Nanobodies specifically binding to transmembrane protein 132a and uses thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a kind of nanobody specifically binding transmembrane protein 132A and application thereof.The amino acid sequence of the nanobody is as shown in SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.24.The present application first develops monoclonal nanobody targeting the mutual area of extracellular region of transmembrane protein 132A and extracellular region of EGFR, solves the bottleneck problem that although existing transmembrane protein 132A antibody targets its extracellular region, the targeted area is not located at the direct interaction interface of transmembrane protein 132A and EGFR, and cannot be used for functional study and targeted therapy, intervenes EGFR signal pathway from the angle of brand-new mechanism of action, can cover more extensive solid tumor patient population, and realizes broad-spectrum antitumor therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nanobody that specifically binds to transmembrane protein 132A and its applications. Background Technology

[0002] Solid tumors (including breast cancer and lung cancer) account for more than 80% of all cancer types. While traditional surgery, radiotherapy, and chemotherapy can control tumor progression to some extent, they suffer from significant side effects, high recurrence rates, and narrow therapeutic windows. Molecular targeted therapy, as an important strategy for cancer treatment, precisely targets tumors by specifically identifying and binding to specific molecular targets on or inside tumor cells, offering higher specificity and lower toxicity compared to traditional treatments. However, current molecular targeted therapies still face numerous challenges, with drug resistance and low treatment response rates being prominent issues. Existing targeted drugs typically only cover a specific tumor type. For example, HER2 monoclonal antibodies are mainly used for HER2-positive breast cancer, while triple-negative breast cancer, which accounts for 15-20% of breast cancers, lacks HER2 expression, rendering current targeted drugs ineffective. EGFR tyrosine kinase inhibitors are effective for lung adenocarcinoma carrying EGFR mutations, but have limited efficacy against EGFR mutation-negative but highly EGFR-expressing squamous cell carcinoma of the lung. Although EGFR-targeting drugs such as cetuximab and panitumumab have been approved for use in colorectal cancer and head and neck squamous cell carcinoma, their benefit population is limited, they are ineffective against most solid tumors, and they have significant adverse reactions. The development of drug resistance and low treatment response rates also severely restrict the clinical application and effectiveness of existing targeted drugs.

[0003] Transmembrane protein 132A (TMEM132A) exhibits abnormally high expression in various malignant tumors. Specifically, bioinformatics analysis has shown that TMEM132A expression levels are significantly abnormal in various solid tumors such as breast cancer, lung cancer, gastric cancer, kidney cancer, and bladder cancer, and is closely related to overall survival, suggesting its potential as a pan-cancer biomarker. However, current research on TMEM132A is limited to correlation analysis; its specific biological functions and molecular mechanisms in tumors have not been systematically elucidated, and there is a lack of specific tools and therapeutic approaches targeting this protein. Existing TMEM132A antibodies are polyclonal antibodies, and although they target the extracellular region, no antibody labeling is available for use in immunoprecipitation experiments to capture TMEM132A in mixed proteins. Furthermore, no existing antibodies have been reported for targeted therapy.

[0004] Nanobodies (also known as single-domain antibodies or VHH antibodies) consist of only a single heavy chain variable region. Compared to traditional monoclonal antibodies, they possess unique advantages such as small molecular weight, high stability, strong affinity, easy penetration of tumor tissue, and the ability to target epitopes that are difficult for traditional antibodies to access. They can effectively overcome the barrier of the dense microenvironment of solid tumors and precisely bind to targets, perfectly meeting the needs of broad-spectrum targeted therapy for solid tumors, and have become a research hotspot in the field of cancer targeted therapy. Currently, several nanobody-related drugs have been approved for marketing or have entered clinical research stages, demonstrating broad application prospects in cancer treatment.

[0005] However, there are currently no reports on nanobodies targeting TMEM132A. More importantly, TMEM132A expression is restricted in normal tissues, mainly distributed in the cerebral cortex and esophagus. Its tumor-specific high expression can minimize off-target side effects on normal tissues. Therefore, developing nanobodies specifically targeting TMEM132A is of great significance for novel broad-spectrum targeted therapy of solid tumors in clinical practice. Summary of the Invention

[0006] Objective of the Invention: The technical problem to be solved by this invention is the limitation of existing tumor-targeted therapies, such as limited target sites, limited beneficiaries, and significant toxic side effects. This invention provides a nanobody that specifically binds to transmembrane protein 132A and its applications. The nanobody described in this invention can specifically recognize the recombinant TMEM132A protein, block the specific binding of the extracellular region of TMEM132A to the extracellular region of EGFR, and effectively inhibit the 3D proliferation of tumor cells such as breast cancer and kidney cancer.

