MDX2003, a novel tetraspecific T cell engager-expander targeting CD19xCD20xCD3xCD28, demonstrates potent preclinical activity against B cell malignancies

Abstract Background: CD19 and CD20 directed therapies, including CAR-T cells, monoclonal antibodies, and bispecific T cell engagers have demonstrated clinical efficacy in B cell malignancies. However, current agents are often limited by cytokine release syndrome (CRS), target antigen heterogeneity, and inadequate efficacy durability. MDX2003 is a first-in-class tetraspecific T cell engager-expander designed to address these challenges by simultaneously targeting CD19 and CD20 on B cells, while co-engaging CD3 and CD28 on T cells. This dual targeting strategy is intended to mitigate antigen loss or downmodulation, while the CD28 co-stimulation promotes robust T cell activation, survival, and sustained cytotoxicity. Methods MDX2003 was evaluated in preclinical in vitro and in vivo preclinical models. In vitro studies assessed antigen binding affinity, T cell activation, B cell killing, cytokine production (IFN-γ, IL-6), and anti-tumor cytotoxic potency using primary human PBMCs co-cultured with lymphoma derived tumor cells expressing varying levels of CD19 and CD20. In vivo anti-tumor activity was tested in a xenograft non-Hodgkin B cell lymphoma (B-NHL) humanized mouse model. Autologous B cell depletion and tolerability were assessed in NSG mice reconstituted with human CD34+ cord blood cells. Results MDX2003 was selected from a combinatorial library of tetraspecific constructs based on its optimized functional potency, antigen selectivity, and ability to eliminate malignant B cells while minimizing cytokine induction. The molecule demonstrated high binding affinity to CD19 and CD20, with comparatively lower affinities for CD3 and CD28, supporting targeted and balanced T cell activation. MDX2003 induced potent, dose-dependent T cell activation and cytotoxicity across B cell derived tumor lines with diverse levels of CD19 and/or CD20, with EC₅₀ values in low picomolar to sub-nanomolar range. MDX2003 retained activity in CD19- or CD20-deficient tumor cell lines, confirming the ability to overcome single-antigen escape. Co-engagement of CD3 and CD28 significantly enhanced T cell activation (CD69↑, CD25↑), proliferation (Ki67↑), and especially survival (Bcl-xL↑) relative to CD3 only targeting. In a humanized mouse model,MDX2003 achieved complete tumor growth inhibition at low doses and was well tolerated with minimal serum cytokine release and no observable toxicity. In vivo, MDX2003 effectively depleted both peripheral and tissue-resident B cells without associated weight loss or significant cytokine release post first dose. Inclusion of CD28 co-engagement contributed to more profound and more durable B cell clearance. These data suggest MDX2003 may serve as an off-the-shelf alternative to CAR T cell therapy. Conclusions MDX2003 is a differentiated tetraspecific T cell engager-expander demonstrating potent anti-tumor efficacy, favorable safety, and a mechanistic rationale for use in B cell malignancies. Its ability to sustain cytotoxicity, overcome antigen heterogeneity, and minimize cytokine-associated toxicities supports continued development in relapsed/refractory B cell malignancies. IND-enabling studies are ongoing, with initial clinical evaluation planned in B-cell lymphoma including patients relapsed after prior CD19-targeted therapy.