Use of CHAD-targeting neutralizing antibodies in the preparation of antitumor drugs

Abstract

The application of CHAD-targeting neutralizing antibodies in the preparation of antitumor drugs relates to the field of biomedical technology. This invention is the first to discover that CHAD is highly expressed in CRPC tissues and confirms that CHAD promotes CRPC progression, thus serving as a novel therapeutic target. Anti-CHAD antibodies can specifically bind to the CHAD protein and effectively inhibit tumor growth and improve tumor invasion CD8 in a mouse xenograft model. + The anti-tumor function of T cells. This invention provides a new strategy for the immunotherapy of tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of a neutralizing antibody targeting CHAD in the preparation of antitumor drugs. Background Technology

[0002] Prostate cancer (PCa) is a very common malignant tumor in men. For patients with advanced prostate cancer (APC), androgen deprivation therapy (ADT) is one of the main treatment methods and is often used as an adjunct to local treatment for high-risk prostate cancer. Although most patients respond well to ADT in the early stages and can effectively control the tumor, almost all patients eventually develop castration-resistant prostate cancer (CRPC). CRPC has a poor prognosis, and there is currently a lack of effective treatments. Therefore, elucidating the mechanisms of its occurrence and development and exploring new effective treatments are of great significance for improving the survival rate of CRPC patients.

[0003] The tumor microenvironment is a crucial component of tumor tissue, characterized by a low-oxygen, weakly acidic environment containing stromal cells and non-cellular components. Stromal cells include tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and tumor-infiltrating lymphocytes (TILs); non-cellular components include the extracellular matrix, intercellular fluid, and extracellular cytokines, chemokines, and metabolites. The tumor microenvironment not only provides the "soil" for tumor cell growth and metastasis but also plays a vital role in tumor immune escape and chemotherapy resistance.

[0004] Chondroadherin (CHAD) is an extracellular matrix protein belonging to the small leucine-rich proteoglycan family. It is characterized by a helical domain composed of leucine repeat sequences, which can stably bind to collagen and integrin receptors. In cartilage tissue, CHAD promotes chondrocyte adhesion, matrix remodeling, and activation of key signaling pathways, thereby maintaining tissue integrity. However, CHAD-deficient mice did not exhibit significant cartilage abnormalities or osteoarthritis phenotypes, suggesting that CHAD is not essential for cartilage homeostasis and disease development. Besides cartilage, CHAD is also associated with various malignant tumors, including leiomyosarcoma, clear cell ovarian carcinoma, and breast cancer. Notably, a cyclic polypeptide derived from CHAD (cyc-CHAD) can inhibit bone metastasis and tumor growth in breast cancer, suggesting that CHAD may play an important role in tumor biology. However, the exact function of CHAD in tumor progression remains unclear, particularly its expression characteristics, biological role, and potential as a therapeutic target in castration-resistant prostate cancer, which have not yet been reported. Summary of the Invention

[0005] The present invention aims to provide the use of an antibody capable of specifically binding to the CHAD protein in the preparation of antitumor drugs (such as CHAD-related cancers like castration-resistant prostate cancer), and an antitumor pharmaceutical composition comprising said antibody. The antibody inhibits the biological activity of the CHAD protein in promoting tumor growth. In cases where the tumor is castration-resistant prostate cancer, the antibody also enhances tumor invasion of CD8. + The anti-tumor function of T cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides the use of an antibody capable of specifically binding to the CHAD protein in the preparation of an antitumor drug, wherein the CHAD protein has the amino acid sequence shown in SEQ ID NO: 1, or has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2.

[0008] The tumor in question is a CHAD-related cancer, such as castration-resistant prostate cancer.

[0009] The antibody can inhibit the biological activity of CHAD protein in promoting tumor growth.

[0010] The antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof. For example, the antibody is a monoclonal antibody.

[0011] The antibody can enhance tumor-infiltrating CD8. +The anti-tumor function of T cells.

[0012] In a second aspect, the present invention provides an antitumor pharmaceutical composition comprising an antibody capable of specifically binding to a CHAD protein, and a pharmaceutically acceptable carrier, wherein the CHAD protein has the amino acid sequence shown in SEQ ID NO: 1, or has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2.

[0013] The tumor in question is a CHAD-related cancer, such as castration-resistant prostate cancer.

[0014] The antibody can inhibit the biological activity of CHAD protein in promoting tumor growth.

