Use of bispecific single-chain antibody fap1v2 targeting pd-l1 and vegfr2

Abstract

The application provides application of a bispecific single-chain antibody FAP1V2 in preparation of a medicine, the medicine can activate an immune system, enhance an anti-tumor immune response, the bispecific single-chain antibody FAP1V2 includes a nanobody for recognizing a target point PD-L1 and a nanobody for recognizing a target point VEGFR2, and the nanobody monomers are connected by a linker.The application activates T cell function and inhibits cancer cell metastasis by simultaneously blocking PD-L1 and inhibiting VEGFR2 signal pathways, activates an immune system, and significantly promotes memory inhibition of the immune system to tumors.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more particularly to the application of FAP1V2, a bispecific single-chain antibody targeting PD-L1 and VEGFR2. Background Technology

[0002] Cancer treatment failure is typically attributed to two main obstacles: ineffective drug therapy and insufficient immune response. Cancer cells often evade drug-mediated cytotoxicity by developing drug resistance mechanisms, while immune cells, especially T cells, are often unable to effectively eliminate tumor cells. This failure of immune clearance is partly attributed to T cell exhaustion, a state in which T cells are impaired in function and proliferate in the tumor microenvironment. Furthermore, the number of tumor-infiltrating cytotoxic immune cells, particularly T cells, and the anti-tumor immune response are often significantly reduced, limiting their ability to effectively target and kill tumor cells. Therapeutic strategies that can effectively activate the immune system to fight cancer are a promising research direction. One promising strategy is to target immune checkpoints, molecules expressed on immune cells that act as "brakes" on the immune response. By using monoclonal antibodies to block these checkpoints, the immune system can be freed to attack tumor cells more effectively. This strategy has achieved significant success in a variety of cancers, particularly melanoma, lung cancer, and bladder cancer. Examples of checkpoint inhibitors include anti-CTLA-4 antibodies, such as ipilimumab and tremelimumab, and anti-PD-1 antibodies, such as nivolumab and durvalumab. By increasing the number and activity of tumor-infiltrating T cells, combined with vaccines and immune checkpoint inhibitors, antitumor efficacy can be further enhanced. Although there is evidence that therapies using anti-PD-1 antibodies (such as sintilimab or cadonilimab) or bispecific antibodies targeting PD-1 and cytotoxic T lymphocyte antigen-4 can promote T cell infiltration and activity in the tumor microenvironment [1], these therapies may lead to a decrease in neutrophil, platelet, and erythrocyte levels, resulting in anemia or leukopenia [2,3], possibly due to blocking targets on these immune cells that have potentially important functions. However, currently used clinical antibodies contain a large number of heterologous regions, such as crystal fragments (Fc), leading to side effects and rapid clearance from the bloodstream.

[0003] Currently, the roles of nanobody-mediated T cell activation and direct T cell recognition of tumor antigens remain unclear. For example, the lack of a light chain variable region (V... L Whether single-chain nanobodies in the Fc and Tc regions can support the binding of endogenous T cell receptors (TCRs) to tumor antigens and induce T cell activation, particularly after transient expression on tumor cells, remains to be demonstrated. Researching these key aspects will be crucial for optimizing current treatments and developing new, effective, and durable cancer therapeutic strategies.

[0004] PD-1 is an inducible protein expressed on activated T cells and B cells, and can also be expressed on NK cells, monocytes, and myeloid-derived dendritic cells after antigen stimulation. Prior to activation, T cells express almost no PD-1, which gradually increases after antigen stimulation. PD-L1 is a functional ligand of PD-1 and may act as a molecular “barrier” protecting PD-L1+ tumor cells from CD8+ T cell-mediated tumor cell clearance, which helps tumor cells evade the immune system. PD-L1 binds to PD-1 on tumor-infiltrating lymphocytes (TILs), transmitting immunosuppressive signals and inhibiting the activation and function of antigen-specific cytotoxic T cells. This interaction thus weakens the immune response, allowing tumors to evade recognition. Recent studies have found that the interaction between PD-1 and its ligands (PD-L1 and PD-L2) leads to attenuation of T cell activation. PD-1 binding to its ligands induces T cell differentiation into exhausted T cells, which exhibit impaired proliferation, cytokine production, and cytotoxicity.

[0005] Vascular endothelial growth factor (VEGF) and its homologous receptor (VEGFR) play crucial roles in tumor angiogenesis (or vascularization). Upregulation of VEGF in tumor blood vessels is a key factor in malignant tumor growth, promoting tumorigenesis, development, and metastasis. VEGFR2 possesses potent tyrosine kinase activity, binding to VEGF and activating multiple downstream signaling pathways, including protein kinase B (AKT), ERK1 / 2, and MAPK. Blocking VEGFR2 signaling has been reported to inhibit tumor cell migration and suppress angiogenesis. Currently, there is no information on how intracellular antibodies targeting both PD-L1 and VEGFR2 affect high expression of PD-1 (PD-1). hi Reports on the activation of immune cells and T cells.

[0006] References

[0007] 1. Petitprez F, Meylan M, de Reyniès A, Sautès-Fridman C, Fridman WH. Thetumor microenvironment in the response to immune checkpoint blockadetherapies. Front Immunol. 2020; 11:784.

[0008] 2. Gao XY, Ji K, Jia YN, Shan F, Chen Y, Xu N, et al. Cadonilimab withchemotherapy in HER2-negative gastric or gastroesophageal junctionadenocarcinoma: the phase 1b / 2COMPASSION-04trial. Nat Med. 2024; 30:1943-51.

