CD158d molecules, antibodies thereof, and uses thereof

Abstract

The invention belongs to the field of biological engineering and biological treatment, and discloses a CD158d molecule, a neutralizing antibody thereof and application. The invention finds that HS3ST3B1 positive tumor-associated fibroblasts can mediate killing lymphocyte death, and further finds a new immune checkpoint molecule CD158d. As the CD158d molecule can mediate tumor fibroblast mediated killer lymphocyte death, the neutralizing antibody capable of neutralizing the CD158d molecule is obtained in the invention. On the basis, the invention develops an application of the CD158d molecule as a target spot in screening or preparing a medicine for inhibiting tumor immune escape, and an application of the CD158d neutralizing antibody in preparing a medicine for inhibiting tumor immune escape.

Description

CD158d molecules, neutralizing antibodies therein, and applications

[0001] This application claims priority to a prior application with patent application number 202210886386.5, entitled “CD158d molecules, neutralizing antibodies therein, and applications”, filed with the State Intellectual Property Office of China on July 26, 2022. The entire text of the prior application is incorporated into this application by reference.

[0002] The invention relates to CD158d molecules, neutralizing antibodies therein and applications, and belongs to the fields of bioengineering and biotherapy.

[0003] Tumor is one of the most serious diseases threatening human life and health. Human understanding and research on tumors has gone through a long process. At present, the treatment of tumors mainly covers a comprehensive treatment strategy including surgery, chemotherapy, radiotherapy and targeted therapy. In recent years, through the rational use of these comprehensive treatment strategies, the survival of cancer patients has been significantly improved. However, distant tumor metastasis, recurrence and treatment tolerance are still the problems that many cancer patients will eventually face, and are also the dilemma of the current treatment of malignant tumors.

[0004] The immune system is an important defense barrier of the body. It identifies and eliminates "non-self" components in the body and maintains the stability of the internal environment through immune defense, immune surveillance and immune self-stabilization. In the occurrence and development of malignant tumors, a dynamic interaction process is formed between them and the systemic immune system. Scholars have proposed the "Cancer Immunoediting" hypothesis, pointing out that the interaction between tumors and the immune system can be divided into three states, including "immune elimination" (Elimination), "immune balance" (Equilibration) and "immune escape" (Escape). In recent years, with the continuous deepening of anti-tumor immunity research, tumor immunotherapy has made major breakthroughs. Emerging immunotherapy strategies such as monoclonal antibody therapy targeting the PD-1 / PD-L1 pathway have achieved significant therapeutic effects in the treatment of malignant tumors such as melanoma, lung cancer, and bladder cancer, which has greatly encouraged researchers' enthusiasm for tumor immunity research. Therefore, restoring the body's normal anti-tumor immunity is considered to be a radical treatment strategy for malignant tumors.

[0005] With the release of a large number of immunotherapy clinical trial results, the therapeutic effects of immune checkpoint inhibitors targeting PD-1 / PD-L1 have become more and more different between different cancers and between different individuals with the same cancer. Even in traditionally considered immune "hot" tumors such as melanoma, lung cancer, and bladder cancer, there are still a large number of patients who are insensitive to immune checkpoint inhibitors. In anti-PD-L1 treatment, even if the patient's PD-L1 is evaluated before treatment and then the sensitivity of PD-L1-positive patients to immunotherapy is analyzed, more than 50% of patients are tolerant to treatment, and even some patients who show good responsiveness to immunotherapy at the beginning have tumor progression during treatment. The former is called "primary immunotherapy tolerance" and the latter is called "secondary immunotherapy tolerance". In the clinical exploration of immunotherapy for traditionally considered immune "cold" tumors such as breast cancer and pancreatic cancer, the researchers first conducted a stratified analysis of different molecular subtypes of the potential possibility of tumor immunotherapy based on tumor mutation load and lymphocyte infiltration. For example, in triple-negative and HER2-positive breast cancer, researchers found that they had relatively higher tumor mutation loads and more lymphocyte infiltration, and the tumor mutation load and lymphocyte infiltration levels in patients with the same type of tumors were associated with their good prognosis, suggesting that immunotherapy has certain therapeutic potential even in immune-cold tumors. As a result, a number of combined immunotherapy clinical trials around the world have been approved for the treatment of immune-cold tumor patient cohorts such as breast cancer. Among them, patients did not have significant survival benefits in the immune checkpoint inhibitor monotherapy model (phase II KEYNOTE-086 study). In terms of combined treatment strategy trials, the IMpassion130 phase III randomized clinical controlled trial analyzed the efficacy of paclitaxel combined with PD-L1 monoclonal antibody or not in breast cancer patients. The interim analysis results showed that there was no significant difference in the overall survival of patients between the PD-L1 monoclonal antibody combination therapy group and the paclitaxel monotherapy group, but in the stratified analysis, it was found that patients in the PD-L1 monoclonal antibody combined with paclitaxel treatment group showed better survival benefits in the treatment of patients with positive immune cell PD-L1. However, the overall benefit of patients is still limited.

