Single-chain antibody FAP1 targeting PD-L1 and applications thereof

Abstract

The application provides a single-chain antibody FAP1 targeting PD-L1 and application thereof, an amino acid sequence of the single-chain antibody FAP1 is shown as SEQ ID NO:1, or a sequence with at least 80 percent identity with the sequence shown as SEQ ID NO:1. The application activates T cell function and inhibits cancer cell metastasis by blocking the PD-L1 signal pathway, activates the immune system, and significantly promotes the memory inhibition of the immune system on the tumor.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to FAP1, a single-chain antibody targeting PD-L1, and its applications. Background Technology

[0002] Cancer cells evade the cytotoxic effects of drugs through various mechanisms, and T cells in the immune system, especially in the tumor microenvironment, often fail to effectively eliminate tumor cells. This inadequate immune response is partly attributed to T cell exhaustion, a phenomenon characterized by the loss of T cell function and decreased proliferative capacity in the tumor microenvironment. Furthermore, the number and function of immune cells in the tumor microenvironment are significantly weakened, particularly the reduction of cytotoxic T cells, limiting the immune system's effective attack on tumors. Therefore, finding therapeutic strategies that can effectively activate the immune system, especially enhancing T cell function, is a key research direction in cancer treatment.

[0003] Currently, immune checkpoint inhibition therapy has emerged as a promising treatment strategy. Immune checkpoint molecules act as "brakes" on the surface of immune cells; tumor cells, by expressing these molecules (such as PD-L1), can suppress the immune response, preventing the immune system from attacking tumor cells. By blocking these immune checkpoints, the suppression of the immune response can be relieved, thereby enhancing the ability of T cells to attack tumor cells. Anti-CTLA-4 and anti-PD-1 antibodies have achieved significant results in the clinical treatment of various cancers, particularly showing good efficacy in melanoma, non-small cell lung cancer, and bladder cancer. Representative drugs include ipilimumab, tremelimumab, nivolumab, and durvalumab.

[0004] However, despite the clinical efficacy of anti-PD-1 antibodies (such as sintilimab and cadonilimab) and bispecific antibodies combining anti-PD-1 and CTLA-4, these treatments still face numerous challenges. A major issue is that these therapies may cause a decrease in the number of immune cells (such as neutrophils, platelets, and erythrocytes), leading to side effects such as anemia or leukopenia. More critically, existing immune checkpoint inhibitory antibodies often contain heterologous regions (such as Fc fragments), which may not only trigger a strong immune response but also lead to rapid antibody clearance, affecting efficacy. Furthermore, their poor tissue penetration limits their effectiveness at tumor sites.

[0005] Nanobodies, as an emerging immunotherapeutic tool, have become a new direction in cancer treatment due to their small molecular weight, low immunogenicity, and ease of engineering. Nanobodies do not contain light chain variable regions (VL) and Fc regions, thus exhibiting high specificity and low immunogenicity. Despite the great potential of nanobodies in targeted therapy, their mechanisms of action in T cell activation and tumor antigen recognition are still not fully understood. In particular, whether nanobodies can promote the binding of endogenous T cell receptors (TCRs) to tumor antigens and activate T cells requires further investigation. Furthermore, the effects of transient expression of nanobodies on tumor cells have not been systematically studied.

[0006] The interaction between PD-1 and its ligand PD-L1 is considered a key factor in tumor immune escape. After PD-L1 binds to PD-1 on tumor-infiltrating lymphocytes (TILs), it inhibits the activation and function of anti-tumor T cells by transmitting immunosuppressive signals, thereby enabling tumor cells to evade immune surveillance. Although existing PD-L1 antibodies (mostly IgG type) have achieved some success in blocking the PD-L1 / PD-1 interaction, they also face problems such as strong immunogenicity, short half-life, and poor tissue penetration. The Fc region in IgG antibodies may not only cause side effects but also lead to rapid drug clearance from the body, affecting their efficacy in tumor treatment.

