An antibody targeting VSIG4 protein and its application

Abstract

The present invention relates to the field of antibody technology and discloses an antibody targeting the VSIG4 protein and its application. The antibody comprises heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID Nos. 1 to 3. The antibody of the present invention has enhanced specificity and high affinity for tumor cells, and is expected to become a candidate for CAR-T drugs for lung cancer, glioma, multiple myeloma, and other diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an antibody targeting VSIG4 protein and its application. Background Art

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

[0003] Traditional hybridoma technology is inefficient and time-consuming to screen for anti-VSIG4 antibodies. Antibodies obtained using phage display technology often lack affinity due to differences in protein modification between prokaryotic expression systems and mammalian systems. Yeast surface display systems combine the capabilities of eukaryotic protein post-translational modification with high-throughput screening, preserving the native conformation of antibodies and improving the human compatibility of screened molecules. Furthermore, peptide-based screening technologies (such as phage-displayed peptide libraries or chemically synthesized peptide libraries) are also widely used to develop candidate molecules targeting VSIG4. These peptides can mimic VSIG4's natural ligand-binding epitopes, blocking its immunosuppressive function or serving as lead compounds for antibody development.

[0004] VSIG4 inhibits the cytotoxicity of CD8 T cells after binding to its ligand and is another potential target for tumor immunosuppression in the B7 family. VSIG4-specific antibodies and peptides have demonstrated the ability to repolarize macrophages and induce immune activation in both in vitro and in vivo models. Tumor growth inhibition has been observed in mouse models when VSIG4 antibodies or peptides are used alone or in combination with anti-PD-1. Currently, there are several clinical drugs targeting VSIG4 under development, mainly for the treatment of solid tumors. Therefore, VSIG4 will become a new target for the treatment of inflammatory diseases or various tumors, and the study of antibodies and peptides with high affinity activity for VSIG4 is of great significance. Summary of the Invention

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

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

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

[0008] On the basis of the above technical solution, preferably, the antibody further comprises a linker, and the amino acid sequence of the linker is shown in SEQ ID No.7.

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

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

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

[0012] In a fourth aspect, the present invention also provides the use of antibodies, isolated nucleic acids or expression vectors in the preparation of drugs 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:

[0016] The antibodies of the present invention have higher specificity and high affinity for tumor cells, and are expected to become CAR-T drug candidates for lung cancer, glioma, multiple myeloma, etc., and can be used as indicators for the diagnosis and prognosis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The flow cytometry results of the monoclonal yeast obtained in the examples of the present invention are as follows;

[0019] Figure 2 The SDS-PAGE gel image identification results of the antibodies obtained in the examples of the present invention, M is M5 Prestained Protein Ladder (10-180 kDa), lane 1 is cell-R3-4C9;

[0020] Figure 3 EC50 of the antibody obtained in the examples of the present invention binding to tumor cells;

[0021] Figure 4 This is the specific IP identification of the antibodies obtained in the examples of the present invention and lymphoma cell lines. M is M5 Prestained Protein Ladder (10-180 kDa), lane 1 is HCT 116 cell lysate, lane 2 is VSIG4 protein, and lane 3 is magnetic beads + Cell-R3-4C9 + VSIG4 cell lysate;

[0022] Figure 5 The results are a comparison of the binding activity of the flow cytometry antibody obtained in the examples of the present invention with that of known antibodies. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] The antibody preparation method of the present invention comprises the following steps: A: immunization with an immunogen for screening antibodies; B: construction of a yeast display library after immunization; C: screening of the yeast display library; D: expression of recombinant antibodies; E: IP identification of recombinant antibodies and tumor cell lines; and F: in vitro verification of the recombinant antibodies.

[0025] The following describes the invention through examples.

[0026] Example 1 Rabbit Immunization

[0027] The amino acid sequence (20-283) of the human VSIG4 extracellular domain was obtained through the global public database UniProt (Q9Y279).

