CD39单克隆抗体的开发及其在临床前研究中增强嵌合抗原受体T细胞对卵巢癌的疗效

研究背景：CD39是一种关键的胞外核苷酸酶，驱动腺苷的产生，在癌症中作为一种重要的免疫抑制性检查点发挥作用。虽然其作为治疗靶点展现出潜力，但临床试验表明需要更具效力的靶向策略。这一需求推动创新，开发新型抗体并探索与多种免疫疗法的战略性联合方法。方法：采用生物层干涉技术、ELISA和流式细胞术筛选并检测抗CD39纳米抗体的亲和力和结合能力。测定抗体对可溶性和膜结合型CD39的阻断能力。利用免疫荧光检测抗体的内吞作用。通过CFSE标记的T细胞增殖、CD25表达和IFN-γ分泌评价抗CD39抗体逆转T细胞功能的效果。在体内动物模型中进一步验证其抗肿瘤作用，并检测抗体给药后肿瘤组织、引流淋巴结和外周血中免疫细胞的表型变化。将抗体序列整合到嵌合抗原受体（CAR）构建中，以诱导MSLN CAR-T细胞分泌抗CD39抗体，在卵巢癌异种移植模型中评估其疗效。结果：利用VHH库筛选出针对人类CD39的单抗，开发出具有高亲和力和强结合及阻断能力的单一表位抗CD39纳米抗体，命名为huCD39 mAb。该抗体在时间依赖性内吞过程中表现出高效性。在体外研究中，huCD39 mAb显著增强T细胞增殖和功能。在体内，huCD39 mAb在免疫完整的小鼠模型中表现出明显的抗肿瘤效果。流式细胞分析显示抗体处理后免疫细胞中的CD39表达下降。我们还发现卵巢癌组织和激活的CAR T细胞中CD39表达升高。随后，开发出一种分泌huCD39 mAb的MSLN CAR-T细胞，在卵巢肿瘤异种移植中表现出有效的清除或抑制作用。结论：开发出具有强阻断作用的创新型huCD39 mAb，有效抑制肿瘤生长。此外，基于该抗体的CAR-T细胞也展现出优异的抗卵巢癌效果，为优化卵巢癌及其他恶性肿瘤的免疫治疗提供了有前景的策略。

Rationale: CD39, a key ectonucleotidase that drives adenosine production, acts as a critical immunosuppressive checkpoint in cancer. Although it has shown promise as a therapeutic target, clinical trials are demonstrating the need for more potent targeting approaches. This need is driving innovation towards the development of novel antibodies and the exploration of strategic combinations with a range of immunotherapies. Methods: An anti-CD39 nanobody was screened and tested for its affinity and binding ability using biolayer interferometry, ELISA and flow cytometry. Blocking ability against soluble and membrane-bound CD39 was measured after CD39 blockade. Internalization was detected using immunofluorescence. The reversal of T-cell function by the anti-CD39 antibody was assessed by CFSE-based T-cell proliferation, CD25 expression and IFN-γ secretion. The in vivo function of tumor growth inhibition was further tested in a mouse model and we also tested the phenotype of immune cells after CD39 antibody administration from tumor tissue, draining lymph nodes and peripheral blood. We inserted the antibody sequence into the chimeric antigen receptor (CAR) construct to induce MSLN CAR-T cells to secret the CD39 antibody, and the efficacy was measured in xenograft models of ovarian cancer. Results: We screened human CD39 antibodies using a VHH library and developed a single-epitope anti-CD39 nanobody, named huCD39 mAb, with high affinity and potent binding and blocking ability. The huCD39 mAb was internalized in a time-dependent manner. The in vitro study revealed that the huCD39 mAb was highly effective in enhancing T-cell proliferation and functionality. In vivo, the huCD39 mAb showed significant anti-tumor efficacy in an immunocompetent mouse model. Flow cytometry analysis demonstrated downregulated CD39 expression in immune cells after antibody administration. We also observed increased CD39 expression in ovarian cancer tissue and in activated CAR T cells. Subsequently, we developed a type of MSLN CAR-T cells secreting huCD39 mAb which showed effective eradication or inhibition in ovarian tumor xenografts. Conclusions: A novel huCD39 mAb with strong blocking ability against human CD39 and potent inhibition of tumor growth has been developed. Furthermore, a modified huCD39 mAb-secreting CAR-T cell has been generated, exhibiting superior efficacy against ovarian cancer. This provides a promising strategy for optimizing immunotherapies in ovarian cancer and potentially other malignancies.