1287 Preclinical characterization of MDX2001, a novel tetraspecific T cell engager (CD3/CD28) targeting TROP2 and c-MET in solid tumor malignancies

<h3>Background</h3> T cell engagers have demonstrated success in treating hematologic malignancies, but they still face limitations for the treatment of solid tumors. ModeX has developed a novel platform termed Lymphocyte Activator and Survival Enhancement Receptor (LASER) antibodies. MDX2001 is a first-in-class tetraspecific LASER recognizing CD3 and CD28 on human T cells and c-MET and TROP2 on tumor cells. It is designed to 1) optimize T cell activation/survival by dual signaling through CD3 and CD28, and 2) overcome target expression heterogeneity and treatment escape/resistance by binding to two different tumor antigens. MDX2001 is engineered with knob-into-hole mutations to promote Fc heterodimerization, and an Fc-null format to prevent triggering of Fc-dependent immune activation. Here, we evaluate the pharmacology of MDX2001 and provide proof of concept for the proposed mechanism of action. <h3>Methods</h3> Binding affinity of MDX2001 was determined using biolayer interferometry. Tumor cytolytic activity was assessed by Real-Time Cell Analysis in co-cultures of human peripheral blood mononuclear cells (PBMCs) and tumor cells. T cell activation was evaluated by flow cytometry and multiplex cytokine analysis.<i> In vivo</i> anti-tumoral efficacy was assessed using a humanized mouse tumor model. <h3>Results</h3> MDX2001 binds to human CD3, CD28, c-MET and TROP2 with nanomolar affinity but does not bind to the mouse, rat, or dog orthologs. It binds to non-human primate CD28, c-MET and TROP2 but not CD3. MDX2001 demonstrates abrogated or significantly reduced binding to Fc-γ receptors while retaining the physiologic pH-dependent binding dynamic with human neonatal Fc receptor. When incubated with human PBMCs and tumor cells expressing TROP2 and c-MET, MDX2001 induced T cell activation as measured by secretion of interleukin (IL)-2, interferon-γ, and IL-6 at concentrations as low as 15 pM. In contrast, MDX2001 induced only marginal T cell activation and cytokine release in human PBMCs in the absence of tumor cells. MDX2001 induced potent <i>in vitro</i> anti-tumor cytolytic activity by human PBMCs against a panel of 10 human tumor cell lines of different tissue origins expressing various levels of TROP2 and c-MET, including cell lines with low TROP2 or low c-MET expression. In a humanized mouse breast cancer tumor model, MDX2001 significantly inhibited tumor growth. <h3>Conclusions</h3> MDX2001 is novel multispecific LASER antibody that demonstrates potent antitumor activity <i>in vitro</i> and <i>in vivo</i>, with minimal T cell activation in the absence of tumor cells. The biochemical and functional properties of MDX2001 support clinical development of this candidate for the treatment of solid tumor malignancies. <h3>Ethics Approval</h3> Animal study conducted at Charles River Laboratories (CRL) in compliance with CRL IACUC under IACUC No. I033.