Nanometer antibody fragment capable of targeted recognition of Nectin-4 protein and application thereof

Abstract

The invention relates to the technical field of nano antibodies, and particularly discloses a nano antibody fragment capable of targeted recognition of Nectin-4 protein and application of the nano antibody fragment, the nano antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2 and CDR3, the preparation method of the nano antibody fragment comprises the following steps: immunizing alpaca, collecting alpaca blood to analyze titer, constructing a bacteriophage library, producing an antibody, and detecting characterization. The prepared nano antibody is smaller in molecular weight and easy to express, and the affinity can adapt to the use of cell therapy; the CAR-T prepared from the antibody has a specific killing function on Nectin4 positive target cells, and has a good application prospect in cellular immunotherapy. The antibody can specifically recognize a human Nectin-4 antigen, has good specificity on human Nectin-4 expression positive cells, and has good application prospects of antibody drugs, antibody coupling drugs and cell therapy drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanobodies, in particular to a nanobody fragment capable of targeting and recognizing Nectin-4 protein and application thereof. BACKGROUND

[0002] Nectin-4 (Poliovirus receptor-like 4, PVRL4) belongs to the immunoglobulin superfamily and is the fourth subtype of the Nectin cell adhesion molecule family. Its molecular structure contains an extracellular variable region (V-type domain), two constant regions (C2-type domain), and a transmembrane region and intracellular tail region. During embryonic development, Nectin-4 participates in key physiological processes such as neural tube closure and organ formation by activating the PI3K / Akt and MAPK signaling pathways. Notably, the expression of Nectin-4 in adult healthy tissues is epigenetically regulated (such as promoter methylation), and only a few parts such as the placenta and skin appendages maintain basic expression. This spatiotemporal specific expression pattern suggests that it may have a potential risk window for tumor transformation.

[0003] Clinical studies have shown that Nectin-4 is significantly overexpressed in malignant tumors such as urothelial carcinoma, breast cancer, pancreatic cancer, triple-negative breast cancer, and bladder cancer. It activates proliferation signals, forms heterodimers with EGFR, enhances ERK1 / 2 phosphorylation, promotes metastasis and invasion, upregulates MMP-9 expression and disrupts E-cadherin-mediated cell junctions, and induces increased PD-L1 expression to participate in the multi-pathway regulation of pro-cancer functions, thereby promoting tumor malignancy progression.

[0004] Currently, there are two anti-Nectin-4 ADC drugs in the clinical stage worldwide: Enfortumab vedotin (Padcev®), a phase III EV-301 study showed that the median OS was 12.9 months (vs. 8.9 months for the chemotherapy group); and the Chinese self-developed drug MRG003, which has an ORR of 38.5% in advanced TNBC. However, existing antibody drugs have the defects of large molecular weight leading to poor tumor penetration, Fc-mediated ADCC effect causing non-specific toxicity, and the like, and there is an urgent need to develop new antibody drugs that can effectively avoid the above problems.

[0005] However, Nectin-4 is overexpressed in various tumor cells, such as urothelial carcinoma, breast cancer, pancreatic cancer, triple-negative breast cancer, and bladder cancer. Nectin-4 can promote tumor cell proliferation, differentiation, migration, invasion, and the like. Therefore, Nectin-4 has been defined as a tumor-associated antigen with pro-cancer properties in various cancers and plays a key role in tumor biology.

[0006] In order to better identify the target molecule, this patent invented a nanobody fragment against hNectin-4 protein and its application, and developed an anti-sequence for the target Nectin-4 protein of cell therapy. Summary of the Invention

[0007] The purpose of this invention is to provide a nanobody fragment capable of targeting and recognizing Nectin-4 protein and its application, in order to solve the problems mentioned above.

[0008] The objective of this invention can be achieved through the following technical solutions: In a first aspect of the invention, a nanobody fragment capable of targeting and recognizing Nectin-4 protein is provided, wherein the nanobody fragment V H The complementarity-determining region (CDR) of the chain has an amino acid sequence selected from the following group of CDRs: CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4.

[0009] SEQ ID NO: 2 (CDR1 sequence): AIG SEQ ID NO: 3 (CDR2 sequence): CISSSDGSTYYADSVKG SEQ ID NO: 4 (CDR3 sequence): DRGPVEGPLAVQAMCAMGRDLADY In a second aspect of the invention, a nanobody fragment capable of targeting and recognizing Nectin-4 protein is provided, wherein V H The amino acid sequence of the chain is shown in SEQ ID NO: 1.

