Bispecific antibody targeting intracellular ny-ESO-1 and use thereof

Abstract

A bispecific antibody targeting intracellular NY-ESO-1 and the use thereof, which relate to the technical field of biomedicine. A bispecific antibody targeting intracellular NY-ESO-1 is designed, in which the symmetrical form enables the complete molecular structure of a CD3 monoclonal antibody to be retained, and meanwhile, a nanobody sequence against NY-ESO-1157-165 / A02 is added at the N terminus, so that two different antigens can be specifically recognized. The provided bispecific antibody is target-dependent, can significantly induce the upregulation of the early and late activation markers of T cells, and has significant anti-tumor activity in vivo with specificity.

Description

A bispecific antibody targeting intracellular NY-ESO-1 and its application Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bispecific antibody targeting intracellular NY-ESO-1 and applications thereof. Background Art

[0002] In recent years, the incidence of tumors has been increasing, posing a serious threat to human health. Tumor immunotherapy is a new type of therapy that has developed rapidly in the past decade, and is on par with chemotherapy, targeted therapy, surgery, and radiotherapy. Under normal circumstances, the immune system can recognize and eliminate tumor cells. However, tumor cells have the ability to escape the immune system and can inhibit the immune system's effective recognition and killing of tumor cells through different links, thereby producing immune tolerance and even promoting the occurrence and development of tumors. The main target of tumor immunotherapy is the body's immune system rather than tumor cells. Tumor cells are eliminated by enhancing the body's natural immune defense against tumors and reshaping the immune microenvironment. On the one hand, this therapy trains immune cells to recognize and eliminate target cells carrying tumor antigens, thereby enhancing immune-mediated tumor cell lysis. On the other hand, it eliminates or reduces the immunosuppressive signals induced by tumor cells, thereby achieving the effect of tumor treatment.

[0003] Bispecific antibodies (BiAbs) are artificial antibodies composed of two different antibody fragments that can specifically recognize and bind to two different antigens or two different antigenic epitopes. In the complex pathogenesis of cancer, multiple mediators are involved in the activation of cancer-related signaling pathways, which limits the effectiveness of cancer treatment based on single specificity. The reduction of activated lymphocytes in the tumor microenvironment (TME) has been shown to lead to adverse immune responses. The mechanism of action of bispecific antibodies in cancer immunotherapy includes 1) binding to T cells or other immune cells (such as natural killer cells) to specifically eliminate tumor cells, 2) bridging receptors to block or activate synergistic signaling pathways, and 3) targeting multiple tumor antigens or different antigenic epitopes on tumor cells to improve tumor selectivity (Li H, Er SawP, Song E. Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. Cell Mol Immunol. 2020, 17(5): 451-461.)

[0004] NY-ESO-1 is a type of tumor-testis antigen. It was first discovered by Chen et al. through antigen serological identification of patients with esophageal squamous cell carcinoma. It is a type of tumor-shared antigen that can induce humoral immunity. NY-ESO-1 has a high expression level in neuroblastoma, synovial sarcoma, esophageal cancer, lung cancer, etc., and the expression level increases with the progression of the disease. It is expressed at a very low level or even not expressed in rectal cancer, lymphoma, etc., but its expression is limited in normal tissues. In principle, it is classified as a tumor-associated antigen. However, since the testicle / placenta does not express HLA alleles, the polypeptide produced by NY-ESO-1 processing cannot be presented to the cell surface. Therefore, NY-ESO-1 can be considered a tumor-specific antigen. Studies have found that NY-ESO-1 can induce both humoral and humoral immunity. The positive IgG in the serum is associated with the production of CD8 against the antigen. + It is positively correlated with T cells and is the most immunogenic tumor-specific antigen discovered to date (Raza A, Merhi M, Inchakalody VP, et al. Unleashing the immune response to NY-ESO-1 cancer testis antigen as a potential target for cancer immunotherapy. J Transl Med, 2020, 18(1): 140.)

