Background Therapeutic antibodies inhibiting PD-1/PD-L1 have demonstrated clinical efficacy though only a fraction of patients respond. Combinations are being explored to enhance responses including anti-PD-1/PD-L1 IgG antibodies with IL-15-pathway stimulating agents to remove PD-1 immunosuppressive signaling and enhance anti-tumor NK and memory CD8 T cell expansion and survival. We have engineered an anti-PD-L1 pentameric high affinity, high avidity IgM, to target low PD-L1 expressing tumors, with an IL-15 superagonist fused to the joining (J) chain.
Methods An anti-PD-L1 IgM was generated by grafting heavy chain variable regions of a high affinity IgG onto the IgM heavy chain framework and co-expressed with the light chains. The IL-15 superagonist fused to the J chain generated PDL1-ISA. Anti-PD-L1 binding was performed using recombinant antigen ELISAs and on cells by FACS. Reporter assays and PBMCs were used for potency testing. Cytokines were evaluated by CBA assays. In vitro cytotoxicity assays used luciferase tagged MDA-MB-231 cells with PBMCs, NK or CD8 T cells. Pharmacodynamic and efficacy studies were conducted in syngeneic and humanized mouse models.
Results The parental anti-PD-L1 IgM antibody bound recombinant and cellular PD-L1 more potently than an IgG antibody with the same binding domain. In functional PD-L1 and PD-1 blocking studies the anti-PD-L1 IgM was as efficacious as the IgG. PDL1-ISA provided a potent proliferation signal to primary human NK and CD8 T cells in vitro with little/no impact on regulatory or CD4 T cells. Limited cytokines were detected following 3–4 days culture with human PBMCs. PDL1-ISA had similar potencies for both human and cynomolgus CD8 T cells, and a 2–3-fold lower potency for mouse cells. Pharmacodynamic studies in humanized and BALB/c mice showed transient and dose-dependent increases in circulating NK and CD8 T cells. PDL1-ISA enhanced in vitro killing of PD-L1 positive MDA-MB-231-Luc cells by human PBMCs, CD8 T and NK cells compared to the anti-PD-L1 IgM (no IL-15). PDL1-ISA also demonstrated efficacy in a hPD-L1-CT26 HuCELL mouse model, with most treated animals having complete tumor regressions. Durable anti-tumor immune memory responses were observed upon tumor re-challenge.
Conclusions We have engineered an IL-15 immunostimulatory anti-PD-L1 IgM antibody that binds PD-L1 more potently than an IgG, stimulates NK and CD8 expansion in vitro and in vivo and induces complete tumor regressions in mouse models. This approach may enhance tumor localization of immunostimulatory cytokine IL-15 though the high affinity and high avidity binding to PD-L1 to improve anti-tumor responses and minimize toxicity.
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