[0010] SEQ ID NO: 1 (Anti-Nectin-4 VHH sequence): EVQLVESGGGLVQPGGSLRLSCAASGLDYYAIGWFRQAPGKEREGVSCISSDGSTYYA DSVKGRFSISRDNAKNTVYLQMNSLKPEDTAVYYCAADRGPVEGPLAVQAMCAMGRDLA DYWGQGTQVTVSS In this invention, the amino acid sequence of the VHH-hFc fusion protein comprises the nanobody sequence described in this invention (e.g., SEQ ID NO: 1) and the human IgG1 Fc sequence (e.g., SEQ ID NO: 1). NO: 5 shown: EVQLVESGGGLVQPGGSLRLSCAASGLDYYAIGWFRQAPGKEREGVSCISSDGSTYYADSVKGRFSISRDNAKNTVYLQMNSLKPEDTAVYYCAADRGPVEGPLAVQAMCAMGRDLADYWGQGTQVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Furthermore, the nanobody fragment that can target and recognize Nectin-4 protein can specifically bind to human Nectin-4 protein and has cross-reactivity with mouse Nectin-4 protein.

[0011] Furthermore, the nanobody fragment that can target and recognize Nectin-4 protein has a molecular weight of 12-15 kDa and exists in monomeric form under non-reducing conditions.

[0012] In a third aspect of the invention, a pharmaceutical composition is provided comprising a nanobody fragment capable of targeting and recognizing the Nectin-4 protein, and the dosage form of the pharmaceutical composition includes an injection, a lyophilized powder for injection, or a sustained-release formulation.

[0013] In a fourth aspect of the invention, an application of a nanobody fragment capable of targeting and recognizing the Nectin-4 protein is provided, including the application of the nanobody fragment in the preparation of antibody drugs, polyclonal antibodies, antibody-drug conjugates, and cell therapy drugs.

[0014] The beneficial effects of this invention are: I. In this invention, the prepared nanobodies have smaller molecular weights, are easier to express, and have affinity suitable for cell therapy. Second, in this invention, the nanobody can specifically recognize humanNectin-4 protein and has dual species specificity in humans and mice. The efficacy and toxicity of the dual specificity in mice are more similar to those in humans, which makes it easier to map the response in animals to humans and thus obtain some guiding information for clinical trials. Third, in this invention, the nanobody has good cell-level specificity, recognizing only cell lines expressing Nectin-4 molecules and not recognizing blank cells, thus showing good prospects for detection applications; IV. In this invention, the antibody can specifically recognize the human Nectin-4 antigen and has good specificity for human Nectin-4 expressing positive cells, binding only to Nectin-4 positive cells; it has good application prospects as an antibody drug, polyclonal antibody drug, antibody-drug conjugate and cell therapy drug. Attached Figure Description

[0015] The invention will now be further described with reference to the accompanying drawings.

[0016] Figure 1 This is a diagram of the nanobody ELISA binding experiment in this invention; Figure 2 This is an experimental diagram of the nanobody ELISA binding to mouseNectin-4 in this invention; Figure 3 This is a flow cytometry diagram of the nanobody binding humanNectin-4 and CHOK1 cells in this invention; Figure 4 This is a flow cytometry diagram of the nanobody binding to mouseNectin-4 and CHOK1 cells in this invention; Figure 5 This is a graph showing the affinity test results of the nanobody binding to human Nectin-4 protein in this invention; Figure 6 This is a graph showing the affinity test results of the nanobody binding to human Nectin-4 protein in this invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Coldspring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0018] Example 1 (Preparation of Nanobodies) 1. Animal Immunization: Alpaca (item number M147) were used for immunization. The immunizing agents were recombinant human Nectin-4 / hFc (purchased from Biointron, catalog number B21629407) and mouse Nectin-4 / His (purchased from Biointron, catalog number 00FNQ6001) protein. The immunization program consisted of 5 immunizations. The first immunization used complete Freund's adjuvant, followed by incomplete Freund's adjuvant and manganese adjuvant [MnJ(β)]. After the 5th immunization, serum titers were measured. The antiserum showed a H+L titer of 1:64000 and a VHH titer of 1:8000 against human Nectin-4, meeting the requirements for library construction.

