WO2024022357A1 - CD158d分子、其中和抗体，及应用 - Google Patents

CD158d分子、其中和抗体，及应用 Download PDF

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WO2024022357A1
WO2024022357A1 PCT/CN2023/109181 CN2023109181W WO2024022357A1 WO 2024022357 A1 WO2024022357 A1 WO 2024022357A1 CN 2023109181 W CN2023109181 W CN 2023109181W WO 2024022357 A1 WO2024022357 A1 WO 2024022357A1
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tumor
cd158d
hs3st3b1
cells
molecule
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宋尔卫
陈嘉宁
曾文锋
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中山大学孙逸仙纪念医院
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to CD158d molecules, neutralizing antibodies and applications, and belongs to the fields of bioengineering and biotherapy.
  • Tumor is one of the most serious diseases threatening human life and health. Humanity's understanding and research of tumors has gone through a long process.
  • the current treatment of tumors mainly covers comprehensive treatment strategies including surgery, chemotherapy, radiotherapy and targeted therapy.
  • comprehensive treatment strategies that rationally use these treatment methods, the survival conditions of cancer patients have been significantly improved.
  • distant metastasis, recurrence and treatment resistance are still difficult problems that many cancer patients will eventually face, and are also the current dilemmas in the treatment of malignant tumors.
  • the immune system is an important defense barrier for the body. It identifies and removes "non-self" components in the body and maintains the stability of the internal environment through immune defense, immune surveillance and immune homeostasis. During the occurrence and development of malignant tumors, a dynamic interaction process is formed between them and the systemic immune system.
  • Scholars have therefore proposed the "Cancer Immunoediting" hypothesis, pointing out that the interaction between tumors and the immune system can be divided into There are three states, including “Elimination”, “Equilibration” and "Escape”. In recent years, with the research on anti-tumor immunity With the continuous deepening of research, major breakthroughs have been made in tumor immunotherapy.
  • Immunotherapy Tolerance In the clinical exploration of immunotherapy for tumors that are traditionally considered immune "cold" tumors such as breast cancer and pancreatic cancer, researchers first conducted an analysis of different molecular subtypes on the potential of tumor immunotherapy based on tumor mutation load and lymphocyte infiltration. Stratified analysis. For example, in triple-negative and HER2-positive breast cancer, researchers have found that they have relatively higher tumor mutation burdens and more lymphocyte infiltration, and the tumor mutation burden and lymphocyte infiltration levels in patients with the same type of tumors are associated with good prognosis. Related, this suggests that immunotherapy has certain therapeutic potential even in immunologically cold tumors.
  • immune cell reinfusion therapy and tumor vaccines are two other important forms of immunotherapy.
  • the anti-tumor immune cells produced by the immune cells injected back into the patient or stimulated by the tumor vaccine are rapidly depleted or die when they reach the tumor, greatly reducing their anti-tumor effect. , thus greatly hindering its actual clinical translation and application.
  • tumor-immunity 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 the antigens to T cells, (3) T Activation of cells in draining lymph nodes, (4) activated T cells migrate to the tumor bed, (5) activated T cells infiltrate into the tumor through blood vessels, (6) activated effector T cells recognize tumor cells, (7) T cells kill tumor cells. Abnormalities in any of the above cycles can lead to anti-tumor immune evasion. However, the PD-1 and PD-L1 antagonists currently used in clinical practice only target the above steps (7).
  • the tumor microenvironment in addition to the tumor cells themselves, the tumor microenvironment also includes endothelial cells, fibroblasts, and pericytes in the stroma, as well as infiltrating immune cells such as lymphocytes, macrophages, granulocytes, myeloid-derived suppressor cells, and Extracellular matrix components and a large number of cytokines, chemokines, etc.
  • endothelial cells such as lymphocytes, macrophages, granulocytes, myeloid-derived suppressor cells, and Extracellular matrix components and a large number of cytokines, chemokines, etc.
  • each component affects anti-tumor immunity, starting from multi-angle and multi-dimensional analysis such as the activation and recruitment of anti-tumor immune cells and the regulation of the tumor microenvironment on the function and survival status of anti-tumor immune cells to explore solid tumors Screening and identifying the key regulatory node molecules in the regulatory network of anti-tumor immunity, thereby providing new targets for immunotherapy, will be an important direction and goal for future tumor immunotherapy research.
  • the present invention starts from the perspective of the interaction between the stromal cell components of the tumor microenvironment and killer lymphocytes that mainly perform anti-tumor immune functions, and analyzes the specific fibroblast subpopulation in the microenvironment that mediates killer lymphocytes.
  • Key regulatory nodes of incompetence identify new immune checkpoint molecules, and block the death of killer lymphocytes mediated by fibroblasts by targeting CD158d immune checkpoint molecules or HS3ST3B1-positive tumor-associated fibroblasts, thereby restoring lymphocytes The ability of cells to kill and eliminate tumor cells.
  • one aspect of the present invention provides the use of HS3ST3B1-positive tumor-associated fibroblasts in preparing a kit for detecting killer lymphocyte death.
  • Another aspect of the present invention provides the use of HS3ST3B1-positive tumor-associated fibroblasts in preparing a kit for detecting tumor immune evasion.
  • Another aspect of the present invention provides the use of HS3ST3B1 as a target in screening or preparing drugs that inhibit tumor immune evasion.
  • Another aspect of the invention provides an immune checkpoint molecule CD158d that mediates tumor fibroblast-mediated killer lymphocyte death.