[0015] The antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof. For example, the antibody is a monoclonal antibody.

[0016] The antibody can enhance tumor-infiltrating CD8. + The anti-tumor function of T cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention first discovered that chondrocyte adhesion protein (CHAD) is highly expressed in castration-resistant prostate cancer (CRPC) tissues. Through transcriptomics analysis, Western blot and immunohistochemical detection, the abnormal expression characteristics of CHAD in CRPC were clarified, providing a molecular basis for its use as a therapeutic target for CRPC.

[0019] (2) The present invention has demonstrated the promoting effect of CHAD on the progression of CRPC through in vivo experiments: In a mouse subcutaneous tumor model, knocking down CHAD expression can reduce tumor volume and weight and significantly inhibit the development of CRPC tumors, indicating that inhibiting CHAD has anti-tumor potential.

[0020] (3) The anti-CHAD antibody of the present invention has significant therapeutic effects in vivo: In the CRPC mouse subcutaneous xenograft model, treatment with the antibody can effectively inhibit tumor growth, and CD8 infiltrates the tumor tissue after treatment. + The anti-tumor function of T cells was significantly improved.

[0021] (4) This invention reveals that CHAD regulates immune cells in the tumor microenvironment and inhibits CD8. + The novel mechanism by which T cells recognize and kill tumor cells, thereby promoting tumor immune escape, provides a new strategy for the immunotherapy of CRPC and has great application prospects. Attached Figure Description

[0022] Figure 1 The diagram shows the results of significantly high CHAD expression in CRPC tumor tissues; where A and B are the transcriptome sequencing results of primary prostate cancer (PPC) and castration-resistant prostate cancer (CRPC) tumor tissues collected from mouse models; C and D are the results of RT-qPCR and Western blot analysis of CHAD expression levels in mouse PPC and CRPC tissues; and E is the result of immunohistochemical (IHC) analysis of CHAD protein expression in human PPC and CRPC tissues.

[0023] Figure 2 The results of CHAD knockdown inhibiting CRPC development are shown in the figure; where A is the Western blot result of Myc-CaP-Control and Myc-CaP-CHAD-KD stable cell lines; B is the result of detecting the proliferation of Myc-CaP-Control and Myc-CaP-CHAD-KD stable cell lines in activated carbon-treated medium (n=3); C, D, and E are the results of subcutaneous tumor formation of 1×10⁶ FVB castrated male rats. 6 Tumor growth curves and tumor weight plots after Myc-CaP-Control and Myc-CaP-CHAD-KD (n=6).

[0024] Figure 3 The diagram shows the preparation process of anti-CHAD neutralizing antibodies. A is the antibody preparation flowchart; B shows the results of SDS-PAGE detection of CHAD protein expression and purification, ELISA determination of serum titers in CHAD-immunized Balb / c mice, and antibody purification; C shows the affinity of the purified antibody to mCHAD protein (with an affinity constant of 2.16 × 10⁻⁶) detected by surface plasmon resonance (SPR) technology. -9 M) Results diagram; D represents the affinity between the antibody and hCHAD protein (its affinity constant is 1.07 × 10⁻⁶). -9 M) Result graph;

[0025] Figure 4 The results show that anti-CHAD neutralizing antibody treatment can significantly inhibit the development of CRPC; where A is a schematic diagram of a mouse model of CRPC treated with neutralizing antibody; B, C, and D are the tumor growth curves and tumor weight graphs of FVB mice after subcutaneous tumor formation and treatment with IgG and anti-CHAD, respectively (n=5). Detailed Implementation

[0026] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0027] Cancer treatment remains a significant challenge in clinical practice. Castration-resistant prostate cancer (CRPC) has a poor prognosis, and currently lacks effective clinical treatments. A key characteristic of CRPC development is its ability to evade the immune system, meaning cancer cells can escape its surveillance and elimination. This is one of the main reasons why current immune checkpoint inhibitors are ineffective in treating CRPC.

[0028] Based on the aforementioned clinical challenges, this invention, through systematic research, has for the first time discovered that chondrocyte adhesion protein (CHAD) plays a crucial role in the development of chronic prostate cancer (CRPC) and identified CHAD as a novel therapeutic target for CRPC. Building upon this, this invention has developed an antibody that specifically binds to CHAD protein and inhibits its biological activity (i.e., a neutralizing antibody specifically targeting CHAD, hereinafter referred to as "anti-CHAD neutralizing antibody" or simply "anti-CHAD antibody"). Experimental results in a mouse xenograft treatment model show that treatment with this anti-CHAD neutralizing antibody significantly inhibits the development of prostate cancer, and that CD8 infiltrates the treated tumor tissue. + The anti-tumor function of T cells was significantly improved. These results indicate that this invention provides a new approach and method for the treatment of CRPC, and has excellent application prospects.

[0029] The following specific examples will provide further explanation.

[0030] The meanings of each sequence number are as follows:

[0031] SEQ ID NO: 1 — Amino acid sequence of the CHAD protein;

[0032] SEQ ID NO: 2 — The nucleotide sequence encoding the CHAD protein shown in SEQ ID NO: 1 (i.e., the CDS region of the CHAD gene);

[0033] SEQ ID NO: 3 — Nucleotide sequence of forward primer EcoRI-F;

[0034] SEQ ID NO: 4 — Nucleotide sequence of reverse primer BamHI-R.

[0035] Example 1: Construction of hCHAD-pET28a recombinant plasmid

[0036] 1. PCR amplification was performed on the template of the CHAD gene CDS region sequence, and the product was recovered by gel electrophoresis.

[0037] 2. Select ECoRI and BamHI as the restriction enzyme sequences, and design the primers as follows:

[0038] EcoRI-F (SEQ ID NO:3): CGGAATTCATGGTCCGCCCAATGCTCT

[0039] BamHI-R (SEQ ID NO:4): CGGGATCCATGGCGGCCAGCTTTCTTGG

[0040] 3. The CDS region sequence of the CHAD gene was obtained by PCR amplification, and its nucleotide sequence is as follows (SEQ ID NO:2):

[0041]

[0042] The amino acid sequence is as follows (SEQ ID NO:1):

[0043] MVRPMLLLSLGLLAGLLPALAACPQNCHCHSDLQHVICDKVGLQKIPKVSEKTKLLNLQRNNFPVLAANSFRAMPNLVSLHLQHCQIREVAAGAFRGLKQLIYLYLSHNDIRVLRAGAFDDLTELTYLYLDHNKVTELPRGLLSPLVNLFILQLNNNKIRELRAGAFQGAKDLRWLYLS ENALSSLQPGALDDVENLAKFHVDRNQLSSYPSAALSKLRVVEELKLSHNPLKSIPDNAFQSFGRYLETLWLDNTNLEKFSDGAFLGVTTLKHVHLENNRLNQLPSNFPFDSLETLALTNNPWKCTCQLRGLRRWLEAKASRPDATCASPAKFKGQHIRDTDAFRSCKFPTKRSKKAGRH

[0044] Glue running and product recycling.

[0045] 4. The pET28a vector and the PCR product from step 3 were simultaneously digested with ECoRI and BamHI, respectively. The mixture was then run on a gel, and the vector and the digested PCR product fragments were recovered.

[0046] 5. Ligate the fragment recovered in step 4 and the vector using T4 ligase.

[0047] 6. Convert the ligation product into DH5α competent cells.

[0048] 7. Pick several clones from the transformed plate and inoculate them into LB medium containing Amp. Incubate at 37°C and 200 rpm for 2 hours with shaking. Then perform PCR identification on each bacterial culture and select positive clones for sequencing identification.

[0049] 8. Extract the target plasmid using the Omega Bio-Tek Plasmid Mini-Prep Kit (catalog number D6943-01), determine the concentration, and store at -20℃ for later use.

[0050] Example 2: Preparation of Monoclonal Antibodies

[0051] 1. Protein expression and purification

[0052] (1) The target plasmid (hCHAD-pET28a recombinant plasmid) of Example 1 was transformed into Rossetta competent cells and plated. The next day, single clones were picked and positive clones were identified by PCR. The bacterial strains were stored in a -80℃ freezer with 50% glycerol for later use.

[0053] (2) Protein expression conditions: Take 200 mL of bacterial culture for protein expression. When the bacterial turbidity reaches OD 600nm When the concentration was 0.6, IPTG (0.5 mM) was added to induce protein expression, and the mixture was cultured at 37°C and 170 rpm for 4 h.

[0054] (3) Bacterial protein extraction:

[0055] Collect the bacterial precipitate by centrifugation. Add lysis buffer to the bacterial precipitate at a ratio of 100µL of bacterial lysis buffer per 10mL of bacterial culture. Vortex to mix and dissolve. Incubate on ice for 30min. Then, sonicate at 150W for 8 cycles (5s / cycle). Add PMSF at a volume ratio of 1:100 and incubate on ice for 5-10min. Centrifuge at 4℃ and 13000rpm for 10-20min.