[0005] The recruitment of effector cells is crucial in tumor immunotherapy. One arm of a bispecific antibody targets a receptor on the surface of tumor cells, while the other arm binds to CD3ε in the TCR complex, recruiting and activating T cells and further inducing highly potent and selective cytotoxicity. When both T cells and tumor cells are simultaneously bound by the bispecific antibody, a cytolytic synapse is formed between the T cells and the tumor cells. This cytolytic synapse contains perforin and cytotoxic granzyme B released by the T cells, leading to tumor cell destruction. The main functions of the CD3 molecule are to stabilize the TCR structure and transmit T cell activation signals. After the TCR specifically recognizes and binds to an antigen, CD3 participates in signal transduction into the T cell cytoplasm. As the first signal to induce T cell activation, it plays a crucial role in T cell antigen recognition and the generation of an immune response. Bispecific antibodies targeting CD3 are rapidly becoming a revolutionary approach in cancer immunotherapy, accounting for over 50% of bispecific antibody drugs used in global clinical trials for cancer treatment. Summary of the Invention

[0006] In view of this, the present invention provides a bispecific antibody targeting intracellular NY-ESO-1 and its application. The present invention designs a bispecific antibody targeting intracellular NY-ESO-1, which retains the complete molecular structure of CD3 monoclonal antibody in symmetrical form and adds anti-NY-ESO-1 at the N-terminus. 157-165 The nanoantibody sequence of / A02 specifically recognizes two different antigens, targeting immune effector cells to tumor cells, thereby increasing the effectiveness of immune effector cells in killing tumor cells.

[0007] The present invention provides a bispecific antibody targeting intracellular NY-ESO-1, wherein the bispecific antibody comprises:

[0008] a first antibody that specifically binds to a first antigen,

[0009] and a second antibody that specifically binds to a second antigen;

[0010] The first antigen is CD3 and the second antigen is the tumor-specific antigen NY-ESO-1;

[0011] The first antibody is a full-length antibody, comprising two heavy chains and two light chains,

[0012] The second antibody is a VHH of a nanobody, and the second antibody is connected to one end of the heavy chain or light chain of the first antibody.

[0013] Tumor-specific antigen NY-ESO-1 is specifically tumor-specific antigen NY-ESO-1 157-165 / A02.

[0014] Preferably, the bispecific antibody comprises one or more VHHs, wherein the VHHs are connected to the N-terminus of the heavy chain of the first antibody via a linker, wherein the amino acid sequence of the linker is GGGGSGGGGSGGGGS.

[0015] More preferably, the bispecific antibody comprises one VHH, and the amino acid sequence of the VHH is shown in SEQ ID No.9.

[0016] The first antibody is monoclonal antibody OKT3 or monoclonal antibody V9.

[0017] Specifically, when the first antibody is the monoclonal antibody OKT3, the heavy chain amino acid sequence is shown in SEQ ID No. 1, and the light chain amino acid sequence is shown in SEQ ID No. 2;

[0018] When the first antibody is monoclonal antibody V9, the heavy chain amino acid sequence is shown as SEQ ID No. 3, and the light chain amino acid sequence is shown as SEQ ID No. 4.

[0019] The present invention also provides a gene encoding the bispecific antibody, wherein the VHH encoding gene sequence is shown in SEQ ID No. 10.

[0020] When the first antibody is the monoclonal antibody OKT3, the gene sequence encoding the heavy chain is shown in SEQ ID No. 5, and the gene sequence encoding the light chain is shown in SEQ ID No. 6;

[0021] When the first antibody is the monoclonal antibody V9, the gene sequence encoding the heavy chain is shown as SEQ ID No. 7, and the gene sequence encoding the light chain is shown as SEQ ID No. 8.

[0022] The present invention also provides a method for preparing the bispecific antibody, comprising the following steps:

[0023] (1) constructing a vector expressing the gene encoding the heavy chain of the first antibody, the gene encoding the light chain, and the gene encoding the VHH;

[0024] (2) Transfecting the vector encoding the heavy chain and light chain of the first antibody expressed in step (1) and the VHH encoding gene into mammalian cells, culturing and then purifying the protein to obtain the bispecific antibody.

[0025] Preferably, the vector in step (1) is pcDNA3.1(+); and the mammalian cell in step (2) is HEK293F cell.

[0026] The present invention also provides the use of the bispecific antibody in the preparation of anti-tumor or anti-autoimmune disease drugs.

[0027] The present invention also provides the use of the gene or cells containing the gene in the preparation of anti-tumor or anti-autoimmune disease drugs.

[0028] Beneficial effects of the present invention:

[0029] The present invention designs a bispecific antibody targeting intracellular NY-ESO-1, which retains the complete molecular structure of CD3 monoclonal antibody in a symmetrical form and adds anti-NY-ESO-1 at the N-terminus. 157-165 The nanobody sequence of / A02 can specifically recognize two different antigens. The bispecific antibody provided by the present invention is target-dependent, significantly upregulating both early and late T cell activation markers, and exhibiting significant specific in vivo anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of a bispecific antibody; wherein ab represents two forms of bispecific antibodies, respectively named VHH-CD3 and VHH-CD3V9.

