively.

[0083] The VH and VL amino acid sequences of TIGIT-M011 are shown in SEQ ID NO.26 and SEQ ID NO.27, respectively.

[0084] The VH and VL amino acid sequences of TIGIT-M013 are shown in SEQ ID NO.28 and SEQ ID NO.29, respectively.

[0085] The amino acid sequences of the heavy chain constant regions of the six antibodies are all identical, as shown in SEQ ID NO. 30.

[0086] The amino acid sequences of the constant regions of the light chains of the six antibodies are all identical, as shown in SEQ ID NO. 31.

[0087] Furthermore, the nucleotide sequence encoding the antibody is shown below:

[0088] Nucleotide sequence encoding the variable region of the antibody heavy chain (underlined sequences are signal peptide encoding sequences):

[0089] The VH and VL nucleotide sequences of TIGIT-M001 are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively.

[0090] The VH and VL nucleotide sequences of TIGIT-M003 are shown in SEQ ID NO.34 and SEQ ID NO.35, respectively.

[0091] The VH and VL nucleotide sequences of TIGIT-M006 are shown in SEQ ID NO.36 and SEQ ID NO.37, respectively.

[0092] The VH and VL nucleotide sequences of TIGIT-M007 are shown in SEQ ID NO.38 and SEQ ID NO.39, respectively.

[0093] The VH and VL nucleotide sequences of TIGIT-M011 are shown in SEQ ID NO.40 and SEQ ID NO.41, respectively.

[0094] The VH and VL nucleotide sequences of TIGIT-M013 are shown in SEQ ID NO.42 and SEQ ID NO.43, respectively.

[0095] The six antibodies encode the same nucleotide sequence for the heavy chain constant region, as shown in SEQ ID NO. 44.

[0096] The six antibodies encode identical nucleotide sequences for the light chain constant region, and the nucleotide sequences for the heavy chain constant region are shown in SEQ ID NO. 45.

[0097] Table 1 shows the VHCDR1-3 and VLCDR1-3 sequences described in this paper;

[0098] Table 1. Sequence List of VHCDR1-3 and VLCDR1-3

[0099]

[0100] Table 2. Antibody sequence listings for TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011 and TIGIT-M013

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Example 3 Antibody Expression, Purification and Identification

[0111] HEK293 cells were transiently transfected simultaneously with the same antibody light and heavy chain expression vectors. The culture supernatant was collected 6-7 days post-transfection and purified using a protein A purification column to obtain the purified antibody. The antibody molecular weight was predicted using a protein molecular weight calculator, and the purity and molecular weight were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0112] SDS-PAGE electrophoresis detection procedure: Take 5 μg of purified antibody and perform electrophoresis separation, staining, and photographing under non-reducing conditions (no β-mercaptoethanol added to protein electrophoresis loading buffer, no boiling of samples) and reducing conditions (β-mercaptoethanol added to protein electrophoresis loading buffer, boiling of samples for 5 min).

[0113] SDS-PAGE test results (see) Figure 1 The predicted molecular weights of the six antibody heavy chains are approximately 49 kDa, the light chains are approximately 24 kDa, and the total molecular weight of the IgG antibody is approximately 146 kDa. SDS-PAGE electrophoresis confirmed that the protein molecular weights were close to the predicted molecular weights, and the antibody purity was good, around 95%.

[0114] Example 4: Identification of antibody specificity and sensitivity

[0115] 4.1 Antibody Specificity Identification

[0116] 4.1.1 Identification of TIGIT antibody specificity by ELISA

[0117] The specificity of TIGIT antibodies was identified using recombinant proteins of human TIGIT (Hu-TIGIT-His), cynomolgus monkey TIGIT (Cyn-TIGIT-hFc), mouse TIGIT (Ms-TIGIT-hFc), Hu-PD-1-His, Hu-PD-L1-His, Hu-CTLA4-His, Hu-CD137-His, and Hu-TIM-3-His.

[0118] ELISA assay procedure: Coat Hu-TIGIT-His, Cyn-TIGIT-hFc, Ms-TIGIT-hFc, Hu-PD-1-His, Hu-PD-L1-His, Hu-CTLA4-His, Hu-CD137-His, and Hu-TIM-3-His recombinant proteins with coating buffer at 1 μg / ml and incubate overnight at 4°C. The next day, wash three times with working wash buffer using a plate washer, then add 5% skim milk powder to block non-specific binding sites (200 μl / well) and incubate at 37°C for 2 hours. After washing three times with a plate washer, add 100 μl of TIGIT antibody (TIGIT antibody control A15153G and TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011 and TIGIT-M013) diluted to 1 μg / ml with 5% skim milk powder, and incubate at 37℃ for 2 h. After washing five times, add 1:5000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (H+L) and incubate at 37℃ for 1 h. After washing five times, add 100 μl of TMB substrate solution, and incubate at 37℃ for 30 min. Add 50 μl of stop solution, and measure OD using a microplate reader. 450 Value. Graphpad software analyzes the detection results.

