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Background Cancer immunotherapy has demonstrated significant clinical success; however, only a subset of patients benefit and show durable responses. Limited efficacy is often attributed to suboptimal anti-tumor T cell generation and impaired development of immunological memory. MDX2004, a first-in-class trispecific antibody-fusion protein recognizing CD3, CD28, and 4-1BB (CD137) on human T cells, is a novel immunotherapeutic candidate designed to overcome such hurdles by increasing stem and memory T cell responses, thereby rejuvenating the immune system and enhancing tumor immunity. Here, we characterize the pharmacologic properties of MDX2004 and provide proof of concept data supporting its proposed mechanism of action.Methods Binding affinity of MDX2004 was determined using biolayer interferometry. T cell responses were studied in vitro using human peripheral blood mononuclear cells (PBMCs) and in vivo in naïve non-human primates (NHPs), and evaluated by flow cytometry and multiplex cytokine analysis. In vitro tumor cytolytic activity was assessed by Real-Time Cell Analysis. In vivo anti-tumoral efficacy was studied in humanized mouse tumor models. Tumor infiltration was analyzed using immunohistochemistry.Results MDX2004 binds to human CD3, CD28 and 4-1BB with nanomolar affinity but lacks cross-reactivity in mouse, rat, dog, or NHP. Consistent with its design, MDX2004 demonstrated markedly reduced or abrogated binding to human Fc-γ receptors while retaining pH-dependent binding to human neonatal Fc receptor. In vitro, MDX2004 induced dose-dependent T cell activation, proliferation, and preferential expansion of memory and stem-like T cell subsets, particularly within the CD8 T cell compartment. MDX2004 also stimulated expansion of viral antigen-specific T cells, and substantial in vitro cytolytic activity in cocultures of antigen-experienced T cells and tumor cells. In vivo, a surrogate MDX2004 with reactivity to the cognate antigens in NHP was well tolerated and induced proliferation of stem-like and memory T cells consistent with its activity in vitro. Furthermore, MDX2004 exerted potent antitumor activity in humanized mouse models of breast and colon cancer. Treatment inhibited tumor growth and was associated with increased intratumoral clustering of proliferating CD8+ T cells.Conclusions MDX2004 is a novel multispecific immune rejuvenator that induces robust T cell responses, including the expansion and activation of stem and memory CD8 T cells, and stimulates tumor regression with acceptable tolerability in animal models. The preclinical activities of MDX2004 support its clinical development for the treatment of cancer.Ethics Approval Mouse study conducted at Charles River Laboratories (Worcester, MA, USA) in compliance with CRL IACUC under IACUC No. I041. . Mouse study conducted at ModeX Therapeutics (Weston, MA, USA) in compliance with IACUC protocol 2022-MOX-02. . NHP study conducted at ALtasciences (Everett, WA, USA). The Testing Facility is accredited by AAALAC International, has an Animal Welfare Assurance with the Office of Laboratory Animal Welfare (OLAW), is registered with the United States Department of Agriculture (USDA), and has an Institutional Animal Care and Use Committee (IACUC) responsible for the Testing Facility’s compliance with applicable laws and regulations concerning the humane care and use of laboratory animals.