  • Another aspect of the invention provides the use of the CD158d molecule as an immune checkpoint that mediates tumor fibroblast-mediated killer lymphocyte death.
  • Another aspect of the present invention provides the use of CD158d molecules as targets in screening or preparing drugs that inhibit tumor immune evasion.
  • Another aspect of the invention provides a neutralizing antibody that binds to CD158d molecules, which neutralizes CD158d to inhibit tumor immune evasion.
  • the neutralizing antibody is a monoclonal antibody or a polyclonal antibody.
  • Another aspect of the invention provides the use of the neutralizing antibody of the invention as an immune checkpoint inhibitor.
  • Another aspect of the present invention provides the use of the neutralizing antibody in preparing drugs that inhibit tumor immune evasion.
  • the present invention found that HS3ST3B1-positive tumor-associated fibroblasts can mediate killer lymphocyte death.
  • the present invention further discovered a new immune checkpoint molecule CD158d, which can mediate the death of killer lymphocytes mediated by tumor fibroblasts.
  • the present invention developed the application of CD158d molecule and HS3ST3B1 as targets in screening or preparing drugs that inhibit tumor immune evasion.
  • the present invention has obtained a neutralizing antibody that can neutralize the CD158d molecule. After neutralizing the CD158d molecule, the antibody can significantly restore the killing of tumors by CTLs. effect.
  • the present invention provides the use of CD158d neutralizing antibodies in the preparation of drugs that inhibit tumor immune escape.
  • Figure 1 is a trend diagram showing that silencing HS3ST3B1 in HS3ST3B1-positive tumor-associated fibroblasts inhibits its induced cytotoxic lymphocyte death.
  • the red numbers represent the proportion of AnnexinV-positive cells (second and fourth quadrants), that is, the proportion of dead cells.
  • Figure 2 is a schematic diagram of enhancing tumor immunity in vivo after silencing HS3ST3B1 in HS3ST3B1-positive tumor-associated fibroblasts.
  • A Schematic diagram of mouse xenograft tumors inoculated with primary tumor cells mixed with HS3ST3B1 + CAFs treated with different treatments.
  • Figure 3 is a schematic diagram showing the termination of the death trend mediated by HS3ST3B1 + CAFs after CD158d was knocked out by CTLs or CD158d was neutralized using neutralizing antibodies.
  • the red numbers represent the proportion of AnnexinV-positive cells.
  • Figure 4 is a schematic diagram of using neutralizing antibodies to target CD158d in vivo to promote tumor immunity.
  • Example 1 HS3ST3B1 + CAFs induce the death of CTLs by expressing HS3S3ST3B1
  • Tumor-associated fibroblasts CAFs and normal breast fibroblasts NBFs were isolated from primary tissues of breast cancer patients or breast tissue of patients undergoing breast reduction surgery.
  • CAFs were divided into HS3ST3B1-negative and HS3ST3B1-positive groups using flow sorting technology.
  • sorting HS3ST3B1-positive tumor-associated fibroblasts from tumors mainly includes the following steps:
  • step 2 Cut the tissue in step 1 with sterile scissors to 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;
  • step 2 Incubate the mixture in step 2 in a 37°C incubator for 2 hours;
  • step 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;
  • step 6 Use a flow cell sorter to sort the cell suspension in step 5 to obtain HS3ST3B1-positive tumor-associated fibroblasts.
  • the construction of a co-culture model of fibroblasts and lymphocytes mainly includes the following steps:
  • fibroblasts are seeded in the lower chamber of the transwell, and cytotoxic lymphocytes are located in the upper chamber;
  • Example 2 HS3ST3B1 + CAFs promote tumor cell immune evasion through HS3ST3B1 in vivo
  • primary tumor cells were isolated from breast cancer tissue, and tumor cells and differently treated HS3ST3B1 + CAFs were mixed and inoculated into the fourth pair of mammary fat pads of immunodeficient mice.
  • monocytes were isolated from the patient's peripheral blood, induced to differentiate into dendritic cells, and then loaded with tumor antigens.
  • the CD8 T lymphocytes isolated from the peripheral blood were then treated with dendritic cells loaded with tumor antigens, thereby constructing in vitro Target the tumor-specific CTLs and perform tail vein reinfusion when the mouse tumor reaches a diameter of 2 mm.
  • xenograft tumors formed by HS3ST3B1 + CAFs mixed tumor cells will resist the immune killing effect of adoptively reinfused CTLs, which is manifested in that the transplanted tumors still show significant growth after adoptive reinfusion of CTLs. long, see Figure 2B.
  • the expression of HS3ST3B1 in this group of CAFs is silenced, the function of adoptive immunity can be significantly restored. This suggests that HS3ST3B1 + CAFs promote tumor cell immune evasion through HS3ST3B1 in vivo.
  • the in vivo verification model of immune evasion mediated by HS3ST3B1-positive fibroblasts mainly includes the following steps:
  • the fibroblasts from the HS3ST3B1 knockout group and the non-knockout group were mixed with primary tumor cells and transplanted into the fat pad of immunodeficient mice;
  • the cell pellet is collected and frozen and thawed 5 times;
  • Mononuclear cells are isolated from the patient’s peripheral blood and induced into DC cells by adding IL4 and GMSCF cytokines;
  • mice 6. Observe tumor progression in mice.
  • Example 3 HS3ST3B1 + CAFs mediate the death of CTLs through CD158d molecules on CTLs
  • CD158d neutralizing antibody can significantly inhibit the death of CTLs induced by HS3ST3B1 + CAFs, see Figure 3 E and F. This indicates that HS3ST3B1 + CAFs mediate the death of CTLs through the CD158d molecule on CTLs, which is a checkpoint molecule that mediates immune suppression.
