Antibody targeting VSIG4 protein and application thereof

Abstract

The invention relates to the technical field of antibodies, and provides a VSIG4 protein targeting antibody and application thereof. The antibody comprises a heavy chain variable region CDR-H1, a heavy chain variable region CDR-H2 and a heavy chain variable region CDR-H3, wherein the amino acid sequences of the CDR-H1, the CDR-H2 and the CDR-H3 are as shown in SEQ ID No. 1 to SEQ ID No. 3. The antibody provided by the invention has higher specificity and high affinity to tumor cells, and is expected to become a CAR-T drug candidate drug for lung cancer, glioma, multiple myeloma and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and particularly relates to an antibody targeting VSIG4 protein and its application. Background Art

[0002] VSIG4 (V-set and immunoglobulin domain-containing protein 4) is an immune regulatory molecule, belonging to the members of the B7 family, and is highly expressed in macrophages and regulatory T cells in the tumor microenvironment. It inhibits T cell activity by binding to receptors, promotes immune escape, and is an important potential target for cancer immunotherapy.

[0003] The efficiency of screening anti-VSIG4 antibodies by traditional hybridoma technology is low and the cycle is long; the antibodies obtained by phage display technology are prone to insufficient affinity due to the differences in prokaryotic expression systems and mammalian protein modifications; the yeast surface display system has both the ability of eukaryotic post-translational modification and high-throughput screening characteristics, can retain the natural conformation of antibodies, and improve the human applicability of screening molecules. In addition, peptide-based screening technologies (such as phage display peptide libraries or chemically synthesized peptide libraries) have also been widely used in the development of candidate molecules targeting VSIG4. These peptide molecules can block their immunosuppressive functions by mimicking the natural ligand-binding epitopes of VSIG4, or serve as lead compounds for antibody development.

[0004] After VSIG4 binds to its ligand, it inhibits the cytotoxic effect of CD8 T cells, which is another potential target for tumor immunosuppression in the B7 family. VSIG4-specific antibodies and peptides have shown the ability to re-polarize macrophages and induce immune activation in vitro and in vivo models, and tumor growth inhibition has been observed in mouse models when using VSIG4 antibodies or peptides alone, as well as in combination with anti-PD-1. Currently, there are several clinical drugs targeting VSIG4 under research, mainly for the treatment of solid tumors. Therefore, VSIG4 will become a new target for the treatment of inflammatory diseases or various tumors, and it is of great significance to study antibodies and peptides with high affinity and activity for VSIG4. Summary of the Invention

[0005] In view of this, the present invention provides an antibody targeting VSIG4 protein with high affinity for tumor cells, which can be used as an indicator for the diagnosis and prognosis of cancer, and is expected to become a candidate drug for CAR-T drugs such as lung cancer, glioma, and multiple myeloma.

[0006] The technical solution of the present invention is realized as follows: In the first aspect, the present invention provides an antibody targeting VSIG4 protein, including CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are as shown in SEQ ID No.1~3.

[0007] Based on the above technical solutions, preferably, the antibody further comprises light chain variable region CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID No. 4-6.

[0008] Based on the above technical solutions, preferably, the antibody further comprises a Linker, and the amino acid sequence of the Linker is as shown in SEQ ID No. 7.

[0009] Based on the above technical solutions, preferably, the amino acid sequence of the antibody is as shown in SEQ ID No. 8.

[0010] In a second aspect, the present invention also provides an isolated nucleic acid encoding the above antibody.

[0011] In a third aspect, the present invention also provides an expression vector comprising the above isolated nucleic acid.

[0012] In a fourth aspect, the present invention also provides the use of the antibody, isolated nucleic acid, or expression vector in the preparation of a medicament for treating VSIG4-mediated related diseases.

[0013] Based on the above technical solutions, preferably, the VSIG4-mediated related diseases include inflammatory diseases and tumors.

[0014] Based on the above technical solutions, preferably, the tumors include lung cancer, glioma, multiple myeloma, and colon cancer.