[0007] To address the aforementioned technical problems, this invention discloses a nanobody that specifically binds to transmembrane protein 132A and its applications. The specific technical solution is as follows: In a first aspect, the present invention provides a nanobody that specifically binds to and targets transmembrane protein 132A, said nanobody comprising a complementarity-determining region, said complementarity-determining region comprising the amino acid sequences CDR1, CDR2 and CDR3 as shown below: The amino acid sequences are as shown in SEQ ID NO. 5 for CDR1, as shown in SEQ ID NO. 7 for CDR2, and as shown in SEQ ID NO. 9 for CDR3; or, The amino acid sequence is as shown in SEQ ID NO. 18 for CDR1, as shown in SEQ ID NO. 20 for CDR2, and as shown in SEQ ID NO. 22 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.12 for CDR1, as shown in SEQ ID NO.14 for CDR2, and as shown in SEQ ID NO.16 for CDR3.

[0008] The nanobody further includes a framework region, which comprises the amino acid sequences FR1, FR2, FR3, and FR4 as shown below: The amino acid sequences are as shown in SEQ ID NO. 4 for FR1, SEQ ID NO. 6 for FR2, SEQ ID NO. 8 for FR3, and SEQ ID NO. 10 for FR4; or, The amino acid sequences are as shown in SEQ ID NO. 17 for FR1, SEQ ID NO. 19 for FR2, SEQ ID NO. 21 for FR3, and SEQ ID NO. 10 for FR4; or, The amino acid sequences are as shown in SEQ ID NO.11 for FR1, SEQ ID NO.13 for FR2, SEQ ID NO.15 for FR3, and SEQ ID NO.10 for FR4.

[0009] The amino acid sequences of the nanobodies are shown in SEQ ID NO.23, SEQ ID NO.25, or SEQ ID NO.24. The amino acid sequence shown in SEQ ID NO.23 is named nanobodies #1, the amino acid sequence shown in SEQ ID NO.25 is named nanobodies #3, and the amino acid sequence shown in SEQ ID NO.24 is named nanobodies #2. In some embodiments of the present invention, nanobodies #1 exhibit the strongest binding affinity to the polypeptide sequence shown in SEQ ID NO.2, while nanobodies #3 and #2 exhibit the strongest binding affinity to the polypeptide sequence shown in SEQ ID NO.3. The polypeptide sequences shown in SEQ ID NO.1 and SEQ ID NO.3 contain a site of TMEM132A that specifically interacts with the extracellular region of EGFR. This amino acid sequence is located in the extracellular region of TMEM132A. Using this polypeptide, nanobodies can be screened as a screening antigen to effectively block the interaction between TMEM132A and EGFR, thereby reducing the stability of EGFR on the surface of cancer cells.

[0010] In a second aspect, the present invention provides a method for preparing the nanobody described in the first aspect, comprising the following steps: The phage library was screened for 3-8 rounds using polypeptide sequences with amino acid sequences as shown in SEQ ID NO. 1-3 to obtain the nanobodies. In some embodiments of the present invention, the following steps are specifically included: (1) Using a polypeptide segment containing the extracellular region of TMEM132A (a total of 3 different polypeptide segments, the amino acid sequences of which are shown in SEQ ID NO.1~3), mixed with a trillion-level CamNano phage library, allowing specifically bound phages to adsorb onto the antigen (polypeptide), while non-specifically bound phages are removed by a washing step; (2) Specifically bound phages are eluted from the antigen using an acidic elution buffer. The eluted phages are then used to infect host bacteria, amplified, and prepared for the next round of screening. (3) To improve the affinity and specificity of the nanobodies, six rounds of specific screening of peptide antigens were performed. Monoclonal phages were picked by limiting dilution or cloning, and crude extracts of recombinant nanobodies were obtained; (4) Enzyme-linked immunosorbent assay (ELISA) was used to verify the binding efficiency of crude extracts of soluble recombinant nanobodies from monoclonal bacteriophages to polypeptide fragments to ensure their specificity. Monoclonal clones with positive ELISA results (positive well read / negative well read ≥ 3, positive well read - negative well read ≥ 1) were selected. Positive monoclonal clones were sequenced, and sequencing analysis yielded the correct antibody sequence.

[0011] Thirdly, the present invention provides a fusion protein, which includes the nanobody described in the first aspect and also includes the immunoglobulin Fc region.

[0012] The immunoglobulin is IgG. In some embodiments of the present invention, the Fc region of the immunoglobulin is the Fc region of mouse IgG, and its nucleotide sequence is shown in SEQ ID NO.29. This makes the nanobody easier to purify subsequently. Purification is performed using a protein G magnetic bead gravity column, which is simple and efficient.

[0013] Fourthly, the present invention provides a nucleic acid molecule encoding the nanobody described in the first aspect or the fusion protein described in the third aspect. The nucleotide sequence of the nucleic acid molecule encoding the nanobody described in the first aspect is shown in SEQ ID NO.26, SEQ ID NO.28, or SEQ ID NO.27.

[0014] In some embodiments of the present invention, the present invention also provides a recombinant vector comprising the nucleic acid molecules described in the fourth aspect.