[0056] Transfer the supernatant to a new EP tube for storage; add an appropriate amount of inclusion body denaturing solution to the precipitate, vortex mix until completely dissolved, and if not completely dissolved, add more inclusion body denaturing solution until dissolved.

[0057] Add 5× loading buffer, denature at 100℃ for 10 min, perform SDS-PAGE, and then recover the gel.

[0058] 2. Titer determination of immunized Balb / c female rats and mice

[0059] (1) Select 8-week-old female Balb / c mice (mother mice), and generally immunize 3-4 mice.

[0060] (2) Mix the purified protein and Freund's adjuvant at a volume ratio of 1:1 and emulsify. Use Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the second and third immunizations; the immunization dose for each mouse is 5-150 mg of protein; inject the emulsified protein into the mice via intraperitoneal injection.

[0061] (3) Mice were immunized for the first time 14 days, the second time 14 days, the third time 14 days, and booster immunization 3-5 days.

[0062] (4) ELISA determination of mouse serum titer:

[0063] (4.1) Coat each well of the ELISA plate with 500 ng of purified protein, with a volume of 100 µL per well. Calculate the required amount of protein based on the number of wells. Then, mix the purified protein with PBS and place it in the sample well. Add the protein to the ELISA plate using a pipette. Coat overnight at 4°C (12-18 h).

[0064] (4.2) Remove the coating solution and blot the plate; block with 5% skim milk at 37°C for 1-2 h, 100 µL per well; then remove the skim milk and blot the plate; take serum from mouse tail tip blood and dilute it at volume ratios of 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600 and add it to the ELISA plate, with 3 replicates per group, and set up a negative control (serum from unimmunized mice) and a positive control (target antibody); incubate at 37°C for 1 h;

[0065] Wash the plate three times with PBST (discard the primary antibody, add 100 µL of PBST to each well, and wash for 3 min at 200 rpm on a shaker), then tap the plate; add the secondary antibody of the corresponding species, incubate at 37℃ for 30 min-1 h; wash the plate three times, then tap the plate.

[0066] Add 100 µL of TMB chromogenic solution to each well and incubate in the dark for 10–15 min; add 25 µL of 4 M HCl or 2 M H₂SO₄ to each well; measure the OD using a microplate reader. 450 nm The results were recorded, and mice with high titers were selected.

[0067] 3. Fusion hybridoma

[0068] (1) Culture and passage SP20 cells one week in advance; two days in advance, take mouse feeder cells (inject 5 mL of pre-cooled DMEM into the peritoneum of the mouse and rub the abdomen 66 times, use a syringe to extract the liquid again, transfer it to a 15 mL centrifuge tube for counting, and seed 10,000 feeder cells in each well of a 96-well plate, for a total of 5 96-well plates); preheat 50% PEG1450 (cell fusion agent) and DMEM at 37°C in advance.

[0069] (2) Select mice with high titers, anesthetize them, and collect blood by gouging out the eyes (the blood was allowed to stand at 37°C for 30 min, centrifuged at 1800g for 10 min, and the serum was mixed with glycerol at a volume ratio of 1:1 and stored at -20°C as a positive control for ELISA detection). The spleen of the mice was removed using sterile instruments in the operating table, placed in PBS, ground, and filtered through a 70µm filter membrane. The red blood cells were lysed and counted, and 1 x 10⁻⁶ cells were collected. 8 Fusing splenocytes, 2 x 1020 SP20 cells were used. 7 The cells were fused and high-valence monoclonal hybridoma cells were screened.

[0070] 4. Monoclonal antibody preparation

[0071] (1) Eight-week-old female Balb / c mice were sensitized by intraperitoneal injection of 400µL of ascites adjuvant per mouse.

[0072] (2) Fifteen days after sensitization, monoclonal hybridoma cells in the logarithmic growth phase (1×10⁻⁶ cells) were injected intraperitoneally. 6 (One mouse per mouse); observe the mice for abdominal distension after one week. If distension is observed, observe daily until the abdomen is significantly swollen. Drain the ascites fluid using a 1mL syringe every two days. After the third drainage, sacrifice the mice. The ascites fluid can be temporarily stored at 4°C. Centrifuge all ascites fluid samples at 1800g for 10 minutes and collect the clear liquid layer in the middle.

