CD39 纳米抗体的开发及其在临床前研究中增强 CAR-T 细胞抗卵巢癌疗效的作用

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- 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.