Preclinical Characterization of an ANTI-CD38/CD3 T CELL-Redirecting Bispecific Antibody

Introduction: Multiple Myeloma (MM) and Non-Hodgkin Lymphoma (NHL) are hematologic malignancies that remain difficult to treat. While autologous CAR-T cell therapies have shown promise in treating these diseases, these therapies are not without issues, including lack of response in many patients, lengthy time to produce CAR-T cells, occasional production failures, as well as high manufacturing costs. As an alternative approach, protein-based T cell engaging and redirecting bispecific antibodies (BsAbs) have been developed. We have generated anti-CD38/CD3 BsAbs to redirect T cells against CD38, a clinically validated antigen in MM and studied their ability to elicit target-dependent tumor cell lysis. The lead molecule is a humanized, stability-engineered CD3-engaging and CD38 antigen affinity-optimized BsAb with reduced effector function to mitigate antigen-independent T cell toxicity. Preclinical data demonstrate potent anti-tumor activity in vitro assays and in vivo studies against CD38-expressing lymphoma and MM cell lines.

Methods: Anti-CD38/CD3 BsAbs were generated by CH3 Fc domain interface engineering for heterodimerization of a CD38-targeting Fab arm and anti-CD3-scFv-Fc fusion chain with hinge mutations for reduced FcR affinities. Novel bispecific molecules that bind to CD38 with various affinities/binding kinetics were evaluated in a series of in vitro and in vivo studies, including target-specific redirected T cell cytotoxicity (RTCC) against cancer cell lines. T cell response profiles, and cytokine release. The lead CD38/CD3 BsAb was selected and further evaluated for its ability to inhibit tumor growth and prolong survival in a disseminated luciferase-expressing Raji xenograft mouse model co-implanted with primary human peripheral blood mononuclear cells (hPBMC).

Results: Our lead CD38/CD3 BsAb possesses the desired CD38 and CD3, affinities resulting in effective tumor antigen and T cell engagement for RTCC. The CD38/CD3 BsAb induced potent T cell-dependent lysis of CD38-positive cancer cells in vitro, with the CD38 antigen density positively correlating with the cytotoxicity potency. Antigen dependent and dose-dependent T cell activation and cytokine release were studied in vitro, with the level of T cell activation and cytokine release being indicative of the anti-tumor potency but not necessarily anti-CD3 affinity. In an in vivo study, we evaluated the impact of CD38 affinity of the BsAb on anti-tumor activity of the BsAbs. The data showed that a balanced CD38 vs CD3 affinity was shown to be preferred for T cell stimulation and prolonged anti-tumor activity. In preclinical cytotoxicity assays against a cancer cell line panel using hPBMC from healthy donors, our lead CD38/CD3 BsAb was benchmarked against daratumumab, a marketed anti-CD38 antibody for MM, and demonstrated more potent tumor cell killing. These data suggest a more robust anti-tumor activity exerted by the CD38/CD3 BsAb through RTCC than daratumumab through antibody-dependent cellular cytotoxicity (ADCC). In Raji tumor cell-bearing NSG mice implanted with previously unstimulated hPBMCs, our CD38/CD3 BsAb induced tumor growth inhibition and prolonged survival compared to control BsAb or hPBMCs-only treated animals.

Conclusions: Our preclinical data demonstrate that our lead CD38/CD3 BsAb recruits T cells against CD38-positive tumor MM and lymphoma cells in a potent target and dose-dependent manner in preclinical studies. These preclinical characterizations support the rationale for clinical investigation of the lead BsAb in selected CD38-positive malignancies.