  • the neutralizing antibody construction experiment mainly includes the following steps:
  • the dosage route is the same as above, plus Freund's incomplete adjuvant, with an interval of 3 weeks;
  • mice Take the immunized BALB/c mice, remove the eyeballs, collect blood, and separate the serum as a positive control serum for antibody detection. At the same time, the mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes, fixed on a petri dish, and then opened the left abdominal skin to see the spleen. Use ophthalmic scissors and cut open with sterile surgery in a clean bench. Peritoneum, remove the spleen and place it in a dish containing 10 ml of incomplete culture medium, wash gently, and carefully peel off the surrounding connective tissue.
  • P/N ⁇ 2.1, or P ⁇ N+3SD is considered positive. If the negative control well is colorless or close to colorless, and the positive control well is clearly colored, the result can be observed directly with the naked eye;
  • mice were intraperitoneally inoculated with phytane or liquid paraffin, 0.3-0.5ml per mouse;
  • hybridoma cells diluted in PBS or serum-free medium were intraperitoneally inoculated at 5 ⁇ 10 5 /0.2ml per mouse;
  • the in vitro model application neutralizing antibody experiment mainly includes the following steps:
  • Example 4 In vivo verification that HS3ST3B1 + CAFs can mediate immunosuppression
  • the present invention collected breast cancer tissues with a high proportion of HS3ST3B1 + CAFs derived from breast cancer patients. /SCID mice to construct PDX model.
  • the PDX model refers to an in vivo research model that uses patient-derived tumor tissue to construct xenografts in immunodeficient mice.
  • the present invention simultaneously collects the peripheral blood of patients with paired breast cancer samples, separates CD8 T lymphocytes and DCs in vitro, uses tumor tissue lysates to treat and activate DCs, and co-cultures DC cells and CD8 T lymphocytes from the same source to induce Tumor-specific CTLs.
  • FIG. 4A after PDX tumors were formed, CTLs were reinfused into the tail vein, and some mice were intraperitoneally injected with CD158d neutralizing antibody every 4 days.
  • the treatment of mice with neutralizing antibodies in an in vivo experimental model mainly includes the following steps:
  • Tumor tissue from the same patient was ground and frozen and thawed 5 times;
  • Mononuclear cells are isolated from the patient’s peripheral blood and induced into DC cells by adding IL4 and GMSCF cytokines;

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Abstract

本发明属于生物工程和生物治疗领域，公开了一种CD158d分子、其中和抗体，及应用。本发明通过发现HS3ST3B1阳性的肿瘤相关成纤维细胞可以介导杀伤性淋巴细胞死亡，进而发现了一种新的免疫检查点分子CD158d。针对CD158d分子可以介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡，本发明获得了能够中和CD158d分子的中和抗体。在此基础上，本发明开发了CD158d分子作为靶点在筛选或制备抑制肿瘤免疫逃避的药物中的应用，以及CD158d中和抗体在制备抑制肿瘤免疫逃的药物中的应用。

Description

CD158d分子、其中和抗体，及应用
本申请要求享有于2022年7月26日向中国国家知识产权局提交的，专利申请号为202210886386.5，名称为“CD158d分子、其中和抗体，及应用”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及CD158d分子、其中和抗体，及应用，属于生物工程和生物治疗领域。
背景技术
肿瘤是威胁人类生命健康最重大的疾病之一。人类对肿瘤的认识与研究经历了一个漫长的过程。目前对于肿瘤的治疗主要是涵盖了外科手术、化疗、放疗和靶向治疗在内的综合性治疗策略。近年来，通过合理的运用这些治疗手段的综合治疗策略，肿瘤患者的生存状况得到了显著的改善。然而，肿瘤远处转移、复发以及治疗耐受仍然是众多肿瘤患者最终必将面对的难题，也是目前恶性肿瘤治疗的困境所在。
免疫系统是机体重要的防御屏障，它通过免疫防御、免疫监视和免疫自稳来识别和清除体内出现的“非己”成分并维持内环境的稳定。在恶性肿瘤的发生发展过程中，其与全身免疫系统之间形成了一个动态的互动过程，学者由此提出了“肿瘤免疫编辑”(Cancer Immunoediting)假说，指出肿瘤与免疫系统的相互作用可分为三种状态，包括“免疫清除”(Elimination)、“免疫平衡”(Equilibration)和“免疫逃逸”(Escape)。近年来，随着对抗肿瘤免疫研究 的不断深入，肿瘤免疫治疗取得了重大的突破，针对PD-1/PD-L1通路的单克隆抗体治疗等新兴免疫治疗策略在治疗黑色素瘤、肺癌、膀胱癌等恶性肿瘤中取得了显著疗效，极大地鼓舞了研究人员的肿瘤免疫研究热情。因此，恢复机体正常的抗肿瘤免疫被认为有望成为恶性肿瘤的根治性治疗策略。
随着大量免疫治疗临床试验结果的出炉，靶向PD-1/PD-L1的免疫检查点抑制剂的治疗效果在不同癌肿间以及同癌肿不同个体间的差异性凸显。即使在传统认为的黑色素瘤、肺癌、膀胱癌等免疫“热”肿瘤中，仍然存在极大部分患者对免疫检查点抑制剂不敏感。在抗PD-L1的治疗中，即使在治疗前先对患者的PD-L1进行评估，然后分析PD-L1阳性患者对免疫治疗的敏感性，仍然有超过50％的患者对治疗耐受，而即使是在一开始对免疫治疗表现出良好反应性的患者也存在部分患者在治疗过程中转而呈现为肿瘤进展，前者称之为“原发免疫治疗耐受”，后者称之为“继发免疫治疗耐受”。而在传统认为的乳腺癌、胰腺癌等免疫“冷”肿瘤的免疫治疗临床探索中，研究人员首先针对肿瘤突变负荷以及淋巴细胞浸润情况对肿瘤免疫治疗的潜在可能性进行了不同分子亚型的分层分析。例如在三阴性及HER2阳性型乳腺癌，研究人员发现其相对具有更高的肿瘤突变负荷以及更多的淋巴细胞浸润，而且同类型的肿瘤患者中肿瘤突变负荷及淋巴细胞的浸润水平与其良好预后相关，这提示免疫疗法即使在免疫冷肿瘤中也具有一定的治疗潜能。于是，全球范围内多项联合免疫治疗临床试验在乳腺癌等免疫冷肿瘤患者队列的治疗中得以获批开展。其中，免疫检查点抑制剂单药治疗模型中患者并无显著生存获益(phase II KEYNOTE-086study)。而联合治疗策略试验方面，IMpassion130III期随机临床对照试验分析了紫杉醇联合PD-L1单抗与否在乳腺癌患者中的疗效对比。其中期分析结果显示，在PD-L1单抗联合治疗组与紫杉醇单药治疗组之间，病人的总生存无显著差异，但是在分层分析中发现，在免疫细胞PD-L1阳性的患者的治疗中PD-L1单抗联合紫杉醇治疗组病人显示出更优的生存获益。然而，患者的整体获益仍然有限。
除了免疫检查点抑制剂，免疫细胞回输治疗以及肿瘤疫苗是另外两个重要的免疫治疗形式。然而，由于实体肿瘤复杂肿瘤微环境调控网络的存在，使得回输到患者体内的免疫细胞或者肿瘤疫苗刺激机体产生的抗肿瘤免疫细胞在到达肿瘤局部迅速耗竭或者死亡，大大降低了其抗肿瘤效应，从而极大地阻碍了其实际临床转化运用。
目前恶性肿瘤免疫治疗效果存在巨大个体化差异，究其根源，一方面在于目前基础研究对于恶性肿瘤免疫逃逸机制的认识仍然不足，而另一方面则是新近发现的抗肿瘤免疫调控节点的靶向干预策略仍然未能实现实际的临床转化。
