A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers

Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CART19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti- CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy