基本原理：CD39 是一种驱动腺苷产生的关键核酸外切酶，是癌症中关键的免疫抑制检查点。尽管它已显示出作为治疗靶点的前景，但临床试验表明需要更有效的靶向方法。这种需求正在推动新型抗体的开发以及与一系列免疫疗法的战略组合探索的创新。方法：采用生物层干涉法、ELISA和流式细胞术筛选抗CD39纳米抗体并测试其亲和力和结合能力。 CD39 阻断后测量针对可溶性和膜结合 CD39 的阻断能力。使用免疫荧光检测内化。通过基于 CFSE 的 T 细胞增殖、CD25 表达和 IFN-γ 分泌来评估抗 CD39 抗体对 T 细胞功能的逆转。我们在小鼠模型中进一步测试了肿瘤生长抑制的体内功能，并且我们还测试了从肿瘤组织、引流淋巴结和外周血施用 CD39 抗体后免疫细胞的表型。我们将抗体序列插入嵌合抗原受体（CAR）构建体中，诱导 MSLN CAR-T 细胞分泌 CD39 抗体，并在卵巢癌异种移植模型中测量疗效。结果：我们利用VHH文库筛选了人CD39抗体，并开发了单表位抗CD39纳米抗体，命名为huCD39 mAb，具有高亲和力和有效的结合和阻断能力。 huCD39 mAb 以时间依赖性方式内化。体外研究表明，huCD39 mAb 在增强 T 细胞增殖和功能方面非常有效。在体内，huCD39 mAb 在免疫活性小鼠模型中显示出显着的抗肿瘤功效。流式细胞术分析表明，施用抗体后免疫细胞中 CD39 表达下调。我们还观察到卵巢癌组织和活化的 CAR T 细胞中 CD39 表达增加。随后，我们开发了一种分泌 huCD39 mAb 的 MSLN CAR-T 细胞，该细胞在卵巢肿瘤异种移植物中显示出有效的根除或抑制作用。结论：开发出一种新型huCD39 mAb，对人CD39具有很强的阻断能力，并能有效抑制肿瘤生长。此外，还产生了一种改良的 huCD39 mAb 分泌 CAR-T 细胞，对卵巢癌表现出卓越的功效。这为优化卵巢癌和其他潜在恶性肿瘤的免疫疗法提供了一种有前途的策略。©作者。

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.© The author(s).