[0009] 3. Zeng TM, Yang G, Lou C, Wei W, Tao CJ, Chen XY, et al. Clinical and biomarker analyzes of sintilimab plus gemcitabine and cisplatin as first-linetreatment for patients with advanced biliary tract cancer. Nat Commun. 2023; 14:1340. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides the application of FAP1V2, a bispecific single-chain antibody targeting PD-L1 and VEGFR2.

[0011] In one aspect, this invention provides the application of the bispecific single-chain antibody FAP1V2 in the preparation of a drug, wherein the drug comprises at least one of the following functions:

[0012] (1) It can specifically bind to PD-L1 and VEGFR2;

[0013] (2) Inhibits tumor cell migration;

[0014] (3) Inhibits tumor metastasis in the body;

[0015] (4) Inhibits tumor growth;

[0016] (5) Eliminate or kill tumor cells;

[0017] (6) Enhance anti-tumor immune response;

[0018] (7) Promotes the proliferation and activation of T cells;

[0019] (8) Recruit TCRβ hi T cells or promoting TCRβ hi The generation or activation of T cells;

[0020] (9) Inhibit or reduce immune cells with high PD-1 expression;

[0021] (10) Add CD25 hi The number of T cells may increase CD25 expression levels;

[0022] The bispecific single-chain antibody FAP1V2 includes a nanobody that recognizes the target PD-L1 and a nanobody that recognizes the target VEGFR2, with the nanobody monomers linked together by a linker.

[0023] In one embodiment of the present invention, the amino acid sequence of the nanobody that recognizes the target PD-L1 is as shown in SEQ ID NO:1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:1.

[0024] In one embodiment of the present invention, the amino acid sequence of the nanobody that recognizes the target VEGFR2 is as shown in SEQ ID NO:2 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:2.

[0025] In one embodiment of the present invention, the connector is a flexible connector. In a specific embodiment of the present invention, the connector is (G4S)n, where n = 1-4, such as GGSGG or (G4S)3.

[0026] In one embodiment of the present invention, the amino acid sequence of the bispecific single-chain antibody FAP1V2 is as shown in SEQ ID NO:3 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:3.

[0027] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 further includes an anchoring peptide and a signal peptide. In one embodiment of the present invention, the anchoring peptide is used to anchor the bispecific single-chain antibody FAP1V2 to the cell surface. In a specific embodiment of the present invention, the anchoring peptide is located at the N-terminus or C-terminus of the bispecific single-chain antibody FAP1V2. In a specific embodiment of the present invention, the anchoring peptide is GPI. In a specific embodiment of the present invention, the signal peptide is an IL-2 signal peptide.

[0028] In one embodiment of the present invention, the tumor is a tumor that highly expresses PD-L1 and / or VEGFR2. In a specific embodiment of the present invention, the tumor includes cervical cancer, ovarian cancer, lung cancer, melanoma, head and neck squamous cell carcinoma, breast cancer, bladder cancer, renal cell carcinoma, liver cancer, colon cancer, and rectal cancer. In one embodiment of the present invention, the tumor is non-small cell lung cancer.

[0029] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can be functionalized by chemical modification (such as polyethylene glycolation), post-translational modification (such as glycosylation), targeting molecule modification (such as surface antibody, RGD sequence or folic acid), or coupled with protein tags (His, Flag, GST, MBP, HA, Myc, GFP, etc.), detectable markers (enzymes, radionuclides, fluorescent dyes, luminescent substances, biotin, etc.) or therapeutic agents (anti-tumor / targeted drugs).

[0030] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 is administered orally or by injection.

[0031] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 is in formulation form.

[0032] In one embodiment of the present invention, the formulation is a lyophilized formulation.

[0033] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can be used in combination with chemotherapy drugs or other targeted drugs. In a specific embodiment of the present invention, the bispecific single-chain antibody FAP1V2 is administered in combination with paclitaxel or anti-CTLA-4 antibody.

[0034] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can be used to prepare a drug for treating lung cancer.

[0035] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can specifically bind to lung cancer cells.

[0036] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can selectively bind to PD-L1 or VEGFR2 expressed by lung cancer cells.

[0037] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can block the interaction between PD-L1 / PD-1 and VEGFR2 / VEGF.

[0038] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 has an immunosuppressive effect on the occurrence and growth of lung cancer.

[0039] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 increases TCRβ in lung cancer cells. hi T cell recruitment increases the infiltration of T cells with high TCRβ expression, thereby enhancing the tumor cell clearance capacity.

[0040] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can promote TCRβ in the spleen. hi T cell generation.

[0041] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can promote the growth of CD25 in splenic lymphocytes. hi An increase in the number of T cells.

[0042] In one embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can effectively inhibit the metastasis of lung cancer cells in vivo. In a specific embodiment of the present invention, the bispecific single-chain antibody FAP1V2 can effectively inhibit liver metastasis of lung cancer cells.

[0043] In a second aspect, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament, the medicament comprising at least one of the following effects:

[0044] (1) It can specifically bind to PD-L1 and VEGFR2;

[0045] (2) Inhibits tumor cell migration;

[0046] (3) Inhibits tumor metastasis in the body;

[0047] (4) Inhibits tumor growth;

[0048] (5) Eliminate or kill tumor cells;

[0049] (6) Enhance anti-tumor immune response;

[0050] (7) Promotes the proliferation and activation of T cells;

[0051] (8) Recruit TCRβ hi T cells or promoting TCRβ hi The generation or activation of T cells;

[0052] (9) Inhibit or reduce immune cells with high PD-1 expression;

[0053] (10) Add CD25 hi The number of T cells may increase CD25 expression levels;

[0054] The pharmaceutical composition includes the aforementioned bispecific single-chain antibody FAP1V2.

[0055] In one embodiment of the present invention, the composition further includes a pharmaceutically acceptable excipient or adjuvant.

[0056] In a third aspect, the present invention provides the use of a nucleotide encoding the above-mentioned bispecific antibody, a carrier comprising the nucleotide, or a host cell comprising the carrier in the preparation of a medicament, the medicament comprising at least one of the following effects:

[0057] (1) It can specifically bind to PD-L1 and VEGFR2;

[0058] (2) Inhibits tumor cell migration;

[0059] (3) Inhibits tumor metastasis in the body;

[0060] (4) Inhibits tumor growth;

[0061] (5) Eliminate or kill tumor cells;

[0062] (6) Enhance anti-tumor immune response;

[0063] (7) Promotes the proliferation and activation of T cells;

[0064] (8) Recruit TCRβ hi T cells or promoting TCRβ hi The generation or activation of T cells;

[0065] (9) Inhibit or reduce immune cells with high PD-1 expression;

[0066] (10) Add CD25 hi The number of T cells may increase, or the expression level of CD25 may be increased.

[0067] Compared with the prior art, the present invention has the following beneficial effects:

[0068] (1) This invention activates T cell function and inhibits cancer cell metastasis by simultaneously blocking PD-L1 and inhibiting the VEGFR2 signaling pathway, thereby activating the immune system and significantly promoting the memory inhibition effect of the immune system on tumors.

[0069] The bispecific intracellular antibody FAP1V2 inhibits the biological functions and downstream responses of PD-L1 and VEGFR2 by targeting and blocking both intracellular and extracellular PD-L1 and VEGFR2. It blocks the PD-1 / PD-L1 immune checkpoint by inhibiting the binding of PD-1 (highly expressed by "unactivated" immune cells) to PD-L1. Transient expression of the FAP1V2 intracellular antibody significantly enhanced TCRβhi T cell-mediated specific immunosuppression against LLC tumors, a function significantly enhanced in both primary and secondary tumors. FAP1V2 inhibited tumor cell metastasis. Two rounds of transient expression of FAP1V2 achieved significant tumor suppression in LLC cells, with complete immunosuppression of LLC tumor growth in 1 / 6 of the mice, activation of TCRβhi T cells and increased tumor infiltration, and inhibition of PD-1hi immune cell emergence, indicating prevention of T cell exhaustion. Increased CD25 expression also supported the enhanced immune response, manifested through increased T cell activity in the spleen.

[0070] (2) The nanobody design of this invention eliminates the need for Fc fragments and V L Light chains help minimize immunogenicity and molecular size, enabling engineered chimeric genes to be expressed intracellularly, thus addressing the problem of low intracellular delivery efficiency of traditional antibodies. Due to the single V... H Their small chain size may allow them sufficient flexibility to bind antigens. Furthermore, the specificity of these endogenous antibodies for tumor-associated antigens (TAAs) offers potential for studying signaling pathways regulated by intracellular TAAs.

[0071] (3) The multi-target nanobodies of the present invention achieve intracellular expression and blockade of target proteins in tumor cells, thereby enhancing immunotherapy for mouse LLC tumors and inhibiting metastasis. These multi-target nanobodies can be transferred into engineered bacteria for large-scale production and purification, serving as antigen-blockade therapeutic agents targeting different cellular sites, significantly reducing the economic burden on cancer patients. Attached Figure Description

[0072] Figure 1 Antibody design map for pEGFP-C1-AP1V2.

[0073] Figure 2 This is a Western blot result of dual-target single-chain antibody expression.

[0074] Figure 3 This is a flow cytometry image showing the binding of FAP1V2 to LLC mouse cells.

[0075] Figure 4 A shows the effect of FAP1V2 on cell viability; Figure 4B and 4C show the results of FAP1V2 inhibiting tumor cell migration.

[0076] Experimental data on FAP1V2 inhibiting LLC tumor growth.

[0077] Figure 5 The image shows the results of FAP1V2 inhibiting LLC tumor growth in mice.

[0078] Figure 6 Immunofluorescence and flow cytometry analysis of intracellular antibody-induced immune system activation against LLC tumors. (AH) Immunofluorescence analysis of tumor infiltration by activated T cells (highly TCRβ-expressing) and immune cells highly expressing PD-1. Tumors were formed by LLC cells transiently expressing intracellular antibodies (A) FAP1, (B) FAV2, or (C) FAP1V2. (D) Control tumors were formed by LLC cells transiently expressing EGFP but not expressing intracellular antibodies. Green fluorescence (indicated by yellow arrows for TCRβ) shows the distribution of activated T cells, while red fluorescence (indicated by pink arrows for PD-1) tracks the recruitment and distribution of immune cells highly expressing PD-1 in tumor tissue. (E) to (H) Distribution of activated T cells (Alexa Fluor488-labeled antibody) and PD-1-highly expressing immune cells (CY3-labeled antibody) in the marginal and central regions of the spleen in mice carrying LLC tumors expressing single-chain intracellular antibodies. Spleens were obtained from mice carrying LLC cells transiently expressing (E)FAP1, (F)FAV2, (G)FAP1V2, or (H) only transiently expressing EGFP. Flow cytometry (I) to (L) detected and analyzed the changes in the proportion of CD25-overexpressing immune T cells in mouse spleens 24 days after initial inoculation with tumor cells transiently expressing intracellular antibodies. The left gate shows the total population of CD25-overexpressing T cells, and the right gate shows a population dominated by small CD25-overexpressing T cells.