[0019] 2. Phage Display Library Construction: Peripheral blood lymphocytes were collected from immunized alpacas, and total RNA was extracted using the Trizol method. cDNA was synthesized using PrimeScript™ II reverse transcriptase. The VHH gene fragment (approximately 500 bp) was specifically amplified by nested PCR and purified using a DNA gel extraction kit. The purified VHH gene was digested with SfiI restriction endonuclease and cloned into a phage display vector digested with the same enzymes. Ligation was performed using T4 DNA ligase. The ligation product was transformed into E. coli competent cells via electroporation to construct a primary library. The library size was determined to be 2.4 × 10^8 pfu, and random clone selection and sequencing confirmed a 100% insertion rate. Subsequently, helper phage M13KO7 was used for rescue and packaging to obtain the phage display library.

[0020] 3. Phage panning and screening: Two strategies were employed for phage panning: solid-phase panning (coating recombinant human / mouse Nectin-4 / His protein) and cell-phase panning (using a CHOK1 cell pool overexpressing human / mouse Nectin-4), each performed twice. After each round of panning, the output / input ratio significantly increased, indicating effective enrichment of specific phages. Clones were selected from the final round of panning products and subjected to high-throughput screening using phage ELISA (detecting binding to the antigen protein) and phage FACS (detecting binding to cells expressing the antigen), yielding a total of 283 positive clones. Sequencing of the positive clones ultimately yielded 36 unique VHH sequences.

[0021] 4. Nanobody Expression and Purification: From the unique sequences screened above, the target VHH gene fragment (e.g., the gene encoding SEQ ID NO: 1) was subcloned into a mammalian cell expression vector suitable for CHO cell expression. This vector was designed to fuse the human IgG1 Fc fragment (hFc) at the C-terminus of the VHH sequence to achieve secretory expression and facilitate Protein A purification. The correctly constructed recombinant plasmid was transiently transfected into CHO cells and expressed under culture conditions of 37°C and 5% CO2. The cell culture supernatant was collected, centrifuged, filtered, clarified, and then captured and purified using a Protein A affinity chromatography column, including steps such as loading, washing, low pH elution, and neutralization. The purified protein solution was replaced with PBS buffer and concentrated to obtain high-purity VHH-hFc fusion protein. Purity was detected by SDS-PAGE and SEC-HPLC, and concentration was determined using NanoDrop. Those skilled in the art will understand that this fusion protein is merely an example constructed for ease of detection and purification, and its binding activity is entirely determined by the nanobody portion (VHH) therein. The core of the present invention is the nanobody fragment itself (i.e., the VHH portion, such as the sequence shown in SEQ ID NO: 1 or the sequence defined by its CDR region).

[0022] 5. Characterization of VHH-hFc fusion protein: To facilitate the detection of binding activity and affinity in subsequent embodiments, the purified VHH-hFc fusion protein was prepared into a solution, referred to as the "antibody solution." In the following embodiments, unless otherwise specified, "antibody to be tested," "nanobody of this patent," or "Anti-Nectin-4 VHH" all refer to this VHH-hFc fusion protein solution at the experimental level, but the detection data are used to characterize and demonstrate the binding characteristics of the core nanobody fragment (VHH) of this invention.

[0023] Example 2 (Detection of antibody affinity using ELISA method) 1. Recombinant humanNectin-4 protein (purchased from Biointron, catalog number B21629406, purity >95%, SEC-HPLC purity 99.047%, endotoxin <1 EU / mg, activity EC50 0.6825 nM) was dissolved in buffer to a concentration of 2 μg / mL. 100 μL of the coating was added to each well of a 96-well plate, and the plate was incubated at 25°C for 2 hours. The plate was washed once with 0.05% PBST to prepare a humanNectin-4 protein ELISA detection plate. 2. Recombinant mouseNectin-4 protein (purchased from Biointron, catalog number 00FNQ6001, purity >95%, SEC-HPLC purity 99.363%, endotoxin <1000 EU / mg, activity EC50 7.689) was used. The nM protein was dissolved in buffer to a concentration of 2 μg / mL. 100 μL of the coating was added to each well of a 96-well plate and incubated at 25°C for 2 hours. The plate was then washed once with 0.05% PBST to prepare a mouseNectin-4 protein ELISA detection plate. 3. Prepare an antibody solution from the Anti-Nectin-4 VHH-hFc fusion protein (containing the nanobody sequence shown in SEQ ID NO: 1) obtained according to Example 1 (Preparation of Nanobodies). Serially dilute the antibody solution 4-fold to create five gradients: 100 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM.