[0006] In addition to immune checkpoint inhibitors, immune cell infusion therapy and tumor vaccines are two other important forms of immunotherapy. However, due to the existence of the complex tumor microenvironment regulatory network of solid tumors, the immune cells infused back into the patient's body or the anti-tumor immune cells stimulated by the tumor vaccine are rapidly exhausted or die when they reach the local tumor, greatly reducing their anti-tumor effects, thus greatly hindering their actual clinical transformation and application.

[0007] At present, there are huge individual differences in the effectiveness of immunotherapy for malignant tumors. The root cause is that, on the one hand, current basic research still lacks understanding of the immune escape mechanism of malignant tumors, and on the other hand, the targeted intervention strategies of the recently discovered anti-tumor immune regulatory nodes have not yet achieved actual clinical transformation.

[0008] In the "tumor-immune cycle", the realization of effective anti-tumor immunity requires seven steps: (1) tumor antigens are released and captured by DC cells, (2) DC cells present antigens to T cells, (3) T cells are activated in draining lymph nodes, (4) activated T cells migrate to the tumor bed, (5) activated T cells infiltrate the tumor through blood vessels, (6) activated effector T cells recognize tumor cells, and (7) T cells kill tumor cells. Abnormalities in any link of the above cycle can lead to anti-tumor immune escape. The PD-1 and PD-L1 antagonists currently used in clinical practice only target the above step (7).

[0009] In addition, in solid tumors, the existence of a complex tumor microenvironment has become an important umbrella for tumor immune escape. In addition to the tumor cells themselves, the tumor microenvironment also includes endothelial cells, fibroblasts, pericytes in the stroma, infiltrating immune cells such as lymphocytes, macrophages, granulocytes, myeloid-derived suppressor cells, and extracellular matrix components and a large number of cytokines, chemokines, etc. In recent years, the results of numerous experimental tumor studies and tumor biological behavior prediction models have shown that the tumor microenvironment plays a vital role in the occurrence and development of malignant tumors, distant metastasis, and treatment tolerance. With the continuous deepening of tumor microenvironment research, researchers have gradually realized that with the progression of the tumor or the intervention of different treatment methods, the interaction between the various cellular components in the tumor microenvironment is in a dynamic change process. Moreover, the various cellular components in the microenvironment are highly heterogeneous, and the same cell type may have different tumor-promoting or tumor-suppressing phenotypes at the same time. Therefore, in-depth research on the dynamic changes of anti-tumor immunity in different stages of tumor development and treatment, especially the impact of the interactions between the various components in the tumor microenvironment on anti-tumor immunity, starting from multi-angle and multi-dimensional analysis such as the activation and recruitment of anti-tumor immune cells and the regulatory effect of the tumor microenvironment on the function and survival status of anti-tumor immune cells, exploring the regulatory network of anti-tumor immunity of solid tumors, screening and identifying the key regulatory node molecules therein, and thus providing new targets for immunotherapy will be an important direction and goal of future tumor immunotherapy research.

[0010]

[0011] In view of the above-mentioned directions and objectives, the present invention starts from the perspective of the interaction between the stromal cell components of the tumor microenvironment and the cytotoxic lymphocytes that mainly perform anti-tumor immune functions, analyzes the key regulatory nodes in which specific fibroblast subsets in the microenvironment mediate the disability of cytotoxic lymphocytes, identifies new immune checkpoint molecules, and blocks the fibroblast-mediated cytotoxic lymphocyte death by targeting CD158d immune checkpoint molecules or HS3ST3B1-positive tumor-associated fibroblasts, thereby restoring the lymphocytes' ability to kill and clear tumor cells.