[0028] The amino acid sequence of the human VSIG4 extracellular domain is: RPILEVPESVTGPWKGDVNLPCTYDPLQ GYTQVLVKWLVQRGSDPVTIFLRDSSGDHIQQAKYQGRLHVSHKVPGDVSLQLSTLEMDDRSHYTCEVTWQTPDGNQVVRDKITELRVQKLSVSKPTVTTGSGYGFTVPQGMRISLQCQARGSPPISYIWYKQQTNNQEPIKVATLSTLLFKPAVIADSGSYFCTAKGQVGSEQHSDIVKFVVKDSSKLLKTKTEAPTTMTYPLKATSTVKQSWDWTTDMDGYLGETSAGPGKSLP.

[0029] The pcDNA3.4 expression vector containing the human VSIG4 extracellular domain gene sequence was constructed using molecular cloning methods (purchased from Thermo Fisher Scientific). The VSIG4 extracellular domain sequence was synthesized by Qingke Biotechnology and then constructed into the pcDNA3.4 expression vector.

[0030] The vector encoding human VSIG4 was transfected into mammalian CHO cells using electroporation and expressed for 14 days. After expression was complete, the cell supernatant was harvested and purified using a cationic column.

[0031] Immunization protocol: After fully emulsifying the expressed and purified human VSIG4 extracellular domain protein with the immune adjuvant, use Freund's complete adjuvant (Company: Sigma, Catalog No.: F5881) for the first immunization, followed by Freund's incomplete adjuvant (Company: Sigma, Catalog No.: F5506) for the subsequent two immunizations. Mix the antigen and adjuvant in a 1:1 volume ratio and inject subcutaneously into the back of the rabbit for a total of four immunizations, with each immunization occurring 2-3 weeks apart.

[0032] Example 2 Construction and screening of ScFv antibody yeast display library

[0033] S1: After four immunizations, rabbits were immunized with auricular venous blood and lymphocytes isolated. Total RNA was extracted using the Novozymes RNA Extraction Kit RC112. RNA was reverse-transcribed into cDNA using the Novozymes RNA Reverse Transcription Kit (HiScript II Q Select RT SuperMix for qPCR R232). Next, the heavy and light chain variable region sequences were amplified using rabbit-specific primers (synthesized by Qingke Biotechnology) using the cDNA as a template. Flexible linkers were added to the amplified heavy and light chain variable region sequences by homologous recombination, resulting in recombinant single-stranded clones. The recombinant products were electroporated into competent yeast cells. After amplification and culture, the yeast cells harvested were used to generate the initial ScFv-His antibody displaying yeast library.

[0034] Among them, the rabbit-specific primers for amplifying the heavy chain are as follows:

[0035] b-fabvh1:ccgccggaattccaggagcagctgaaggag;

[0036] rb-fabvh2:ccgccggaattccaggagcagctgrtggag;

[0037] rb-fabvh3:ccgccggaattccaggagcagctggaggagtcc;

[0038] rb-ch1-r: cgccgcggatcccgtgggcttgctgcatgtcg.

[0039] Rabbit-specific primers for light chain amplification are as follows:

[0040] rb-fabvk1: cggcggggtaccgaccctatgctgacccag;

[0041] rb-fabvk2: cggcggggtaccgatgtcgtgatgacccag;

[0042] rb-fabvk3:cggcggggtaccgcagccgtgctgacccag;

[0043] rb-cl-r: gccggctctagattarcagtcacccctrttgaagc.

[0044] S2: The yeast library was panned using the tumor cell line HCT 116 for three rounds. The specific method is as follows:

[0045] Place 50 μL of magnetic beads in a 1.5 mL sterile EP tube, wash once with 1 mL of sterile 1× PBS (PBS will be used hereafter), add 5–10 μg of target antigen (VSIG4 extracellular domain protein), and incubate with rotation overnight at 4°C. Pour the induced yeast into a 50 mL sterile centrifuge tube, centrifuge at 3000 g for 3 minutes to collect the yeast, and discard the supernatant. Wash three times with 50 mL of PBS, resuspend the yeast in 50 mL of 0.1% BSA-PBS, rotate at room temperature, and block for 1 hour. Wash the blocked yeast twice with 50 mL of PBS, and resuspend the yeast in 50 mL of 0.1% BSA-PBS. Meanwhile, place the magnetic beads from the overnight incubation on a magnetic rack, wash twice with 1 mL of PBS, resuspend the beads in 1 mL of PBS, and add them to the yeast. Incubate at room temperature with rotation for 1–2 hours. After incubation, place on a magnetic stand and adsorb for 30 minutes, discard the supernatant, wash five times with 10 mL PBS, and finally resuspend the antigen-bound yeast with 1 mL PBS. -2 , 10 -3 , 10 -4 , spread on SD-Trp solid plates and determine the reservoir capacity. The remaining yeast was spread on 15 cm SD-Trp solid plates and collected for the next round of panning.