[0015] Fifthly, the present invention provides the use of the nanobody described in the first aspect or the fusion protein described in the third aspect in the preparation of drugs for treating solid tumors. The solid tumors include any one of renal cell carcinoma, neuroblastoma, prostate cancer, breast cancer, and lung cancer. The nanobody described in the first aspect or the fusion protein described in the third aspect can inhibit the 3D proliferation of various solid tumor cells, specifically including, but not limited to, renal clear cell carcinoma cells (such as 769-P), neuroblastoma cells (such as SKN-BE2), prostate cancer cells (such as LNCAP-C4-2), breast cancer cells (such as the TNBC PDX cell line), and lung cancer cells (such as A549).

[0016] In some embodiments of the present invention, in 3-D culture, the nanobodies #1 and #3 of the present invention can significantly inhibit the formation and growth of tumor cells at a concentration of 10 μg / mL, with an inhibition rate of over 40%, and an inhibition rate of up to 80% in breast cancer, and the inhibition rate is dose-dependent.

[0017] Sixthly, the present invention provides the application of the nanobody described in the first aspect or the fusion protein described in the third aspect in blocking the interaction between transmembrane protein 132A and EGFR, wherein the application is for purposes other than disease diagnosis and / or treatment. The blocking effect can act on various solid tumor cells, including but not limited to any one or more combinations of breast cancer, clear cell renal cell carcinoma, lung cancer, neuroblastoma, and prostate cancer.

[0018] In a seventh aspect, the present invention also provides the application of the nanobody described in the first aspect or the fusion protein described in the third aspect in the preparation of kits for detecting the expression level of transmembrane protein 132A in vitro and / or kits for detecting transmembrane protein 132A interacting proteins. In some embodiments of the present invention, the nanobody captures TMEM132A in the sample to be tested in vitro; specifically, it can be used in co-immunoprecipitation (Co-IP) experiments, using the specific binding effect of the nanobody described in the present invention to enrich and pull down TMEM132A from biological samples, so as to achieve the separation, extraction and protein interaction analysis of TMEM132A.

[0019] Beneficial effects: This invention is the first to discover and identify the direct interaction interface between the extracellular region of TMEM132A and the extracellular region of EGFR, and to determine the key amino acid sequence involved in the interaction, providing a clear target for intervening in this interaction; and successfully using this key segment to screen and obtain nanobodies. This invention is also the first to develop a monoclonal nanobody targeting the interaction region between the extracellular regions of TMEM132A and EGFR. While existing TMEM132A antibodies target the extracellular region of TMEM132A, the target region is not located at the direct interaction interface between TMEM132A and EGFR. The TMEM132A antibody provided by this invention solves the bottleneck problem that existing antibodies cannot be used for functional studies and targeted therapy. Specifically, this invention has the following advantages compared to the prior art: (1) Novel target strategy: This invention develops nanobodies targeting the interaction region between the extracellular region of membrane protein TMEM132A and the extracellular region of EGFR, intervening in the EGFR signaling pathway from a novel mechanism of action, which can cover a wider range of solid tumor patients and achieve broad-spectrum anti-tumor therapy. (2) Highly efficient blocking of interactions: The nanobody of the present invention can specifically recognize and bind to the interaction region between TMEM132A and EGFR, and efficiently block the protein interaction between TMEM132A and EGFR, thereby inhibiting the abnormal activation of EGFR from the source. (3) Promote EGFR degradation: The nanobody of the present invention can promote EGFR degradation and significantly reduce EGFR protein level by efficiently blocking the protein interaction between TMEM132A and EGFR, thereby blocking the stabilizing effect of TMEM132A on EGFR. (4) Broad-spectrum antitumor activity: The nanobody of the present invention showed significant antiproliferative activity in three-dimensional culture models of various solid tumor cells, including but not limited to breast cancer, renal cancer, lung cancer, prostate cancer, neuroblastoma, etc., and has broad-spectrum therapeutic potential; (5) Good tumor targeting: Since the expression of TMEM132A is limited in normal tissues (especially the cerebral cortex, esophagus, etc.), the nanobody of the present invention has better selectivity for tumor tissues and can minimize off-target toxicity to normal tissues. (6) Safety advantages: Nanobodies have small molecular weight and low immunogenicity, which can effectively break through the barrier of the dense microenvironment of solid tumors and accurately bind to the target, perfectly meeting the needs of broad-spectrum targeted therapy for solid tumors; and Fc fusion protein can realize the simple and efficient purification and expression of nanobodies. (7) Broad application prospects: This invention can provide novel candidate drugs for tumor targeted therapy based on the TMEM132A-EGFR signaling axis. It can be used alone as an anti-tumor drug or in combination with traditional targeted drugs, chemotherapy drugs, immunotherapy drugs, etc., and has broad clinical application prospects. Existing EGFR-targeting monoclonal antibodies (such as cetuximab, panitumumab, etc.) directly target the extracellular III region of EGFR itself, and exert their effects by blocking ligand binding or receptor dimerization. Their target is the EGFR molecule. The nanobody of this invention targets the interaction region between TMEM132A and EGFR, rather than EGFR itself. The target is TMEM132A (the upstream regulatory molecule of EGFR). Due to the different target, this invention may provide new treatment options for the following patients: (a) patients who are unresponsive to or unsuitable for existing EGFR monoclonal antibody therapy (e.g., without high EGFR expression); (b) patients who are resistant to EGFR targeted therapy. Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 This is a map of a recombinant plasmid containing a nanobody-Fc fusion protein.

