Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function

Abstract

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells. Moreover, inhibitory signals from malignant cells could be transformed into stimulatory signals by synthetic fusion molecules with extracellular inhibitory receptors fused to an intracellular stimulatory domain. Here, we designed a variety of different TIM-3-CD28 fusion proteins to turn inhibitory signals derived by TIM-3 engagement into T-cell activation through CD28. In the absence of anti-CD19 CAR, two TIM-3-CD28 fusion receptors with large parts of CD28 showed strongest responses in terms of cytokine secretion and proliferation upon stimulation with anti-CD3 antibodies compared to controls. We then combined these two novel TIM-3-CD28 fusion proteins with first- and second-generation anti-CD19 CAR T cells and found that the fusion receptor can increase proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells. These additionally armed CAR T cells showed excellent effector function. In terms of safety considerations, the fusion receptors showed exclusively increased cytokine release, when the CAR target CD19 was present. We conclude that combining checkpoint fusion proteins with anti-CD19 CARs has the potential to increase T-cell proliferation capacity with the intention to overcome inhibitory signals during the response against malignant cells.

Targeting LAG-3, TIM-3, and TIGIT for cancer immunotherapy

“…Thus, several studies have explored selective blockers of these inhibitory receptors in CAR-T cells. To date, at least two TIM-3-CD28 fusion proteins have been designed to increase the proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR-T cells [ 212 ]. In addition, TIGIT has been identified is a marker for CD19 CAR-T cell dysfunction in experiments involving single-cell RNA sequencing and in analysis of surface protein marker levels before and after CAR-T cell infusion in NHL patients.…”

Targeting TIM-3 for hematological malignancy: latest updates from the 2022 ASH annual meeting

“…These fusion proteins alone demonstrate the strongest response upon stimulation with anti-CD3 antibodies. Importantly, these proteins can increase the proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells [ 10 ]. Thus, combining IC fusion proteins with anti-CD19 CARs has the potential to increase the T cell proliferation capacity.…”

CAR-T cell therapy for hematological malignancies: Limitations and optimization strategies

“…Similar approaches are used for another immune checkpoint T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) by generating TIM-3-CD28 fusion proteins in CD19- or CD138-CAR-T cells. It converts the immunosuppressive TIM-3 signaling to CAR-T cells immunostimulation ( 48 , 49 ).…”