[0119] in:

[0120] The coating solution (BD OptEIA Coating Buffer, 51-2713KC) was 0.1M sodium carbonate at pH 9.5;

[0121] Washing solution (BD OptEIAWash Buffer, 51-9003739) 20× concentrated washing solution, diluted with deionized water or distilled water to make 1× working solution;

[0122] The colorimetric solution A (BD OptEIA Substrate Reagent A, 51-2606KZ) is a buffer solution containing hydrogen peroxide;

[0123] The colorimetric solution B (BD OptEIA Substrate Reagent B, 51-2607KZ) contains 3,3',5,5'-tetramethylbenzidine (TMB) as an organic solvent.

[0124] Stop solution (BD OptEIA Stop Solution, 51-2608KZ): 1M sulfuric acid; 5% milk: 5 grams of milk added to 100 ml of 1×PBS solution.

[0125] Test results (see) Figure 2 All six TIGIT antibodies effectively recognized human TIGIT and cynomolgus monkey recombinant TIGIT proteins. TIGIT-M001, TIGIT-M003, and TIGIT-M013 antibodies showed weak cross-binding with mouse TIGIT protein, while TIGIT-M006, TIGIT-M007, and TIGIT-M011 antibodies did not bind to mouse TIGIT protein. This suggests that TIGIT-M001 / TIGIT-M003 / TIGIT-M013 antibodies recognize and bind to different TIGIT epitopes compared to TIGIT-M006, TIGIT-M007, and TIGIT-M011 antibodies.

[0126] Six antibodies did not bind to human PD-1, PD-L1, CTLA4, CD137, and TIM-3 recombinant proteins, suggesting that TIGIT antibodies have good specificity in recognizing and binding to TIGIT proteins. The commercially available control antibody A15153G only binds to human TIGIT protein and not to cynomolgus monkey or mouse TIGIT proteins, indicating that the TIGIT epitopes recognized and bound by these six antibodies are different from those recognized and bound by A15153G.

[0127] 4.1.2 Western blotting to identify the specificity of TIGIT antibodies

[0128] Western blotting was used to identify the binding characteristics of TIGIT antibodies to recombinant TIGIT protein under both non-reducing and reducing conditions.

[0129] Western blotting procedure: Take 1 μg of TIGIT-His recombinant protein and separate it under both non-reducing (protein electrophoresis loading buffer without β-mercaptoethanol, sample not boiled) and reducing (protein electrophoresis loading buffer with β-mercaptoethanol, sample boiled for 5 min) conditions using a 10% SDS-PAGE gel at 150V for approximately 1 h. After electrophoresis, transfer the separated proteins from the SDS-PAGE gel to a nitrocellulose membrane at 300mA for 1 h. After transfer, block with 5% skim milk powder at room temperature for 2 h. Incubate the membrane with 0.5 μg / ml antibodies (TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, and TIGIT-M013) overnight at 4℃. Wash the membrane three times with PBST solution, 5 min each time. Secondary antibody was added to horseradish peroxidase-labeled goat anti-mouse IgG (H+L) diluted 1:5000, and the reaction was carried out at room temperature for 1 hour. Unbound antibodies were removed by washing with PBST. Chemiluminescent substrate was added, and the results were recorded using a gel imaging system.

[0130] Test results (see) Figure 3 The antibodies TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, and TIGIT-M013 primarily recognize recombinant TIGIT proteins under non-reducing conditions, suggesting that TIGIT antibodies mainly recognize TIGIT protein conformational epitopes; weak bands are observed at the TIGIT protein under reduced conditions under strong exposure conditions.

[0131] 4.1.3 Flow cytometry detection of TIGIT protein on cell membrane surface

[0132] The TIGIT protein expressed on the cell membrane surface after transfection of 293FT cells with the TIGIT-GFP recombinant plasmid was identified by flow cytometry.

