Development Of Off-The-Shelf B7h3 Chimeric Antigen Receptor Nk Cell Therapeutic With Broad Applicability Across Many Solid Tumors.

Abstract Although chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive efficacy in a number of hematological malignancies, successful application of CAR T cells in solid tumor indications has been challenging due to difficulty identifying and targeting suitable tumor associated antigens (TAA) without eliciting significant on-target, off-tumor toxicity. Furthermore, the challenges of tumor heterogeneity and antigen escape need to be addressed when treating bulky solid tumors. Successful development of CAR-targeted cellular therapies for solid tumor indications will consequently require careful selection of target antigen, effector cell, and cell engineering to balance on-tumor efficacy with potential for off-tumor toxicities. In contrast to CAR-T cell therapies, adoptive natural killer (NK) cell immunotherapies have been well tolerated, but conventional, donor-derived strategies have had limited efficacy. B7H3 is an immune checkpoint molecule of the B7 protein superfamily and an ideal TAA; B7H3 has broad expression in cancer and limited expression on healthy tissues. Clinically, upregulation of B7H3 is often associated with metastatic cancer and poor patient prognosis. To target B7H3 with a multifaceted effector cell population, we selected the use of NK cells to leverage their clinical safety profile and versatile innate tumor recognition and cytotoxicity features. Here we describe the optimization and characterization of a novel anti-B7H3 CAR and its application in an induced pluripotent stem cell-derived, off-the-shelf NK (iNK) cell immunotherapy. These anti-B7H3 CAR iNK cells utilize multiplexed genetic engineering to enhance persistence, effector function, and to enable multi-antigen targeting through combinations with monoclonal antibody therapy. Using a novel B7H3 binding domain, CAR constructs were first screened in primary T cells for the optimal spacer length by in vitro and in vivo cytotoxicity assays. Following optimized CAR selection, in vitro proof of concept studies demonstrated broad B7H3 CAR reactivity against an array of tumor cell line targets of both hematologic and solid organ cancers. B7H3 CAR-T cells were highly reactive against cell lines derived from ovarian, prostate, bone, and breast cancers as demonstrated by cytotoxicity and production of IFNγ and TNFα. iNK cells carrying the optimized CAR design were evaluated in vitro for B7H3 specificity using cytotoxicity assays and intracellular cytokine staining. In addition, B7H3 CAR iNK cells synergized with anti-EGFR and anti-HER2 monoclonal antibodies to produce antibody-dependent cellular cytotoxicity, leading to further enhancement of tumor targeting. In summary, these data demonstrate successful targeting of B7H3 using an off-the-shelf, engineered NK cell platform for the effective elimination of multiple tumor types. Citation Format: Ryan Bjordahl, John Goulding, Hui-Yi Chu, Frank Cichocki, Nicholas Zorko, Zachary Davis, Martin Felices, Alex Garcia, Svetlana Gaidarova, Wen-I Yeh, Tom T. Lee, Jeffrey S. Miller, Bahram Valamehr. Development of off-the-shelf B7H3 chimeric antigen receptor NK cell therapeutic with broad applicability across many solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1539.