在“肿瘤-免疫循环”中，有效抗肿瘤免疫的实现需要经历7个步骤：(1)肿瘤抗原释放并被DC细胞捕获，(2)DC细胞将抗原提呈给T细胞，(3)T细胞在引流淋巴结的活化，(4)活化T细胞迁移到肿瘤床，(5)活化T细胞穿过血管浸润到肿瘤中，(6)活化的效应T细胞识别肿瘤细胞，(7)T细胞杀伤肿瘤细胞。而以上循环中任何一个环节出现异常均可导致抗肿瘤免疫逃逸的发生。而目前临床实际运用的PD-1和PD-L1拮抗剂仅仅是针对以上步骤(7)。
此外，在实体肿瘤中，复杂肿瘤微环境存在成为导致肿瘤免疫逃逸的重要保护伞。除了肿瘤细胞本身，肿瘤微环境中还包括了间质中的内皮细胞、成纤维细胞、周细胞，浸润而来的免疫细胞，如淋巴细胞、巨噬细胞、粒细胞、髓源抑制性细胞以及细胞外基质成分和大量的细胞因子、趋化因子等。近年来，众多的肿瘤实验性研究以及肿瘤生物学行为预测模型的研究结果均显示出肿瘤微环境在恶性肿瘤的发生发展、远处转移以及治疗耐受中发挥着至关重要的作用。随着肿瘤微环境研究的不断深入，研究人员逐渐意识到，随着肿瘤的进展或者不同治疗手段的干预，肿瘤微环境中各细胞组分之间的相互作用是处于动态的变化过程中的，而且，微环境中各种细胞成分具有高度异质性，同一种细胞类型可能同时存在不同的促肿瘤或抑肿瘤表型。因此，深入研究抗肿瘤免疫在肿瘤发生发展及治疗的各个不同阶段的动态变化特征，尤其是肿瘤微环境中 各组分之间的相互作用对抗肿瘤免疫的影响，从抗肿瘤免疫细胞的活化、募集以及肿瘤微环境对抗肿瘤免疫细胞功能和生存状态的调控作用等多角度、多维度分析入手，探索实体肿瘤抗肿瘤免疫的调控网络，筛选鉴定出其中的关键调控节点分子，从而为免疫治疗提供新的靶点将是未来肿瘤免疫治疗研究的重要方向和目标。
发明内容
针对上述方向和目标，本发明从肿瘤微环境间质细胞成分与主要执行抗肿瘤免疫功能的杀伤性淋巴细胞相互作用的角度入手，解析微环境中特异成纤维细胞亚群介导杀伤性淋巴细胞失能的关键调控节点，鉴定新的免疫检查点分子，通过靶向CD158d免疫检查点分子或者HS3ST3B1阳性肿瘤相关成纤维细胞从而阻断成纤维细胞所介导的杀伤性淋巴细胞死亡，从而恢复淋巴细胞对肿瘤细胞的杀伤清除能力。
为此，本发明一方面提供了HS3ST3B1阳性的肿瘤相关成纤维细胞在制备检测杀伤性淋巴细胞死亡的试剂盒中的应用。
本发明另一方面提供了HS3ST3B1阳性的肿瘤相关成纤维细胞在制备检测肿瘤免疫逃避的试剂盒中的应用。
本发明另一方面提供了HS3ST3B1作为靶点在筛选或制备抑制肿瘤免疫逃避的药物中的应用。
本发明另一方面提供了一种免疫检查点分子CD158d，其介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡。
本发明另一方面提供了CD158d分子在作为免疫检查点中的应用，其介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡。
本发明另一方面提供了CD158d分子作为靶点在筛选或制备抑制肿瘤免疫逃避的药物中的应用。
本发明另一方面提供了一种结合CD158d分子的中和抗体，其通过中和 CD158d来抑制肿瘤免疫逃避。
在本发明优选的方案中，所述的中和抗体为单克隆抗体或多克隆抗体。
本发明另一方面提供了本发明所述的中和抗体在作为免疫检查点抑制剂中的应用。
本发明再一方面提供了所述的中和抗体在制备抑制肿瘤免疫逃避的药物中的应用。
有益效果
与现有技术相比，本发明发现了HS3ST3B1阳性的肿瘤相关成纤维细胞可以介导杀伤性淋巴细胞死亡。在此基础上，本发明进一步发现了一种新的免疫检查点分子CD158d，其可以介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡。基于上述发现，本发明开发了CD158d分子和HS3ST3B1作为靶点在筛选或制备抑制肿瘤免疫逃避的药物中的应用。
针对CD158d分子可以介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡，本发明获得了能够中和CD158d分子的中和抗体，该抗体中和CD158d分子后，可以显著恢复CTLs对肿瘤的杀伤作用。由此，本发明提供了CD158d中和抗体在制备抑制肿瘤免疫逃的药物中的应用。
附图说明
图1是在HS3ST3B1阳性的肿瘤相关成纤维细胞中沉默HS3ST3B1抑制了其诱导的细胞毒性淋巴细胞死亡的趋势图。
A、流式细胞术检测未处理CTLs(CTRL组)和各组跟不同成纤维细胞共培养后的CTLs中AnnexinV荧光抗体和PI着色情况的代表图。
红色数字代表AnnexinV阳性(第二象限和第四象限)的细胞比例，也即死亡细胞的比例。
B、图1A定量(n＝3)。
C、检测未处理CTLs(CTRL组)和各组跟不同成纤维细胞共培养后的CTLs培养体系中乳酸脱氢酶LDH的释放，以LDH的释放比例代表发生死亡的细胞的比例(n＝3)。
图2是在HS3ST3B1阳性的肿瘤相关成纤维细胞中沉默HS3ST3B1后增强体内肿瘤免疫示意图。
A、原代肿瘤细胞与不同处理的HS3ST3B1+CAFs混合接种的小鼠异种移植瘤模式图。
B、原代肿瘤细胞与不同处理的HS3ST3B1+CAFs混合接种到小鼠后，不处理或者回输CTLs，各种异种移植瘤的生长(n＝8)。