[0007] Therefore, developing a small-molecule, low-immunogenic PD-L1 antibody with good tissue penetration, especially a single-chain antibody that can effectively and persistently block the interaction between PD-L1 and PD-1, has become an important need in cancer immunotherapy. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides FAP1, a single-chain antibody targeting PD-L1, and its applications. This invention proposes a V-based... H Novel single-chain antibodies with structural domains are designed to address the limitations of existing antibody therapies and provide a more efficient and safer cancer immunotherapy approach.

[0009] In one aspect, the present invention provides a single-chain antibody FAP1, the amino acid sequence of which is shown in SEQ ID NO:1, or an amino acid sequence having 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.

[0010] In one embodiment of the present invention, the single-chain antibody FAP1 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).

[0011] In one embodiment of the present invention, the single-chain antibody FAP1 can specifically bind to PD-L1 and block the interaction between PD-L1 and PD-1.

[0012] In one embodiment of the present invention, the single-chain antibody FAP1 can specifically bind to cervical cancer cells. In another embodiment of the present invention, the single-chain antibody FAP1 can selectively bind to PD-L1 expressed by cervical cancer cells.

[0013] In one embodiment of the present invention, the single-chain antibody FAP1 can be used to prepare a drug for treating lung cancer.

[0014] In one embodiment of the present invention, the single-chain antibody FAP1 has an immunosuppressive effect on the occurrence and growth of lung cancer.

[0015] In one embodiment of the present invention, the single-chain antibody FAP1 can promote TCRβ hi T cell generation.

[0016] In one embodiment of the present invention, the single-chain antibody FAP1 promotes increased infiltration of T cells with high TCRβ expression in tumor cells and enhances the tumor cell clearance ability.

[0017] In a second aspect, the present invention provides a nucleotide encoding the single-chain antibody FAP1, a vector containing the nucleotide, and a host cell comprising the vector or expressing the single-chain antibody FAP1.

[0018] In a third aspect, the present invention provides a pharmaceutical composition comprising the above-described single-chain antibody FAP1.

[0019] In one embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient or adjuvant.

[0020] This invention provides, in four aspects, the use of the above-mentioned single-chain antibody FAP1, nucleotide, vector, or host cell in the preparation of a drug, wherein the drug comprises at least one of the following effects:

[0021] (1) It can specifically bind to PD-L1;

[0022] (2) Inhibits tumor cell migration;

[0023] (3) Inhibits tumor metastasis in the body;

[0024] (4) Inhibits tumor growth;

[0025] (5) Eliminate or kill tumor cells;

[0026] (6) Enhance anti-tumor immune response;

[0027] (7) Promotes the proliferation and activation of T cells;

[0028] (8) Recruit TCRβ hi T cells or promoting TCRβ hi The generation or activation of T cells.

[0029] In one embodiment of the present invention, the tumor is a PD-L1 highly expressed tumor. In a specific embodiment of the present invention, the tumor includes cervical cancer, ovarian cancer, lung cancer, and melanoma. In one embodiment of the present invention, the tumor is non-small cell lung cancer.

[0030] In one embodiment of the present invention, the drug can inhibit the occurrence and growth of lung cancer cells, promote the infiltration of T cells with high TCRβ expression in lung cancer cells, and improve the clearance ability of lung cancer cells.

[0031] In one embodiment of the present invention, the single-chain antibody FAP1 is administered orally or by injection.

[0032] In one embodiment of the present invention, the single-chain antibody FAP1 is in formulation form.

[0033] In one embodiment of the present invention, the formulation is a lyophilized formulation.

[0034] In one embodiment of the present invention, the single-chain antibody FAP1 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.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) This invention activates T cell function and inhibits cancer cell metastasis by blocking PD-L1, activates the immune system, and significantly promotes the memory inhibition effect of the immune system on tumors.

[0037] (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... HTheir 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.

[0038] (3) The nanobody gene of the present invention can be transferred into engineered bacteria for large-scale production and purification, which significantly reduces the economic burden on cancer patients. Attached Figure Description

[0039] Figure 1 This is a Western blot result of FAP1 expression in HeLa cells.

