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Introduction: Regulatory CD8 T cells (CD8 Treg) are responsible for the selective killing of self-reactive and pathogenic CD4 T cells. In autoimmune disease, CD8 Treg may accumulate in the peripheral blood but fail to control the expansion of pathogenic CD4 T cells that subsequently cause tissue destruction. This CD8 Treg dysfunction is due in part to the expression of inhibitory killer immunoglobulin-like receptors (KIR; KIR2DL isoforms [KIR2DL1, KIR2DL2, and KIR2DL3]); these molecules serve as autoimmune checkpoints and limit CD8 Treg activation. Methods: Here we describe the pre-clinical characterization of MTX-101, a bispecific antibody targeting inhibitory KIR and CD8. Using human peripheral blood mononuculear cells (PBMC) derived from healthy donors and autoimmune patients, humanized mouse models, and human derived tissue organoids, we evaluated the molecular mechanisms and functional effects of MTX-101. Results: By binding to KIR, MTX-101 inhibited KIR signaling that can restore CD8 Treg ability to eliminate pathogenic CD4 T cells. MTX-101 bound and activated CD8 Treg in human peripheral blood mononuclear cells (PBMC), resulting in increased CD8 Treg cytolytic capacity, activation, and prevalence. Enhancing CD8 Treg function with MTX-101 reduced pathogenic CD4 T cell expansion and inflammation, without increasing pro-inflammatory cytokines or activating immune cells that express either target alone. MTX-101 reduced antigen induced epithelial cell death in disease affected tissues, including in tissue biopsies from individuals with autoimmune disease (i.e., celiac disease, Crohn's disease). The effects of MTX-101 were specific to autoreactive CD4 T cells and did not suppress responses to viral and bacterial antigens. In a human PBMC engrafted Graft versus Host Disease (GvHD) mouse model of acute inflammation, MTX-101 bound CD8 Treg and delayed onset of disease. MTX-101 induced dose dependent binding, increased prevalence and cytolytic capacity of CD8 Treg, as well as increased CD4 T cell death. MTX-101 selectively bound CD8 Treg without unwanted immune cell activation or increase of pro-inflammatory serum cytokines and exhibited an antibody-like half-life in pharmacokinetic and exploratory tolerability studies performed using IL-15 transgenic humanized mice with engrafted human lymphocytes, including CD8 Treg at physiologic ratios. Conclusion: Collectively, these data support the development of MTX-101 for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]