[0073] (3) Monoclonal antibodies were purified using Protein A / G gravity column.

[0074] (4) The concentration of the purified antibody was measured (A280nm), and it was concentrated using an ultrafiltration tube (50kD pore size); the concentrated antibody was mixed with glycerol at a volume ratio of 1:1, the concentration was measured, and it was stored in a -20℃ freezer.

[0075] Example 3: Cell Culture

[0076] 1. Cell resuscitation: Wipe the work surface with disinfectant alcohol. Prepare the waste container, tippers, culture dishes, sterile centrifuge tubes, and other necessary experimental materials and place them in the work area. Irradiate with ultraviolet light for half an hour. Place the RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin, sodium pyruvate solution, and HEPES solution at room temperature half an hour beforehand. Prepare for the experiment after half an hour. Prepare RPMI 1640 medium with a final concentration of 10% fetal bovine serum, 1% penicillin-streptomycin, 1% sodium pyruvate solution, and 1% HEPES solution (all ratios are volume ratios). The cells were then removed from the -80°C freezer or liquid nitrogen and quickly placed in a 37°C water bath. The cryovials were shaken to thaw them quickly. After that, they were placed in a centrifuge and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the cryovial solution was discarded with a pipette. The cells were then resuspended in the prepared complete culture medium with a pipette. The cells were gently mixed by pipetting and then added to a culture dish. Finally, the remaining culture medium was added and the dish was placed flat in a cell culture incubator.

[0077] 2. Cell Medium Change: The Myc-CaP cells used in this experiment were adherent cells. Cells were removed from the cell culture incubator, and the color of the culture medium was observed. Cell growth was observed under an inverted microscope. If the culture medium turned yellow or there was a lot of cell debris, the culture medium was discarded, and the cells were washed twice with PBS. Trypsin was added, and the cells were digested in the cell culture incubator for 3 minutes. Then, an equal volume of complete culture medium was added, and the cells were gently transferred to sterile centrifuge tubes using a pipette. The tubes were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium and seeded into sterile culture dishes. Finally, the cells were placed flat in the cell culture incubator for culture.

[0078] 3. Cell passage: Observe the cell density under an inverted microscope. If the cell confluence reaches 70%, then pass the cells.

[0079] 4. Cell cryopreservation: Observe cell growth under a microscope, then select cells in good condition for cryopreservation. Centrifuge at 1000 rpm for 3 min, discard the supernatant, add serum-free cryopreservation solution according to the cell pellet, gently pipette to mix, and transfer to 2 mL sterile cryovials. Record the cell name, batch, and date of cryopreservation. Finally, seal the cryovials with sealing film and store them at -80℃ or in liquid nitrogen.

[0080] 5. Cell counting: After digesting the cells, use a pipette to transfer the cells into centrifuge tubes. If the cells are too dense, dilute them 10 times and then use a pipette to transfer 10 µL of cells into a cell counting chamber. Insert the chamber into a cell counter and count the cells under a microscope.

[0081] Example 4: Animal Experiment

[0082] 1. Six-week-old FVB mice were used, with at least five mice in each of the experimental and control groups. The testes and epididymis of the mice were surgically removed first, and a subcutaneous tumor formation experiment was performed 3 days later.

[0083] 2. The Myc-CaP-CHAD cell line was used to conduct subcutaneous tumor formation experiments in FVB mice. Three days later, the mice were injected intraperitoneally with 10 mg / kg of anti-CHAD neutralizing antibody (monoclonal antibody prepared in Example 2), and the injection was repeated every 4 days.

[0084] 3. Continuously monitor tumor growth and measure tumor size every 3 days to plot tumor growth curves. When the tumor diameter in the control group reached approximately 1.5 cm, the mice were euthanized, tumor tissue was collected, weighed (and a tumor weight graph was plotted), and a portion of the tumor from each group was digested and analyzed by flow cytometry (refer to Example 5).

[0085] Example 5: Flow Cytometry Analysis

[0086] 1. The tumor tissue from Example 4 was digested, and the resulting single-cell suspension was washed twice with PBS and resuspended with PBS.

[0087] 2. Surface staining: Markers expressed on the cell surface, such as CD45, CD3, and CD8, are stained at a ratio of 10... 6 Add 1 µL of flow cytometry antibody per cell and mix thoroughly. The cell pellet volume is 200 µL (resuspended in PBS). Incubate at 4°C in the dark for 20 min. Then add 1 mL of PBS to resuspend the pellet, centrifuge and discard the supernatant, and then add another 200 µL of PBS to resuspend the cell pellet.