[0012] To this end, the present invention provides, on one hand, the use of HS3ST3B1-positive tumor-associated fibroblasts in the preparation of a kit for detecting cytotoxic lymphocyte death.

[0013] Another aspect of the present invention provides the use of HS3ST3B1-positive tumor-associated fibroblasts in preparing a kit for detecting tumor immune escape.

[0014] Another aspect of the present invention provides the use of HS3ST3B1 as a target in screening or preparing drugs for inhibiting tumor immune escape.

[0015] Another aspect of the present invention provides an immune checkpoint molecule CD158d, which mediates tumor fibroblast-mediated cytotoxic lymphocyte death.

[0016] Another aspect of the present invention provides the use of CD158d molecules as immune checkpoints, which mediate the cytotoxic lymphocyte death mediated by tumor fibroblasts.

[0017] Another aspect of the present invention provides the use of CD158d molecules as targets in screening or preparing drugs for inhibiting tumor immune escape.

[0018] Another aspect of the present invention provides a neutralizing antibody that binds to CD158d molecules, which inhibits tumor immune escape by neutralizing CD158d.

[0019] In a preferred embodiment of the present invention, the neutralizing antibody is a monoclonal antibody or a polyclonal antibody.

[0020] Another aspect of the present invention provides use of the neutralizing antibody of the present invention as an immune checkpoint inhibitor.

[0021] Another aspect of the present invention provides the use of the neutralizing antibody in the preparation of a drug for inhibiting tumor immune escape.

[0022] Compared with the prior art, the present invention discovered that HS3ST3B1-positive tumor-associated fibroblasts can mediate cytotoxic lymphocyte death. On this basis, the present invention further discovered a new immune checkpoint molecule CD158d, which can mediate cytotoxic lymphocyte death mediated by tumor fibroblasts. Based on the above findings, the present invention has developed the use of CD158d molecules and HS3ST3B1 as targets in screening or preparing drugs that inhibit tumor immune escape.

[0023] In view of the fact that CD158d molecules can mediate the death of cytotoxic lymphocytes mediated by tumor fibroblasts, the present invention obtains a neutralizing antibody capable of neutralizing CD158d molecules, which can significantly restore the killing effect of CTLs on tumors after neutralizing CD158d molecules. Therefore, the present invention provides the use of CD158d neutralizing antibodies in the preparation of drugs for inhibiting tumor immune escape.

[0024] FIG. 1 is a trend graph showing that silencing HS3ST3B1 in HS3ST3B1-positive tumor-associated fibroblasts inhibits its-induced cytotoxic lymphocyte cell death.

[0025] A. Representative images of flow cytometry detection of Annexin V fluorescent antibody and PI staining in untreated CTLs (CTRL group) and CTLs in each group after co-culture with different fibroblasts.

[0026] The red numbers represent the proportion of Annexin V-positive cells (quadrant II and quadrant IV), that is, the proportion of dead cells.

[0027] B. Quantification of Figure 1A (n=3).

[0028] C. The release of lactate dehydrogenase LDH in the culture system of untreated CTLs (CTRL group) and CTLs of each group co-cultured with different fibroblasts was detected, and the proportion of LDH release represented the proportion of dead cells (n=3).

[0029] FIG. 2 is a schematic diagram of enhancing tumor immunity in vivo after silencing HS3ST3B1 in HS3ST3B1-positive tumor-associated fibroblasts.

[0030] A. Primary tumor cells and HS3ST3B1 treated with different + Schematic diagram of mouse xenograft tumors inoculated with CAFs.

[0031] B. Primary tumor cells and HS3ST3B1 treated with different methods + After CAFs were mixed and inoculated into mice, the growth of various xenograft tumors was observed without treatment or with reinfusion of CTLs (n=8).

[0032] Figure 3 shows that after CD158d was knocked out in CTLs or CD158d was neutralized by neutralizing antibodies, HS3ST3B1 was terminated. + Schematic diagram of the death trend mediated by CAFs.

[0033] A. Representative images of flow cytometry detection of Annexin V fluorescent antibody and PI staining of CTLs cultured alone and in each group (UT, sgGFP and CD158d knockout groups) after co-culture with different fibroblasts.

[0034] The red numbers represent the proportion of Annexin V-positive cells.

[0035] B. Quantification of Figure 3A (n=3).

[0036] C. The release of LDH was detected after CTLs cultured alone and each group of CTLs (UT, sgGFP and CD158d knockout groups) were co-cultured with different fibroblasts (n=3).