[0133] Flow cytometry assay procedure: 293FT cells were transfected with the TIGIT-GFP recombinant plasmid for 48 h, and single-cell suspensions were prepared by trypsin digestion. Cells were washed once with PBS, and 1 μg / ml of mouse anti-human TIGIT antibody (TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, and TIGIT-M013) was added. The cells were incubated at room temperature for 30 min, washed once with PBS, and then 1:1000 diluted AF647-labeled goat anti-mouse IgG (H+L) antibody was added. The cells were incubated at room temperature in the dark for 30 min. Cells were washed twice with PBS, and resuspended in 200 μl of PBS solution. The assay was performed using BD LSRFortessa. TMThe samples were analyzed using flow cytometry and the results were analyzed using BDFACSDiva software.

[0134] Flow cytometry analysis of TIGIT protein on the surface of 293FT cells transfected with TIGIT-GFP recombinant plasmid (see results) Figure 4 All TIGIT antibodies can bind to the TIGIT protein expressed on the surface of 293FT cells transfected with the TIGIT recombinant plasmid.

[0135] 4.2 Antibody sensitivity identification.

[0136] Detection of TIGIT and GFP double-positive cells using TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011 and TIGIT-M013 antibodies ( Figure 4 The proportions of cells in the upper right quadrant were 17.9%, 18.2%, 16.8%, 19.1%, 17.5%, and 17.0%, respectively, and the proportions of TIGIT-positive cells detected by the six antibodies were basically consistent.

[0137] 4.2.1 Indirect ELISA detection of TIGIT antibody and sensitivity of TIGIT protein

[0138] Detection Procedure: Hu-TIGIT-His and Hu-TIGIT-hFc recombinant proteins were serially diluted 10-fold (1000-0.001 ng / ml) and coated onto 96-well ELISA plates, 100 μl / well, and incubated overnight at 4°C. Primary antibodies (1 μg / ml) of TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, TIGIT-M013, and control antibody (A15153G) were added. The remaining steps were the same as the ELISA detection procedure described above.

[0139] ELISA sensitivity test results (see) Figure 5 Using an indirect ELISA method, the sensitivity of the self-developed TIGIT antibody and control antibody A15153G in detecting TIGIT-His recombinant protein was 100 ng / ml, and the sensitivity in detecting TIGIT-hFc recombinant protein was 10 ng / ml. The sensitivity of the six self-developed TIGIT antibodies was similar to that of the commercially available TIGIT antibody A15153G.

[0140] Example 5: Identification of TIGIT Antibody Affinity

[0141] Antibody affinity was detected using a ForteBio Octet molecular interaction analyzer. The TIGIT-hFc recombinant protein was used as the antigen for detection, employing a FORTEBIO anti-human IgG Fc capture (AHC) biosensor, with the TIGIT antibody A15153G used as a control antibody.

[0142] Affinity assay procedure: The FORTEBIO anti-human IgG Fc capture (AHC) biosensor was pre-wetted in equilibration buffer (0.1% BSA + 0.02% Tween 20 in PBS solution) for 10 minutes. TIGIT-hFc recombinant protein was diluted to 5 μg / mL with equilibration buffer and added to the second column of a light-protected 96-well plate, 200 μl / well. TIGIT antibody was serially diluted from 250 nM to 31.3 nM and added to the fourth column of a light-protected 96-well plate, 200 μl / well. A 0 nM antibody blank control was set up, and 200 μl of equilibration buffer was added. The equilibration buffer was then added to the first and third columns, 200 μl / well. ForteBio Octet molecular interaction analyzer was used for detection. The sensor was equilibrated in the first column for 60 seconds to obtain the baseline equilibration curve, and then antigen was immobilized in the second column for 100 seconds. The antibody is washed for 120 seconds in the third column, then bound to the antibody in the fourth column for 80 or 180 seconds to obtain the binding curve. Finally, the antibody is dissociated in the first column for 300 seconds to obtain the dissociation curve. The ForteBio Octet analysis software is used to fit and analyze the curves to obtain the affinity values.

[0143] Affinity test results (see) Figure 6 Antibody affinity was determined using the ForteBio Octet system, with a buffer solution without added TIGIT protein as a blank control and commercially available TIGIT antibody A15153G as a detection control.

[0144] After data analysis, the antibody's affinity K D The values ​​are 9.28 × 10 -12 M(TIGIT-M001), 2.42×10 -11 M(TIGIT-M003), 1.26×10 -11 M(TIGIT-M006), 3.79×10 -11 M(TIGIT-M007), 1.40×10 -11 M(TIGIT-M011), 8.37×10 -11 M(TIGIT-M013) and 3.51×10 -9 M(A15153G).

[0145] Curve fit goodness R 2The specific affinities were 0.9720 (TIGIT-M001), 0.9783 (TIGIT-M003), 0.9815 (TIGIT-M006), 0.8756 (TIGIT-M007), 0.9368 (TIGIT-M011), 0.8643 (TIGIT-M013), and 0.9442 (A15153G), respectively. The affinity of the independently developed TIGIT antibody was significantly higher than that of the commercially available TIGIT antibody A15153G.