[0079] Figure 7 The image shows the effect of FAP1V2 on tumor metastasis in mice. Detailed Implementation

[0080] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0081] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0082] Materials and Methods:

[0083] The human cervical cancer cell line HeLa (as a model cell line) was purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences) and cultured in 1640 medium containing 10% (v / v) fetal bovine serum (FBS, Gibco, Invitrogen, USA), 100 μg / mL penicillin and 100 μg / mL streptomycin (Gibco, Invitrogen, USA) at 37°C and 5% CO2.

[0084] HeLa cells transfected with the pEGFP-C1 plasmid (named HeLa-EGFP) served as a negative control. HeLa cells were transfected with the pEGFP-C1-AP1V2 (encoding FAP1V2), pEGFP-C1-AP1 (encoding FAP1), or pEGFP-C1-AV2 (encoding FAV2) plasmids, and named HeLa-FAP1V2, HeLa-FAP1, or HeLa-FAV2, respectively.

[0085] Rabbit anti-PD-L1, rabbit anti-VEGFR2, rabbit anti-GFP polyclonal antibodies, rhodamine B 5-isothiocyanate (RBITC)-labeled goat anti-rabbit IgG antibody, and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody were purchased from Sangon Biotech (Shanghai, China). Mouse anti-EGFP / GFP monoclonal antibody, HRP-labeled goat anti-mouse IgG antibody, and normal mouse IgG antibody were purchased from Beyotime (Shanghai, China). Other antibodies used for immunofluorescence analysis were purchased from Servare Biotech Inc. (Wuhan, China). TCRβ hi T cells refer to T cells that highly express TCRβ and CD25. hi T cells refer to T cells that highly express CD25.

[0086] Data collection and statistical analysis

[0087] Immunofluorescence analysis was performed using confocal fluorescence microscopy, acquiring data for each experimental condition under identical settings. All data are expressed as mean ± standard error (Mean ± SEM). Unpaired two-tailed t-tests were used for comparisons between two groups. For comparisons involving multiple groups, one-way ANOVA was performed using Prism 6.0 software (GraphPad), followed by two-way ANOVA. A p-value < 0.05 was considered statistically significant. p-values ​​are summarized as: P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, unless otherwise specified.

[0088] Example 1: Plasmid Construction and Expression

[0089] 1. Plasmid construction

[0090] (1) Containing anti-PD-L1V H (FAP1), EGFP and anti-VEGFR2V H The recombinant bispecific intracellular antibody FAP1V2 gene sequence (FAV2) was obtained using nucleotide polymerization technology (Sangon Biotech Co., Ltd., Shanghai, China). This sequence was cloned into the mammalian expression vector pEGFP-C1, where EGFP, acting as a fluorescent indicator, is linked between FAP1 and FAV2 to track transient (24-96 hours) transgene expression of FAP1V2. H The gene and EGFP are linked via a flexible linker peptide (GGGGS)3. The N-terminus of FAP1 is fused to the IL-2 signal peptide (GenBank: AAD48509.1), and the C-terminus of FAV2 is fused to the GPI anchoring sequence (hPLAP, exon 10, GenBank: M19159.1). This recombinant plasmid contains V antibodies against PD-L1 and VEGFR2. H The sequence was named pEGFP-C1-AP1V2. Figure 1 As shown, the bispecific endogenous antibody FAP1V2 targets PD-L1 and VEGFR2, including IL-2 signaling and anti-PD-L1V. H Region, (GGGGS)3 linker, EGFP segment, (GGGGS)3 linker, anti-VEGFR2V H Regions and GPI anchoring zones ( Figure 1 The IL-2 signaling pathway is designed at the N-terminus to guide FAP1V2 into the extracellular space. A GPI-anchoring peptide is attached to the C-terminus to anchor FAP1V2 to the cell surface, enabling its interaction with surface PD-L1 and VEGFR2 antigens. EGFP is designed as a transient reporter gene for AP1V2 expression and as an indicator for tracking the intracellular location of FAP1V2; it also acts as a linker between AP1 and AV2. For comparison, pEGFP-C1-AP1 plasmid containing only the AP1 gene sequence and pEGFP-C1-AV2 plasmid containing only the AV2 gene sequence were constructed. Anti-PD-L1 antibody (PDB: 5XJ4) and anti-VEGFR2 antibody (GenBank: ACH41918.1) were used to express the FAP1V2 antigen. H The sequences were retrieved from the publicly available database of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ).

[0091] FAP1 amino acid sequence (SEQ ID NO:1):

[0092] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS

[0093] FAV2 amino acid sequence (SEQ ID NO:2):

[0094] LEESGGGLVRPGGSLRLSCAASGFTFSRSAMSWVRQAPGKGLEWVSGIDDDGGSTNYADSVKGRLTISRDNSKNTLFLQVNSPRAEDTAVYYCAKVRDSGYDFAPFDIWGQGTMVTVS

[0095] FAP1V2 amino acid sequence (SEQ ID NO:3):

[0096] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGLRSRAQASNSGGGGSGGGGSGGGGSLEESGGGLVRPGGSLRLSCAASGFTFSRSAMSWVRQAPGKGLEWVSGIDDDGGSTNYADSVKGRLTISRDNSKNTLFLQVNSPRAEDTAVYYCAKVRDSGYDFAPFDIWGQGTMVTVS

[0097] IL-2 signal peptide sequence (SEQ ID NO:4):

[0098] MYRMQLLSCIALSLALVTNS

[0099] GPI anchoring sequence (SEQ ID NO:5):

[0100] GWPLARPGTGRPTRSSYTETVQAMCSRTAPGRMLPRARA

[0101] 2. Cell transfection

[0102] Cell transfection according to LipoFiter TM 3. (HANBIO, Shanghai, China) Follow the manufacturer's instructions. HeLa cells were seeded one day before transfection. When cell confluence reached 70%, plasmid / LipoFiter was added. TM 3. Add the complex to serum-free cell culture medium. (Include plasmid / LipoFiter) TM After co-culturing the 3-complex for 6 hours, the plasmid / LipoFiter complex was removed. TM Serum-free medium containing the 3-complex was added to fresh medium containing 10% FBS serum. Plasmids and LipoFiter... TM The mass ratio of 3 was 1:1 (w / v). The amount of plasmid used in each six-well plate was 4 μg, LipoFiter. TM 3. The dosage is 4 μL. After transfection, the cells were cultured for another 48 hours before subsequent experiments.

