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 constructs

M28zM28z1XXPD1DNR
Target Mesothelin Mesothelin 
Costimulatory domain CD28 CD28 
CD3z No mutations 2 ITAM mutations (1XX) 
T-cell intrinsic checkpoint blockade (PD1DNR) No Yes 
In vitro results 
Human T-cell transduction, range 25%-82% 30%-89% 
PD-1 extracellular domain mRNA expression compared to untransduced, fold 158 
Cytotoxicity, range 
E:T 10:1 35%-45% 25%-51% 
E:T 5:1 28%-44% 20%-38% 
E:T 2:1 17%-32% 14%-24% 
Accumulation, range, fold 110-390 53-622 
Effector cytokines (E:T 1:1, 24 h), range 
IL-2 14-23 ng/mL 9-19 ng/mL 
TNF-α 545-977 pg/mL 380-852 pg/mL 
IFN-γ 8-11 ng/mL 6-15 ng/mL 
In vivo results 
Tumor eradication 26 days 19 days 
Median survival 56 days Not reached 
Tumor progression as measured by bioluminescence imaging following rechallenge 
Rechallenged 3 times over 15 days +1 log +0.2 logs 
Rechallenged 10 times over 52 days +3-4 logs +0.5 logs 
Current status In clinical trial IND submission pending 
M28zM28z1XXPD1DNR
Target Mesothelin Mesothelin 
Costimulatory domain CD28 CD28 
CD3z No mutations 2 ITAM mutations (1XX) 
T-cell intrinsic checkpoint blockade (PD1DNR) No Yes 
In vitro results 
Human T-cell transduction, range 25%-82% 30%-89% 
PD-1 extracellular domain mRNA expression compared to untransduced, fold 158 
Cytotoxicity, range 
E:T 10:1 35%-45% 25%-51% 
E:T 5:1 28%-44% 20%-38% 
E:T 2:1 17%-32% 14%-24% 
Accumulation, range, fold 110-390 53-622 
Effector cytokines (E:T 1:1, 24 h), range 
IL-2 14-23 ng/mL 9-19 ng/mL 
TNF-α 545-977 pg/mL 380-852 pg/mL 
IFN-γ 8-11 ng/mL 6-15 ng/mL 
In vivo results 
Tumor eradication 26 days 19 days 
Median survival 56 days Not reached 
Tumor progression as measured by bioluminescence imaging following rechallenge 
Rechallenged 3 times over 15 days +1 log +0.2 logs 
Rechallenged 10 times over 52 days +3-4 logs +0.5 logs 
Current status In clinical trial IND 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.