[0037] D. Flow cytometry was used to detect the Annexin V fluorescent antibody and PI staining of CTLs cultured alone and in each group (UT, IgG and CD158d neutralizing antibody groups) after co-culture with different fibroblasts (n=3).

[0038] E. The release of LDH was detected after single culture of CTLs and co-culture of CTLs in different groups (UT, IgG and CD158d neutralizing antibody groups) with different fibroblasts (n=3).

[0039] FIG. 4 is a schematic diagram of using neutralizing antibodies to target CD158d in vivo to promote tumor immunity.

[0040] A, Schematic diagram of targeted CD158d combined with adoptive immunization in PDX mice.

[0041] B. PDX tumor growth rate of mice in the non-CTLs infusion group and the CTLs infusion groups with different treatments (PBS, IgG and CD158d neutralizing antibody treatment).

[0042] C. The size of all PDX tumors in mice without CTLs infusion and in different CTLs infusion treatment groups (PBS, IgG and CD158d neutralizing antibody treatment).

[0043] D. Representative images of PDX tumor size detected by PET / CT in mice without CTLs transfusion and in different CTLs transfusion treatment groups (PBS, IgG and CD158d neutralizing antibody treatment).

[0044] In order to better illustrate the purpose and advantages of the present method, the specific implementation content of the present invention is further described in detail in conjunction with the accompanying drawings and specific embodiments.

[0045] Example 1: HS3ST3B1 + CAFs induce CTLs death by expressing HS3S3ST3B1

[0046] Tumor-associated fibroblasts (CAFs) and normal breast fibroblasts (NBFs) were isolated from primary tissues of breast cancer patients or breast tissues of patients undergoing breast reduction surgery.

[0047] CAFs were divided into two groups: HS3ST3B1 negative and positive using flow cytometry.

[0048] The above fibroblasts were co-cultured with cytotoxic lymphocytes (CTLs). Compared with normal breast fibroblasts (NBFs) and HS3ST3B1-negative tumor-associated fibroblasts (HS3ST3B1), the - CAFs, HS3ST3B1-positive tumor-associated fibroblasts HS3ST3B1 + CAFs can significantly induce CTLs death, see A, B, and C in FIG1 .

[0049] When fibroblasts were infected with lentivirus and shRNAs were used to silence the expression of HS3S3ST3B1 in fibroblasts, HS3ST3B1 + The CTLs death tendency induced by CAFs was inhibited, see A, B, and C in FIG1 .

[0050] The above results show that HS3ST3B1 + CAFs induce the death of CTLs by expressing HS3S3ST3B1.

[0051] In this embodiment, HS3ST3B1-positive tumor-associated fibroblasts were isolated from tumors, which mainly included the following steps:

[0052] 1. Collect about 1 cubic centimeter of fresh breast cancer tissue from patients;

[0053] 2. Use sterile scissors to mince the tissue in step 1 into about 1 cubic millimeter, and add 10 ml of culture medium containing 1.5 mg / ml type 1 collagenase and 1.5 mg / ml type 3 collagenase;

[0054] 3. Incubate the mixture in step 2 in a 37°C incubator for 2 hours;

[0055] 4. Filter the tissue culture fluid in step 3 to obtain a primary cell suspension;

[0056] 5. Centrifuge the cell suspension in step 4, resuspend, add anti-PDGFRa flow cytometry antibody and anti-HS3ST3B1 primary antibody and incubate for 30 minutes, centrifuge to remove the supernatant and resuspend, further add fluorescent secondary antibody and incubate for 45 minutes, finally centrifuge to remove the supernatant and resuspend;

[0057] 6. Use a flow cytometer to sort the cell suspension in step 5 to obtain HS3ST3B1-positive tumor-associated fibroblasts.

[0058] In this embodiment, the construction of the fibroblast and lymphocyte co-culture model mainly includes the following steps:

[0059] 1. HS3ST3B1-positive tumor-associated fibroblasts and cytotoxic lymphocytes were co-cultured, with fibroblasts seeded in the lower chamber of the transwell and cytotoxic lymphocytes in the upper chamber;

[0060] 2. Perform cell death detection on the cytotoxic lymphocytes after co-culture (LDH release assay, flow cytometry).