[0031] FIG2 is a diagram showing the SDS-PAGE analysis results of the bispecific antibody VHH-CD3; wherein lane 1 is a non-reduced sample and lane 2 is a reduced sample.

[0032] FIG3 is a diagram showing the SDS-PAGE analysis results of the bispecific antibody VHH-CD3V9; wherein lane 1 is a non-reduced sample and lane 2 is a reduced sample.

[0033] Figure 4 shows the lactate dehydrogenase (LDH) detection results of the bispecific antibody VHH-CD3; Figure a shows the tumor cell A375 NY detection results, and Figure b shows the A375 detection results.

[0034] Figure 5 shows the lactate dehydrogenase (LDH) detection results of the bispecific antibody VHH-CD3V9; Figure a shows the tumor cell A375 NY detection results, and Figure b shows the A375 detection results.

[0035] Figure 6 shows the results of upregulation of T cell activation markers CD69 and CD25 mediated by the bispecific antibody VHH-CD3.

[0036] Figure 7 shows the results of the upregulation of T cell activation markers CD69 and CD25 mediated by the bispecific antibody VHH-CD3V9: wherein ab represents the upregulation of CD69 and CD25, respectively.

[0037] FIG8 is a graph showing the in vivo anti-tumor activity of the bispecific antibody VHH-CD3; ** indicates p<0.01, *** indicates p<0.001.

[0038] Figure 9 shows the results of in vivo anti-tumor activity of the bispecific antibody VHH-CD3V9; * indicates p<0.05, ** indicates p<0.01. DETAILED DESCRIPTION

[0039] Example 1 Expression and purification of bispecific antibodies

[0040] The two bispecific antibody gene sequences of the present invention were synthesized by GenScript Biotech, wherein the VHH encoding gene sequence is shown in SEQ ID No. 10, the heavy chain encoding gene sequence is shown in SEQ ID No. 5, the light chain encoding gene sequence is shown in SEQ ID No. 6, the other heavy chain encoding gene sequence is shown in SEQ ID No. 7, and the light chain encoding gene sequence is shown in SEQ ID No. 8. VHH is connected to the N-terminus of the heavy chain of the first antibody through a linker (GGGGS) 3. The two bispecific antibody genes were inserted into the EcoRI and NotI restriction sites of the vector pcDNA 3.1 (+) to obtain two bispecific antibody expression plasmids. After the above two bispecific antibody expression plasmids were transfected into HEK293F cells for 3-4 days, they were centrifuged at 4000 × g for 20 min, the cell supernatant was taken, filtered through a 0.22 μm filter membrane, and purified using an AKTA protein purifier and a HiTrap protein A affinity column. The purification steps are as follows: a. Turn on the AKTA protein purifier and the connected control computer. After the instrument is connected to the computer, set the pressure parameters (high pressure 0.25 MPa); b. Place the A and B pump heads in pure water filtered through a 0.45 μm filter membrane, set the flow rate (3 mL / min) and the flushing ratio of the AB pump (50% B). After the pure water is flushed to conductivity equilibrium (about 60 mL), connect the HiTrap protein A affinity column (5 mL) to the AKTA purifier and continue to flush with pure water for at least three column volumes; c. Change the flushing ratio of the AB pump to 0% B, and replace the A pump with 50 mM Tris-HCl (PH7.4) loading buffer. After conductivity equilibrium, replace the A pump with the supernatant of the culture medium to be purified. After the loading is completed, replace the A pump with the loading buffer until conductivity equilibrium is achieved; then change the ratio of the AB pump to 100% B, and replace the B pump with 1 mol / L sodium acetate (PH 3.0) Elution buffer was added to obtain the target protein by elution. The AKTA purifier was flushed with pure water to conductivity equilibrium and the entire system was preserved with 20% ethanol (v / v).

[0041] The different fusion proteins were ultrafiltered using a 50 kDa pore size ultrafiltration membrane. After the solvent was replaced with PBS buffer, the protein concentration was determined using a Nanodrop ND-1000. Aliquots were then stored frozen at -80°C until further use. Protein purity was analyzed by SDS-PAGE, as shown in Figures 2 and 3.

[0042] Example 2 Bispecific Antibody-Mediated Killing of Tumor Cells by PBMC

[0043] Lactate dehydrogenase (LDH) detection.