[0088] 3. Cell viability staining: Add 0.3 µL of cell viability stain to each tube of cells and incubate at room temperature in the dark for 10 min. Then resuspend the cells in 1 mL of PBS, centrifuge, and discard the supernatant.

[0089] 4. Resuspend the cells in 500 µL of fixative and fix at room temperature in the dark for 20 min. Then add an equal volume of permeabilization buffer and mix well. Centrifuge and discard the supernatant. Resuspend the cells in 200 µL of permeabilization buffer and add flow cytometry antibodies expressing intracellular markers (GZMB, Perforin, and LAG3, etc.). Then add 1 mL of permeabilization buffer to resuspend, centrifuge, and discard the supernatant.

[0090] 5. The analysis was performed using a Beckman flow cytometer.

[0091] Example 6: CHAD is highly expressed in castration-resistant prostate cancer (CRPC) tissues.

[0092] To investigate the development mechanism of CRPC, this embodiment uses a mouse prostate cancer Myc-CaP allogeneic xenograft model, which can simulate the process of castration resistance development in human prostate cancer. The Myc-CaP cell line is derived from primary prostate cancer in c-Myc transgenic mice and can form tumors in immunocompetent male FVB mice. Its xenograft cells express androgen receptors and exhibit androgen-dependent growth. After castration treatment in mice, the tumor shrinks, but subsequently enlarges again and develops into AR-positive castration-resistant prostate cancer.

[0093] like Figure 1 As shown in Figure A, based on the tumor development stage, this embodiment classifies tumors before castration as PPC and tumors that shrink after castration and then grow back as CRPC. This embodiment is based on the above-mentioned animal model of castration-resistant prostate cancer and transcriptome sequencing of clinical PPC and CRPC samples. Figure 1 According to (A), Figure 1As shown in Figure B, CHAD expression was significantly upregulated in CRPC, suggesting that CHAD may play an important role in CRPC development. To further investigate the role of CHAD in CRPC development, this example measured CHAD expression in collected mouse model PPC and CRPC samples. Figure 1 As can be seen from C and D, CHAD expression in CRPC tumor tissue is significantly higher than its expression in PPC tissue. Similarly, according to Figure 1 As shown in Figure E, similar results were obtained from clinically collected PPC and CRPC tumor samples. This indicates that CHAD is highly expressed in CRPC and may be closely related to the development of CRPC.

[0094] Example 7: Knockdown of CHAD expression inhibits CRPC tumor growth

[0095] To investigate whether CHAD plays an important role in the development of CRPC, this embodiment used lentiviral particles carrying CHAD 3'-UTR-guided shRNA interference sequences or blank vector sequences to infect prostate cancer cells Myc-CaP, establishing stable CHAD knockdown cell lines and control cell lines, named Myc-CaP-CHAD-KD and Myc-CaP-control.

[0096] Following this, a mouse xenograft experiment was also conducted in this embodiment. Castrated male wild-type FVB mice aged 6-8 weeks were subcutaneously inoculated with 1×10⁻⁶ mol / L thiocarcinoma cells. 6 In vivo tumorigenesis experiments were conducted using Myc-CaP-CHAD-KD cell lines and corresponding control cell lines. Figure 2 As shown in C to E, knockdown of CHAD significantly inhibited the development of CRPC tumors. This indicates that knockdown of CHAD can suppress the development of CRPC.

[0097] Example 8: Anti-CHAD neutralizing antibody inhibits CRPC tumor growth and enhances CD8. + T-cell anti-tumor function

[0098] Prostate cancer is often described as an immune "cold" tumor, with a relatively small number of immune cells and a weak immunogenic response in its tumor microenvironment (TME). This makes prostate cancer, especially castration-resistant prostate cancer (CRPC), difficult to treat effectively with conventional immune checkpoint inhibitors (such as anti-PD-1 antibodies). Although immune checkpoint inhibitors have shown significant anti-tumor effects in some tumor types, their effects are not significant in CRPC, suggesting that CRPC may exhibit immune escape characteristics. Due to the urgent need to develop new immune checkpoint inhibitors, the high-affinity anti-CHAD neutralizing antibody prepared in this embodiment shows promise as a novel treatment for CRPC immunotherapy. This antibody has a strong affinity for both human and mouse CHAD proteins. Figure 3 ).

