CD45 sequestration lowers the signaling threshold in lymphocytes and enhances anti-tumor immunity

CD45 plays a central role in immune signal regulation by controlling the spatial dynamics of phosphatase activity through steric segregation of its bulky rigid extracellular domain. To modulate CD45 activity, here we develop and characterize protein engineering approaches to induce multivalent clustering of CD45, effectively mimicking the endogenous local receptor sequestration during immune synapse formation. In doing so, we engineer a biologic that enables precise, tunable control over CD45 surface localization and activity. CD45 sequestration exhibited striking synergy when administered in combination with intratumorally anchored IL-12 therapy, markedly delaying tumor progression and extending survival in syngeneic murine melanoma and carcinoma models. Immune profiling revealed that CD8⁺ T cells are essential mediators of this synergistic antitumor response. Mechanistically, IL-12 initiates a wave of antigen generation and T cell priming, while CD45 sequestration subsequently enhances tumor-specific CD8⁺ T cell activation, expansion, and functional states within the tumor-draining lymph node. These findings suggest that CD45 sequestration lowers the activation threshold of T cells, broadens the tumor-reactive T cell repertoire, and therefore promotes more robust tumor-specific T cell responses. Altogether, we establish CD45 as a promising novel target for cancer immunotherapy, capable of potentiating strong anticancer immune responses. Mice bearing B16F10 tumors, left untreated, treated with alum IL-12, or treated with alum IL-12 + CD45 XL (n = 4-5/group) were harvested 48 h. after the completion of treatment and sorted for CD8+ T cells specific for the immunodominant p15E retroviral antigen in the tdLN for downstream scRNA-seq. Tumor-draining inguinal lymph nodes (tdLN) were harvested, manually dissociated, and filtered through a 5 mL round-bottom tube with cell-strainer cap (Falcon) using the blunt rubber end of a 1mL syringe plunger (BD Falcon). All lymph node material was used in FACS sorting. Viability was assessed with Zombie Aqua dye (BioLegend, 1:1000) in PBS for 20 minutes at room temperature. Subsequent washes and surface staining were performed in PBS supplemented with 1% bovine serum albumin (Sigma). Samples were resuspended in buffer containing Fc block (clone 93, eBioscience, 1:50) and AlexaFlour 647 labeled p15E tetramer (H2-Kb MuLV env 574-581, NIH Tetramer Core) at 1:100 for 30 min at RT. Extracellular staining was performed at 4oC for 30 min. Cells were also stained with Totalseq C anti-mouse hashing antibodies (BioLegend, 1:100) before FACS. For p15E+ CD8+ T cell sorting cells were sorted on a Sony MA900 and gated on FSC and SSC, Live, CD45+, CD8+, CD44+, and p15E Tetramer+. Cells were subsequently processed according to the 10x Genomics 5` Immune Profiling v3 protocol.