[0046] The results showed that the first round of screening library capacity was 2.3×10 5 , the second round of screening library capacity: 6.2×10 6 , the third round storage capacity: 8.8×10 6 .

[0047] S3: Monoclonal yeast flow cytometry screening of positive clones. After three rounds of panning, 96 individual yeast colonies were picked into a 96-well round-bottom culture dish containing 100 μL of SD-Trp medium. The dish was incubated at 30°C and 200 rpm for 12 hours. The dish was then centrifuged at 2000 g for 3 minutes, the supernatant discarded, and fresh induction medium (SG-Trp) was added to the dish. The dish was incubated at 20°C and 200 rpm for 36 hours. 25 μL of the culture was transferred to a new 96-well U-bottom plate and centrifuged at 2000 g for 3 minutes. The supernatant discarded, and the yeast pellet was blocked with 200 μL of BSA for 30 minutes. The plate was washed twice with PBSA. The prepared His-tag fluorescent-conjugated secondary antibody (diluted in BSA) was added and incubated at 4°C with shaking for 30 minutes. The plate was washed twice with PBSA. Meanwhile, prepare the required HCT116 tumor lymphocytes. Wash the cells twice with PBSA and resuspend them in a fluorescent dye (HY-D0938, 1:8000 dilution) diluted in BSA. Incubate at 4°C for 30 min. Wash twice with PBS and resuspend the cells in PBSA. Add 100 μL / well of the cells to a yeast culture dish labeled with a His-tag fluorescent secondary antibody, mix thoroughly, and incubate at 4°C with shaking for 30 min. Flow cytometry analysis is then performed.

[0048] In this example, the constructed ScFv yeast display library was subjected to three rounds of panning and then to monoclonal yeast flow cytometry screening to obtain a candidate antibody with a strong signal, which was named cell-R3-4C9.

[0049] Figure 1 This is a positive result from flow cytometry screening of a single yeast clone, indicating binding of the yeast to HCT 116 cells. The positive yeast was sent to Qingke for sequencing, and the complete ScFv sequence was obtained. The amino acid sequence is shown in SEQ ID No. 8.

[0050] The amino acid sequence of antibody cell-R3-4C9 is: QSVKESGGRLVTPGASLTLTCKASGFS FSSGYDMCWVRQAPGKGLEWIACIYAGSSENTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARQYISSRGYWLWGPGTLVTVSS-linker(GGGGSGGGGSEAAAKGGGGSGG GGS)-EFQPVLTQTPSPVSAAVGGTVSISCQSSQSLYYNKYLSWYQQKPGQPPKLLIYWASTLASGVPSRFKGSGSGTEFTLTISGVQCDDAATYYCAGDGSTTESDIVFGGGTEVVVKGDP. (SEQ ID No.8)

[0051] Heavy chain variable region:

[0052] CDR-H1-IMGT: SGYDMC; (SEQ ID No. 1)

[0053] CDR-H2-IMGT: CIYAGSSENTYYASWAKG; (SEQ ID No. 2)

[0054] CDR-H3-IMGT: QYISSRGYWL. (SEQ ID No.3)

[0055] Light chain variable region:

[0056] CDR-L1-IMGT: QSSQSLYYNKYLS; (SEQ ID No. 4)

[0057] CDR-L2-IMGT: WASTLAS; (SEQ ID No. 5)

[0058] CDR-L3-IMGT: AGDGSTTESDIV. (SEQ ID No.6)

[0059] Linker: (GGGGS)2-(EAAAK)1-(GGGGS)2. (SEQ ID No.7)

[0060] Example 3 Recombinant Antibody Expression

[0061] Yeast clones that were positive for flow cytometry 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 cells (ATCC, CBP60437) using polyethyleneimine (PEI). The transfected mammalian cells were cultured in suspension for 5 days. The cell culture supernatant was then collected by centrifugation at 2000 rpm for 10 minutes. The antibody was purified using an affinity chromatography column and analyzed by SDS-PAGE electrophoresis.

