Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies

Abstract

Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB co-stimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was demonstrated in both patients and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel anti-self antibodies. These data demonstrate the potential of utilizing mRNA engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope-spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors.

Figure 1. Antitumor activity of CARTmeso cells

(A) CT imaging of MPM patient 17510-105 showing the patient’s dominant mesothelioma mass prior to receiving CARTmeso infusion on Schedule 1 (baseline) and 35 days after receiving the first CARTmeso infusion on Schedule 2 (day +84). (B) Whole body FDG PET/CT imaging of PDA patient 21211-101 obtained at baseline, day +34 after completing intravenous CARTmeso cell infusions and day +77 after completing intra-tumoral CARTmeso cell infusions. (C) Analysis of maximum standardized uptake value (SUVmax) and mean metabolic volumetric product (MVPmean) at baseline, day +34 and day +77 is shown for all lesions (Total) and individual sites of disease (Peritoneal, Liver, and Pancreas) for PDA patient 21211-101. (D) Shown is a representative flow cytometry plot of ascites from PDA patient 21211-101 analyzed with isotype control antibodies versus anti-mesothelin and anti-c-met antibodies to identify mesothelin⁺ c-met⁺ tumor cells. Flow cytometry findings were quantified to determine the total number of cells/mL and tumor cells/μL in the ascites which is shown in the bar graph with comparison between day +3 and day +15 after beginning intravenous CARTmeso cell infusions.

Figure 2. In vivo persistence of CARTmeso cells and trafficking to primary and metastatic tumor sites

RNA was isolated from whole blood on the indicated day before (Pre) and after (Post) CARTmeso cell infusion (black arrows). The relative amount of CARTmeso and CD3ε transcripts with normalization to the housekeeping gene PP1B was determined. Shown is the ratio of CARTmeso transgene transcripts to CD3ε transcripts × 100 for MPM patient 17510-105 during (A) Schedule 1. The arrows indicate injection of 10⁸ and 10⁹ CARTmeso cells on day 0 and day 7. (B) The ratio of CARTmeso transgene transcripts to CD3ε transcripts × 100 within whole blood for PDA patient 21211-101 during Schedule 3 is shown. The arrows indicate injection of 3×10⁸ CARTmeso cells i.v. (C) The ratio of CARTmeso transgene transcripts to CD3ε transcripts × 100 within ascites on Day +3 and +25 after intravenous CARTmeso cell infusion, within ascites on Day +70 after intratumoral CARTmeso cell injection, within primary pancreatic tumor tissue prior to intratumoral injection with CARTmeso cells on day +35 (Tumor, Pre IT1) and day +57 (Tumor, Pre IT2) and within peripheral blood mononuclear cells before (Pre IT2 PBMC) and after (Post IT2 PBMC) intratumoral injection with CARTmeso cells on day +57 is shown. See Supplemental figure S1 for details on cell injection dose and schedule.

Figure 3. Serum cytokines and chemokines after CARTmeso cell infusion

Fold change in serum cytokines and chemokines in PDA patient 21211-101 on Day 0 at 1 hour and 24 hours later after the first intravenous CARTmeso cell infusion. Baseline levels (pg/mL) of cytokines/chemokines are shown below each corresponding soluble factor.

Figure 4. CARTmeso cell induction of antitumor antibodies

Sera obtained from patients pre-and post-CARTmeso therapy were analyzed for the presence of antibodies reactive with mesothelin and with cell extracts from autologous and allogeneic tumor cell lines. (A) Sera from MPM patient 17510-105 obtained at day −3 and day +64 were analyzed by immunoblotting for reactivity against purified mesothelin protein (MESO), SV40 large T antigen protein (SV), and lysates obtained from seven mesothelioma cell lines (REN, 208, 213, 302, 307, M30 and M60). New bands at ~70 kD (red boxes) and ~45 kD (blue boxes) were seen after CARTmeso cell infusion. (B) Sera from PDA patient 21211-101 obtained at days −7, +57 and +92 were analyzed by immunoblotting for reactivity against mesothelin protein (MESO), SV40 large T antigen protein (SV), lysate from a PDA cell line (Panc1) and lysate from a tumor cell line derived from the patient’s own ascites (21211 Tumor). A strong anti-mesothelin band was noted in the Day +57 sample (blue arrow) and new bands were seen after receiving CARTmeso cell infusion at ~65–80 kD (red boxes) for the autologous 21211 cell line and for the Panc1 cell lines (blue boxes).