[0061] Example 2: HS3ST3B1 + CAFs promote tumor cell immune evasion via HS3ST3B1 in vivo

[0062] As shown in FIG2A , primary tumor cells were isolated from breast cancer tissues and the tumor cells were mixed with HS3ST3B1 treated with different + CAFs were mixed and inoculated into the fourth pair of mammary fat pads of immunodeficient mice. At the same time, monocytes were isolated from the patient's peripheral blood, induced to differentiate into dendritic cells, and then loaded with tumor antigens. The CD8T lymphocytes isolated from the peripheral blood were then treated with dendritic cells loaded with tumor antigens, thereby constructing specific CTLs for the tumor in vitro, and the tumor was reinfused into the tail vein when the diameter of the mouse tumor reached 2mm.

[0063] The present invention discovered HS3ST3B1 + Xenografts formed by CAFs mixed with tumor cells resist the immune killing effect of adoptively infused CTLs, as shown by the significant growth of the transplanted tumors after adoptive infusion of CTLs, as shown in Figure 2B. However, silencing the expression of HS3ST3B1 in this group of CAFs can significantly restore the function of adoptive immunity. This indicates that HS3ST3B1 + CAFs promote tumor cell immune evasion via HS3ST3B1 in vivo.

[0064] In this example, the in vivo validation model of HS3ST3B1-positive fibroblast-mediated immune escape mainly includes the following steps:

[0065] 1. Sorted HS3ST3B1-positive tumor-associated fibroblasts, and knocked out the HS3ST3B1 gene of fibroblasts using CRISPR-Cas9 technology;

[0066] 2. Primary tumor cells are obtained by sorting from patient tumor tissues;

[0067] 3. Fibroblasts from the HS3ST3B1 knockout group and the non-knockout group were mixed with primary tumor cells and implanted into the fat pads of immunodeficient mice;

[0068] 4. Construction of primary tumor cell-specific cytotoxic lymphocytes in vitro:

[0069] (1) After primary tumor cells were expanded, cell pellets were collected and repeatedly frozen and thawed five times;

[0070] (2) Centrifugation at 14,000 rpm and collection of the supernatant (tumor cell lysate);

[0071] (3) Isolate monocytes from the patient's peripheral blood and add IL4 and GMSCF cytokines to induce them into DC cells;

[0072] (4) adding tumor lysate to DC cell culture medium;

[0073] (5) Add T lymphocytes from the same source and co-culture for 5 days, and sort CD8-positive CTL cells;

[0074] 5. Infuse CTL cells into the tail vein of the immunodeficient mouse model constructed in step 3;

[0075] 6. Observe the tumor progression in mice.

[0076] Example 3: HS3ST3B1 + CAFs mediate the death of CTLs through the CD158d molecule on CTLs

[0077] When CD158d expression in CTLs was silenced using CRISPR-CAS9, HS3ST3B1 + The CTLs death tendency induced by CAFs was inhibited, see A, B, and C in FIG. 3 .

[0078] Furthermore, neutralizing antibodies against CD158d were constructed in vitro and added to the co-culture system of CAFs and CTLs. It was found that the addition of neutralizing antibodies against CD158d could significantly inhibit the expression of HS3ST3B1. + CAFs induced CTLs death, see Figure 3 E, F. This indicates that HS3ST3B1 +CAFs mediate the death of CTLs through the CD158d molecule on CTLs, which is a checkpoint molecule that mediates immunosuppression.

[0079] In this embodiment, the neutralizing antibody construction experiment mainly includes the following steps:

[0080] 1. In vitro recombinant human CD158d full-length protein (Ag), the protein sequence is shown in SEQ ID NO: 1.

[0081] 2. Mouse immunization:

[0082] (1) For the first immunization, Ag 50ug / animal, with Freund's complete adjuvant, injected subcutaneously at multiple points, usually 1.5ml, at intervals of 3 weeks;

[0083] (2) Second immunization, with the same dosage and route as above, plus Freund's incomplete adjuvant, at an interval of 3 weeks;

[0084] (3) The third immunization, with the same dose as above, without adjuvant, was intraperitoneally injected, and blood was collected 7 days later to measure the titer and immune effect, with an interval of 3 weeks;

[0085] (4) Booster immunization, dose 50 μg, intraperitoneal injection;

[0086] (5) After 3 days, the spleen was harvested for fusion;

[0087] 3. Cell fusion:

[0088] (1) Preparation of feeder cells:

[0089] Preparation of mouse peritoneal macrophages:

[0090] ① Use 6-10 week old BALB / c mice;

[0091] ② Pull the neck to kill, soak in 75% alcohol, disinfect for 3 minutes, cut the skin with sterile scissors to expose the peritoneum. Use a pipette to inject 6ml of culture medium, rinse repeatedly, and suck out the rinsing fluid;

[0092] ③Put into a 10ml centrifuge tube and centrifuge at 1200rpm for 5min.;

[0093] ④Suspend with 20% calf serum culture medium and adjust the cell number to 1×10 5 / ml, add to 96-well plate, 100ul / well; place in 37 degree incubator for culture;

[0094] (2) Preparation of myeloma cells:

[0095] ① Expand the culture of myeloma cells 48-36 hours before fusion;

[0096] ② On the day of fusion, use an elbow dropper to gently blow the cells off the bottle wall and collect them in a 50ml centrifuge tube or fusion tube;

[0097] ③ Centrifuge at 1000r / min for 5-10 minutes and discard the supernatant;

[0098] ④ Add 30 ml of incomplete culture medium and centrifuge to wash once. Then resuspend the cells in 10 ml of incomplete culture medium and mix well;

[0099] ⑤ Take the myeloma cell suspension, add 0.4% trypan blue dye to count the live cells and set aside;

[0100] (3) Preparation of spleen cells

[0101] Take the immunized BALB / c mice, remove the eyeballs to collect blood, and separate the serum as the positive control serum for antibody detection. At the same time, kill the mice by cervical dislocation, soak in 75% alcohol for 5 minutes, fix on the culture dish and lift the skin on the left side of the abdomen to see the spleen. Change to ophthalmic scissors and forceps, use sterile surgical scissors to open the peritoneum in the clean bench, remove the spleen and place it in a dish containing 10ml of incomplete culture medium, wash gently, and carefully peel off the surrounding connective tissue. Place on the stainless steel sieve in the dish, grind into a cell suspension with a syringe needle, and count, so that the spleen cells enter the incomplete culture medium in the dish. Use a pipette to blow several times to make a single cell suspension. Usually 1×10 8 -2.5×10 8 splenocytes;

[0102] (4) Cell fusion

[0103] ① 1×10 8 Splenocytes and 1×10 7 Myeloma cells SP2 / 0 were mixed in a 50 ml fusion tube, incomplete medium was added to 30 ml, and mixed thoroughly;

[0104] ② Centrifuge at 1000r / min for 5-10 minutes and aspirate the supernatant as much as possible;

[0105] ② Tap the bottom of the fusion tube lightly with your palm to make the precipitated cells loose and even;

[0106] ③ Use a 1ml pipette to add 1ml of preheated 50% PEG within 30s, stirring gently while adding;

[0107] ④ Let the suction tube stand for 1 minute;

[0108] ⑥ Add preheated incomplete culture medium to terminate the PEG action, and add 1ml, 2ml, 3ml, 4ml, 5ml, and 10ml every 2 minutes;

[0109] ⑦800 rpm, 5 minutes, discard the supernatant;

[0110] ⑧ Add 5 ml of complete culture medium, gently pipette the precipitated cells to suspend and mix, then add complete culture medium to 40-50 ml, dispense into 96-well cell culture plates, 100 ul per well, and then place the culture plates at 37°C, 5% CO 2 Culture in an incubator;

[0111] ⑨Add selection medium after 6 hours, 50ul per well, and half of the medium is replaced with selection medium after 3 days;

[0112] ⑩ Frequently observe the growth of hybridoma cells. When they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection;

[0113] (5) Selection of hybridoma cells:

[0114] ① Dilute the antigen to 10ug / ml with coating solution;

[0115] ② Add 100ul / well to the wells of the ELISA plate and place at 4℃ overnight or at 37℃ for 2 hours;

[0116] ③Discard the liquid in the wells, wash with detergent 3 times, 3 minutes each time, and pat dry;

[0117] ④Add 100ul blocking solution to each well and block at 37℃ for 1 hour;

[0118] ⑤ Wash 3 times with detergent;

[0119] ⑥ Add 100ul of the culture supernatant of the hybridoma cells to be tested to each well, set up positive, negative and blank controls, incubate at 37℃ for 1 hour, wash and pat dry;

[0120] ⑥Add enzyme-labeled secondary antibody, 100ul per well, incubate at 37℃ for 1 hour, wash, and pat dry;

[0121] ⑦ Add substrate solution. Add 100ul of freshly prepared substrate solution to each well and incubate at 37℃ for 20 minutes.