[0022] Figure 2 The image shows the SDS-PAGE results of the purified nanobody-Fc fusion protein.

[0023] Figure 3 The image shows the experimental results of TMEM132A capture in cells by antibody co-immunoprecipitation (Co-IP), where EV represents cells stably expressing the empty vector (pHAGE-CMV-3×Flag); and Flag-TMEM132A represents cells stably expressing the TMEM132A vector (pHAGE-CMV-TMEM132A-3×Flag).

[0024] Figure 4 The results of the co-immunoprecipitation (Co-IP) experiment show that the antibody blocks the interaction between EGFR and TMEM132A in vitro.

[0025] Figure 5 This is an experiment to test the stability of EGFR.

[0026] Figure 6 This is a diagram showing the results of a 3D proliferation inhibition experiment on PDX cells for breast cancer.

[0027] Figure 7The images show the results of 3D proliferation inhibition experiments on other tumor cells. In the images, A represents A549 cells, B represents SKN-BE2 cells, C represents 769-P cells, and D represents LNCAP-C4-2 cells.

[0028] Figure 8 The graph shows the results of in vivo safety testing of body weight and biochemical indicators. In the graph, A is body weight, B is total protein (TP), C is blood glucose (GLU), D is creatinine (Crea), E is alkaline phosphatase (ALP), F is alanine aminotransferase (ALT), and G is aspartate aminotransferase (AST).

[0029] Figure 9 HE staining results of mouse heart, liver, spleen, lung and kidney tissue sections.

[0030] Figure 10 This is an overview diagram of the experimental process for this application. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0032] Example 1 Screening of nanobodies This embodiment first designs and synthesizes antigenic peptides for subsequent immune screening. Based on the interaction region analysis between TMEM132A and EGFR, the following peptide antigens were synthesized, with the specific peptide sequences as follows: SEQ ID NO.1: CWWRRLRASLR; SEQ ID NO.2: CAPLLPLRIELT; SEQ ID NO. 3: CVDFWWRRLRASL.

[0033] Next, phage screening of nanobodies is performed, which includes the following steps: (1) After mixing three different polypeptide segments (SEQ ID NO.1~3), they were mixed with a trillion-level CamNano phage library. The phages that specifically bind to the antigen (polypeptide) are adsorbed, and the non-specifically bound phages are washed away.

[0034] (2) The specifically bound phages are eluted from the antigen using an acidic elution buffer. The eluted phages are then used to infect host bacteria, amplified, and prepared for the next round of screening.

[0035] (3) In order to improve the affinity and specificity of nanobodies, multiple rounds of panning are usually required (a total of 6 rounds of specific panning of peptide antigens are performed). Monoclonal phages are picked by limiting dilution or cloning, and crude extracts of recombinant nanobodies are obtained.

[0036] (4) Enzyme-linked immunosorbent assay (ELISA) was used to verify the binding efficiency of crude extracts of soluble recombinant nanobodies from monoclonal bacteriophages to polypeptide fragments to ensure their specificity. ELISA results showed that a positive well read / negative well read ratio (PE-ELISA ratio) ≥3 and a positive well read / negative well read difference (PE-ELISA difference) ≥1 indicated a positive monoclonal cell. The positive monoclonal cells were sequenced to obtain the amino acid sequence of the nanobodies.

[0037] (5) A total of 3 positive clones were obtained. The PE-ELISA and sequencing results are shown in Table 1. The antibody with clone number #1 had the strongest binding affinity to peptide SEQ ID NO.2, while the antibodies with clone numbers #2 and #3 both bound to peptide SEQ ID NO.3.

[0038] Table 1 ELISA Results

[0039] The sequencing results were compared using DNASTAR, and the nanobodies were classified according to their CDR3 region sequences. The CDR partitions of nanobodies #1, #2, and #3 (according to the IMGT numbering scheme) are as follows: The specific sequence partitions of nanobody #1 include: the amino acid sequences of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are shown in SEQ ID NO.4~10 respectively; the amino acid sequence of nanobody #1 is shown in SEQ ID NO.23, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.26.

[0040] The sequence partition of nanobody #2 includes: the amino acid sequences of FR1, CDR1, FR2, CDR2, FR3 and CDR3 are shown in SEQ ID NO.11~16 respectively, the amino acid sequence of FR4 is shown in SEQ ID NO.10; the amino acid sequence of nanobody #2 is shown in SEQ ID NO.24, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.27.