[0103] 3. Transient intracellular expression of FAP1V2 antibody

[0104] Forty-eight hours after transfection with the recombinant plasmid, HeLa cells were collected and lysed, and Western blot analysis was performed to analyze EGFP expression to report single-target or dual-target EGFP. H Intracellular antibody expression was assessed using rabbit anti-GFP polyclonal antibody as the primary antibody and HRP-labeled goat anti-rabbit IgG as the secondary antibody. Results are as follows: Figure 2 As shown. Western blot analysis results indicate that these fusion V H Successful expression of endogenous antibodies in HeLa cells.

[0105] Example 2: Detection of the binding of FAP1V2 to LLC mouse cells (expressing PD-L1 and VEGFR2)

[0106] 1. 293T mouse cells were subjected to a 3×10⁻⁶ ppm incubation. 5Cells were seeded at 100 cells / well in 6-well plates and cultured overnight. Afterward, plasmids pEGFP-FAP1, pEGFP-FAV2, and pEGFP-FAP1V2 were transfected using lipid transfection reagent (HANBIO, Shanghai, China), which was endotoxin-free. Cells were lysed using RIPA lysis buffer containing a mixture of protease inhibitors (Selleck) and a mixture of phosphatase inhibitors II (MCE), and centrifuged at 12,000 × g (4 °C) for 15 min, collecting the supernatant.

[0107] 2. LLC cells in logarithmic growth phase were fixed with 4% paraformaldehyde for 30 minutes at room temperature, centrifuged at 2,000×g for 2 minutes, and the supernatant was removed. Cells were blocked with 5% BSA (dissolved in PBS) at room temperature for 1 hour, centrifuged again at 2,000×g for 2 minutes, and the supernatant was removed. Cells were incubated with 293T cell lysis buffer containing FAP1, FAV2, or FAP1V2 at room temperature for 30 minutes, and then incubated with commercially available rabbit anti-mouse PD-L1-APC or anti-mouse VEGFR2-APC (bound to phycocyanin (APC)) (Elabscience, Wuhan, China) at room temperature for 30 minutes. After centrifugation at 2,000×g for 4 minutes, the supernatant was removed, 200 μL of fresh culture medium was added, and flow cytometry analysis was performed (using the APC detection channel). This experiment was independently repeated three times. Data are expressed as Mean MFV ± SEM, based on the three mean fluorescence values ​​(MFV) of the cell population in each sample reported by flow cytometry. The fluorescence signal detected by flow cytometry originated from the APC-A channel. *, **, and *** indicate statistically significant differences compared to the control group at P < 0.05, P < 0.01, and P < 0.001, respectively; #, ##, and ### indicate significant differences between treatment groups at the P < 0.05, P < 0.01, and P < 0.001 levels.

[0108] The results are as follows Figure 3 As shown. Figure 3 A shows LLC cells under four conditions, from left to right: Control group: not incubated with 293T lysis buffer or commercial antibody; Anti-PD-L1 antibody group: incubated with commercial anti-mouse PD-L1 antibody; Single-target antibody (FAP1) + anti-PD-L1 antibody group: incubated with 293T lysis buffer expressing single-target antibody FAP1, followed by the addition of commercial anti-mouse PD-L1 antibody; Dual-target antibody (FAP1V2) + anti-PD-L1 antibody group: incubated with 293T lysis buffer expressing dual-target antibody FAP1V2, followed by the addition of commercial anti-mouse PD-L1 antibody. 3B bar chart represents... Figure 3 The average fluorescence value shown in Figure A.

[0109] Figure 3C shows LLC cells under four conditions, from left to right: Control group: not co-incubated with 293T lysis buffer or commercial antibody; Anti-VEGFR2 antibody group: co-incubated with commercial anti-mouse VEGFR2 antibody; Single-target antibody (FAV2) + anti-VEGFR2 antibody group: co-incubated with 293T lysis buffer expressing the single-target antibody FAV2, followed by the addition of commercial anti-mouse VEGFR2 antibody; Dual-target antibody (FAP1V2) + anti-VEGFR2 antibody group: co-incubated with 293T lysis buffer expressing the dual-target antibody FAP1V2, followed by the addition of commercial anti-mouse VEGFR2 antibody. A 3D bar chart is presented. Figure 3 The average fluorescence value shown in C is the result.

[0110] (A) through (D) flow cytometry and antibody competition experiments showed that intracellular antibodies FAP1 and FAP1V2, or FAV2 and FAP1V2, can selectively bind to PD-L1 or VEGFR2 expressed in LLC cells.

