Control of Metastases via Myeloid CD39 and NK Cell Effector Function.

Natural killer (NK) cell protection from tumor metastases is a critical feature of the host immune response to cancer, but various immunosuppression mechanisms limit NK cell effector function. The ectoenzyme, CD39, expressed on tumor-infiltrating myeloid cells, granulocytes, and lymphocytes, including NK cells, converts extracellular ATP (eATP) into AMP and, thus, potentially suppresses eATP-mediated proinflammatory responses. A CD39-targeting monoclonal antibody (mAb) that inhibits the mouse ectoenzyme CD39 suppressed experimental and spontaneous metastases in a number of different tumor models and displayed superior antimetastatic activity compared with the CD39 inhibitor POM1 and inhibitors and mAbs that block other members of the adenosinergic family (e.g., A2AR and CD73). The antimetastatic activity of anti-CD39 was NK cell and IFNγ dependent, and anti-CD39 enhanced the percentage and quantity of IFNγ produced and CD107a expression in lung-infiltrating NK cells following tumor challenge and anti-CD39 therapy. Using conditional Cd39 gene-targeted mouse strains and adoptive NK cell transfers, we showed that CD39 expressed on bone marrow-derived myeloid cells was essential for anti-CD39's antimetastatic activity, but NK cell expression of CD39 was not critical. The eATP receptor P2X7 and the NALP3 inflammasome, including downstream IL18, were critical in the mechanism of action of anti-CD39, and the frequency of P2X7 and CD39 coexpressing lung alveolar macrophages was specifically reduced 1 day after anti-CD39 therapy. The data provide a mechanism of action involving NK cells and myeloid cells, and anti-CD39 combined with anti-PD-1, NK cell-activating cytokines IL15 or IL2, or an inhibitor of A2AR to effectively suppress tumor metastases.

Dual targeting of RANKL and PD-1 with a bispecific antibody improves anti-tumor immunity.

OBJECTIVES: The addition of RANKL/RANK blockade to immune checkpoint inhibitors (ICIs) such as anti-PD-1/PD-L1 and anti-CTLA4 antibodies is associated with increased anti-tumor immunity in mice. Recent retrospective clinical studies in patients with advanced melanoma and lung cancer suggest the addition of anti-RANKL antibody to ICI increases the overall response rate relative to ICI treatment alone. Based on this rationale, we developed a novel bispecific antibody (BsAb) co-targeting RANKL and PD-1. METHODS: We characterized target binding and functional activity of the anti-RANKL/PD-1 BsAb in cell-based assays. Anti-tumor activity was confirmed in experimental lung metastasis models and in mice with established subcutaneously transplanted tumors. RESULTS: The anti-RANKL/PD-1 BsAb retained binding to both RANKL and PD-1 and blocked the interaction with respective counter-structures RANK and PD-L1. The inhibitory effect of anti-RANKL/PD-1 BsAb was confirmed by demonstrating a complete block of RANKL-dependent osteoclast formation. Monotherapy activity of anti-RANKL/PD-1 BsAb was observed in anti-PD-1 resistant tumors and, when combined with anti-CTLA-4 mAb, increased anti-tumor responses. An equivalent or superior anti-tumor response was observed with the anti-RANKL/PD-1 BsAb compared with the combination of parental anti-RANKL plus anti-PD-1 antibodies depending upon the tumor model. DISCUSSION: Mechanistically, the anti-tumor activity of anti-RANKL/PD-1 BsAb required CD8+T cells, host PD-1 and IFNγ. Targeting RANKL and PD-1 simultaneously within the tumor microenvironment (TME) improved anti-tumor efficacy compared with combination of two separate mAbs. CONCLUSION: In summary, the bispecific anti-RANKL/PD-1 antibody demonstrates potent tumor growth inhibition in settings of ICI resistance and represents a novel modality for clinical development in advanced cancer.

CD96 targeted antibodies need not block CD96-CD155 interactions to promote NK cell anti-metastatic activity.

CD96 is a transmembrane glycoprotein Ig superfamily receptor, expressed on various T cell subsets and NK cells, that interacts with nectin and nectin-like proteins, including CD155/polio virus receptor (PVR). Here, we have compared three rat anti-mouse CD96 mAbs, including two that block CD96-CD155 (3.3 and 6A6) and one that does not block CD96-CD155 (8B10). Using flow cytometry, we demonstrated that both mAbs 3.3 and 6A6 bind to the first Ig domain of mouse CD96 and compete with CD155 binding, while mAb 8B10 binds to the second Ig domain and does not block CD155. While Fc isotype was irrelevant concerning the anti-metastatic activity of 3.3 mAb, in four different experimental metastases models and one spontaneous metastasis model, the relative order of anti-metastatic potency was 6A6 > 3.3 > 8B10. The metastatic burden control of all of the anti-CD96 clones was highly dependent on NK cells and IFN-γ. Consistent with its inability to block CD96-CD155 interactions, 8B10 retained anti-metastatic activity in CD155-deficient mice, whereas 3.3 and 6A6 lost potency in CD155-deficient mice. Furthermore, 8B10 retained most of its anti-metastatic activity in IL-12p35-deficient mice whereas the activity of 3.3 and 6A6 were partially lost. All three mAbs were inactive in CD226-deficient mice. Altogether, these data demonstrate anti-CD96 need not block CD96-CD155 interactions (ie. immune checkpoint blockade) to promote NK cell anti-metastatic activity.