[0041] The sequence partition of nanobody #3 includes: the amino acid sequences of FR1, CDR1, FR2, CDR2, FR3 and CDR3 are shown in SEQ ID NO.17~22 respectively, the amino acid sequence of FR4 is shown in SEQ ID NO.10; the amino acid sequence of nanobody #3 is shown in SEQ ID NO.25, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.28.

[0042] Example 2 Construction and expression of nanobody-Fc fusion protein Using the pcDNA3.4 eukaryotic expression vector as a backbone, recombinant vector sequences for expressing the three nanobodies screened in Example 1 were designed, resulting in the construction of three recombinant expression plasmids. The transcriptional unit elements of each recombinant expression plasmid, from 5' to 3', are: the CMV promoter (inherent in pcDNA3.4), the interleukin-2 (IL-2) signal peptide coding sequence (SEQ ID NO. 30), the nanobodies coding sequence obtained from the screening, and the mouse IgG1 Fc fragment coding sequence (SEQ ID NO. 29). The IL-2 signal peptide and the nanobodies, as well as the nanobodies and the mouse IgG1 Fc gene sequences, are directly tandem, as shown below. Figure 1 As shown, the full-length recombinant plasmid sequences were all synthesized artificially using whole-genome chemical synthesis. The synthesized products were verified to have no mutations by sequencing and alignment, resulting in three target recombinant eukaryotic expression plasmids.

[0043] The obtained recombinant plasmid was transfected into HEK293F cells for expression, and purified by Protein G affinity chromatography to obtain nanobody-Fc fusion proteins. The amino acid sequences of nanobody #1-Fc fusion protein are shown in SEQ ID NO.31; nanobody #2-Fc fusion protein are shown in SEQ ID NO.32; and nanobody #3-Fc fusion protein are shown in SEQ ID NO.33. The transfection process was as follows: HEK293F cells were digested and cultured in T175 flasks at a cell density of 1.2 × 10⁻⁶ cells / year. 6Cells / mL, cultured overnight, with fresh medium replaced 1 hour before transfection. The transfection mixture was prepared according to the following ratios: 25 μg recombinant plasmid DNA was mixed with 1 mL Opti-MEM to obtain the first mixture; 75 μL PEI solution (1 mg / mL, PEI Max, Polysciences, 24765-1, MW 58.44) was mixed with 1 mL Opti-MEM to obtain the second mixture; the second mixture was slowly added dropwise to the first mixture (plasmid DNA:PEI solution = 1:3 mg / mL), mixing by pipetting, and incubated at room temperature for 15 min to obtain the final mixture. The final mixture was added dropwise to HEK293F cells, and the cells were mixed by cross-linking in the culture flask before being returned to the incubator for transfection. On day 5 post-transfection, the culture medium was collected once, centrifuged at 1500 rpm for 5 min, and the cells were removed. The supernatant was used for protein purification to obtain the nanobody-Fc fusion protein. The specific method for protein purification is as follows: Take a gravity chromatography column of appropriate specifications, install the lower gasket, add an appropriate amount of pure water to rinse the column and gasket, and close the lower outlet. Mix Protein G Beads thoroughly, use a pipette tip to draw an appropriate amount of slurry and add it to the gravity column, and open the lower outlet to drain the protective solution. Add an appropriate amount of pure water to rinse the medium, and after the liquid in the column has drained by gravity, close the lower outlet. Add 5 column volumes of equilibration buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.0) to equilibrate, so that the packing material is in the same buffer system as the target protein, and repeat 3 times. Add the sample to the equilibrated gravity column, retain the sample for at least 2 minutes, and collect the eluent. Repeated loading can increase the binding efficiency. Wash with 10-15 column volumes of washing buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.0) to remove non-specifically adsorbed contaminating proteins, and collect the washing buffer. Elute with 5–10 column volumes of eluent (0.1 M glycine, pH 3.0), collect fractions, one tube for each column volume, and determine A. 280 Read the value until elution fraction A. 280 The reading is 0. The elution fraction is neutralized immediately with 1 / 10 the volume of neutralizing buffer (1M Tris-HCl, pH 8.5). The purification effect is then assessed using SDS-PAGE of the obtained sample (including the elution fraction, wash fraction, and elution fraction) and the original sample, as needed. Figure 2 As shown.