[0015] The antibody targeting VSIG4 protein and its application of the present invention have the following beneficial effects compared with the prior art: The antibody of the present invention has higher specificity and high affinity for tumor cells, and is expected to become a CAR-T drug candidate for lung cancer, glioma, multiple myeloma, etc., and can be used as an indicator for cancer diagnosis and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figure 1 It is the flow cytometry identification result of the monoclonal yeast obtained in the embodiment of the present invention; Figure 2The identification result of the antibody SDS-PAGE gel diagram obtained in the embodiment of the present invention. M is M5 Prestained Protein Ladder (10-180 kDa), and lane 1 is cell-R3-4C9; Figure 3 The EC50 of the antibody obtained in the embodiment of the present invention binding to tumor cells; Figure 4 The specific IP identification of the antibody obtained in the embodiment of the present invention with lymphoma cell lines. M is M5 Prestained Protein Ladder (10-180 kDa), lane 1 is the HCT 116 cell lysate, lane 2 is the VSIG4 protein, and lane 3 is the magnetic bead + Cell-R3-4C9 + VSIG4 cell lysate; Figure 5 The comparison result of the binding activity of the flow cytometry-detected antibody obtained in the embodiment of the present invention with the known antibody. Specific implementation manners

[0018] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] The method for preparing the antibody of the present invention includes the following steps: A: Immunization with the immunogen for screening the antibody; B: Construction of the yeast display library after immunization; C: Screening of the yeast display library; D: Expression of the recombinant antibody; E: IP identification of the recombinant antibody with tumor cell lines; F: In vitro verification of the recombinant antibody.

[0020] The following will be described in detail through examples.

[0021] Example 1 Rabbit immunization The amino acid sequence (20-283) of the extracellular domain of human VSIG4 was obtained from the global public database UniProt (Q9Y279).

[0022] Amino acid sequence of the extracellular domain of human VSIG4: RPILEVPESVTGPWKGDVNLPCTYDPLQ GYTQVLVKWLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVSHKVPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQVVRDKITELRVQKLSVSKPTVTTGSGYGFTVPQGMRISLQCQARGSPPISYIWYKQQTNNQEPIKVATLSTLLFKPAVIADSGSYFCTAKGQVGSEQHSDIVKFVVKDSSKLLKTKTEAPTTMTYPLKATSTVKQSWDWTTDMDGYLGETSAGPGKSLP。

[0023] Using the method of molecular cloning, a pcDNA3.4 expression vector containing the gene sequence of the extracellular domain of human VSIG4 (purchased from Thermo Fisher Scientific) was constructed. The extracellular domain sequence of VSIG4 was synthesized by Tsingke Biotechnology and then constructed into the pcDNA3.4 expression vector.

[0024] Using the method of electroporation, the vector encoding human VSIG4 was transfected into mammalian cells CHO for expression for 14 days. After the expression was completed, the cell supernatant was harvested and purified using a cation column.

[0025] Immunization protocol: After the extracellular domain protein of human VSIG4 was fully emulsified with an immunoadjuvant, Freund's complete adjuvant (company: sigma, product number: F5881) was used for the primary immunization, and Freund's incomplete adjuvant (company: sigma, product number: F5506) was used for the next two immunizations. According to the volume ratio of antigen to adjuvant of 1:1, rabbits were immunized by subcutaneous injection in the back, and a total of 4 immunizations were carried out, with an interval of 2-3 weeks each time.

[0026] Example 2 Construction and screening of ScFv antibody yeast display library S1: After immunizing 4 times, draw blood from the marginal ear vein of rabbits and isolate lymphocytes. Use the RNA extraction kit RC112 from Novoprotein to extract total RNA. Reverse transcribe the RNA into cDNA using the Novoprotein RNA reverse transcription kit (HiScript II Q Select RT SuperMix for qPCR R232). Next, using the cDNA as a template, amplify the variable region sequences of the heavy chain and light chain respectively with rabbit - specific primers (synthesized by Tsingke Biological Company). Add a flexible linker to the amplified variable region sequences of the heavy chain and light chain by homologous recombination and form a recombinant single - strand, which is cloned into the yeast vector Pct302 (modified by our own laboratory). The recombinant product is electrotransformed into yeast competent cells, and the yeast collected after amplification culture is the initial yeast library displaying ScFv - His antibody.