[0111] Example 3 Cell viability detection

[0112] 3×10 3 LLC cells were seeded into 96-well plates and transfected according to step 2 of Example 1. The plasmids used for transfection were pEGFP-C1, pEGFP-C1-AP1, pEGFP-C1-AV2, and pEGFP-C1-AP1V2. Forty-four hours after transfection, the old medium was replaced with fresh medium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Beyotime, China) was added. The cells were then cultured at 37°C for another 4 hours. The medium was then removed, and DMSO was added at 37°C to dissolve the purple crystals. Finally, Synergy was used... TM The H4 multi-functional microplate reader (BioTek, USA) measures absorbance at a wavelength of 490 nm.

[0113] Cell viability analysis showed ( Figure 4 A) FAP1, FAV2, and FAP1V2 had no significant effect on the viability of tumor cells.

[0114] Example 4: Determination of Cell Migration and Metastasis

[0115] HeLa and LLC cancer cells simultaneously expressing high levels of PD-L1 and VEGFR-2 were seeded into 6-well plates and transfected according to step 2 of Example 1. The plasmids used for transfection were pEGFP-C1 (control group), pEGFP-C1-AP1, pEGFP-C1-AV2, and pEGFP-C1-AP1V2; untransfected cells were used to assess background signal. 24 hours after transfection, vertical wells were scratched using a 200 μL pipette tip (Titan, China), and the scratching time was recorded as 0 hours. Subsequently, the cells were carefully washed 2-3 times with PBS. Then, the original medium was replaced with low-serum (2%) medium, and photographs were taken. Cell culture was performed at 37°C and 5% CO2, with observation and photography every 12 hours until the gaps between untransfected cells healed. Images were processed using ImageJ software, and seven horizontal lines were randomly drawn and the migration distance was calculated. The migration distance was calculated using the formula D = (A... initial -A measured The calculation is ) / W, where D represents the migration distance, and A initial A represents the initial unhealed area (before healing); measured The area of ​​the unhealed portion at the time of measurement is represented by denoted as ; W represents the width of the photograph taken at the same magnification. This experiment was repeated at least three times. The results are as follows: Figure 4 Figures B and 4C show the migration images of HeLa and LLC cells transiently transfected with different single-chain antibody genes at different time points (showing effective expression of the exogenous gene). Orange boxes indicate unhealed areas, black dashed lines indicate the edges of attached cells, and green double arrows indicate the distance between the edges. Figure 4C shows the inhibitory effect of the antibody on the migration scratch assay of HeLa and LLC cells. Untransfected cells (HeLa-Blank or LLC-Blank) and cells transfected only with the vector plasmid (HeLa-EGFP or LLC-EGFP) served as controls. *, **, *** indicate significant differences compared to the control group at the P<0.05, P<0.01, and P<0.001 levels, respectively.

[0116] The results showed that in HeLa or LLC cells, cells expressing FAV2 or FAP1V2 exhibited the most significant inhibition of cell migration compared to untransfected cells. FAP1 expression also had some inhibitory effect on cell migration, but its inhibitory effect was weaker than that of FAV2 or FAP1V2 expression. Therefore, we hypothesize that the antibodies FAP1V2 and FAV2 primarily inhibit cancer cell migration by blocking the key regulator of cell migration, VEGFR2, rather than by inhibiting cell viability.

[0117] Example 4: Detection of Immune-Resistant LLC Tumors in Mice

[0118] C57BL / 6 mice were randomly divided into 4 groups, with 6 mice in each group. LLC cells were transfected with plasmids containing FAP1, FAV2, and FAP1V2 genes, as well as a blank plasmid, according to the manufacturer's instructions, using a lipid transfection reagent (HANBIO, Shanghai, China). Transfected LLC cells (1×10⁷) were subcutaneously injected into the right axilla of C57BL / 6 mice to establish a tumor model. Four days later, transfected LLC cells (1×10⁷) were re-inoculated into the left axilla of each group of mice. Transgenic expression was maintained for approximately 7-8 days to promote an immune response. Throughout the experiment, mice in different groups were individually labeled and tracked. Tumor size and mouse weight were recorded daily, and tumor volume was calculated using the following formula: V 肿瘤 = Length × Width × Height × π / 6. After 4 weeks of experimentation, mice were euthanized by dislocation, tumors were dissected and photographed. In addition, vital organs (heart, liver, spleen, lungs, kidneys, and stomach) were cut into small pieces, fixed in 4% formaldehyde, and embedded in paraffin. Then, paraffin-embedded tissue sections were prepared, H&E stained, and histopathological analysis of the organs was performed. PD-1 in tumors and spleen was analyzed using immunofluorescence microscopy. hi and TCRβ hi Distribution and levels of immune cells. After grinding the spleen and treating it with collagenase, hyaluronidase, and DNase I (Tansoole platform, Fuzhou, China), activated T cells (CD25APC) in the mouse spleen were detected by flow cytometry analysis using anti-mouse CD25APC (clone: ​​PC61.5). hi The amount of VEGFR2 (overexpressed in LLC cells) was determined in the lungs and liver of each mouse model group using VEGFR2 as a biomarker.

[0119] Experimental results: (1) such as Figure 5 As shown, 5A presents the timeline of transient FAP1V2 expression-mediated anticancer therapy, including immune activation and anti-metastatic effects. 5B shows the tumor size changes in mice injected with LLC cancer cells and transiently expressing antibodies to FAP1, FAV2, or FAP1V2. Mice transfected with a blank plasmid (pEGFP-C1) served as controls. Tumor size data in the same axilla were expressed as mean tumor size ± SEM in mice of 6 per group. 5C shows images of tumors isolated from the right and left axillas at day 28 when the mice were sacrificed.

