Abstract 1148: 1XX mutations slow CAR T cell signaling and increase in vivo persistence
Authors: Bamdad Cynthia C. , Smagghe Benoit J. , Carter Mark G. , Walkley Danica M. , Nash Jac-Leen S. , Yi Kevin R. , Grant Trevor J. , Reale Laura M. , Nash Michael J. , Riley Gregory L. , Stewart Andrew K. 
Published: June 2023

Abstract
Purpose: Develop CAR T cells that have increased persistence in vivo and kill low antigen expressing solid tumor cancer cells.
Experimental: Three CARs were generated that were all targeted to the tumor with the same antibody fragment, huMNC2, that recognizes a cryptic epitope on the MUC1 cleavage product called MUC1* (muk 1 star). One construct comprises a 41BB co-stimulatory domain, while the second construct has a CD28 co-stimulatory domain. The first 2 CARs have a wild-type CD3z, whereas the 3rd bears Michel Sadelain’s “1XX” mutations in CD3z. These are 2 Tyrosine to Phenylalanine mutations in ITAM 2 and ITAM 3. The 1XX mutations were designed to slow signaling to eliminate the problem of CAR T cell exhaustion. Human T cells were separately transduced with the 3 CARs and tested in NSG mice bearing human breast tumors. The tumors were heterogeneous tumors comprising parent cells that fluoresce red and express medium to low amounts of the antigen, MUC1*, and cells that fluoresce green wherein the parent cells have been transduced to express more MUC1*. The percent of the tumor that expressed high levels of MUC1* was varied from 7.5% to 15% to 30%. 150 female NSG mice were implanted with 500,000 tumor cells. The animals received a single CAR T cell injection of one of the three CAR T cells at an Effector to Target ratio of 10:1, 5:1, or 1:1. The experiment was allowed to proceed for 96 days, with periodic IVIS measurements to track tumor growth with a focus on tumor recurrence. After sacrifice, residual tumors were excised, dissociated and analyzed to determine if tumor recurrence was being driven by the high antigen cells or the low antigen cells. CAR T cells were recovered from the blood and the spleen. They were enumerated and analyzed by flow cytometry for the presence of markers of exhaustion.
Unpublished Results: Tumor recurrence occurred in mice treated with CAR T cells bearing wild-type CD3z, but essentially did not recur in the mice treated with CAR T cells bearing the 1XX mutations. Four hundredfold more CAR T cells were recovered from the mice treated with the CAR-1XX cells compared to CARs with wild-type CD3z, and the CAR-1XX cells appeared not to be exhausted even after 96 days. CAR-1XX T cells administered at a 1:1 E:T ratio, with only 7.5% of the tumor comprised of high antigen expressing cells, were effectively killed with no significant tumor recurrence. In contrast, the CARs with wild-type CD3z had no effect on tumor growth in this group. Surprisingly, analysis of excised tumors showed that tumor recurrence in mice treated with CAR wild-type CD3z T cells was driven by the low antigen density cells, whereas the CAR-1XX T cells killed high antigen and low antigen density cells at the same rate.
Conclusions: 1XX mutations in CD3z greatly increase CAR T persistence in vivo and unexpectedly enable the killing of low antigen tumor cells.
Citation Format: Cynthia C. Bamdad, Benoit J. Smagghe, Mark G. Carter, Danica M. Walkley, Jac-Leen S. Nash, Kevin R. Yi, Trevor J. Grant, Laura M. Reale, Michael J. Nash, Gregory L. Riley, Andrew K. Stewart. 1XX mutations slow CAR T cell signaling and increase in vivo persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1148.