[0122] ⑧2mol / LH 2 SO 4 The reaction was terminated and the OD value was read on an enzyme-linked immunosorbent reader;

[0123] ⑩Result determination: P / N ≧ 2.1, or P ≧ N + 3SD is considered positive. If the negative control well is colorless or nearly colorless, and the positive control well is clearly colored, the result can be directly observed with the naked eye;

[0124] (6) Cloning of hybridoma cells (limiting dilution method)

[0125] ①Prepare mouse spleen cells as feeder cells;

[0126] ② Prepare the hybridoma cell suspension to be cloned and dilute it to three different dilutions of 5, 10 and 20 cells per ml using HT medium containing 20% ​​serum;

[0127] ③ Add 5×10 4 -1×10 5 The ratio of cells was determined by adding peritoneal macrophages to the above hybridoma cell suspensions;

[0128] ④ Each hybridoma cell is dispensed into a 96-well plate, with 100ul per well;

[0129] 5. 37°C, 5% CO 2 After 6 days of culture, antibodies can be detected when clones visible to the naked eye appear. Observe under an inverted microscope, mark the wells where only a single clone grows, and take the supernatant for antibody detection;

[0130] ⑥ Take the cells from the antibody-positive wells for expansion culture and freeze;

[0131] 4. Identification of Ig class and subclass of monoclonal antibodies

[0132] (1) Coat the ELISA plate with 10 μg / ml antigen, 50 μl / well, at 4°C overnight;

[0133] (2) After washing, add the monoclonal antibody sample to be tested, 100ul / well, and incubate at 37°C for 1 hour. Set up negative and positive control wells;

[0134] (3) After washing, add HRP-labeled anti-mouse Ig antibody reagent, 100ul / well, and color development at 37℃ in the dark for 20 minutes. 2 SO 4 After the reaction is terminated, the subtype of the antibody is determined based on the color;

[0135] 5. Production and purification of monoclonal antibodies

[0136] (1) Production of monoclonal antibodies in animals

[0137] ① Adult BALB / c mice were intraperitoneally inoculated with pristane or liquid paraffin, 0.3-0.5 ml per mouse;

[0138] ② After 7-10 days, intraperitoneal inoculation of hybridoma cells diluted with PBS or serum-free medium, 5×10 per mouse 5 / 0.2ml;

[0139] ③After an interval of 5 days, observe the ascites production of the mice every day. If the abdomen is obviously swollen and the skin feels tense when touched, the ascites can be collected. Usually 3 ml of ascites can be collected from each mouse;

[0140] ④ Centrifuge the ascites (2000r / min for 5 minutes) to remove cell components and other precipitates, collect the supernatant, determine the antibody titer, aliquot, and freeze at -70℃ for later use;

[0141] (2) Purification of monoclonal antibodies (octanoic acid-ammonium sulfate precipitation method)

[0142] ① Centrifuge the ascites at 4°C, 12,000 rpm for 15 min to remove impurities;

[0143] ② Take 1 part of ascites and add 2 parts of 0.06mol / L pH5.0 acetate buffer, add 33ul of octanoic acid per ml of diluted ascites, add octanoic acid dropwise under stirring at room temperature, and mix at room temperature for 30 minutes;

[0144] ③Stand at 4℃ for 2 hours, centrifuge at 12000g for 30 minutes, and discard the precipitate;

[0145] ④ The supernatant was filtered through a nylon mesh and dialyzed in 50 volumes of 0.01M pH 7.4 PBS at 4°C for 6 h;

[0146] ⑤ Add an equal volume of saturated ammonium sulfate solution to the supernatant after dialysis;

[0147] ⑥Stand at 4℃ for more than 1h, centrifuge at 10000g for 30 minutes, and discard the supernatant;

[0148] ⑦ The precipitate was dissolved in an appropriate amount of PBS (containing 137mmol / L NaCl, 2.6mol / L KCl, 0.2mmol / L EDTA) and dialyzed overnight in 50-100 times the volume of PBS;

[0149] ⑧ Take a small amount of dialyzed sample and dilute it appropriately, then use UV spectrophotometer to detect protein content, and use SDS-PAGE and WB to detect antibody purity.