[0044] A375 is an endogenously expressed NY-ESO-1 157-165 / A02 melanoma cell line, low antigen expression, A375 NY is a fluorescent marker constructed by stably transferring EGFP-ubiquitin-SLLMWITQC into A375 cells and NY-ESO-1 157-165 / A02 high-expressing tumor cell lines.

[0045] The destruction of cell membranes caused by apoptosis or necrosis leads to the release of enzymes from the cytoplasm into the culture medium, including the relatively stable enzyme lactate dehydrogenase (LDH). Quantitative analysis of cytotoxicity can be achieved by measuring the activity of LDH released into the culture medium from cells with ruptured plasma membranes.

[0046] PBMC were purchased from Shanghai Saili Biotechnology Co., Ltd. PBMC and NY-ESO-1 157-165 A375 and A375NY pMHC-positive tumor cells were mixed at a 4:1 effector-target ratio and plated in 96-well plates. A gradient of bispecific antibody concentrations was added to a final volume of 100 μl (n=3). Cells were cultured in phenol red-free 1640 medium containing 1% FBS to prevent interference with the LDH assay. After incubation for 24 or 48 hours in a 5% CO2, 37°C cell culture incubator, LDH levels released by tumor cell death were measured using an LDH assay kit (Tongren Chemical, CK12) to characterize tumor cell mortality and calculate IC50 values. The anti-tumor activity of different bispecific antibody formats was then compared.

[0047] The bispecific antibody VHH-CD3 exhibited dose-dependent cytotoxic effects against both the naturally NY-ESO-1-expressing cell line A375 and the cell line with high A375 expression (Figure 4). The bispecific antibody VHH-CD3V9 exhibited dose-dependent cytotoxic effects against both the naturally NY-ESO-1-expressing cell line A375 and the cell line with high A375 expression, while the control monoclonal antibody exhibited weak cytotoxic activity, indicating that the cytotoxic effect of the bispecific antibody was not due to nonspecific killing caused by activated T cells but was target-dependent (Figure 5).

[0048] Example 3 Bispecific Antibody-Mediated T Cell Activation Detection

[0049] Tumor A375 cells and PBMCs were mixed at a 4:1 effector-target ratio and plated in 48-well plates. A series of bispecific antibodies diluted in RPMI-1640 complete medium were added to a final volume of 300 μL (n=3) and incubated in a cell culture incubator for 40 h. Cells were harvested by centrifugation at 600 × g for 5 min, washed once with 1× PBS, and resuspended in 100 μL of 1× PBS. 5 μL of FITC-conjugated mouse anti-human CD3 antibody (BD, 555339), 5 μL of APC-conjugated mouse anti-human CD69 antibody (BD, 555533), and 1 μL of PE-conjugated mouse anti-human CD25 antibody (BD, 555432) were added and incubated at 4°C in the dark for 30 min. After washing twice with 1× PBS, cells were resuspended in 200 μL of 1× PBS and lysed using an ACEA NovoCyte TM The proportions of CD69- and CD25-positive T cells were detected by flow cytometry.

[0050] The bispecific antibody VHH-CD3 significantly upregulated CD69, an early T cell activation marker, and CD25, a late T cell activation marker (Figure 6). The bispecific antibody VHH-CD3V9 significantly upregulated CD69, an early T cell activation marker, and CD25, a late T cell activation marker, while the control CD3 monoclonal antibody did not induce T cell activation (Figure 7).

[0051] Example 4 In vivo anti-tumor activity of bispecific antibody VHH-CD3

[0052] Jicui Yaokang purchased 15 severely immunodeficient NOD-SCID mice aged 5-7 weeks and weighing about 20 g, and inoculated A375 cells (2×10 6 cells / mouse), and wait until the tumor grows to 100 mm 3 The mice were randomly divided into three groups: PBS group, VHH-CD3 group, and CD3 monoclonal antibody (OKT3 monoclonal antibody, heavy chain amino acid sequence is shown in SEQ ID No. 11, light chain amino acid sequence is shown in SEQ ID No. 2) group. Each mouse was intraperitoneally injected with human PBMC (1×10 7 cells / mouse), a transient humanized tumor-bearing mouse model of immune cells was constructed. Subsequently, except for the PBS group, the other two groups of mice were administered 0.5 mg / kg of control drugs and experimental drugs by intraperitoneal injection, once every 3 days, for a total of 3 times. The tumor size of the mice (measuring the length and width of the tumor, tumor size = length × width × width / 2) and body weight were measured every 3 days. After three doses, the tumor volume of the VHH-CD3 group showed significant differences from the PBS group (P < 0.001) and also showed significant differences from the CD3 monoclonal antibody group (P < 0.01) (Figure 8), indicating that the bispecific antibody VHH-CD3 has obvious specific in vivo anti-tumor activity.