[0146] Example 6: Flow cytometry identification of TIGIT protein on the surface of T cells by TIGIT antibody recognition.

[0147] Collect 200 μl of human peripheral blood, add 2 ml of erythrocyte lysis buffer, and lyse at room temperature for 10 min. Centrifuge at 1500 rpm for 5 min and discard the supernatant. Wash the pellet once with PBS, then vortex to resuspend the cell pellet. Add 1 μg of TIGIT antibody (TIGIT-M001, TIGIT-M003, TIGIT-M006, TIGIT-M007, TIGIT-M011, and TIGIT-M013) and react at room temperature for 30 min; prepare a blank control tube without antibody. Wash once with PBS, then add 1:1000 diluted AF647-labeled goat anti-mouse IgG (H+L) antibody and react at room temperature in the dark for 30 min. Wash twice with PBS, then add CD45 Percp, CD3 BV605, CD4 FITC, and CD8 APC-H7 antibodies and react at room temperature for 30 min. Wash twice with PBS, and resuspend the cells in 200 μl of PBS solution. Utilize BD LSRFortessa... TM The samples were analyzed using flow cytometry and the results were analyzed using BDFACSDiva software.

[0148] Results of flow cytometry detection of TIGIT protein on the surface of T cells ( Figure 7 ): Analysis selected CD45 + CD3 + Further analysis of T cells was performed. Results showed that all TIGIT antibodies could bind to CD8. + T cells and CD8 — T cells (mainly CD4) + TIGIT proteins bind to the surface of T cells. TIGIT antibodies can be used to detect TIGIT proteins on the surface of endogenous immune cell membranes.

Claims

1. An anti-human TIGIT monoclonal antibody TIGIT-M003 or its antigen-binding fragment, wherein the anti-human TIGIT monoclonal antibody TIGIT-M003 comprises complementarity-determining region sequences of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region complementarity-determining region respectively contain CDR1, CDR2 and CDR3; wherein, The amino acid sequence of VHCDR1 is SEQ ID NO.1, the amino acid sequence of VHCDR2 is SEQ ID NO.3, and the amino acid sequence of VHCDR3 is SEQ ID NO.7; the amino acid sequence of VLCDR1 is SEQ ID NO.9, the amino acid sequence of VLCDR2 is SEQ ID NO.13, and the amino acid sequence of VLCDR3 is SEQ ID NO.

15. 2.The anti-human TIGIT monoclonal antibody TIGIT-M003 or an antigen binding fragment thereof of claim 1, characterized in that, The VH and VL amino acid sequences of the anti-human TIGIT monoclonal antibody TIGIT-M003 are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively. 3.The anti-human TIGIT monoclonal antibody TIGIT-M003 or an antigen binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment includes an antibody constant region Fc, which comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from any one of IgD, IgE, IgM, IgA, IgG1, IgG2a, IgG2b, IgG3, and IgG4; the light chain constant region is a κ-type or λ-type light chain constant region. 4.The anti-human TIGIT monoclonal antibody TIGIT-M003 or an antigen binding fragment thereof of claim 1, wherein, The antigen-binding fragment includes a functional fragment for antigen binding, wherein the functional fragment is selected from one or more of Fab, Fab', F(ab')2, Fv or scFv.

5. A nucleic acid molecule encoding the anti-human TIGIT monoclonal antibody TIGIT-M003 as described in claim 1, or an antigen-binding fragment thereof; said nucleic acid molecule encoding VH and VL of TIGIT M003; the nucleotide sequences of VH and VL of TIGIT M003 are shown in SEQ ID NO.34 and SEQ ID NO.35, respectively.

6. A vector or host cell comprising a nucleic acid molecule encoding the anti-human TIGIT monoclonal antibody TIGIT-M003 as described in claim 1 or an antigen-binding fragment thereof.

7. A composition comprising the anti-human TIGIT monoclonal antibody TIGIT-M003 as claimed in claim 1 or its antigen-binding fragment, the nucleic acid molecule as claimed in claim 5, or the vector or host cell as claimed in claim 6.

8. A kit for detecting TIGIT protein, the kit comprising instructions and a detection reagent, the detection reagent comprising an anti-human TIGIT monoclonal antibody TIGIT-M003 or its antigen-binding fragment as described in any one of claims 1-4, a nucleic acid molecule as described in claim 5, or a vector or host cell as described in claim 6.

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