Anti-tigit-pd1 bispecific antibodies and uses thereof

Abstract

The application discloses an anti-TIGIT-PD1 bispecific antibody and application thereof, and belongs to the technical field of tumor treatment. The bispecific antibody comprises an immunoglobulin antibody lgG of anti-PD1 and a single-chain antibody scFv of anti-TIGIT. The single-chain antibody scFv comprises a variable region VH and a variable region VL. The amino acid sequences of complementary determining regions CDR-H1 to CDR-H3 in the variable region VH are SEQ ID NO. 5-7. The amino acid sequences of complementary determining regions CDR-L1 to CDR-L3 in the variable region VL are SEQ ID NO. 8-10. The bispecific antibody has a brand-new bispecific sequence and bispecific activity, and thus has a more excellent tumor treatment effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an anti-TIGIT-PD1 bispecific antibody and application thereof. BACKGROUND

[0002] TIGIT (T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9) is a member of the poliovirus receptor (PVR) / nectin family. It is composed of an extracellular immunoglobulin variable (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a canonical immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoglobulin tyrosine-based inhibitory motif (ITT) motif. TIGIT is expressed in lymphocytes and is a co-inhibitory receptor that is highly expressed in tumor-infiltrating lymphocytes, particularly in effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells, and natural killer (NK) cells. CD155 (also known as PVR, Necl5, or Tage4) is a high-affinity ligand for TIGIT. Once the tumor surface highly expresses CD155 and binds to TIGIT on the surface of NK and T cells, their killing effect on tumor cells is inhibited. TIGIT and PVR are widely expressed in different types of solid tumors, indicating that the TIGIT-PVR signaling pathway may be a major tumor immune escape mechanism and a new type of immune checkpoint after PD-1 / PD-L1.

[0003] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein composed of 288 amino acids, and PD-L1 is a ligand of PD-1. After PD-1 binds to PD-L1, an inhibitory signal can be provided to induce T cell apoptosis and inhibit T cell activation and proliferation. In other words, the PD-1 monoclonal antibody can help the immune cells (T cells) in the body to recognize and kill tumor cells, thereby playing an anti-cancer role.

[0004] A bispecific monoclonal antibody (BsAb) is a special antibody artificially made to bind to two different antigens or antigen epitopes at the same time. The main mechanisms of action of bispecific antibodies include: mediating immune cell killing, double-target signal blocking, and promoting the formation of a functional complex of proteins. This makes bispecific antibodies have unique advantages compared to traditional monoclonal antibodies.

[0005] The present application is dedicated to elucidate an anti-TIGIT-PD1 bispecific antibody, which has a brand-new bispecific sequence and bispecific activity, and has a more excellent tumor treatment effect. SUMMARY

[0006] In view of the above, the present application provides a bispecific antibody capable of specifically binding to TIGIT and PD1 with a brand-new sequence, and further provides an application of the bispecific antibody.

[0007] To achieve the above object, the present application adopts the following technical solutions.

[0008] The present application provides an anti-TIGIT-PD1 bispecific antibody, which comprises an immunoglobulin antibody lgG against PD1 and a single-chain antibody scFv against TIGIT, wherein the single-chain antibody scFv comprises a variable region VH and a variable region VL, the variable region VH is connected with the variable region VL through a peptide linker L1, and the carboxyl end of each single-chain antibody scFv is connected with the amino end of the heavy chain of the immunoglobulin antibody lgG through a peptide linker L2.

[0009] The variable region VH comprises a complementarity determining region CDR-H1, a CDR-H2 and a CDR-H3, wherein the amino acid sequence of CDR-H1 is SEQ ID NO. 5, the amino acid sequence of CDR-H2 is SEQ ID NO. 6, and the amino acid sequence of CDR-H3 is SEQ ID NO. 7.

[0010] The variable region VL comprises a complementarity determining region CDR-L1, a CDR-L2 and a CDR-L3, wherein the amino acid sequence of CDR-L1 is SEQ ID NO. 8, the amino acid sequence of CDR-L2 is SEQ ID NO. 9, and the amino acid sequence of CDR-L3 is SEQ ID NO. 10.

[0011] Further, the single-chain antibody scFv is a murine antibody or a humanized antibody.

[0012] Preferably, the single-chain antibody scFv is a murine antibody, wherein the heavy chain amino acid sequence of the single-chain antibody scFv is SEQ ID NO. 1, and the light chain amino acid sequence thereof is SEQ ID NO. 3.