[0120] Experimental results showed that after two transient expression cycles of the fusion antibody (lasting 24–96 hours each), the anti-PD-L1-anti-VEGFR2 chimeric fusion antibody FAP1V2 exhibited superior immunosuppressive activity against LLC cell tumorigenesis and growth in C57BL / 6 mice. FAP1 showed milder inhibition, while FAV2 expression provided moderate inhibition, with tumor growth rate reduced by approximately 50% compared to the control group without fusion antibody expression. Secondary tumors established four days later (tumors transiently expressing the fusion antibody) showed significantly enhanced immunosuppression compared to the primary tumor, with intracellular expression of FAP1V2 in LLC cells exhibiting the best tumor growth inhibition. In one of six mice transfected with FAP1V2-transfected LLC cells, no LLC tumor growth was detected. Mice without fusion antibody expression served as the control group (see...). Figure 5 (A-5C). (A) to (C) show the role of FAP1V2 anti-PD-L1-anti-VEGFR2 chimeric antibody in enhancing immunosuppression of LLC cell tumorigenesis and growth.

[0121] (2) TCRβ is crucial for T cell activation and specific immune responses. Immunofluorescence microscopy was used to analyze the high expression of TCRβ (TCRβ...). hi T cells and high expression of PD-1 (PD-1) hi The recruitment of immune cells in tumors. Tumors from the FAP1, FAV2, and FAP1V2 groups showed increased infiltration of T cells with high TCRβ expression and improved tumor cell clearance, with the FAP1V2 treatment group showing the most significant effect. Figure 6 (A to 6C).

[0122] In tumors treated with the FAP1V2 dual-targeting antibody, almost no PD-1-overexpressing immune cells were observed. In contrast, some PD-1-overexpressing immune cells were present in the FAP1 treatment group, while the FAV2 treatment group recruited even more PD-1-overexpressing immune cells. However, in tumor tissues without transient antibody expression, PD-1... hi More immune cells invaded this group than in the other group; TCRβ was detected in this group. hi There are fewer T cells and more tumor cells. Figure 6 D).

[0123] Overall, tumor tissues expressing the single-chain antibody PD-L1 at an early stage, including FAP1V2 and FAP1, are more effective at recruiting TCRβ. hi T cells, not PD-1 hi Immune cells, suggesting that with high expression of TCRβ, T cell activation and specific immune responses may lead to specific immunosuppression against LLC tumors.

[0124] Mice inoculated with LLC tumor cells that transiently express antibodies showed TCRβ in the spleen. hi T cell production increased. Conversely, the control and FAV2 groups showed the highest levels of PD-1 in the spleen. hi Immune cells ( Figure 6 EH). This indicates that transient expression of antibodies, particularly the bispecific antibody FAP1V2, significantly enhances the immune response to these tumors.

[0125] (3) As the site of T cell activation and a reservoir of T cells, the spleen plays a crucial role in T cell activation, storage, and immune response. Since CD25 (IL-2 receptor α chain) is highly expressed on activated T cells, the levels of CD25 in the spleen of mice transiently expressing antibodies were measured. hi Changes in T cells.

[0126] Flow cytometry analysis of the fluorescence intensity-forward scattering area (FSC-A) curves of splenic lymphocytes (mainly B cells and T cells) revealed an increase in the number of immune cells expressing medium to high levels of CD25 across all antibody-expressing groups. The FAP1V2 and FAP1 groups showed the largest increase, rising from 17.7% to 23.4%. In all treatment groups, CD25... hi The number of T cells increased significantly, and their size was smaller, with the increase being particularly pronounced in the FAP1V2 group, approximately 2.2 times that of the control group. Figure 6 IL). Since CD25 is induced by high levels of TCR ligands and is highly expressed on activated T cells (mainly effector T cells and regulatory T cells), it represents active immune regulation and immune response, and when combined with TCRβ... hi Increased T cell production further demonstrates the enhanced immune response in the FAP1V2 transient expression group. Simultaneously, smaller CD25 cells were observed in splenic lymphocytes. hi An increase in the number of T cells (which may be more capable of penetrating tumor tissue) may suggest systemic regulation and optimization in the anticancer immune response. Elevated CD25 levels are associated with several physiological effects, one of which is its binding to the IL-2Rβ and IL-2Rγ chains to form the complete IL-2 receptor complex. This complex transmits IL-2 signals to T cells, leading to cell activation and proliferation, thereby guiding tumor suppression mechanisms.

[0127] (4) Detection of tumor metastasis in mice

[0128] The results are as follows Figure 7As shown, 7A illustrates the invasion of mouse liver tissue by LLC tumor cells transiently expressing a single-chain antibody. VEGFR2 serves as a biomarker protein for tumor cells. 7B shows the transient expression of VEGFR2. H The invasion of mouse lung tissue by antibody-infected LLC cells. Lung sections were obtained from mice inoculated with LLC tumor cells, the tumor cells transiently expressing FAP1 (a), FAV2 (b), FAP1V2 (c), and cells transfected with the vector plasmid (d).

[0129] In vivo analysis showed that intact LLC tumor cells were not detected in mouse biopsies in the FAV2 or FAP1V2 (anti-VEGFR2) antibody groups. Similar hepatocyte migration was observed in the FAP1 (anti-PD-L1) and control groups (without transient antibody expression). Figure 7 A). In lung tissue, no intact LLC tumor cells were detected in the FAP1V2 group. Cell migration was most severe in the control group, followed by the FAP1 group, and then the FAV2 group. Figure 7 B). The results showed that, as a bifunctional antibody, FAP1V2 could effectively inhibit the migration of LLC cells in the experimental mouse model.