[0053] Example 5 In vivo anti-tumor activity of the bispecific antibody VHH-CD3V9

[0054] Jicui Yaokang purchased 20 severely immunodeficient NOD-SCID mice aged 5-7 weeks and weighing about 20 g, and inoculated A375 cells (2×10 6 cells / mouse), and wait until the tumor grows to 40 mm. 3 The mice were randomly divided into 4 groups: PBS group, VHH-CD3V9 0.1 mg / kg group, VHH-CD3V9 1 mg / kg group, and UCHT1 V9 monoclonal antibody (heavy chain amino acid sequence is shown in SEQ ID No. 12, light chain amino acid sequence is shown in SEQ ID No. 4) 1 mg / kg group. Each mouse was intraperitoneally injected with human PBMC (1×10 7 cells / mouse), a transient humanized tumor-bearing mouse model of immune cells was constructed. Subsequently, except for the PBS group, the other three groups of mice were intraperitoneally injected with 0.1 mg / kg or 1 mg / kg of control drugs and experimental drugs, once every 3 days, for a total of 6 times. The tumor size of the mice (measuring the length and width of the tumor, tumor size = length × width × width / 2) and body weight were measured every 3 days. After six doses, the tumor volume of the VHH-CD3V9 1 mg / kg group showed significant differences from the PBS group (P<0.001) and also showed significant differences from the V9 monoclonal antibody 1 mg / kg group (P<0.01) (Figure 9), indicating that the bispecific antibody VHH-CD3V9 has obvious specific in vivo anti-tumor activity.

Claims

1. A bispecific antibody targeting intracellular NY-ESO-1, characterized in that: The bispecific antibody comprises: a first antibody that specifically binds to a first antigen, and a second antibody that specifically binds to a second antigen; The first antigen is CD3 and the second antigen is the tumor-specific antigen NY-ESO-1; The first antibody is a full-length antibody, including two heavy chains and two light chains, The second antibody is a VHH of a nanobody, and the second antibody is connected to one end of the heavy chain or light chain of the first antibody.

2. The bispecific antibody according to claim 1, characterized in that The bispecific antibody comprises one or more VHHs, and the VHHs are connected to the N-terminus of the heavy chain of the first antibody via a linker.

3. The bispecific antibody according to claim 2, characterized in that The amino acid sequence of the VHH is shown in SEQ ID No.

9.

4. The bispecific antibody according to claim 1, characterized in that The first antibody is monoclonal antibody OKT3 or monoclonal antibody V9; When the first antibody is the monoclonal antibody OKT3, the heavy chain amino acid sequence is shown in SEQ ID No.1, and the light chain amino acid sequence is shown in SEQ ID No.2; When the first antibody is monoclonal antibody V9, the heavy chain amino acid sequence is shown as SEQ ID No.3, and the light chain amino acid sequence is shown as SEQ ID No.

4.

5. A gene encoding the bispecific antibody according to any one of claims 1 to 4, wherein the VHH encoding gene sequence is shown in SEQ ID No. 10, When the first antibody is the monoclonal antibody OKT3, the heavy chain encoding gene sequence is shown in SEQ ID No.5, and the light chain encoding gene sequence is shown in SEQ ID No.6; When the first antibody is monoclonal antibody V9, the gene sequence encoding the heavy chain is shown as SEQ ID No.7, and the gene sequence encoding the light chain is shown as SEQ ID No.

8.

6. The method for preparing a bispecific antibody according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) constructing a gene vector expressing the first antibody heavy chain encoding gene, the light chain encoding gene and the VHH encoding gene; (2) Transfecting the vector expressing the gene encoding the heavy chain of the first antibody, the gene encoding the light chain and the gene encoding the VHH in step (1) into mammalian cells, culturing and purifying the protein to obtain the bispecific antibody.

7. The preparation method according to claim 6, characterized in that: The vector in step (1) is pcDNA3.1(+); the mammalian cell in step (2) is HEK293F cell.

8. Use of the bispecific antibody according to any one of claims 1 to 4 in the preparation of anti-tumor or anti-autoimmune disease drugs.

9. Use of the gene according to claim 5 or a cell comprising the gene in the preparation of an anti-tumor or anti-autoimmune disease drug.

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