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Archive > Volume 30 Issue 5 > 2023,30(5):380-386. DOI:10.3872/j.issn.1007-385X.2023.05.003 Prev Next
Basic Research
Evaluation of anticancer activity of PD-1/CTLA-4 bispecific antibody and its lgGl isotype based on target humanized mice
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Abstract:
Objective: To construct programmed death-1 (PD-1)/cytotoxic T lymphocyte antigen-4 (CTLA-4) bispecific antibody (BsAb) based on the target humanized mice, evaluate the anticancer activity of BsAb and its IgG1 isotype and investigate its potential working mechanism. Methods: Different formats and isotypes of PD-1/CTLA-4 antibodies BsAb1, BsAb2 and BsAb3 were constructed, expanded and purified. The target affinity of purified BsAbs were tested by surface plasmon resonance (SPR), and the biological activity of antibodies were tested by luciferase reporter gene assay and FCM. The efficacy of BsAbs was evaluated in vivo based on the MC38-hPD-L1 colon carcinoma cell transplant tumor model in humanized B-hPD-1-hPD-L1-hCTLA-4 mice. The working mechanism of PD-1/CTLA-4 BsAbs in transplant tumor tissues was investigated by tumor-infiltrating lymphocyte (TIL) analysis. Results: Successfully prepared BsAb1, BsAb2, and BsAb3 all demonstrated relatively strong specific affinity to checkpoint targets PD-1 and CTLA-4, showed different levels of blocker efficacy to target pathways, and significantly suppressed the growth of transplant tumors (P<0.05 or P<0.01). BsAb with IgG1 isotype was more potent in vivo than other isotypes (P<0.01). Tumor-infiltrating lymphocyte analysis revealed that BsAb2-IgG1 significantly increased the percentage of cytotoxic T lymphocytes (CTLs) (P<0.05) and significantly decreased the percentage of tumor-infiltrating regulatory T (Treg) cells (P<0.01), thus making the tumor immune microenvironment more conducive in killing tumor cells. However, the ADCC-enhanced Fc mutation isotype of BsAb2-SI can not further improve anti-tumor activity. Conclusion: The IgG1 PD-1/CTLA-4 antibody with Fc effector function has more potent in vivo anticancer efficacy, because it can better remove Treg cells in tumor-infiltrating lymphocytes.
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