[0130] Vascular endothelial growth factor (VEGF) binds to VEGFR2, inducing the formation of VEGFR2 receptor dimers and activating downstream signaling pathways, playing a crucial role in angiogenesis, vascular development, and cancer metastasis. Therefore, FAP1V2 directly inhibits VEGFR2, leading to the suppression of cancer metastasis.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of a bispecific single chain antibody FAP1V2 for the preparation of a medicament, characterized in that: The drug has at least one of the following effects: (1) It can specifically bind to PD-L1 and VEGFR2; (2) Inhibits tumor cell migration; (3) Inhibits tumor metastasis in the body; (4) Inhibits tumor growth; (5) Eliminate or kill tumor cells; (6) Enhance anti-tumor immune response; (7) Promotes the proliferation and activation of T cells; (8) recruiting TCRβ hi T cell or promoting TCRβ hi generation or activation of a T cell; (9) Inhibit or reduce immune cells with high PD-1 expression; (10) increasing CD25 hi the number of T cells or increasing CD25 expression levels; The bispecific single-chain antibody FAP1V2 includes a nanobody that recognizes the target PD-L1 and a nanobody that recognizes the target VEGFR2, with the nanobody monomers linked together by a linker.

2. Use of a bispecific single chain antibody FAP1V2 according to claim 1 for the preparation of a medicament, characterized in that: The amino acid sequence of the nanobody that recognizes the target PD-L1 is as shown in SEQ ID NO:1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:

1. Or the amino acid sequence of the nanobody that recognizes the target VEGFR2 is as shown in SEQ ID NO:2 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:2; Alternatively, the connector may be a flexible connector; preferably, the connector is (G4S)n, n = 1-4, such as GGSGG or (G4S)3; Or the amino acid sequence of the bispecific single-chain antibody FAP1V2 is as shown in SEQ ID NO:3 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:

3.

3. Use of a bispecific single chain antibody FAP1V2 according to claim 1 for the preparation of a medicament, characterized in that: The bispecific single-chain antibody FAP1V2 also includes an anchoring peptide and a signal peptide; Preferably, the anchoring peptide sequence is as shown in SEQ ID NO:5, and the signal peptide sequence is as shown in SEQ ID NO:

4.

4. The application of the bispecific single-chain antibody FAP1V2 as described in claim 1 in the preparation of a drug, characterized in that: The bispecific single-chain antibody FAP1V2 is chemically modified, post-translational modified, targeted molecule modified, conjugated with protein tags, detectable markers or therapeutic agents; Alternatively, the bispecific single-chain antibody FAP1V2 may be administered orally or by injection; Alternatively, the bispecific single-chain antibody FAP1V2 may be in formulation form; Alternatively, the bispecific single-chain antibody FAP1V2 may be used in combination with chemotherapy drugs or other targeted drugs.

5. The application of the bispecific single-chain antibody FAP1V2 as described in claim 1 in the preparation of a drug, characterized in that: The bispecific single-chain antibody FAP1V2 can be used to prepare drugs for treating lung cancer.

6. The application of the bispecific single-chain antibody FAP1V2 as described in claim 1 in the preparation of a drug, characterized in that: The bispecific single-chain antibody FAP1V2 can specifically bind to lung cancer cells; Preferably, the bispecific single-chain antibody FAP1V2 can selectively bind to PD-L1 or VEGFR2 expressed by lung cancer cells; Preferably, the bispecific single-chain antibody FAP1V2 has an immunosuppressive effect on the occurrence and growth of lung cancer; The bispecific single-chain antibody FAP1V2 increases TCR beta in lung cancer cells hi Recruitment of T cells, increased infiltration of T cells with high TCR beta expression, increased ability to clear tumor cells.

7. The application of the bispecific single-chain antibody FAP1V2 as described in claim 1 in the preparation of a drug, characterized in that: The bispecific single chain antibody FAP1V2 can facilitate TCRβ hi Generation of T cells; or the bispecific single chain antibody FAP1V2 can promote an increase in the number of CD25 hi T cells in the spleen.

8. The application of the bispecific single-chain antibody FAP1V2 as described in claim 1 in the preparation of a drug, characterized in that: The bispecific single-chain antibody FAP1V2 can effectively inhibit liver metastasis of lung cancer cells.

9. The use of a pharmaceutical composition in the preparation of a drug, characterized in that: The drug has at least one of the following effects. (1) It can specifically bind to PD-L1 and VEGFR2; (2) Inhibits tumor cell migration; (3) Inhibits tumor metastasis in the body; (4) Inhibits tumor growth; (5) Eliminate or kill tumor cells; (6) Enhance anti-tumor immune response; (7) Promotes the proliferation and activation of T cells; (8) recruiting TCRβ hi T cell or promoting TCRβ hi generation or activation of a T cell; (9) Inhibit or reduce immune cells with high PD-1 expression; (10) increasing CD25 hi the number of T cells or increasing CD25 expression levels; The pharmaceutical composition comprises the bispecific single-chain antibody FAP1V2 according to any one of claims 1-8.

10. The use of a nucleotide encoding the bispecific antibody of any one of claims 1-8, a carrier comprising said nucleotide, or a host cell comprising said carrier in the preparation of a medicament, said medicament comprising at least one of the following effects: (1) It can specifically bind to PD-L1 and VEGFR2; (2) Inhibits tumor cell migration; (3) Inhibits tumor metastasis in the body; (4) Inhibits tumor growth; (5) Eliminate or kill tumor cells; (6) Enhance anti-tumor immune response; (7) Promotes the proliferation and activation of T cells; (8) recruiting TCRβ hi T cell or promoting TCRβ hi generation or activation of a T cell; (9) Inhibit or reduce immune cells with high PD-1 expression; (10) increasing CD25 hi the number of T cells or increasing CD25 expression levels.

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