[0040] Figure 2 The graph shows the results of FAP1 competing with commercial antibodies to bind to the target antigen of HeLa.

[0041] Figure 3 This is a diagram from a cell compatibility experiment of FAP1.

[0042] Figure 4 The image shows the results of FAP1 inhibiting LLC tumor growth in mice.

[0043] Figure 5 Immunofluorescence is an immunofluorescence of the immune system activation against LLC tumors induced by intracellular antibodies. Detailed Implementation

[0044] 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.

[0045] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0046] Materials and Methods:

[0047] 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.

[0048] HeLa cells transfected with the pEGFP-C1 plasmid (named HeLa-EGFP) served as a negative control. HeLa cells were transfected with the pEGFP-C1-AP1 plasmid (encoding FAP1) and named HeLa-FAP1.

[0049] 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 were purchased from Beyotime (Shanghai, China). Other antibodies used for immunofluorescence analysis were purchased from Servare Biotech Inc. (Wuhan, China).

[0050] Data collection and statistical analysis

[0051] 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.

[0052] Example 1: Plasmid Construction and Expression

[0053] 1. Plasmid construction

[0054] (1) Containing anti-PD-L1 V H The single-chain antibody sequence of the FAP1 gene (EGFP) 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 was used as a fluorescent indicator to track transient (24-96 hours) transgenic expression of FAP1. HThe gene and EGFP are linked via a flexible linker peptide (GGGGS)3. The N-terminus of FAP1 is fused to an IL-2 signal peptide (GenBank: AAD48509.1), and the C-terminus of EGFP is fused to a GPI anchoring sequence (hPLAP, exon 10, GenBank: M19159.1), used to anchor FAP1 to the cell surface, thereby enabling it to interact with the surface PD-L1 antigen. This recombinant plasmid contains an anti-PD-L1 V... H The sequence was named pEGFP-C1-AP1. The V of the anti-PD-L1 antibody (PDB:5XJ4) H The sequences were retrieved from the publicly available database of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ).

[0055] FAP1 amino acid sequence (SEQ ID NO:1):

[0056]

[0057] 2. Cell transfection

[0058] 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.

[0059] 3. Transient intracellular expression of FAP1V2 antibody

[0060] 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 as a reporter single-target vaccine. 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 1 As shown in the figure. Western blot analysis results indicate that the single-chain endogenous antibody was successfully expressed in HeLa cells.

[0061] Example 2: Detection of FAP1 binding to HeLa target antigen

[0062] 1. HeLa cells at 2×10 5 Cells were seeded at a density of [missing value] mL in 6-well plates. The plasmids used were pEGFP-C1 (control) and pEGFP-C1-AP1. Forty-eight hours post-transfection, cells were treated with trypsin, collected, and fixed in 4% paraformaldehyde, then washed three times with PBS. Cells were incubated with primary antibodies at room temperature for 1–2 hours. The major antibodies used were rabbit anti-PD-L1 and rabbit anti-VEGFR2. After washing three times with PBS, cells were incubated with RBITC-labeled goat anti-rabbit IgG (secondary antibody) at room temperature for 1 hour. After washing three times with PBS, the fluorescence of PD-L1 on the surface of HeLa cells was detected by the PE-A assay channel of flow cytometry.

[0063] The results are as follows Figure 2 As shown. Flow cytometry and antibody competition assays demonstrated that the intracellular antibody FAP1 selectively binds to PD-L1 expressed in HeLa cells, blocking the binding of commercially available anti-PD-L1 antibodies to PD-L1 on the cell surface.

[0064] Example 3 Cell viability detection

[0065] 3×10 3 HeLa cells were seeded into 96-well plates and transfected according to step 2 of Example 1. The plasmids used for transfection were pEGFP-C1 and pEGFP-C1-AP1; untransfected HeLa cells served as a blank control. Forty-four hours after transfection, the old culture 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 culture 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.

[0066] Cell viability analysis showed ( Figure 3 FAP1 had no significant effect on the viability of tumor cells.

