RG6076 (CD19-4-1BBL): CD19-Targeted 4-1BB Ligand Combination with Glofitamab As an Off-the-Shelf, Enhanced T-Cell Redirection Therapy for B-Cell Malignancies

Synthetic T cell redirecting therapies, using chimeric antigen receptor (CAR)-T cells or CD3-bispecific antibodies targeting B-cell surface antigens such as CD19 and CD20, currently in clinical development, are emerging as promising, potential therapeutic approaches for the treatment of non-Hodgkin lymphomas (NHL). CD3-bispecific antibodies and first generation CAR-T cells only provide T cell receptor stimulation, so-called "signal 1", to the redirected T cells, but lack costimulatory, so-called "signal 2", support of those T cells. Agonism of costimulatory receptors on T cells, such as CD28 and/or 4-1BB, can increase the strength and durability of a T cell-mediated response via multiple mechanisms. Co-stimulation can enhance T cell specific cytotoxicity, proliferation, secretion of Th1-polarizing cytokines, recruitment of additional T cells via increased chemokine secretion, T cell metabolic fitness, and resistance to T-cell exhaustion and to activation-induced T-cell death. Indeed, 2^nd generation CAR-T cells that incorporate CD28 or 4-1BB co-stimulation have replaced 1^st generation ones in clinical development. However, complex manufacturing logistics and the need of specialized clinical centers for the administration of CAR-T cells significantly limit their broad application.

In order to provide an off-the-shelf, synthetic T cell redirection approach delivering both signals 1 and 2 to T cells, CD3-bispecific antibodies would need combination with systemically administered T-cell costimulatory agonists. Yet, clinical development of 1^st generation costimulatory agonists has not been successful to date due to on-target, off-tumor immune-mediated toxicity, such as hepatotoxicity. To overcome this limitation, we have generated a novel 4-1BB costimulatory agonist, CD19-targeted 4-1BBL (CD19-4-1BBL, RG6076, RO7227166), and are developing it in combination with a potent CD20xCD3 T cell bispecific antibody, CD20-TCB (RG6026 or glofitamab). CD19-4-1BBL consists of a trimeric, human 4-1BBL fused to a monovalent CD19-targeting IgG1 antibody with an engineered Fc region devoid of FcgR binding. As effective agonism of 4-1BB receptor requires crosslinking of more than three receptor units on a T cell, CD19-4-1BBL is systemically inactive unless it binds to CD19 and clusters on the surface of targeted B-cells to hyper-crosslink multiple 4-1BB receptors on redirected T cells. In our off-the-shelf, combination approach, glofitamab binds to CD20 on B-cells and engages CD3 on redirected T cells, providing signal 1 and inducing the expression of 4-1BB on those T cells. CD19-4-1BBL can then target those activated T cells and provide them with signal 2.

In preclinical experiments, we show that CD19-4-1BBL can boost glofitamab-mediated cytokine release by activated T cells in healthy donor as well as DLBCL patient-derived PBMCs. Using a human diffuse large B cell lymphoma (DLBCL) tumor-bearing (WSU-DLCL2) fully humanized mouse model, we observed a CD19-4-1BBL dose-dependent, synergistic combination effect with glofitamab, leading to strongly increased T cell accumulation in tumors, tumor growth inhibition and regression. Importantly, CD19-4-1BBL was also able to prevent tumor escape to glofitamab monotherapy at late treatment time points in a fully humanized mouse model bearing large OCI-Ly18 human DLBCL tumors.

Glofitamab monotherapy has recently demonstrated encouraging activity in relapsed/refractory NHL patients with reported complete response rates in DLBCL in the same range as those of 2^nd generation CAR-T cells that already incorporate both T cell signals 1 and 2. The preclinical data we report here provide a strong rationale for adding CD19-4-1BBL-mediated T cell signal 2 to glofitamab in the clinic to further boost treatment efficacy and deliver an off-the-shelf, enhanced T cell redirection approach alternative to CAR-T cell therapy. CD19-4-1BBL is currently in clinical trials (NCT04077723).