[0044] Example 3: Co-immunoprecipitation (Co-IP) of TMEM132A captured by nanobody-Fc fusion protein. This embodiment verifies the capture effect of the nanobody (#1, #2 and #3)-Fc fusion protein prepared in Example 2 on TMEM132A in cell lysate. The specific method is as follows: 769-P cells were infected with either a virus expressing the empty vector (pHAGE-CMV-3×Flag) or a lentivirus expressing the TMEM132A vector (pHAGE-CMV-TMEM132A-3×Flag) at an MOI of 1. 72 hours post-infection, cells were selected using puromycin (2 μg / mL) until no cells survived in the blank wells, yielding the empty vector control cell line (EV) and the stable overexpression cell line (Flag-TMEM132A). Cell pellets were collected and lysed on ice for 30 min with EBC lysis buffer containing protease and phosphatase inhibitors (0.1 mM EDTA, 50 mM Tris-HCl, 120 mM NaCl, 0.5% v / v NP-40, and 10% v / v glycerol). The cells were then sonicated on ice for 30 s (5 s on, 5 s off, 50% power). After sonication, the cells were centrifuged at 4 °C and 8228 g for 10 min, and the supernatant was collected. A... 280 The absorbance value was used to determine the concentration of the cell lysis buffer. The cell lysis buffer was diluted to 1 mg / mL using EBC lysis buffer. Nanobody (#1, #2, and #3)-Fc fusion protein (final concentration 2 μg / mL) was added to the lysis buffer of the stable overexpressing cell line (Flag-TMEM132A), and the mixture was incubated overnight at 4 °C to capture the overexpressed TMEM132A in the cells. Co-IP experiments were then performed. 10 μL of protein G magnetic beads were added, and the mixture was incubated at 4 °C for 4 h. The mixture was then separated using a magnetic rack to capture all antibody complexes in the mixture. SDS-PAGE analysis was then performed, and the Flag antibody was used to detect TMEM132A in the cell lysis buffer.

[0045] The results are as follows Figure 3 As shown, antibodies #1, #2, and #3 all successfully captured Flag-TMEM132A from the total protein in cell lysates. This means that the nanobody (#1, #2, and #3)-Fc fusion proteins provided by this invention can enrich TMEM132A in biological samples, enabling the separation, extraction, and protein-protein interaction analysis of TMEM132A. Therefore, this invention solves the current problem of the ineffective application of TMEM132A antibodies in IP experiments, providing a reliable tool for related research.

[0046] Example 4: Experiment to block the interaction between TMEM132A and EGFR This embodiment uses the stable overexpression cell line 769-P-Flag-TMEM132A obtained in Example 3 and the empty vector control cell line 769-P-EV to verify the effect of implementing nanobody (#1, #2 and #3)-Fc fusion proteins on the interaction between TMEM132A and EGFR. The specific method is as follows: Co-immunoprecipitation (Co-IP) experiment: Cell lysate with a concentration of 1 mg / mL was prepared according to the method described in Example 3. Nanobody (#1, #2 and #3)-Fc fusion protein (final concentration 10 μg / mL) was added to the lysate of the stable overexpressing cell line (Flag-TMEM132A). The mixture was incubated overnight at 4 °C to block the interaction between the overexpressed TMEM132A in the lysate and the endogenous EGFR in the cells in vitro. Then, the Co-IP experiment was performed (the amount of EGFR protein bound to TMEM132A was detected using EGFR antibody) to detect the inhibitory effect of different nanobodies on the formation of TMEM132A-EGFR complex.

[0047] The results are as follows Figure 4 As shown, antibodies #1 and #3 significantly inhibited the formation of the TMEM132A-EGFR complex; while antibody #2 could not block the formation of the complex.

[0048] Example 5: EGFR Stability Testing Experiment This embodiment examines the effect of the prepared nanobody (#1 and #3)-Fc fusion proteins on EGFR stability. The specific method is as follows: Tumor cell lines (renal carcinoma 786-O cells) were treated with 0, 5, 10, or 20 μg / mL nanobody-Fc fusion protein for 96 h. Cells were then collected, and changes in EGFR protein levels were detected. Results are as follows: Figure 5 As shown, both #1 and #3 treatments resulted in a decrease in EGFR protein levels in cells, exhibiting a dose-dependent effect.

[0049] Example 6: 3D Proliferation Inhibition Experiment of Breast Cancer PDX Cells Breast cancer PDX cell lines were seeded into soft agar, and culture medium containing 0, 5, or 10 μg / mL of nanobody-Fc fusion protein was added. Fresh culture medium containing the corresponding concentration of nanobody-Fc fusion protein was added every 2 days. After 3-4 weeks of culture, INT staining was performed, and the morphology and size of the spheroids were recorded by photography.

[0050] Experimental results are as follows Figure 6 As shown, #1 and #3 nanobodies can inhibit the formation of 3D spheroids in breast cancer PDX tumor cells at a concentration of 5 μg / mL. When the concentration reaches 10 μg / mL, the inhibition rate of 3D spheroids is more than 70%, which is dose-dependent.

[0051] Example 7: Tumor Cell 3D Proliferation Inhibition Experiment Tumor cells (including any one of the following: renal cell carcinoma 769-P, neuroblastoma SKN-BE2, prostate cancer LNCAP-C4-2, and KRAS-mutant lung cancer cells A549) were seeded in soft agar, and medium containing 10 μg / mL nanobody-Fc fusion protein (hereinafter referred to as nanobody) was added. Fresh medium containing 10 μg / mL nanobody was added every 2 days. After culturing for 3-4 weeks, INT staining was performed, and the morphology and size of the spheroids were photographed and recorded. The experiment is as follows. Figure 7 As shown, #1 and #3 nanobodies at a concentration of 10 μg / mL can inhibit the formation of 3D spheres in various tumor cells, with an inhibition rate of over 50%.