[0013] Preferably, the single-chain antibody scFv is a humanized antibody, wherein the single-chain antibody scFv comprises an amino acid sequence combination mode of one of the following schemes: a combination of SEQ ID NO. 11 and SEQ ID NO. 13 (VH1 / VL1), a combination of SEQ ID NO. 11 and SEQ ID NO. 14 (VH1 / VL2), a combination of SEQ ID NO. 12 and SEQ ID NO. 13 (VH2 / VL1), and a combination of SEQ ID NO. 12 and SEQ ID NO. 14 (VH2 / VL2).

[0014] In a further aspect, the constant region of the immunoglobulin antibody lgG is from a human antibody.

[0015] In a further aspect, the heavy chain constant region sequence of the immunoglobulin antibody lgG is the heavy chain constant region of human lgG1, and the light chain constant region sequence is the light chain constant region of human lgG1.

[0016] In a further aspect, the heavy chain amino acid sequence of the bispecific antibody is SEQ ID NO. 15, and the light chain amino acid sequence is SEQ ID NO. 17.

[0017] The present application further provides a nucleotide molecule encoding the bispecific antibody as described above.

[0018] The present application further provides an expression vector containing the nucleotide molecule as described above.

[0019] The present application further provides a host cell containing the expression vector as described above.

[0020] The present application further provides a pharmaceutical composition containing the bispecific antibody as described above and any one or more than one pharmaceutically acceptable adjuvant.

[0021] The present application also provides the use of the pharmaceutical composition as described above in the preparation of a medicament for preventing and / or treating tumors.

[0022] The present application has the following advantages:

[0023] The anti-TIGIT-PD1 bispecific antibody has a brand-new bispecific sequence. On the one hand, the bispecific antibody can bind to tumor cells with high expression of TIGIT antigen to inhibit tumor proliferation. On the other hand, it can block the signal pathway of PD-1 and PD-L1. PD-L1 is expressed on tumor cells, and PD-1 is expressed on T cells. The binding of PD-1 and PD-L1 can inhibit the proliferation and activation of T cells. By blocking the signal pathway of PD-1 and PD-L1, the immune killing function of T cells can be restored. The bispecific antibody can simultaneously bind to TIGIT and PD1 antigens, block the signal pathway, and activate T cells, and has more excellent bispecific activity and can better inhibit tumor cells.

[0024] In addition, the bispecific antibody is composed of the lgG molecule of the anti-PD1 antibody and the scFv form of the domain of the anti-TIGIT antibody. Such a bispecific structure can effectively improve the stability of the entire fusion protein while retaining the affinity of the lgG molecule itself. And based on the preparation of the bispecific antibody of the lgG molecule, the stability and immunogenicity uncertainty caused by excessive changes in molecular structure can be effectively reduced, which has significant advantages. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Purification results of 8A4 hybridoma antibody in Example 1;

[0026] Figure 2 Test results of 8A4 hybridoma antibody binding activity with TIGIT-His antigen in Example 1;

[0027] Figure 3 Test results of 8A4 hybridoma antibody competition activity with PVR-hFc ligand in Example 1;

[0028] Figure 4 Purification results of H006-8A4 humanized antibody in Example 1;

[0029] Figure 5 Test results of H006-8A4 humanized antibody binding activity with TIGIT-His antigen in Example 1;

[0030] Figure 6 Results of FACS detection of H006-8A4 humanized antibody 293F-TIGIT cell binding activity in Example 1;

[0031] Figure 7 Results of ELISA detection of H006-8A4 humanized antibody competition activity with PVR-mFc in Example 1;

[0032] Figure 8 Results of FACS detection of H006-8A4 humanized antibody competition activity with PVR-mFc in Example 1;

[0033] Figure 9 Fluorescent signal of H006-8A4 series humanized antibody thermal stability detection in Example 1;

[0034] Figure 10 H006-8A4 humanized antibody induced IFN-γ secretion in cell supernatant in Example 1;

[0035] Figure 11 Pharmacodynamics of 8A4 antibody in Hepa1-6 cell mouse xenograft model in Example 2;

[0036] Figure 12 Purification results of anti-TIGIT-PD1 bispecific antibody in Example 4;

[0037] Figure 13 ELISA determination of PD1 / TIGIT bispecific antibody binding activity with TIGIT-His and PD-1-His in Example 5;

[0038] Figure 14 Test results for the competition activity of the PD1 / TIGIT bispecific antibody with PD-L1-mFc in the ELISA assay in Example 5. DETAILED DESCRIPTION

[0039] Embodiments of the present application are described in detail below, the embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0041] The first aspect of the present application provides an anti-TIGIT-PD1 bispecific antibody, the theoretical basis of the TIGIT-PD1 bispecific antibody is that effector T cells are the main force to kill tumors, mainly generated by stem cell-like memory T cells, and stem cell-like memory T cells express PD-1 and TIGIT, and do not express other negative regulators (such as Tim-3), which makes the TIGIT-PD1 bispecific antibody can "activate" stem cell-like memory T cells, and produce a continuous supply of effector T cells.