[0150] In this embodiment, the in vitro model application neutralizing antibody experiment mainly includes the following steps:

[0151] 1. Adding neutralizing antibodies to the co-culture model of fibroblasts and cytotoxic lymphocytes;

[0152] 2. Detection of cytotoxic lymphocyte death.

[0153] Example 4: In vivo validation of HS3ST3B1 + CAFs can mediate immunosuppression

[0154] To further validate HS3ST3B1 in vivo +CAFs can mediate immunosuppression, and CD158d is an immunosuppression-related checkpoint molecule. The present invention collects HS3ST3B1 from breast cancer patients. + Breast cancer tissues with a high proportion of CAFs were used to construct PDX models in immunodeficient NOD / SCID mice. PDX models refer to in vivo research models in which patient-derived tumor tissues are used to construct xenografts in immunodeficient mice.

[0155] The present invention simultaneously collects peripheral blood from patients with breast cancer samples, separates CD8T lymphocytes and DCs in vitro, activates DCs by treating tumor tissue lysates, and co-cultures DC cells and CD8T lymphocytes from the same source to induce tumor-specific CTLs. As shown in Figure 4A, after PDX tumors are formed, CTLs are reinfused into the tail vein, and some mice are intraperitoneally injected with CD158d neutralizing antibodies every 4 days.

[0156] The results showed that HS3ST3B1 + PDX constructed from breast cancer tissue with a high proportion of CAFs can resist the killing of tumor-specific CTLs, see B, C, and D of Figure 4, and neutralization of CD158d with neutralizing antibodies can significantly restore the killing of PDXs by CTLs, see B, C, and D of Figure 4). This shows that HS3ST3B1 + Enrichment of CAFs leads to immunosuppression, while targeting CD158d can significantly restore tumor immunity.

[0157] In this embodiment, the in vivo experimental model of mice is treated with neutralizing antibodies, which mainly includes the following steps:

[0158] 1. Construction of immunodeficient mouse PDX model

[0159] (1) Use sterile scissors to cut fresh tumor tissue into 1 cubic millimeter tissue particles;

[0160] (2) implanting tissue particles into mouse fat pads;

[0161] (3) Continued passage of tumor tissue after it has formed;

[0162] 2. Construction of tumor-specific cytotoxic T lymphocytes in vitro

[0163] (1) Grind the tumor tissue from the same patient and freeze-thaw it 5 times;

[0164] (2) Centrifuge at 14,000 rpm and collect the supernatant;

[0165] (3) Isolate monocytes from the patient's peripheral blood and add IL4 and GMSCF cytokines to induce them into DC cells;

[0166] (4) adding tumor lysate to DC cells;

[0167] (5) Add T lymphocytes from the same source and co-culture for 5 days, and sort CD8-positive CTL cells;

[0168] 3. Adoptive immunization of PDX model, injection of CTL cell suspension into tail vein;

[0169] 4. Inject the constructed neutralizing antibody intraperitoneally every 3 days and measure the tumor size.

[0170] The above is only a preferred embodiment of the present invention, and the present invention should not be limited to the contents disclosed in the embodiment and the drawings. Any equivalent or modification completed without departing from the spirit disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

Application of HS3ST3B1-positive tumor-associated fibroblasts in the preparation of a kit for detecting cytotoxic lymphocyte death. Application of HS3ST3B1-positive tumor-associated fibroblasts in the preparation of a kit for detecting tumor immune evasion. Application of HS3ST3B1 as a target in screening or preparing drugs that inhibit tumor immune evasion. An immune checkpoint molecule, CD158d, that mediates cytotoxic lymphocyte death mediated by tumor fibroblasts. The CD158d molecule is used as an immune checkpoint to mediate cytotoxic lymphocyte death mediated by tumor fibroblasts. Application of CD158d molecule as a target in screening or preparing drugs that inhibit tumor immune evasion. A neutralizing antibody that binds to the CD158d molecule, which inhibits tumor immune evasion by neutralizing CD158d. The neutralizing antibody according to claim 7, which is a monoclonal antibody or a polyclonal antibody. Use of the neutralizing antibody according to claim 7 or 8 as an immune checkpoint inhibitor. Use of the neutralizing antibody according to claim 7 or 8 in the preparation of a drug for inhibiting tumor immune escape.

Images