Background4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T cells and NK cells, by enhancing proliferation, survival, cytolytic activity and IFN-γ production. Its ability to induce potent anti-tumor CD8+ and NK cell activity makes 4-1BB an attractive target for designing novel therapeutics for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel 4-1BB x 5T4 bispecific antibody that stimulates 4-1BB function only when co-engaged with 5T4, a tumor-associated antigen. The combined preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic.MethodsALG. APV-527 was built based the ADAPTIR™ platform with binding domains to 4-1BB and 5T4 generated using the ALLIGATOR-GOLD® human scFv library. ALG.APV-527 was tested using primary cells in the presence or absence of cells expressing 5T4. Cell Trace-labelled PBMC sub-optimally stimulated with anti-CD3, to induce 4-1BB expression, cells were gated using flow cytometry. T cell cytotoxicity was assessed by quantifying cell death in CD8+ T cell/tumor cell co-cultures, and images were obtained using a cell live imaging system (Cytation 5). For tumor inhibition studies, human 4-1BB knock-in mice were injected subcutaneously with MB49 cells transfected with human 5T4. Cured mice were subsequently used in a toxicity study and liver pathology was evaluated.ResultsIn vitro, ALG.APV-527 enhances primary CD8+ T cell and NK cell function and proliferation in the presence of 5T4-expressing cells. Using imaging, ALG.APV-527 in combination with a bispecific T cell engager caused increased cell death in T cell/tumor cell co-cultures. ALG.APV-527 inhibited growth of established tumors at doses as low as 2 µg/mouse in a syngeneic bladder cancer model. Following recovery, mice exhibited a memory response when rechallenged with tumor. In a high dose safety study in human 4-1BB knock-in mice, ALG.APV-527 did not cause significant systemic immune activation, whereas urelumab analogue treated mice induced dermatitis, elevated serum cytokines, CD8+ T-cell liver infiltration and systemic T-cell proliferation.ConclusionsALG. APV-527 induces potent CD8+ T cell and NK cell co-stimulation and T-cell cytotoxicity and has potent in vivo anti-tumor activity, without inducing systemic toxicity. Based on preclinical data, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of a variety of 5T4-expressing solid tumors.Ethics ApprovalAll studies were review and approved by the Internal Animal Care and Use Committee (IACUC) of Aptevo Therapeutics
Antigen Presented by Tumors In vivo Determines the Nature of CD8+ T-Cell Cytotoxicity