[0042] The bispecific antibody can specifically bind to TIGIT and PD-1, the bispecific antibody includes an immunoglobulin antibody lgG against PD1 and a single-chain antibody scFv against TIGIT, specifically, two identical single-chain antibodies scFv and an immunoglobulin antibody lgG are connected. Each of the single-chain antibodies scFv includes a variable region VH and a variable region VL, the variable region VH is connected to the variable region VL through a peptide structure L1, and the carboxyl end of each single-chain antibody scFv is connected to the amino acid of the lgG heavy chain through a peptide linker L2.

[0043] The "bispecific antibody" described herein refers to a bispecific antibody having two different antigen binding sites, which contains two anti-TIGIT single-chain antibodies scFv and an anti-PD1 immunoglobulin antibody lgG connected thereto, the C-terminus (i.e. the carboxyl end) of each single-chain antibody scFv is connected to the N-terminus of the heavy chain of the immunoglobulin antibody lgG through a peptide linker L2, thereby forming a heavy chain fusion protein of the bispecific antibody, wherein the variable region VH of each single-chain antibody scFv is connected to its variable region VL through a peptide linker L1. It can be understood that the peptide linker L1 and the peptide linker L2 described herein can adopt the commonly used connecting fragments in the art, and such connecting fragments can be (GGGGS)n, wherein n is a positive integer, which can be selected according to actual needs, and preferably n is 2, 3, 4, 5, etc.

[0044] The bispecific antibody in the form of lgG-scFv used herein is composed of an anti-PD1 lgG molecule and an anti-TIGIT scFv form, and such a double antibody structure can effectively improve the stability of the entire fusion protein while retaining the affinity of the lgG molecule itself. And based on the preparation of the lgG molecule-based double antibody, it can effectively reduce the uncertainty of stability and immunogenicity caused by excessive changes in molecular structure, which has significant advantages.

[0045] Further, it is well known in the art that the binding region of an antibody generally includes a heavy chain variable region and a light chain variable region, and each variable region includes three complementarity determining region CDR domains. The CDR domains of the heavy chain and the light chain of the antibody are referred to as CDR-H and CDR-L, respectively.

[0046] In the present application, the variable region VH of the single-chain antibody scFv includes the complementarity determining region CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of CDR-H1 is SEQ ID NO. 5, the amino acid sequence of CDR-H2 is SEQ ID NO. 6, and the amino acid sequence of CDR-H3 is SEQ ID NO. 7.

[0047] The variable region VL of the single-chain antibody scFv includes the complementarity determining region CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of CDR-L1 is SEQ ID NO. 8, the amino acid sequence of CDR-L2 is SEQ ID NO. 9, and the amino acid sequence of CDR-L3 is SEQ ID NO. 10.

[0048] Further, the single-chain antibody scFv described herein can be a murine antibody or a humanized antibody.

[0049] In one exemplary embodiment of the present application, the single-chain antibody scFv is a murine antibody, wherein the heavy chain amino acid sequence of the single-chain antibody scFv is SEQ ID NO. 1, the nucleotide sequence encoding the heavy chain is SEQ ID NO. 2; the light chain amino acid sequence of the single-chain antibody scFv is SEQ ID NO. 3, the nucleotide sequence encoding the light chain is SEQ ID NO. 4.

[0050] In another exemplary embodiment of the present application, the single-chain antibody scFv is a humanized antibody, wherein the single-chain antibody scFv comprises one of the following combinations of amino acid sequences: the combination of SEQ ID NO. 11 and SEQ ID NO. 13 (VH1 / VL1), the combination of SEQ ID NO. 11 and SEQ ID NO. 14 (VH1 / VL2), the combination of SEQ ID NO. 12 and SEQ ID NO. 13 (VH2 / VL1), the combination of SEQ ID NO. 12 and SEQ ID NO. 14 (VH2 / VL2).

[0051] In a further aspect, the constant region of the immunoglobulin antibody lgG is from a human antibody.

