Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial
Authors: Kiesgen Stefan , Linot Camille , Quach Hue T. , Saini Jasmeen , Bellis Rebecca , Banerjee Srijita , Hou Zhaohua , Chintala Navin K. , Sadelain Michel , Adusumilli Prasad S. 
Published: September 2020

Abstract
Background: We previously established the safety and antitumor efficacy of regionally delivered mesothelin-targeted M28z chimeric antigen receptor (CAR) T cells combined with programmed death-1 (PD-1) antibody (NCT02414269). As a next step, we developed next-generation CAR T cells equipped with a modified CD3z signaling domain with loss-of-function mutations within 2 of 3 ITAM motifs (1XX), and a PD-1 dominant negative receptor (PD1DNR) that provides T-cell intrinsic checkpoint blockade (M28z1XXPD1DNR CAR T cells). Herein, we provide evidence of the preclinical safety and enhanced antitumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells.
Methods: Comparative cytotoxicity, proliferation, and cytokine secretion of human T cells engineered to express M28z or M28z1XXPD1DNR CAR were assessed by chromium-release, accumulation, and Luminex assays, respectively. The antitumor efficacy of a single dose (1x105 CAR T cells; E:T 1:1000) of intrapleurally administered M28z or M28z1XXPD1DNR CAR T cells was investigated in NSG mice with orthotopic pleural mesothelioma by serial bioluminescence imaging and by comparing survival. Following tumor eradication, functional persistence of CAR T cells was tested by repeated tumor challenge (increasing doses of 2x106 to 10x106 tumor cells).
Results: In vitro, both M28z and M28z1XXPD1DNR CAR T cells exhibited antigen-specific cytotoxicity, accumulation, and effector cytokine secretion (table). In vivo, a single dose of M28z1XXPD1DNR CAR T cells led to tumor eradication, mice exhibited enhanced survival with weight gain, and resistance to tumor reestablishment upon 10 tumor rechallenges (table) versus a single dose of M28z CAR T cells.
Table.In vitro and in vivo characteristics of M28z and M28z1XXPD1DNR CAR T-cell constructsM28zM28z1XXPD1DNRTargetMesothelinMesothelinCostimulatory domainCD28CD28CD3zNo mutations2 ITAM mutations (1XX)T-cell intrinsic checkpoint blockade (PD1DNR)NoYesIn vitro resultsHuman T-cell transduction, range25%-82%30%-89%PD-1 extracellular domain mRNA expression compared to untransduced, fold4158Cytotoxicity, rangeE:T 10:135%-45%25%-51%E:T 5:128%-44%20%-38%E:T 2:117%-32%14%-24%Accumulation, range, fold110-39053-622Effector cytokines (E:T 1:1, 24 h), rangeIL-214-23 ng/mL9-19 ng/mLTNF-α545-977 pg/mL380-852 pg/mLIFN-γ8-11 ng/mL6-15 ng/mLIn vivo resultsTumor eradication26 days19 daysMedian survival56 daysNot reachedTumor progression as measured by bioluminescence imaging following rechallengeRechallenged 3 times over 15 days+1 log+0.2 logsRechallenged 10 times over 52 days+3-4 logs+0.5 logsCurrent statusIn clinical trialIND submission pending
Conclusion: Supported by the safety, tumor eradication, and functional persistence, M28z1XXPD1DNR CAR T cells will advance to IND submission and initiation of a phase I clinical trial in patients with pleural mesothelioma, and further extend our investigation to other mesothelin-expressing solid tumors.
Citation Format: Stefan Kiesgen, Camille Linot, Hue T. Quach, Jasmeen Saini, Rebecca Bellis, Srijita Banerjee, Zhaohua Hou, Navin K. Chintala, Michel Sadelain, Prasad S. Adusumilli. Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-378.