[0052] Example 8: Safety Testing To assess the in vivo safety of antibodies #1 and #3 in mice, an orthotopic breast tumor model was established using the mouse breast cancer 4T1 cell line. The test antibodies were administered via intratumoral injection, and their effects on the function of major organs and tissue morphology were examined. Logarithmically growing mouse breast cancer 4T1 cells were resuspended in sterile phosphate-buffered saline (PBS) and inoculated under the fourth pair of mammary fat pads (mammary pads) of female BALB / c mice (2 × 10⁻⁶ cells). 6 (cells / each), until the tumor volume grows to approximately 100-200 mm. 3 Mice were randomly divided into three groups of five each: a control group (PBS), a nanobody #1-Fc fusion protein group, and a nanobody #3-Fc fusion protein group. The administration route was intratumoral injection at a dose of 50 mg / kg (based on tumor tissue weight), administered every three days for five consecutive days. The control group received the same volume of PBS solution via the same method. Mouse body weight and tumor volume were measured every three days.

[0053] On day 17 after drug administration, mice in each group were anesthetized, peripheral blood was collected from the heart, serum was separated, and the levels of biochemical indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA), total protein (TP), and blood glucose (GLU) were measured to assess the potential effects of the test antibody on liver, heart, and systemic cell damage. The experiment is as follows... Figure 8 As shown in A, after injection of nanobodies #1 and #3 into mice, there was no significant change in mouse body weight. Furthermore, the biochemical results are as follows: Figure 8As shown in B~G, after antibody injection, the levels of biochemical indicators such as total protein (TP), blood glucose (GLU), creatinine (Crea), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) remained unchanged, indicating that antibodies #1 and #3 have no potential impact on the liver, heart, etc., and have good safety characteristics.

[0054] In addition, mice were euthanized, and the heart, liver, spleen, lungs, and kidneys were dissected and completely removed. After rinsing with physiological saline, the tissues were fixed in 4% paraformaldehyde solution for histopathological examination. The fixed heart, liver, spleen, lung, and kidney tissues were dehydrated with graded ethanol, embedded in paraffin, sectioned (approximately 4-5 μm thick), and then stained with hematoxylin and eosin (HE) as usual. Morphological changes in each organ tissue were observed under a light microscope, and photographs were taken and recorded to compare whether there were significant pathological changes between the treatment group and the control group. Figure 9 As shown, compared with the control group, the histopathological results of #1 and #3 nanobodies injected into mice showed no obvious pathological damage to the heart, liver, spleen, lungs and kidneys.

[0055] The overall overview of the experimental design described in this invention is as follows: Figure 10 As shown.

[0056] This invention provides a nanobody that specifically binds to transmembrane protein 132A and its application, along with a conceptual approach and method. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A nanobody that specifically binds to transmembrane protein 132A, characterized in that, The nanobody includes a complementarity-determining region, which comprises the amino acid sequences CDR1, CDR2, and CDR3 as shown below: The amino acid sequences are as shown in SEQ ID NO. 5 for CDR1, as shown in SEQ ID NO. 7 for CDR2, and as shown in SEQ ID NO. 9 for CDR3; or, The amino acid sequence is as shown in SEQ ID NO. 18 for CDR1, as shown in SEQ ID NO. 20 for CDR2, and as shown in SEQ ID NO. 22 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.12 for CDR1, as shown in SEQ ID NO.14 for CDR2, and as shown in SEQ ID NO.16 for CDR3.

2. The nanobody according to claim 1, characterized in that, The nanobody includes a framework region comprising the amino acid sequences FR1, FR2, FR3, and FR4 as shown below: The amino acid sequences shown are as follows: CDR1 (SEQ ID NO. 5), CDR2 (SEQ ID NO. 7), and CDR3 (SEQ ID NO. 9), corresponding to FR1 (SEQ ID NO. 4), FR2 (SEQ ID NO. 6), FR3 (SEQ ID NO. 8), and FR4 (SEQ ID NO. 10); or, The amino acid sequences described are as follows: CDR1 as shown in SEQ ID NO. 18, CDR2 as shown in SEQ ID NO. 20, and CDR3 as shown in SEQ ID NO. 22, corresponding to the amino acid sequences FR1 as shown in SEQ ID NO. 17, FR2 as shown in SEQ ID NO. 19, FR3 as shown in SEQ ID NO. 21, and FR4 as shown in SEQ ID NO. 10; or, The amino acid sequences are CDR1 as shown in SEQ ID NO.12, CDR2 as shown in SEQ ID NO.14, and CDR3 as shown in SEQ ID NO.16, corresponding to FR1 as shown in SEQ ID NO.11, FR2 as shown in SEQ ID NO.13, FR3 as shown in SEQ ID NO.15, and FR4 as shown in SEQ ID NO.