[0052] In a further aspect, the constant region of the immunoglobulin antibody lgG is from a human antibody.

[0053] In one exemplary embodiment of the present application, the heavy chain amino acid sequence of the bispecific antibody is SEQ ID NO. 15, and the light chain amino acid sequence of the bispecific antibody is SEQ ID NO. 17.

[0054] The second aspect of the present application provides a nucleotide molecule encoding the bispecific antibody according to the first aspect of the present application. It is understood that the nucleotide molecule can be prepared by conventional methods in the art, such as PCR method or artificial sequence synthesis, which will not be elaborated herein.

[0055] The third aspect of the present application provides an expression vector comprising the nucleotide molecule according to the second aspect of the present application. The expression vector can be of conventional types in the art, which will not be elaborated herein, and preferably, the expression vector is a plasmid.

[0056] The fourth aspect of the present application provides a host cell containing the expression vector of the third aspect of the present application. It is understood that the host cell can be selected according to conventional methods in the art, as long as the expression vector of the third aspect of the present application can be stably self-replicated, and the nucleotide molecule carried can be effectively expressed. Specifically, the host cell can be a eukaryotic expression cell or a prokaryotic expression cell, and the transformation method of the host cell is a conventional method in the art, which will not be described in detail.

[0057] The fifth aspect of the present application provides a pharmaceutical composition containing the bispecific antibody of the first aspect of the present application and any one or more pharmaceutically acceptable adjuvants. It is understood that the bispecific antibody in the pharmaceutical composition is in a therapeutically effective amount, specifically, the effective amount herein refers to an amount sufficient to prevent or treat the symptoms or diseases of medical conditions. After being used in a specific patient or medical subject, the following changes can be produced: the condition to be treated is improved, the overall health of the patient / subject is improved. The effective amount can also be a dosage regimen below the maximum dose to avoid significant side effects or toxicity. The pharmaceutically acceptable adjuvants include carriers, excipients, diluents, etc., and the specific types can be adjusted according to the type of the pharmaceutical composition, which will not be described in detail herein.

[0058] The sixth aspect of the present application provides the use of the pharmaceutical composition of the fifth aspect of the present application in the preparation of a medicament for preventing and / or treating tumors. The prevention and / or treatment of tumors described herein mainly refers to the effect of preventing and / or treating tumors, specifically including the delay of the development of symptoms associated with tumors and / or the reduction of the severity of these symptoms, also including the alleviation of symptoms associated with existing tumors and the prevention of the occurrence of other symptoms, and also including the reduction or prevention of tumor metastasis, etc.

[0059] The tumors that can be prevented and / or treated by the medicament described herein specifically include but are not limited to liver cancer, gastric cancer, lung cancer, colorectal cancer, etc.

[0060] The present application will be described below by specific examples, it should be noted that the specific examples below are only for illustrative purposes, and do not limit the scope of the present application in any way, it is understood that the following examples do not include detailed description of conventional methods, which are well known to those skilled in the art and are described in many existing publications.

[0061] The reagents, experimental materials and their sources used in the following examples are specifically described as follows:

[0062] Experimental reagents:

[0063] TIGIT mFc: purchased from Sino Biological, Cat# 10917-H38H;

[0064] TIGIT-His: purchased from Sino Biological, Cat# 10917-H08H;

[0065] PVR-hFc: purchased from Sino Biological, Cat# 10109-H02H;

[0066] PVR-mFc: Lsbio LS-G138916;

[0067] PD1-His: purchased from Sino Biological, Cat# 10377-H08H-B;

[0068] PD-L1-mFc: purchased from Sino Biological, Cat# 10084-H05H;

[0069] Experimental materials:

[0070] Bal B / C mice: purchased from Shanghai Xipu-Bike Experimental Animal Co., Ltd;

[0071] 293F-TIGIT cells: prepared by using lentivirus infection in the laboratory;

[0072] CD hybridoma culture medium Green features, Cat: 11279023;

[0073] C57L / J mice: purchased from Shanghai Xipu-Bike Experimental Animal Co., Ltd.

[0074] It should be noted that the following examples focus on the preparation of anti-TIGIT single chain antibody scFv, while the anti-PD-1 immunoglobulin antibody lgG is prepared by using the technology known in the art, and the specific method can be referred to the disclosure of Chinese patent application No. CN105175544A, and the anti-PD-1 immunoglobulin antibody lgG used herein is the anti-PD-1 humanized monoclonal antibody AbB8 disclosed in Chinese patent application No. CN105175544A.