[0027] Among them, the rabbit - specific primers for amplifying the heavy chain are as follows: b - fabvh1: ccgccggaattccaggagcagctgaaggag; rb - fabvh2: ccgccggaattccaggagcagctgrtggag; rb - fabvh3: ccgccggaattccaggagcagctggaggagtcc; rb - ch1 - r: cgccgcggatcccgtgggcttgctgcatgtcg.

[0028] The rabbit - specific primers for amplifying the light chain are as follows: rb - fabvk1: cggcggggtaccgaccctatgctgacccag; rb - fabvk2: cggcggggtaccgatgtcgtgatgacccag; rb - fabvk3: cggcggggtaccgcagccgtgctgacccag; rb - cl - r: gccggctctagattarcagtcacccctrttgaagc.

[0029] S2: Use the tumor cell line HCT 116 to perform 3 rounds of panning on the yeast library. The specific method is as follows: Take 50 μL of magnetic beads into a 1.5 mL sterile EP tube, wash once with 1 mL of sterile 1×PBS (PBS will be used hereinafter), add 5 - 10 μg of the target antigen (VSIG4 extracellular domain protein), and incubate with rotation at 4°C overnight. Pour the induced yeast into a 50 mL sterile centrifuge tube, centrifuge at 3000 g for 3 min to collect the yeast, and discard the supernatant. Wash 3 times with 50 mL of PBS, resuspend the yeast with 50 mL of 0.1% BSA-PBS, and rotate at room temperature for 1 h for blocking. Wash the blocked yeast 2 times with 50 mL of PBS, and resuspend the yeast with 50 mL of 0.1% BSA-PBS. Meanwhile, place the magnetic beads incubated overnight on a magnetic stand, wash 2 times with 1 mL of PBS, resuspend the magnetic beads with 1 mL of PBS and add them to the yeast, and incubate with rotation at room temperature for 1 - 2 h. After incubation, place on a magnetic stand, adsorb for 30 min, discard the supernatant, wash 5 times with 10 mL of PBS, and finally resuspend the yeast bound with the antigen with 1 mL of PBS. Dilute the panned yeast to 10 -2 、10 -3 、10 -4 , spread on SD-Trp solid plates to determine the library size. Spread the remaining yeast on a 15 cm SD-Trp solid plate, collect it, and perform the next round of panning.

[0030] The results showed that the library size in the first round of screening: 2.3×10 5 , the library size in the second round of screening: 6.2×10 6 , and the library size in the third round: 8.8×10 6 .

[0031] S3: Flow cytometry screening of monoclonal yeast for positive clones. After three consecutive rounds of panning, 96 individual yeast colonies were picked into a 96-well round-bottom culture dish containing 100 μL of SD-Trp medium. Incubate continuously at 30 °C and 200 rpm for 12 h, then centrifuge the culture dish at 2000 g for 3 min, discard the supernatant, and supplement the culture dish with fresh induction medium SG-Trp. Incubate continuously at 20 °C and 200 rpm for 36 h. Take 25 μL of the culture to a new 96-well plate (U-bottom), centrifuge at 2000 g for 3 min, discard the supernatant, block the yeast pellet with 200 μL of BSA for 30 min, wash twice with PBSA, add the prepared His-tag fluorescently labeled secondary antibody (diluted with BSA), and incubate with shaking at 4 °C for 30 min. Wash twice with PBSA. Meanwhile, prepare the required tumor lymphocytes HCT116, wash the cells twice with PBSA, resuspend the cells with a fluorescent dye diluted with BSA (HY-D0938, diluted 1:8000), incubate at 4 °C for 30 min, wash twice with PBS, resuspend the cells with PBSA, add 100 μL / well to the yeast culture dish labeled with the His-tag fluorescently labeled secondary antibody and mix well, and incubate with shaking at 4 °C for 30 min. Then perform flow cytometry analysis on the machine.

[0032] In this example, after three rounds of panning and screening of the constructed ScFv yeast display library, monoclonal yeast flow cytometry screening was performed to obtain a candidate antibody with strong signal, named cell-R3-4C9.

[0033] Figure 1 It is a positive figure determined for monoclonal yeast flow cytometry screening, showing the binding of yeast to HCT 116 cells. The yeast corresponding to this positive was sent to Qingke for sequencing to obtain the complete ScFv sequence, and the amino acid sequence is shown in SEQ ID No.8.

[0034] The amino acid sequence of antibody cell-R3-4C9 is: QSVKESGGRLVTPGASLTLTCKASGFS FSSGYDMCWVRQAPGKGLEWIACIYAGSSENTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARQYISSRGYWLWGPGTLVTVSS-linker(GGGGSGGGGSEAAAKGGGGSGGGGS)-EFQPVLTQTPSPVSAAVGGTVSISCQSSQSLYYNKYLSWYQQKPGQPPKLLIYWASTLASGVPSRFKGSGSGTEFTLTISGVQCDDAATYYCAGDGSTTESDIVFGGGTEVVVKGDP. (SEQ ID No.8) Heavy chain variable region: CDR-H1-IMGT: SGYDMC; (SEQ ID No.1) CDR-H2-IMGT: CIYAGSSENTYYASWAKG; (SEQ ID No.2) CDR-H3-IMGT: QYISSRGYWL. (SEQ ID No.3) Light chain variable region: CDR-L1-IMGT: QSSQSLYYNKYLS; (SEQ ID No.4) CDR-L2-IMGT: WASTLAS; (SEQ ID No.5) CDR-L3-IMGT: AGDGSTTESDIV. (SEQ ID No.6) Linker: (GGGGS)2-(EAAAK)1-(GGGGS)2. (SEQ ID No.7) Example 3 Recombinant antibody expression The yeast clones that were positive by flow cytometry verification were sent to Qingke for sequencing. After sequence analysis, the ScFv sequence was fused with a His tag and cloned into the mammalian expression vector pTT5. The constructed vector was transfected into HEK293F (ATCC, CBP60437) using polyethyleneimine (PEI). The transfected mammalian cells were cultured in suspension for 5 days. Then, the supernatant of the cell culture was collected by centrifugation at 2000 rpm for 10 min. The antibody was purified using an affinity chromatography column and analyzed by SDS-PAGE electrophoresis.

[0035] After sequencing the candidate antibody, the antibody sequence was fused with a His tag to construct a eukaryotic expression vector. The antibody was purified after transfection of HEK293F cells, and the results are as Figure 2 shown.

[0036] Example 4 Recombinant antibody identification S1, Identification of recombinant antibody and tumor cell line IP Cell lysate preparation: Take 5×10 6 cells (HCT 116), wash 3 times with PBS, add cell lysate (E-IR-IP004) to resuspend the cells, and lyse for 2 min. Centrifuge at 18000g at 4°C for 15 min to remove the precipitate, and the supernatant is the cell lysate.

[0037] IP experiment: Take 50 μL of magnetic beads conjugated with His-tag antibody into a 1.5 mL centrifuge tube. Place the centrifuge tube on a magnetic stand for 2 min, discard the supernatant, wash 3 times with 1 mL of PBS, add 10 μg of recombinant antibody, and incubate with rotation overnight at 4°C. Next, place the centrifuge tube on the magnetic stand for 2 min, discard the supernatant, wash 3 times with 1 mL of PBS, add 200 μL of cell lysate, and incubate with rotation for 1 h at 4°C. Next, place the centrifuge tube on the magnetic stand for 2 min, discard the supernatant, wash 5 times with 1 mL of PBS, resuspend the precipitate with 20 μL of PBS, add 20 μL of reducing buffer and mix well, incubate at 100°C for 10 min, centrifuge at 8000 g for 1 min, and then take 5 μL of the supernatant for SDS-PAGE gel electrophoresis. Analyze the protein bands by WB.