[0062] After sequencing the candidate antibody, the antibody sequence was fused with the His tag to construct a eukaryotic expression vector, and the antibody was purified after transfection into HEK293F cells. Figure 2 shown.

[0063] Example 4 Identification of recombinant antibodies

[0064] S1, IP identification of recombinant antibodies and tumor cell lines

[0065] Preparation of cell lysate: Take 5×10 6Wash HCT 116 cells three times with PBS, resuspend in cell lysis buffer (E-IR-IP004), and lyse for 2 minutes. Centrifuge at 18,000 g for 15 minutes at 4°C to remove the precipitate. The supernatant is the cell lysate.

[0066] IP experiment: Transfer 50 μL of His-tag antibody-coupled magnetic beads to a 1.5 mL centrifuge tube. Place the tube on a magnetic rack for 2 minutes, discard the supernatant, wash three times with 1 mL of PBS, add 10 μg of recombinant antibody, and incubate with rotation at 4°C overnight. Next, place the tube on a magnetic rack for 2 minutes, discard the supernatant, wash three times with 1 mL of PBS, add 200 μL of cell lysis buffer, and incubate at 4°C with rotation for 1 hour. Next, place the tube on a magnetic rack for 2 minutes, discard the supernatant, wash five times with 1 mL of PBS, resuspend the pellet in 20 μL of PBS, add 20 μL of reducing buffer, mix thoroughly, incubate at 100°C for 10 minutes, centrifuge at 8000 g for 1 minute, and load 5 μL of the sample onto an SDS-PAGE gel. Protein bands were analyzed by Western blotting.

[0067] S2, flow cytometry detection of EC50 of recombinant antibody binding to tumor cells

[0068] A concentration gradient of human VSIG4 extracellular domain protein in PBS was prepared from 0.1 nM to 10 μM. Recombinant antibody (0.5 μM) was added to each gradient to a total volume of 100 μL. After incubation at room temperature for 30 minutes, the cells were bound to 5 × 10^5 HCT 116 tumor cells and incubated at 4°C for 30 minutes. The cells were then added with the prepared fluorescently labeled anti-His antibody (diluted 1:400 in BSA) and incubated at 4°C for 30 minutes. The cells were centrifuged at 200 g for 5 minutes, washed twice with PBSA, and analyzed by flow cytometer.

[0069] The binding of recombinant antibodies to the tumor cell line HCT 116 was detected by flow cytometry. Figure 3 As shown, the antibody can specifically bind to tumor cells with an EC50 of 35 nM.

[0070] IP experiments such as Figure 4 As shown, a single band appeared after the antibody bound to the cell lysate (HCT 116), indicating that the antibody specifically bound to the cell surface VSIG4 extracellular domain protein.

[0071] S4, Comparison of recombinant antibody and Anti-VSIG4 mAb 12A12

[0072] The recombinant antibody and control antibody Anti-VSIG4 mAb 12A12 (Sazinsky S, Zafari M,Klebanov B, Ritter J, Nguyen PA, Phennicie RT, Wahle J, Kauffman KJ, RazlogM, 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 were taken and mixed with 5×10 5 HCT 116 tumor cells were bound and incubated at 4°C for 30 minutes. Anti-His fluorescently labeled antibody (1:400 diluted in BSA) was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 200 g for 5 minutes, washed twice with PBSA, and analyzed by flow cytometer.

[0073] Figure 5The flow cytometry analysis of HCT 116 tumor cells was compared between the recombinant antibody and known antibodies reported in the literature (see 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. Epub2024 Jun 18. PMID: 38888519; PMCID: PMC11336900.). The results showed that the recombinant antibody had better binding activity and is expected to become a new generation of candidate antibodies for tumor treatment and detection.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

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

2. The antibody targeting VSIG4 protein according to claim 1, wherein: The antibody further comprises a linker, and the amino acid sequence of the linker is shown in SEQ ID No.

7.

3. The antibody targeting VSIG4 protein according to claim 1, wherein: The amino acid sequence of the antibody is shown in 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 to 3.

5. An expression vector, characterized in that: The expression vector comprises the isolated nucleic acid of claim 4.

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