[0075] Example 1 Preparation of anti-TIGIT single chain antibody scFv

[0076] 1. Construction of anti-TIGIT murine antibody

[0077] Mouse immunization

[0078] (1) Primary immunization: Two Bal B / C mice (8-week-old female) numbered 54 and 55 were selected, and the mice were immunized with TIGIT mFc antigen mixed with Freund's complete adjuvant at 1:1, 4-5 point subcutaneous injection was adopted to make the mice produce B cells capable of secreting specific target protein antibodies; after the serum titer of the mice was measured and the condition titer was greater than 8K, the mice were boosted once, and the serum ELISA titer in this embodiment was 1:810000;

[0079] (2) Booster immunization: 30 days after the primary immunization, the 54th and 55th mice were boosted in the same way as the primary immunization, and the results showed that the two mice all met the titer (more than 2 times of the negative control and more than 0.25 OD450 value corresponding to the dilution value was the titer of the antibody, and the titer greater than or equal to 8K met the requirements), and the mice were sacrificed 3 days later, and the spleen was taken out, and the spleen cell population was obtained after grinding.

[0080] Hybridoma fusion

[0081] (1) Cell fusion

[0082] The spleen cells of the 54th and 55th mice after death were taken out, and the immune mouse cell fusion was carried out according to the cell fusion standard operating procedure according to the principle of SP2 / 0 myeloma cells:spleen cells = 1:5, and the fusion mark was H006 fusion cell plate.

[0083] (2) Fusion screening

[0084] The fusion cells were detected by ELISA method, TIGIT-his was used for coating, 31 wells with strong binding to TIGIT-his and strong competition with PVR-hFc ligand (i.e. wells with OD value greater than 2) were selected for subcloning.

[0085] (3) Subcloning

[0086] The subcloned cells were detected, and the positive cell clones were cloned, and the cells were further purified. After two cycles of subcloning, ELISA detection showed that the clone number marked as 8A4 clone had strong binding to TIGIT-his (OD450=1.520) and strong competition with PVR-hFc (OD450=0.207), and the clone was established and detected.

[0087] Murine antibody purification

[0088] The hybridoma cells were expanded, and the cell supernatant was collected, the antibody was captured by protein A magnetic beads, eluted with 0.1M, pH 3.0 glycine, replaced with PBS elution buffer, and ultrafiltered with an ultrafiltration tube to obtain the final high-purity antibody, and the results were as follows Figure 1As shown in the results, the purified mouse antibody has a purity of greater than 90%.

[0089] Hybridoma antibody activity testing

[0090] (1) ELISA method for detecting the binding activity of 8A4 mouse antibody to Tigit-his

[0091] The binding affinity of 8A4 mouse antibody to target antigen TIGIT-his was detected by ELISA method, with a sample concentration gradient of 1:3 and a concentration gradient of 0.00001-9 μg / mL. The test results are shown in Table 1. Figure 2 As shown in the results, the 8A4 mouse antibody has strong binding to the antigen (EC50=0.09276), with clear upper and lower platforms on the curve, a large window, and good activity.

[0092] (2) ELISA method for detecting the competition activity of 8A4 mouse antibody to PVR-hFc

[0093] The test plate was coated with 2.0 μg / ml of TIGIT-his, and a sample concentration gradient of 1:3 and a concentration gradient of 0.00001-9 μg / mL were set to determine the OD value for detecting the competition activity of 8A4 mouse antibody to 3.0 μg / ml of PVR-hFc ligand. The test results are shown in Table 2. Figure 3 As shown in the results, the 8A4 mouse antibody has a clear trend in OD value, with clear upper and lower platforms on the curve, and strong competition activity (IC50=0.08234).

[0094] 2. Humanization of anti-TIGIT mouse antibody

[0095] Hybridoma antibody sequencing

[0096] The hybridoma cells were cultured at 37°C in 5% carbon dioxide, and the culture medium was CD hybridoma culture medium. A small amount of purified high-purity RNA was obtained, which was reverse transcribed into cDNA as a template for in vitro amplification of the target fragment using specific PCR primers. The positive PCR product was ligated to a T vector for plasmid recombination and transformation. Positive clones were selected by blue-white spot screening and PCR verification, cultured, and sent for sequencing. The sequencing results were analyzed, and the correct sequence was determined, completing the sequencing of the antibody.

[0097] The heavy chain amino acid sequence of the anti-TIGIT mouse antibody is SEQ ID NO. 1, and the nucleotide sequence encoding the heavy chain is SEQ ID NO. 2. The CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the heavy chain variable region are SEQ ID NO. 5-7, respectively.

[0098] The light chain amino acid sequence of the anti-TIGIT murine antibody is SEQ ID NO. 3, and the nucleotide sequence encoding the light chain is SEQ ID NO. 4; wherein the CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region have the amino acid sequences of SEQ ID NO. 8-10, respectively.

[0099] Antibody humanization

[0100] Using the method of 3D modeling, including using alphafoldII to predict the structure of the variable region of the antibody, database alignment, and mutating the original murine sequence to a human sequence.

[0101] In this embodiment, the original sequence of the 8A4 antibody is designed as the following multiple humanized antibody sequences:

[0102] H006_8A4_huVH1, which has the amino acid sequence of SEQ ID NO. 11;

[0103] H006_8A4_huVH2, which has the amino acid sequence of SEQ ID NO. 12;

[0104] H006_8A4_huVL1, which has the amino acid sequence of SEQ ID NO. 13;

[0105] H006_8A4_huVL2, which has the amino acid sequence of SEQ ID NO. 14.

[0106] The designed sequences are combined into humanized antibodies, and the specific forms are as follows:

[0107] huVL1-huVH1 (VH1 / VL1);

[0108] huVL2-huVH1 (VH1 / VL2);

[0109] huVL1-huVH2 (VH2 / VL1);

[0110] huVL2-huVH2 (VH2 / VL2).

[0111] The above humanized antibodies are transfected in the Expi 293 mammalian expression system, and after 7-10 days of culture, the supernatant is obtained by centrifugation, and the expression of the antibodies is carried out.

[0112] Humanized antibody purification

[0113] The above humanized antibodies expressed in Expi 293 cells were collected, and the supernatant was used to capture the antibodies using protein A magnetic beads, eluted with 0.1 M, pH 3.0 glycine, replaced with PBS elution buffer, and ultrafiltered using an ultrafiltration tube to obtain the final high-purity antibody. The results are shown in Table 1. Figure 4 As shown in Table 1, the experimental results show that the purity of the purified humanized antibodies is greater than 90%.

[0114] 2. Activity detection of humanized antibodies

[0115] (1) ELISA method for detecting the binding activity of H006-8A4 humanized antibodies to TIGIT-his

[0116] By ELISA method, the detection plate was coated with 0.5 μg / ml TIGIT-his, a sample concentration gradient of 1:3 was set, the concentration gradient was 0.00001-9 μg / mL, and the binding activity OD value was measured to detect the binding affinity of H006-8A4 series humanized antibodies to target antigen. The results are shown in Table 2. Figure 5 As shown in Table 2, the data show that compared with the positive antibody, the H006-8A4 series humanized antibodies have stronger binding to the antigen, the curve has clear upper and lower platforms, the window is large, and the activity is better.

[0117] (2) FACS method for detecting the binding activity of H006-8A4 humanized antibodies to 293F-TIGIT cells

[0118] By FACS method, 293F-TIGIT cells (laboratory self-made) were uniformly laid in a 96-well cell plate, a sample concentration gradient of 1:3 was set, the concentration gradient was 0.00001-9 μg / mL, and the cells were combined, then 3 μg / ml of Alex488 labeled goat anti-mouse secondary antibody / Alex488 labeled goat anti-human secondary antibody was added to each well, and the binding activity OD value was measured after binding to detect the binding strength of H006-8A4 series humanized antibodies to 293F-TIGIT cells. The results are shown in Table 3. Figure 6 As shown in Table 3, the data show that the H006-8A4 series antibodies have relatively strong binding to 293F-TIGIT cells.

[0119] (3) ELISA method for detecting the competition activity of H006-8A4 humanized antibodies to PVR-mFc

[0120] The detection plate was coated with 2.0 μg / ml TIGIT-his, a sample concentration gradient of 1:3 was set, the concentration gradient was 0.00001-9 μg / mL, and the OD value was measured to detect the competition activity of H006-8A4 series humanized antibodies to 9.0 μg / ml PVR-mFc ligand. The results are shown in Table 4. Figure 7As shown in the figure, the data shows that the antibody OD value has a clear trend, and the curve has a clear up-and-down platform, with strong competitive activity.