[0067] Example 4: Determination of Cell Migration

[0068] HeLa cancer cells simultaneously expressing high levels of PD-L1 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) and pEGFP-C1-AP1; 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 to calculate the migration distance. The migration distance was calculated using the formula D = (A... initial -A measured The calculation is performed using ) / W, where D represents the migration distance and A initial A represents the initial unhealed area (before healing); measured The area of ​​unhealed tissue at the time of measurement is shown; W represents the width of the photograph at the same magnification. This experiment was repeated at least three times. The results showed that, in HeLa cells, cells expressing FAP1 exhibited the most significant inhibition of cell migration compared to untransfected cells.

[0069] Example 4: Detection of Immune-Resistant LLC Tumors in Mice

[0070] C57BL / 6 mice were randomly divided into two groups of six each. LLC cells were transfected with a plasmid containing FAP1, the gene, and 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 the tumors was analyzed using immunofluorescence microscopy. hi and TCRβ hi Distribution and level of immune cells.

[0071] Experimental results: (1) such as Figure 4The image shows the tumor size in the right axilla of mouse tumor-bearing mice two weeks after transient expression of dual-target antibodies in the right axilla (4-8 days). Mice without expressed antibodies served as the control group. The results demonstrate the role of FAP1 in enhancing immunosuppression of LLC cell tumorigenesis and growth.

[0072] (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 cell recruitment in tumors. Tumors from FAP1 showed increased infiltration of T cells with high TCRβ expression and improved tumor cell clearance. The FAP1 treatment group was significantly more effective than the blank tumor-bearing mouse control group. Figure 5 ).

[0073] 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. A single-chain antibody FAP1, characterized in that: Its amino acid sequence is shown in SEQ ID NO:1, or has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:

1.

2. The single-chain antibody FAP1 as described in claim 1, characterized in that: The single-chain antibody FAP1 includes modified or conjugated single-chain antibodies.

3. The single-chain antibody FAP1 as described in claim 1, characterized in that: The single-chain antibody FAP1 can specifically bind to PD-L1 and block the interaction between PD-L1 and PD-1.

4. The single-chain antibody FAP1 as described in claim 1, characterized in that: The single-chain antibody FAP1 can specifically bind to cervical cancer cells; preferably, the single-chain antibody FAP1 can selectively bind to PD-L1 expressed by cervical cancer cells.

5. The single-chain antibody FAP1 as described in claim 1, characterized in that: The single-chain antibody FAP1 can be used to prepare drugs for treating lung cancer; Preferably, the single-chain antibody FAP1 has an immunosuppressive effect on the occurrence and growth of lung cancer.

6. The single-chain antibody FAP1 as described in claim 1, characterized in that: The single-chain antibody FAP1 can promote TCRβ hi T cell generation.

7. The single-chain antibody FAP1 as described in claim 1, characterized in that: The single-chain antibody FAP1 promotes increased infiltration of T cells with high TCRβ expression in tumor cells and enhances the tumor cell clearance ability.

8. A nucleotide encoding the single-chain antibody FAP1 according to any one of claims 1-7, a vector containing the nucleotide, or a host cell comprising the vector or expressing the single-chain antibody FAP1.

9. A pharmaceutical composition comprising the single-chain antibody FAP1 according to any one of claims 1-7.

10. The use of the single-chain antibody FAP1 according to any one of claims 1-7, the nucleotide, vector, or host cell according to claim 8, in the preparation of a medicament, wherein the medicament comprises at least one of the following effects: (1) It can specifically bind to PD-L1; (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) Recruit TCRβ hi T cells or promoting TCRβ hi The generation or activation of T cells; Preferably, the tumor is a PD-L1 highly expressed tumor; preferably, the tumor includes cervical cancer, ovarian cancer, lung cancer, and melanoma; more preferably, the tumor is non-small cell lung cancer. Preferably, the drug can inhibit the occurrence and growth of lung cancer cells, promote the infiltration of T cells with high TCRβ expression in lung cancer cells, and improve the clearance ability of lung cancer cells.

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