图3是在CTLs敲除了CD158d或者利用中和抗体中和了CD158d后，终止了HS3ST3B1+CAFs所介导的死亡趋势示意图。
A、流式细胞术检测单独培养CTLs和各组CTLs(UT，sgGFP和敲除CD158d组)跟不同成纤维细胞共培养后AnnexinV荧光抗体和PI着色情况的代表图。
红色数字代表AnnexinV阳性的细胞比例。
B、图3A定量(n＝3)。
C、检测单独培养CTLs和各组CTLs(UT，sgGFP和敲除CD158d组)跟不同成纤维细胞共培养后LDH的释放(n＝3)。
D、流式细胞术检测单独培养CTLs和各组CTLs(UT，IgG和CD158d中和抗体组)跟不同成纤维细胞共培养后AnnexinV荧光抗体和PI着色情况(n＝3)。
E、检测单独培养CTLs和各组CTLs(UT，IgG和CD158d中和抗体组)跟不同成纤维细胞共培养后LDH的释放(n＝3)。
图4是利用中和抗体体内靶向CD158d促进肿瘤免疫的示意图。
A、PDX小鼠上进行靶向CD158d联合过继性免疫的模式图。
B、未回输CTLs组和不同处理的回输CTLs组(PBS，IgG和CD158d中 和抗体处理)小鼠PDX肿瘤生长速度。
C、未回输CTLs组和不同处理的回输CTLs组(PBS，IgG和CD158d中和抗体处理)小鼠所有PDX肿瘤的大小。
D、PET/CT检测未回输CTLs组和不同处理的回输CTLs组(PBS，IgG和CD158d中和抗体处理)小鼠PDX肿瘤大小的代表图。
具体实施方式
为了更好的说明本方法的目的和优点，结合附图及具体实施例对本发明具体实施内容做进一步详细说明。
实施例1：HS3ST3B1+CAFs通过表达HS3S3ST3B1诱导CTLs的死亡
从乳腺癌患者原代组织或缩乳手术患者乳腺组织中分离肿瘤相关成纤维细胞CAFs和乳腺正常成纤维细胞NBFs。
利用流式分选技术将CAFs分成HS3ST3B1阴性和阳性两群。
将上述成纤维细胞和细胞毒性淋巴细胞CTLs进行共培养，相比乳腺正常成纤维细胞NBFs和HS3ST3B1阴性的肿瘤相关成纤维细胞HS3ST3B1-CAFs，HS3ST3B1阳性的肿瘤相关成纤维细胞HS3ST3B1+CAFs可以显著诱导CTLs死亡，参见附图1的A、B、C。
当利用慢病毒感染成纤维细胞转录shRNAs沉默成纤维细胞中HS3S3ST3B1的表达之后，HS3ST3B1+CAFs诱导的CTLs死亡趋势被抑制，参加附图1的A、B、C。
上述结果表明，HS3ST3B1+CAFs通过表达HS3S3ST3B1诱导CTLs的死亡。
本实施例中，从肿瘤中分选出HS3ST3B1阳性的肿瘤相关成纤维细胞，主要包括以下步骤：
1、收集患者乳腺癌新鲜组织约1立方厘米；
2、将步骤1中的组织用无菌剪刀剪碎至约1立方毫米，加入含有1.5mg/ml 1型胶原酶和1.5mg/ml 3型胶原酶的培养基10ml；
3、将步骤2中的混合物在37℃培养箱中孵育2小时；
4、对步骤3中的组织培养液进行过滤从而获得原代细胞悬液；
5、对步骤4中的细胞悬液进行离心，重悬后加入抗PDGFRa的流式抗体和抗HS3ST3B1的一抗孵育30分钟，离心去掉上清液后重悬，进一步加入荧光二抗孵育45分钟，最后离心去掉上清液后重悬；
6、利用流式细胞分选仪对步骤5中细胞悬液进行分选，从而获得HS3ST3B1阳性的肿瘤相关成纤维细胞。
本实施例中，成纤维细胞和淋巴细胞共培养模型的构建，主要包括以下步骤：
1、将HS3ST3B1阳性的肿瘤相关成纤维细胞和杀伤性淋巴细胞共培养，成纤维细胞种于transwell下室，细胞毒性淋巴细胞位于上室；
2、对共培养后的细胞毒性淋巴细胞进行细胞死亡检测(LDH释放实验，流式检测)。
实施例2：HS3ST3B1+CAFs在体内通过HS3ST3B1促进肿瘤细胞免疫逃避
如附图2A所示，从乳腺癌组织中分离出原代肿瘤细胞，将肿瘤细胞和不同处理的HS3ST3B1+CAFs混合接种到免疫缺陷小鼠第四对乳腺脂肪垫中。同时，从病人外周血分离单核细胞，诱导其向树突状细胞分化以后负载肿瘤抗原，再用负载了肿瘤抗原的树突状细胞处理外周血分离所得的CD8T淋巴细胞，从而在体外构建出针对该肿瘤的特异性CTLs，并在小鼠肿瘤形成直径达到2mm时进行尾静脉回输。
本发明发现HS3ST3B1+CAFs混合肿瘤细胞形成的异种移植瘤会抵抗过继性回输的CTLs的免疫杀伤作用，表现为CTLs过继回输后移植瘤仍然显著生 长，参见附图2B。而当沉默这一群CAFs中HS3ST3B1的表达后，可以显著恢复过继性免疫的功能。这表明HS3ST3B1+CAFs在体内通过HS3ST3B1促进了肿瘤细胞免疫逃避。
本实施例中，HS3ST3B1阳性成纤维细胞介导的免疫逃避的体内验证模型，主要包括以下步骤：
1、分选得到的HS3ST3B1阳性的肿瘤相关成纤维细胞，利用crispr-cas9技术敲除成纤维细胞HS3ST3B1的基因；
2、从患者肿瘤组织分选得到原代肿瘤细胞；
3、将敲除HS3ST3B1组，未敲除组的成纤维细胞分别混合原代肿瘤细胞种植于免疫缺陷小鼠脂肪垫；
4、体外构建原代肿瘤细胞特异的细胞T毒性淋巴细胞：
(1)原代肿瘤细胞扩增后收集细胞沉淀，反复冻融5次；
(2)14000转/分离心，收集上清(肿瘤细胞lysate)；
(3)从患者外周血分离单核细胞，并加入IL4，GMSCF细胞因子诱导成DC细胞；
(4)将肿瘤裂解物加入DC细胞培养液中；