[0121] (4) FACS method for detecting the competitive activity of H006-8A4 humanized antibody and PVR-mFc

[0122] By FACS method, 293F-TIGIT cells were uniformly plated into a 96-well cell plate, a constant concentration of PVR-mFc of 3 μg / ml was set, and the concentration gradient of the sample to be tested was set, and then combined with the cells, and then 3 μg / ml of Alex488 labeled goat anti-mouse secondary antibody was added to each well. After binding, the competitive activity OD value was detected to detect the strength of the competition between H006-8A4 humanized antibody and PVR-mFc. The results are shown in Figure 8 As shown in the figure, the data shows that the H006-8A4 series of antibodies have relatively strong competitive activity.

[0123] 3. Thermal stability experiment of humanized antibody

[0124] Using Protein Thermal Shift TM Dye Reagent Kit, the fluorescence signal intensity was detected by real-time fluorescence quantitative PCR method to monitor the thermal stability of the protein, and the results are shown in Table 1 and Figure 9 The Tm values of the H006-8A4 series of humanized antibodies are lower than the parent, among which H006-8A4-huVH1 / L1, H006-8A4-huVH2 / L1, H006-8A4-huVH2 / L2 have similar thermal stability to the parent, which is slightly lower than the parent; the thermal stability of H006-8A4-huVH1 / L2 is significantly lower than the parent.

[0125] Table 1 Tm values of H006-8A4 series of humanized antibodies

[0126] Protein name Tm value 1 Tm value 2 Average H006-8A4-Hwt / Lwt 92.14 92.07 92.11 H006-8A4-huVH1 / L1 89.1 88.95 89.03 H006-8A4-huVH1 / L2 70.27 70.33 70.30 H006-8A4-huVH2 / L1 88.22 88.17 88.20 H006-8A4-huVH2 / L2 88.26 88.22 88.24

[0127] 4. MLR experiment of humanized antibody

[0128] Set the concentration gradient of all samples, incubate with completely induced differentiated cells, and use an ELISA kit to detect the OD value to detect the secretion of IFN-γ in the cell supernatant. The test results are shown in Figure 10 The data shows that H006-8A4-huVH1 / VL2 and H006-8A4-huVH2 / VL2 have a promoting effect on the secretion of IFN-γ by cells.

[0129] Example 2 Pharmacodynamic evaluation of H006-8A4-huVH2 / VL2 antibody in Hepa1-6 mouse hepatoma subcutaneous tumor model

[0130] Hepa1-6 subcutaneous tumor model was established, 5 million tumor cells were subcutaneously inoculated, 100 μl BD matrix glue was injected at the same time, and when the tumor volume grew to ~ 120 mm 3 When the mice were randomly divided into 4 groups, 8 mice in each group, the average tumor volume was 119 mm 3 after grouping. Each mouse was given 100 μg, 4 times a day, the control group was normal saline, and the experimental group was antibody, etc.

[0131] The tumor growth curves of the control group and each experimental group are shown in Figure 11 : On the 21st day after grouping, i.e. at the end of the experiment, the average tumor volume of Group-1 (Vehicle) control group was 2118.7 mm 3 ; the average tumor volume of Group-2 (8A4) was 1410.9 mm 3 , the tumor volume inhibition rate TGI TV (%) was 35.4%; the average tumor volume of Group-3 (PD-1 antibody) was 1027.1 mm 3 , the tumor volume inhibition rate TGI TV (%) was 60.5%; the average tumor volume of Group-4 (8A4+PD-1 antibody) was 519.5 mm 3 , the tumor volume inhibition rate TGI TV (%) was 80.0%.

[0132] The above results show that the tumor inhibition effect of H006-8A4-huVH2 / VL2 antibody in this embodiment is strong, and the antibody activity is good.

[0133] Example 3 Anti-TIGIT-PD1 bispecific antibody sequence

[0134] In this embodiment, an anti-TIGIT single-chain antibody scFv and an anti-PD-1 immunoglobulin antibody lgG are connected in series to construct an anti-TIGIT-PD1 bispecific antibody, and the specific form is scFv-L2-lgG. Among them, the single-chain antibody scFv adopts the molecular form of VL-L1-VH, the heavy chain variable region VH2 (SEQ ID NO. 12) and the light chain variable region VL2 (SEQ ID NO. 14) of the humanized antibody sequence are connected through the peptide linker L1 (GGGGSGGGGSGGGGSGGGGS) to obtain the single-chain antibody scFv against TIGIT. The heavy chain of the anti-TIGIT-PD1 bispecific antibody is obtained by connecting the single-chain antibody scFv and the heavy chain of the immunoglobulin antibody lgG against PD-1 using the peptide linker L2 (GGGGSGGGGSGGGGS).