[0038] S2. Detection of the EC50 of the binding of the recombinant antibody to tumor cells by flow cytometry Prepare PBS solutions with a concentration gradient from 0.1 nM to 10 μM for human VSIG4 extracellular domain protein. Add 0.5 μM of the recombinant antibody to each gradient to make the total volume 100 μL. Incubate at room temperature for 30 min, then bind to 5 × 10^5 tumor cells HCT 116, incubate at 4°C for 30 min, add the prepared anti-His fluorescently labeled antibody (diluted with BSA, 1:400), and incubate at 4°C for 30 min. Centrifuge at 200 g for 5 min, wash 2 times with PBSA, and detect by machine.

[0039] Flow cytometry was used to detect the binding of the recombinant antibody to the tumor cell line HCT 116, as Figure 3 shown. The antibody can specifically bind to tumor cells, and its EC50 is 35 nM.

[0040] As shown in the IP experiment Figure 4 shown, a separate band appeared after the antibody bound to the cell lysate (HCT 116), indicating that the antibody specifically binds to the VSIG4 extracellular domain protein on the cell surface.

[0041] S4. Comparison of the recombinant antibody with Anti-VSIG4 mAb 12A12 antibody Take the recombinant antibody and the control antibody Anti-VSIG4 mAb 12A12 (Sazinsky S, Zafari M, Klebanov B, Ritter J, Nguyen PA, Phennicie RT, Wahle J, Kauffman KJ, Razlog M, Manfra D, Feldman I, Novobrantseva T. Antibodies Targeting Human or Mouse VSIG4 Repolarize Tumor-Associated Macrophages Providing the Potential of Potent and Specific Clinical Anti-Tumor Response Induced across Multiple Cancer Types. Int J Mol Sci. 2024 Jun 3;25(11):6160. doi: 10.3390 / ijms25116160. PMID: 38892347; PMCID: PMC11172757.) 0.2 μg and bind them to 5×10 5 tumor cells HCT 116 respectively, incubate at 4°C for 30 min, add the prepared anti-His fluorescently labeled antibody (diluted with BSA, 1:400), and incubate at 4°C for 30 min. Centrifuge at 200 g for 5 min, wash twice with PBSA, and detect on the machine.

[0042] Figure 5For the flow cytometry comparison of the recombinant antibody and the known antibody reported in the literature against HCT 116 tumor cells (refer to Xu X, Ding Y, Dong Y, Yuan H, Xia P, Qu C, Ma J, Wang H, Zhang X, Zhao L, LiZ, Liang Z, Wang J. Nanobody-Engineered Biohybrid Bacteria Targeting Gastrointestinal Cancers Induce Robust STING-Mediated Anti-Tumor Immunity. Adv Sci (Weinh). 2024 Aug;11(31):e2401905. doi: 10.1002 / advs.202401905. Epub 2024 Jun 18. PMID: 38888519; PMCID: PMC11336900.), the results show that this antibody has better binding activity and is expected to become a candidate antibody for a new generation of tumor treatment, detection, etc.

[0043] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antibody targeting the VSIG4 protein, characterized in that: It includes heavy chain variable region CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 are shown as SEQ ID No.1-3; The antibody further includes light chain variable region CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 are shown as SEQ ID No.4-6.

2. The antibody targeting VSIG4 protein according to claim 1, characterized in that: The antibody further includes a Linker, and the amino acid sequence of the Linker is shown as SEQ ID No.

7.

3. The antibody targeting VSIG4 protein according to claim 1, wherein: The amino acid sequence of the antibody is shown as SEQ ID No.

8.

4. An isolated nucleic acid, characterized in that: The isolated nucleic acid encodes the antibody according to any one of claims 1-3.

5. An expression vector, characterized in that: The expression vector contains the isolated nucleic acid according to claim 4.

6. Use of the antibody according to any one of claims 1-3, or the isolated nucleic acid according to claim 4, or the expression vector according to claim 5 in the preparation of a medicament for treating VSIG4-mediated related diseases.

7. The application according to claim 6, characterized in that: The VSIG4-mediated related diseases include inflammatory diseases and tumors.

8. The application according to claim 7, characterized in that: The tumors include lung cancer, glioma, multiple myeloma and colon cancer.

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