[0099] To further examine the efficacy of anti-CHAD neutralizing antibody therapy in the development of CRPC, this study conducted an anti-CHAD neutralizing antibody therapy experiment in a CRPC-bearing mouse model. First, Myc-CaP-CHAD cells were subepithelially transplanted into castrated 6-week-old male mice. Three days later, the mice were intraperitoneally injected with 10 mg / kg of anti-CHAD neutralizing antibody, administered every 4 days until the tumor volume reached 500 mm². 3 Mice were then sacrificed and tumors were collected. Results showed that the tumor volume and weight of mice treated with antibodies were significantly smaller than those in the control group. Figure 4 (A to D). This indicates that anti-CHAD antibodies can effectively inhibit the development of CRPC.

[0100] Furthermore, this embodiment utilizes flow cytometry to counteract CD8-infiltrating CRPC tumors after CHAD antibody treatment. + T cell analysis showed that, compared with the control group, the tumor tissue of the anti-CHAD antibody treatment group had higher levels of CD8+. + T cell infiltration was significantly increased. This finding suggests that treatment with anti-CHAD antibodies can promote CD8 cell infiltration. + T cells infiltrated the tumor site more effectively and exerted their anti-tumor effects. Further analysis revealed that CD8+ cells were more effective after antibody treatment. + The significant increase in perforin and GZMB secreted by T cells indicates that anti-CHAD antibody treatment can enhance CD8. + The killing function of T cells. Meanwhile, CD8... + The expression of the exhaustion-related molecule LAG3 on T cells was significantly reduced, which may mean that anti-CHAD antibody treatment can inhibit CD8. + The expression of T cell exhaustion-related molecules was reduced, restoring their anti-tumor activity. These results indicate that intervention with anti-CHAD antibodies can significantly increase CD8 expression.+ T's ability to kill tumor cells.

[0101] In summary, the results of the above examples indicate that: (1) transcriptomics, Western blot, and immunohistochemical analyses showed that chondroitin adherent protein (CHAD) was highly expressed in castration-resistant prostate cancer (CRPC) tissues, suggesting that it could serve as a therapeutic target for CRPC; (2) in a mouse subcutaneous tumorigenesis model, knockdown of CHAD significantly inhibited the development of CRPC tumors; and (3) in a CRPC tumor-bearing mouse model, anti-CHAD neutralizing antibody treatment effectively inhibited tumor growth and enhanced tumor infiltration CD8. + The anti-tumor function of T cells.

[0102] The above results indicate that CHAD plays a promoting role in CRPC progression, and its mechanism may be related to the inhibition of CD8. + T cells are involved in recognizing and killing tumor cells and promoting tumor immune escape. Anti-CHAD neutralizing antibodies can inhibit the above-mentioned effects of CHAD, significantly improve the treatment effect of CRPC, and provide a new strategy for the immunotherapy of CRPC.

[0103] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present technical solution are within the protection scope of the present invention.

Claims

1. The use of an antibody capable of specifically binding to the CHAD protein in the preparation of antitumor drugs, wherein, The CHAD protein has the amino acid sequence shown in SEQ ID NO: 1, or has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:

2.

2. The use according to claim 1, wherein, The tumor is castration-resistant prostate cancer.

3. The use according to claim 1 or 2, wherein, The antibody can inhibit the biological activity of CHAD protein in promoting tumor growth.

4. The use according to claim 1 or 2, wherein, The antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof.

5. The use according to claim 1 or 2, wherein, The antibody can enhance tumor-infiltrating CD8. + The anti-tumor function of T cells.

6. An antitumor pharmaceutical composition comprising an antibody capable of specifically binding to the CHAD protein, and a pharmaceutically acceptable carrier, wherein, The CHAD protein has the amino acid sequence shown in SEQ ID NO: 1, or has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:

2.

7. The antitumor pharmaceutical composition according to claim 6, wherein, The tumor is castration-resistant prostate cancer.

8. The antitumor pharmaceutical composition according to claim 6 or 7, wherein, The antibody can inhibit the biological activity of CHAD protein in promoting tumor growth.

9. The antitumor pharmaceutical composition according to claim 6 or 7, wherein, The antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof.

10. The antitumor pharmaceutical composition according to claim 6 or 7, wherein, The antibody can enhance tumor-infiltrating CD8. + The anti-tumor function of T cells.

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