Abstract PO074: Logic-gating HER2 CAR-T to the tumor microenvironment mitigates on-target, off-tumor toxicity without compromising cytotoxicity against HER2-over-expressing tumors
Authors: Zhang Wei , He Qun , Lopez Benjamin , Hu Jianfang , Kundu Anirban , Andraza Michelle C. , Kerner Alissa R. , Schreiber Gregory H. , Shepard H. Michael , Frost Gregory I. 
Published: February 2021

Abstract
Chimeric antigen receptor (CAR) modified T cells have demonstrated promising anti-tumor effects in hematologic cancers. Because CD19 expression is restricted to B cells, CD19 CAR-T B cell aplasia is a tolerable on-target, off-tumor toxicity. However, antigens found in solid tumors, such as HER2, are also expressed in many critical tissues. HER2 overexpression/amplification occurs in many malignancies, including breast, gastric, lung, ovarian and pancreas. With its elevated receptor copy number and relatively homogeneous expression following gene amplification, HER2 represents an attractive antigen to target via CAR-T. Unfortunately, severe toxicity related to off-tumor binding of the CAR-T to HER2 present in normal tissue may limit the use of CAR-T therapy. To circumvent this issue, we designed a “logic-gated” HER2-targeted CAR-T that preferentially recognizes HER2 in the tumor microenvironment (TME), thereby limiting on-target toxicity of low HER2 levels expressed in normal tissue. HER2 scFvs with pH-restricted binding towards physiologic levels of HER2 were screened as CARs in primary T cells and demonstrated pH dependent cytotoxicity and cytokine release in vitro; the pH-dependence was also preserved in the context of HER2 CAR-Ts vs. ungated HER2 CARs. Antitumor activity and cellular kinetics were assessed in NSG mice bearing human HER2-amplified xenografts. Logic-gated HER2 CARs were capable of regressing established gastric (NCI-N87), breast (BT-474), and ovarian (SK-OV-3) tumors with HER2 amplification. Importantly, logic-gated HER2 CAR-T cells were also capable of completely regressing large established gastric carcinoma xenografts that had progressed on prior trastuzumab therapy. Anti-tumor activity and cellular kinetics were dose dependent, with robust in vivo expansion to peak levels of 190,000 copies/µg gDNA. On-target, off-tumor safety of the CAR-Ts was assessed in NSG mice with enforced expression of human HER2 and luciferase in hepatocytes using a hydrodynamic gene delivery (HGD) model. Compared to ungated HER2 CAR-T constructs, logic-gated HER2 CAR-Ts did not eliminate hepatocyte luciferase expression with human HER2 +1 staining in mouse livers as determined by Herceptest scoring of livers at necropsy. In conclusion, these results demonstrate that a logic-gated HER2-targeted CAR-T is capable of eliminating established HER2-amplified malignancies in a xenograft model, while mitigating potential on-target, off-tumor toxicity.
Citation Format: Wei Zhang, Qun He, Benjamin Lopez, Jianfang Hu, Anirban Kundu, Michelle C. Andraza, Alissa R. Kerner, Gregory H. Schreiber, H. Michael Shepard, Gregory I. Frost. Logic-gating HER2 CAR-T to the tumor microenvironment mitigates on-target, off-tumor toxicity without compromising cytotoxicity against HER2-over-expressing tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO074.