10.

3. The nanobody according to claim 2, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.

24.

4. A fusion protein, characterized in that, The fusion protein includes the nanobody according to any one of claims 1 to 3, and further includes an immunoglobulin Fc region, wherein the immunoglobulin Fc region is the mouse IgG Fc region, and its nucleotide sequence is shown in SEQ ID NO. 29; the N-terminus of the immunoglobulin Fc region is attached to the C-terminus of the nanobody.

5. A nucleic acid molecule, characterized in that, It encodes the nanobody as described in any one of claims 1 to 3 or the fusion protein as described in claim 4.

6. The use of the nanobody according to any one of claims 1 to 3 or the fusion protein according to claim 4 in the preparation of a kit for detecting the expression level of transmembrane protein 132A in vitro.

7. The use of the nanobody according to any one of claims 1 to 3 or the fusion protein according to claim 4 in the preparation of an immunoprecipitation reagent, characterized in that, The nanobody or the fusion protein is used to capture transmembrane protein 132A in the sample to be tested in vitro.

8. The application of a nanobody that specifically binds to transmembrane protein 132A in the preparation of a drug for treating solid tumors, wherein the solid tumor is any one of clear cell renal cell carcinoma, neuroblastoma, prostate cancer, breast cancer, and lung cancer; The nanobody includes a complementarity-determining region, which comprises the amino acid sequences CDR1, CDR2, and CDR3 as shown below: The amino acid sequences are as shown in SEQ ID NO. 5 for CDR1, as shown in SEQ ID NO. 7 for CDR2, and as shown in SEQ ID NO. 9 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.18 for CDR1, as shown in SEQ ID NO.20 for CDR2, and as shown in SEQ ID NO.22 for CDR3.

9. The application according to claim 8, characterized in that, The nanobody includes a framework region comprising the amino acid sequences FR1, FR2, FR3, and FR4 as shown below: The amino acid sequences shown are as follows: CDR1 (SEQ ID NO. 5), CDR2 (SEQ ID NO. 7), and CDR3 (SEQ ID NO. 9), corresponding to FR1 (SEQ ID NO. 4), FR2 (SEQ ID NO. 6), FR3 (SEQ ID NO. 8), and FR4 (SEQ ID NO. 10); or, The amino acid sequences described are as follows: CDR1 as shown in SEQ ID NO.18, CDR2 as shown in SEQ ID NO.20, CDR3 as shown in SEQ ID NO.22, corresponding to the amino acid sequences as follows: FR1 as shown in SEQ ID NO.17, FR2 as shown in SEQ ID NO.19, FR3 as shown in SEQ ID NO.21, and FR4 as shown in SEQ ID NO.

10.

10. The application according to claim 9, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.23 or SEQ ID NO.

25.

11. The use of a fusion protein in the preparation of a drug for treating solid tumors, wherein the solid tumor is any one of clear cell renal cell carcinoma, neuroblastoma, prostate cancer, breast cancer, and lung cancer; wherein the fusion protein comprises a nanobody that specifically binds to transmembrane protein 132A, and further comprises an immunoglobulin Fc region, wherein the immunoglobulin Fc region is a mouse IgG Fc region, the nucleotide sequence of which is shown in SEQ ID NO. 29; wherein the N-terminus of the immunoglobulin Fc region is attached to the C-terminus of the nanobody; The nanobody includes a complementarity-determining region, which comprises the amino acid sequences CDR1, CDR2, and CDR3 as shown below: The amino acid sequences are as shown in SEQ ID NO. 5 for CDR1, as shown in SEQ ID NO. 7 for CDR2, and as shown in SEQ ID NO. 9 for CDR3; or, The amino acid sequences are as shown in SEQ ID NO.18 for CDR1, as shown in SEQ ID NO.20 for CDR2, and as shown in SEQ ID NO.22 for CDR3.

12. The application according to claim 11, characterized in that, The nanobody includes a framework region comprising the amino acid sequences FR1, FR2, FR3, and FR4 as shown below: The amino acid sequences shown are as follows: CDR1 (SEQ ID NO. 5), CDR2 (SEQ ID NO. 7), and CDR3 (SEQ ID NO. 9), corresponding to FR1 (SEQ ID NO. 4), FR2 (SEQ ID NO. 6), FR3 (SEQ ID NO. 8), and FR4 (SEQ ID NO. 10); or, The amino acid sequences described are as follows: CDR1 as shown in SEQ ID NO.18, CDR2 as shown in SEQ ID NO.20, CDR3 as shown in SEQ ID NO.22, corresponding to the amino acid sequences as follows: FR1 as shown in SEQ ID NO.17, FR2 as shown in SEQ ID NO.19, FR3 as shown in SEQ ID NO.21, and FR4 as shown in SEQ ID NO.

10.

13. The application according to claim 12, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.23 or SEQ ID NO.25.

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