[0024] 4. Add antibody solutions of different concentrations to the ELISA test plate, adding 100 μL of the antibody to each well. Incubate at 25°C for 1 hour, then wash three times with 0.05% PBST. Next, use anti-human IgG-Fc, HRP (GenScript Biotech, A01854-200), adding 100 μL of each well. Incubate at 25°C for 30 minutes, then wash four times with 0.05% PBST. Add 100 μL of substrate to each well.

[0025] 5. After incubating at room temperature for 15 minutes, measure the absorbance at 415 nm using an ELISA reader and record the value.

[0026] Test results as follows Figure 1 , 2 (The horizontal axis represents antibody concentration, and the vertical axis represents OD415 absorbance; a higher absorbance indicates greater antibody binding.) See Tables 1 and 2 for further details. Table 1 [ELISA detection of binding of nanobodies (VHH-hFc form) to human Nectin-4] Antibody Name EC50 (nM) BMax Anti-Nectin-4 VHH-hFc 0.6125 2.53 Enfortumab (positive control) 2.226 2.686 Anti-HEL hlgGl (negative control) No binding Not applicable Table 2 [ELISA detection of binding of nanobodies (VHH-hFc form) to mouse Nectin-4] Antibody Name EC50 (nM) BMax Anti-Nectin-4 VHH-hFc 0.4821 2.173 Human Nectin-1 protein (positive control) Weak binding Not applicable Anti-HEL hlgGl (negative control) No binding Not applicable Results analysis: Table 1 shows that the EC50 of the nanobody of this patent against human Nectin-4 is 0.6125 nM, which is better than the positive control; Table 2 shows that its EC50 against mouse Nectin-4 is 0.4821 nM, indicating that the nanobody has excellent human-mouse cross-reactivity.

[0027] Example 3 (Detection of antibody affinity by flow cytometry) 1. CHOK1-Human Nectin-4 cell pool (purchased from Biointron, catalog number B21629406), CHOK1-Mouse Nectin-4 cell pool (purchased from Biointron, catalog number 00FNQ6001) and blank CHOK1 cells were used for subsequent flow cytometry detection. 2. Seed CHOK1-Human Nectin-4 cells into 96-well plates, with a seeding density of 1.5E+05 cells per well; 3. Seed CHOK1-Mouse Nectin-4 cells into 96-well plates, with a seeding density of 1.5E+05 cells per well; 4. Seed blank CHO-K1 cells into 96-well plates, with a seeding density of 1.5E+05 cells per well; 5. Prepare serially diluted Anti-Nectin-4 VHH-hFc fusion protein antibody solutions using PBS, resulting in 100 nM, 25 mM, 12.5 nM, 6.25 nM, and 3.125 nM solutions. Add 50 μL of each solution to the wells and incubate at 4 °C for 1 h in a chromatography cabinet. Wash twice with MACS buffer (Miltenyi, 130-091-221). 6. Using Alexa Fluor® 647 Affini Pure Goat Anti-Human IgG, Fcγ fragment specific (Jakson / 115-605-071), add 100 μL to each test well, place in a chromatography cabinet, incubate at 4°C for 30 minutes, and wash twice with MACS buffer. 7. Use flow cytometry to detect and record fluorescence intensity.

[0028] Test results as followsFigure 3 , 4 (The horizontal axis represents antibody concentration, and the vertical axis represents the average fluorescence intensity of the M3 signal; a higher intensity indicates greater antibody binding.) See also Tables 3 and 4: Table 3 [Flow cytometry detection of binding of nanobodies (VHH-hFc form) to CHOK1-Human Nectin-4 cells] Antibody Name EC50 (nM) BMax (MFI) Anti-Nectin-4 VHH-hFc 1.353 734332 Enfortumab (positive control) 4.357 725016 Anti-HEL human IgGl (negative control) No binding Not applicable Table 4 [Flow cytometry detection of binding of nanobodies (VHH-hFc form) to CHOK1-Mouse Nectin-4 cells] Antibody Name EC50 (nM) BMax (MFI) Anti-Nectin-4 VHH-hFc 2.978 735567 Human Nectin-1 protein (positive control) 18.29 849966 Anti-HEL human IgGl (negative control) No binding Not applicable Results analysis: Table 3 shows that the EC50 of the proposed nanobody on human Nectin-4 expressing cells was 1.353 nM, which is superior to the positive control; Table 4 shows that its EC50 on mouse Nectin-4 expressing cells was 2.978 nM, further confirming its good human-mouse cross-reactivity. Furthermore, the nanobody did not bind to blank CHOK1 cells, demonstrating good specificity.

