在临床前研究中，CD39纳米病的开发及其增强对卵巢癌的嵌合抗原受体T细胞有效性

理由：CD39是一种驱动腺苷产生的关键核苷酸酶，它是癌症中关键的免疫抑制检查点。尽管它已显示为治疗靶点的希望，但临床试验表明需要采用更有效的靶向方法。这种需求是推动创新新型抗体的开发，并探索与一系列免疫疗法的战略组合。方法：筛选抗CD39纳米机构，并使用Biolayer干涉测量法，ELISA和流式细胞仪测试其亲和力和结合能力。在CD39阻断后测量了针对可溶性和膜结合CD39的阻塞能力。使用免疫荧光检测到内在化。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T细胞功能的反转。在小鼠模型中进一步测试了肿瘤生长抑制的体内功能，我们还测试了CD39抗体从肿瘤组织，排出淋巴结和外周血后的免疫细胞表型。我们将抗体序列插入嵌合抗原受体（CAR）构建体中，以诱导MSLN CAR-T细胞秘密秘密CD39抗体，并在卵巢癌的异种移植模型中测量了功效。结果：我们使用VHH库筛选了人类CD39抗体，并开发了一种单位抗CD39纳米病，称为HUCD39 mAb，具有高亲和力，有效的结合和阻断能力。 HUCD39 mAb以时间依赖的方式内化。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在免疫能力的小鼠模型中显示出明显的抗肿瘤功效。流式细胞仪分析表明，抗体给药后免疫细胞中的CD39表达下调。我们还观察到CD39在卵巢癌组织和活化的CAR T细胞中的表达增加。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：已经开发了一种针对人CD39的强大阻塞能力和对肿瘤生长的有效抑制作用的新型HUCD39 mAb。此外，已经产生了一种改良的HUCD39单元格式CAR-T细胞，对卵巢癌表现出了较高的功效。这为优化卵巢癌和潜在其他恶性肿瘤的免疫疗法提供了有希望的策略。

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.