Background Immune checkpoint blockade with PD-(L)1 antibody has become a new standard of treatment for many cancers for its remarkable clinical responses. But only a minority of cancer patients responded to PD-1/PD-L1 blockade therapy due to varieties of resistance mechanisms. Unmet needs persist for patients with relapsed/refractory tumors after PD-1/PD-L1 blockade therapies. Upregulated PD-L1 expression is observed in a broad range of tumor types while normal expression of PD-L1 is limited primarily to T, B, antigen-presenting cells, and some non-lymphoid tissues. Thus, PD-L1 represents an attractive target for antibody-drug conjugate (ADC) for improving the therapeutic window via antibody targeted chemotherapy.
Methods We discovered an array of anti-PD-L1 antibodies from immunized rabbits through single B cell culture and cloning technology. Binding affinity, specificity, blockade potency and epitope bin were determined using ELISA, FACS and PD-1: PD-L1 blockade assays. To screen for antibody candidates suitable for ADC development, we determined internalization and in vitro ADC killing properties of the antibodies employing FACS, immunofluorescent microscopy and in vitro cytotoxicity assays, using PD-L1 expressing tumor cell lines or engineered cell lines. Furthermore, we determined in vivo immune checkpoint blockade efficacy of the lead candidate antibody in a transgenic mouse colon cancer model as well as ADC killing efficacy in a human lung cancer xenograft tumor model.
Results We demonstrated that antibodies of high affinity and specificity to human and cynomolgus PD-L1 can effectively block the interaction of Jurkat-PD-1 cells with PD-L1 expressed on the surface of engineered cell lines. Among these antibodies, the lead antibody AT-001, binds to a unique epitope and exhibits higher internalization efficiency, compared to benchmark antibody or commercial anti-PD-L1 antibody drugs Imfinzi, Tecentriq or Bavencio. Furthermore, AT-001 showed potent in vivo efficacy in MC38-hPD-L1 syngeneic mouse model, comparable to Imfinzi. When conjugated to vc-MMAE or GGFG-Dxd, AT-001 ADC showed more potent in vitro killing of PD-L1+ cells, more potent and safer in vivo efficacy in human lung cancer xenograft studies, compared to benchmark ADC. In vivo study of AT-001 ADC conjugated via a new linker with improved hydrophilicity, better serum stability and increased safety profile is ongoing.
Conclusions AT-001 ADC demonstrated strong cytotoxic killing potency against PD-L1 expressing cells in in vitro and in vivo efficacy studies. In addition, AT-001 also demonstrated strong in vitro and in vivo PD-1/PD-L1 blockade activities. These data support further preclinical development of AT-001 ADC as a novel bifunctional drug candidate for solid tumor treatment.
Acknowledgements We sincerely thank our collaborator Shanghai Escugen Biotechnology for the preparation of AT-001 ADCs, and staff of the CellMosaic company for the preparation and QC of anti-human IgG-Deruxtecan, as well as Biocytogen Beijing and Shanghai model organisms company for performing the in vivo efficacy studies.
Ethics Approval The study obtained ethics approval by the institutional IACUC committees of Biocytogen Beijing and Shanghai Model Organisms companies (at which the studies were conducted), and followed international, national and institutional guidelines for the humane treatment of animals and complies with relevant legislation
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