(5)加入同一来源的T淋巴细胞共培养5天，分选CD8阳性的CTL细胞；
5、给步骤3中构建的免疫缺陷小鼠模型尾静脉回输CTL细胞；
6、观察小鼠肿瘤进展。
实施例3：HS3ST3B1+CAFs通过CTLs上的CD158d分子介导CTLs的死亡
当利用CRISPR-CAS9沉默了CTLs中的CD158d的表达之后，HS3ST3B1+CAFs诱导的CTLs死亡趋势被抑制，参见附图3的A、B、C。
进一步地，在体外构建了CD158d的中和抗体，并将其加入到CAFs和 CTLs的共培养体系中，发现加入CD158d的中和抗体可以显著抑制HS3ST3B1+CAFs诱导的CTLs的死亡，参见附图3的E、F。这表明HS3ST3B1+CAFs通过CTLs上的CD158d分子介导了CTLs的死亡，CD158d是介导免疫抑制的一个检查点分子。
本实施例中，中和抗体构建实验，主要包括以下步骤：
1、体外重组人的CD158d全长蛋白(Ag)，蛋白序列如序列1所示。
2、小鼠免疫：
(1)初次免疫，Ag 50ug/只，加福氏完全佐剂皮下多点注射，一般1.5ml，间隔3周；
(2)第二次免疫，剂量途径同上，加福氏不完全佐剂，间隔3周；
(3)第三次免疫，剂量同上，不加佐剂，腹腔注射，7天后采血测其效价，检测免疫效果，间隔3周；
(4)加强免疫，剂量50ug，腹腔注射；
(5)3天后，取脾融合；
3、细胞融合：
(1)饲养细胞的制备：
小鼠腹腔巨噬细胞的准备：
①用6-10周龄的BALB/c小鼠；
②拉颈处死，浸泡于75％的酒精，消毒3min，用无菌剪刀剪开皮肤，暴露腹膜。用吸管注入6ml培养液，反复冲洗，吸出冲洗液；
③放入10ml离心管，1200rpm离心5min.；
④用20％小牛血清的培养液混悬，调整细胞数为1╳105/ml，加入96孔板，100ul/孔；放入37度孵箱培养；
(2)骨髓瘤细胞的准备：
①于融合前48-36小时，将骨髓瘤细胞扩大培养；
②融合当天，用弯头滴管将细胞从瓶壁轻轻吹下，收集于50ml离心管或 融合管内；
③1000r/min离心5-10分钟，弃去上清；
④加入30ml不完全培养基，离心洗涤一次。然后将细胞重悬浮于10ml不完全培养基，混匀；
⑤取骨髓瘤细胞悬液，加0.4％台酚蓝染液作活细胞计数后备用；
(3)脾细胞的准备
取已经免疫的BALB/c小鼠，摘除眼球采血，并分离血清，作为抗体检测时的阳性对照血清。同时通过颈脱位致死小鼠，浸泡于75％酒精中5分钟，于培养皿上固定后掀开左侧腹部皮肤，可看到脾脏，换眼科剪镊，在超净台中用无菌手术剪开腹膜，取出脾脏置于已盛有10ml不完全培养基的平皿中，轻轻洗涤，并细心剥去周围结缔组织。置平皿中不锈钢筛网上，用注射器针芯研磨成细胞悬液后计数，使脾细胞进入平皿中的不完全培养基。用吸管吹打数次，制成单细胞悬液。通常每只小鼠1×108-2.5×108个脾细胞；
(4)细胞融合
①将1×108脾细胞与1×107骨髓瘤细胞SP2/0混合于一支50ml融合管中，补加不完全培养基至30ml，充分混匀；
②1000r/min离心5-10分钟，将上清尽量吸净；
②在手掌上轻击融合管底，使沉淀细胞松散均匀；
③用1ml吸管在30s内加入预热的50％PEG 1ml，边加边轻轻搅拌；
④吸入吸管静置1min；
⑥加入预热的不完全培养液，终止PEG作用，连续每2min内分别加入1ml，2ml，3ml，4ml，5ml，10ml；
⑦800rpm，5分钟，弃去上清；
⑧加入5ml完全培养基，轻轻吹吸沉淀细胞，使其悬浮并混匀，然后补加完全培养基至40-50ml，分装96孔细胞培养板，每孔100ul，然后将培养板置37℃，5％CO2培养箱内培养；
⑨6h后补加选择培养基，每孔50ul，3天后用选择培养基半换液；
⑩经常观察杂交瘤细胞生长情况，待其长至孔底面积1/10以上时，吸出上清供抗体检测；
(5)杂交瘤细胞的选择：
①抗原用包被液稀释至10ug/ml；
②以100ul/孔量加入酶标板孔中，置4℃过夜或37℃吸附2小时；
③弃去孔内的液体，同时用洗涤液洗3次，每次3分钟，拍干；
④每孔加100ul封闭液37℃封闭1小时；
⑤洗涤液洗3次；
⑥每孔加100ul待检杂交瘤细胞培养上清，同时设立阳性、阴性对照和空白对照，37℃孵育1小时，洗涤，拍干；
⑥加酶标第二抗体，每孔100ul，37℃孵育1小时，洗涤，拍干；
⑦加底物液，每孔加新鲜配制的底物使用液100ul，37℃20分钟；
⑧以2mol/L H2SO4终止反应，在酶联免疫阅读仪上读取OD值；
⑩结果判定：以P/N≧2.1，或P≧N+3SD为阳性，若阴性对照孔无色或接近无色，阳性对照孔明确显色，则可直接用肉眼观察结果；
(6)杂交瘤细胞的克隆化(有限稀释法)
①制备小鼠脾细胞为饲养细胞；
②制备待克隆的杂交瘤细胞悬液，用含20％血清的HT培养基稀释至每毫升含5、10和20个细胞3种不同的稀释度；
③按每毫升加入5×104-1×105细胞的比例，在上述杂交瘤细胞悬液中分别加入腹腔巨噬细胞；
④每种杂交瘤细胞分装96孔板一块，每孔量为100ul；
⑤37℃、5％CO2培养6天，出现肉眼可见的克隆即可检测抗体，在倒置显微镜下观察，标出只有单个克隆生长的孔，取上清作抗体检测；
⑥取抗体检测阳性孔的细胞扩大培养，并冻存；
4、单克隆抗体的Ig类与亚类的鉴定
(1)以10ug/ml浓度的抗原包被酶标板，50ul/孔，4℃过夜；