[0029] Example 4 (Detection of antibody affinity using surface plasmon resonance method) 1. Affinity testing of the selected antibody was performed using a Biacore 8k instrument. The run buffer was HBS-EP+. The VHH-hFc fusion protein was captured using a Protein A chip (Cytiva, trade number 29127558).

[0030] 2. Prepare the Anti-Nectin-4 VHH-hFc antibody solution and the analytes: human Nectin-4 / His (Biointron, trade number B21629406) and mouse Nectin-4 / His (Biointron, trade number 00FNQ6001). Perform 2-fold serial dilutions of the analytes.

[0031] 3. Capture VHH-hFc to approximately 120-180 RU at a flow rate of 10 μL / min. Binding and dissociation flow rates were 30 μL / min, with a binding time of 120 s and a dissociation time of 200 s. Regeneration was performed using 10 mM Gly-HCl (pH 1.5) at a flow rate of 30 μL / min for 30 s. The fitted model was a 1:1 binding ratio.

[0032] Test results as follows Figure 5 , 6 (X-axis, unit - seconds: represents the change of time during the experiment, usually starting from the baseline, through the binding phase, to the dissociation phase; Y-axis, unit - RU: represents the intensity of the binding reaction, the higher the RU value, the more molecules are bound) and as shown in Tables 5 and 6: Table 5 [SPR assay of affinity of nanobodies (VHH-hFc form) for human Nectin-4] Ligand Capture concentration Analyte Analyte concentration ka (1 / Ms) kd (1 / s) KD (M) Anti-Nectin-4 VHH-hFc 0.5 ug / ml Human Nectin4 / His 1.5625-50 nM (2-fold gradient) 1.77E+06 9.31E-03 5.24E-09 Table 6 [SPR assay of affinity of nanobodies (VHH-hFc form) for mouse Nectin-4] Ligand Capture concentration Analyte Analyte concentration ka (1 / Ms) kd (1 / s) KD (M) ANb1379-M147-5M-CP1R2P1-80 (VHH-hFc format) 0.5 ug / ml Mouse Nectin4 / His 1.5625-50 nM (2-fold gradient) 1.70E+06 3.08E-02 1.81E-08 Results analysis: Table 5 shows that the nanobody of this patent has a nanomolar affinity for human Nectin-4 (KD = 5.24 nM). Table 6 shows that it also maintains a nanomolar affinity for mouse Nectin-4 (KD = 18.1 nM), further demonstrating its effective human-mouse cross-reactivity.

[0033] The foregoing has provided a detailed description of one embodiment of the present invention, but this description is merely a preferred embodiment and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the claims of this invention should still fall within the patent coverage of this invention.

Claims

1. A nanobody fragment capable of targeting and recognizing Nectin-4 protein, characterized in that: The nanobody fragment V H The complementarity-determining region (CDR) of the chain has an amino acid sequence selected from the group consisting of CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO:

4.

2. A nanobody fragment capable of targeting and recognizing Nectin-4 protein, characterized in that... Its V H The amino acid sequence of the chain is shown in SEQ ID NO:

1.

3. The nanobody fragment according to any one of claims 1 or 2, characterized in that: It can specifically bind to human Nectin-4 protein and has cross-reactivity with mouse Nectin-4 protein.

4. The nanobody according to claim 1 or 2, characterized in that: The nanobody has a molecular weight of 12-15 kDa and exists in monomeric form under non-reducing conditions.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the nanobody fragment as described in claim 1 or 2, and the dosage form of the pharmaceutical composition includes an injection, a lyophilized powder for injection, or a sustained-release formulation.

6. An application of a nanobody fragment capable of targeting and recognizing Nectin-4 protein, characterized in that... This includes the use of the nanobody fragments described in claim 1 or 2 in the preparation of antibody drugs, antibody-drug conjugates, and cell therapy drugs.

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