[0135] The heavy chain amino acid sequence of the anti-TIGIT-PD1 bispecific antibody is SEQ ID NO. 15, and the light chain amino acid sequence is SEQ ID NO. 17; the nucleotide sequence encoding the heavy chain is SEQ ID NO. 16, and the nucleotide sequence encoding the light chain is SEQ ID NO. 18.

[0136] Example 4 Purification of anti-TIGIT-PD1 bispecific antibody

[0137] The bispecific antibody in Example 3 was expressed in Expi 293 cells, the cell supernatant was collected, the antibody was captured using protein A magnetic beads, eluted using 0.1M, pH 3.0 glycine, the elution buffer was replaced with PBS, and the final high-purity antibody was obtained by ultrafiltration using an ultrafiltration tube. Antibody purification was performed according to the standard operating procedures for protein purification, and the results are shown in Figure 12 The experimental results show that the purity of the purified antibody is greater than 90%.

[0138] Example 5 Activity detection of anti-TIGIT-PD1 bispecific antibody

[0139] (1) ELISA method for detecting the binding activity of anti-TIGIT-PD1 bispecific antibody to TIGIT-his and PD1-his: The detection plate was coated with 0.5 μg / ml of TIGIT-his, and a sample concentration gradient of 1:3 was set, with a concentration gradient of 0.00001-9 μg / mL; the enzyme-labeled plate was placed in a multifunctional enzyme-labeled instrument to measure the OD value at 450 nm. The data were fitted by four-parameter curve using Graphpad Prism software, and the EC50 value was obtained to determine the strength of the binding of the bispecific antibody to the target antigen. The test results are shown in Figure 13 The results show that the TIGIT-PD1 bispecific antibody has strong binding to Tigit his antigen, the curve has clear upper and lower platforms, the window is large, and the activity is good; when the antibody binds to PD1-his antigen, the OD value is high, and the curve has a high upper platform.

[0140] (2) ELISA method for detecting the competition activity of anti-TIGIT-PD1 bispecific antibody to PD-L1-mFc: The test plate was coated with 2.0 μg / ml of PD-1-his, and a sample concentration gradient of 1:3 was set, with a concentration gradient of 0.00001-9 μg / mL, and the OD value was measured to detect the competition activity of anti-TIGIT-PD1 bispecific antibody to 0.3 μg / ml of PD-L1-mFc. The results are shown in Figure 14 The data show that the OD value trend is obvious, the curve has upper and lower platforms, and has obvious competition activity.

[0141] Any combination of the technical features in the above-described embodiments can be made, and for the sake of brevity, not all possible combinations are described, however, as long as the combination of the technical features does not exist in contradiction, it shall be considered within the scope of the present disclosure.

[0142] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it shall not be understood as a limitation on the patent scope of the present application. It shall be pointed out that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these shall be within the protection scope of the present application. Therefore, the protection scope of the present application patent shall be subject to the appended claims.

Claims

1. An anti-TIGIT-PD1 bispecific antibody, characterized in that, The heavy chain amino acid sequence of the bispecific antibody is SEQ ID NO. 15, and the light chain amino acid sequence is SEQ ID NO.

17.

2. The anti-TIGIT-PD1 bispecific antibody of claim 1, wherein, The constant region of the immunoglobulin antibody lgG is from a human antibody.

3. The anti-TIGIT-PD1 bispecific antibody of claim 1, wherein, The heavy chain constant region sequence of the immunoglobulin antibody lgG is the heavy chain constant region of human lgG1, and the light chain constant region sequence is the light chain constant region of human lgG1.

4. A nucleotide molecule, characterized in that, It encodes the bispecific antibody as claimed in any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein, The nucleotide sequence encoding the heavy chain of the bispecific antibody is shown as SEQ ID NO.

16.

6. The nucleic acid molecule of claim 4, wherein, The nucleotide sequence encoding the light chain of the bispecific antibody is shown as SEQ ID NO.

18.

7. An expression vector, characterized by, It contains the nucleotide molecule as claimed in any one of claims 4-6.

8. A host cell, characterized in that, It contains the expression vector as claimed in claim 7.

9. A pharmaceutical composition, characterized by, It contains the bispecific antibody as claimed in any one of claims 1-3 and any one or more pharmaceutically acceptable adjuvants.

10. Use of a pharmaceutical composition according to claim 9 for the manufacture of a medicament, characterized in that, The drug is used for preventing and / or treating tumors, which are liver cancer, gastric cancer, lung cancer or colorectal cancer.

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