(2)洗涤后，加入待检的单抗样品，100ul/孔，37℃1小时，设阴性、阳性对照孔；
(3)洗涤后，加入HRP标记的抗小鼠类及亚类Ig的抗体试剂，100ul/孔，37℃避光显色20分钟，用2mol/L H2SO4终止反应后，根据颜色判断抗体的亚型；
5、单克隆抗体的生产及纯化
(1)动物体内生产单抗
①成年BALB/c小鼠腹腔接种降植烷或液体石蜡，每只小鼠0.3-0.5ml；
②7-10天后腹腔接种用PBS或无血清培养基稀释的杂交瘤细胞，每只小鼠5×105/0.2ml；
③间隔5天后，每天观察小鼠腹水产生情况，如腹部明显膨大，以手触摸时，皮肤有紧张感，即可采集腹水。通常每只小鼠可采3ml腹水；
④将腹水离心(2000r/min 5分钟)，除去细胞成分和其他的沉淀物，收集上清，测定抗体效价，分装，-70℃冻存备用；
(2)单克隆抗体的纯化(辛酸-硫酸铵沉淀法)
①腹水4℃12000rpm离心15min，去除杂质；
②取1份腹水加2份0.06mol/L PH5.0醋酸缓冲液，按每毫升稀释腹水加33ul辛酸的比例，室温搅拌下逐滴加入辛酸，室温混合30min；
③4℃静置2小时，取出12000g离心30分钟，弃沉淀；
④上清经尼龙筛过滤，于50倍体积的0.01M PH7.4PBS中4℃透析6h；
⑤在透析后的上清中加入等体积饱和硫酸铵溶液；
⑥4℃静置1h以上，10000g离心30分钟，弃上清；
⑦沉淀溶于适量PBS(含137mmol/L NaCl，2.6mol/L KCl，0.2mmol/L EDTA)中，于50-100倍体积的PBS中透析过夜；
⑧取少量透析后样品适当稀释后，以紫外分光光度计检测蛋白含量，SDS-PAGE，WB检测抗体纯度。
本实施例中，体外模型应用中和抗体实验，主要包括以下步骤：
1、成纤维细胞与细胞毒性淋巴细胞共培养模型中加入构建中和抗体；
2、检测细胞毒性淋巴细胞死亡。
实施例4：体内验证HS3ST3B1+CAFs可以介导免疫抑制
为了进一步在体内验证HS3ST3B1+CAFs可以介导免疫抑制，以及CD158d是一个免疫抑制相关的检查点分子，本发明收集了乳腺癌患者来源的HS3ST3B1+CAFs比例高的乳腺癌组织，在免疫缺陷的NOD/SCID小鼠构建PDX模型。PDX模型是指用病人来源的肿瘤组织在免疫缺陷小鼠中构建异种移植物的体内研究模型。
本发明同步收集了乳腺癌样本配对的病人外周血，在体外分别分出CD8T淋巴细胞和DC，利用肿瘤组织裂解物处理激活DC，并将同一来源的DC细胞和CD8T淋巴细胞共培养，诱导出肿瘤特异的CTLs。如附图4A所示，在PDX成瘤后，进行尾静脉回输CTLs，部分小鼠每4天腹腔注射CD158d中和抗体。
结果发现，HS3ST3B1+CAFs比例高的乳腺癌组织构建的PDX可以抵抗肿瘤特异的CTLs的杀伤，参见附图4的B、C、D，而利用中和抗体中和CD158d后，可以显著恢复CTLs对PDXs的杀伤，参见附图4的B、C、D)。这表明HS3ST3B1+CAFs的富集会导致免疫抑制，而靶向CD158d可以显著恢复肿瘤免疫。
本实施例中，小鼠在体实验模型应用中和抗体治疗，主要包括以下步骤：
1、构建免疫缺陷小鼠PDX模型
(1)用无菌剪刀将新鲜肿瘤组织切成1立方毫米大小组织颗粒；
(2)将组织颗粒植入小鼠脂肪垫；
(3)成瘤后的组织继续传代；
2、体外构建肿瘤特异细胞毒性T淋巴细胞
(1)将同一患者来源的肿瘤组织研磨后反复冻融5次；
(2)14000转/分离心，收集上清；
(3)从患者外周血分离单核细胞，并加入IL4，GMSCF细胞因子诱导成DC细胞；
(4)将肿瘤裂解物加入DC细胞；
(5)加入同一来源的T淋巴细胞共培养5天，分选CD8阳性的CTL细胞；
3、PDX模型过继免疫，尾静脉注射CTL细胞悬液；
4、每隔3天腹腔注射构建的中和抗体，并测量肿瘤大小。
以上所述为本发明的较佳实施例而已，本发明不应该局限于该实施例和附图所公开的内容。凡是不脱离本发明所公开的精神下完成的等效或修改，都落入本发明保护的范围。

Claims (10)

  1. HS3ST3B1阳性的肿瘤相关成纤维细胞在制备检测杀伤性淋巴细胞死亡的试剂盒中的应用。
  2. HS3ST3B1阳性的肿瘤相关成纤维细胞在制备检测肿瘤免疫逃避的试剂盒中的应用。
  3. HS3ST3B1作为靶点在筛选或制备抑制肿瘤免疫逃避的药物中的应用。
  4. 一种免疫检查点分子CD158d，其介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡。
  5. CD158d分子在作为免疫检查点中的应用，其介导肿瘤成纤维细胞所介导的杀伤性淋巴细胞死亡。
  6. CD158d分子作为靶点在筛选或制备抑制肿瘤免疫逃避的药物中的应用。
  7. 一种结合CD158d分子的中和抗体，其通过中和CD158d来抑制肿瘤免疫逃避。
  8. 根据权利要求7所述的中和抗体，其为单克隆抗体或多克隆抗体。
  9. 权利要求7或8所述的中和抗体在作为免疫检查点抑制剂中的应用。
  10. 权利要求7或8所述的中和抗体在制备抑制肿瘤免疫逃避的药物中的应用。
PCT/CN2023/109181 2022-07-26 2023-07-25 CD158d分子、其中和抗体，及应用 WO2024022357A1 (zh)

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