Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 29 (5), 614-625

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-Transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy

Affiliations

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-Transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy

Chien-Fu Hung et al. Hum Gene Ther.

Abstract

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 109 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival. No significant off-target toxicities were observed. These data support further investigation of CARMA-hMeso as a potential treatment for ovarian cancer and other mesothelin-expressing cancers.

Keywords: cell therapy; chimeric antigen receptor (CAR); immunotherapy; mesothelin; ovarian cancer.

Conflict of interest statement

These studies were funded by MaxCyte, Inc. C.F.H. and L.A.E. received financial and material support from MaxCyte, Inc. L.L., A.V., C.A., P.N., R.S., and M.V.P. are employees of and have personal financial interests in MaxCyte, Inc.

Figures

<b>Figure 1.</b>
Figure 1.
The structure of three different antigen-specific mRNA chimeric antigen receptors (CARMA): two specific for mesothelin with human single chain variable fragment (scFv; CARMA-αhMeso-H) or murine scFv (CARMA-αhMeso-M), and one specific for CD19 with murine scFv (CARMA-αCD19-M). Isolated peripheral blood mononuclear cells (PBMCs) that have not been further selected, expanded, or activated are electroporated with CARMA using the cGMP-compliant MaxCyte GT® System™, and then either cryopreserved or used fresh in experiments.
<b>Figure 2.</b>
Figure 2.
Biodistribution of CARMA-hMeso cells following intraperitoneal (i.p.) injection in NSG mice. Cryopreserved control peripheral blood lymphocytes (PBLs) or CARMA-hMeso cells at 5 × 107 were injected i.p. into NSG mice on day 0 (D0). (A) On D1, D2, D4, and D7, blood was collected and profiled by staining with anti-human CD3-PE and CD45-FITC and analysis by flow cytometry. On D8, (B) peritoneal washings and (C) splenocytes were stained with anti-human CD3-PE and CD45-FITC and analyzed by flow cytometry. Significant differences were observed in the blood on D2 (p = 0.012) and D4 (p = 0.0028), but there was no statistically significant difference on D7 (p = 0.098). No statistically significant differences were observed in the peritoneum on D7 (p = 0.0603) or in the spleen on D8.
<b>Figure 3.</b>
Figure 3.
Injection i.p. of CARMA-hMeso cells controlled tumor growth in a mouse hMeso-expressing ovarian tumor model in nude mice. Mouse ovarian tumors expressing hMeso were initiated by i.p. injection of 3 × 105 Defb29 Vegf-luc/Hmeso into nude mice on D0. On D3, the baseline luminescence intensity from the mouse abdomen was imaged using IVIS. The CARMA-hMeso cells were thawed and injected i.p. on D4, and the mice were imaged once a week. (A) Luminescence intensity of mouse imaging of increasing doses of the CARMA-hMeso cells used. Phosphate-buffered saline (PBS) and non-specific CARMA cells (CD19 CARMA) were used as controls. Cells were injected immediately after thawing or rested in a 37°C incubator for 3 h. (B) Bar graph of luminescence intensity over a period of 13 days. Student's t-tests were done to compare each group with the PBS group. Only significant values are shown.
<b>Figure 4.</b>
Figure 4.
Intravenous injection of CARMA-hMeso cells did not have treatment effects on ovarian cancer in vivo. Defb29 Vegf-luc/Hmeso cells (1 × 104) were injected i.p. into nude mice on D0. On D3, the baseline luminescence intensity from the mouse abdomen was imaged using IVIS. The CARMA-hMeso cells were thawed and injected on D4 by i.p. or retro-orbital (r.o.) injection with different cell numbers, and treated mice were imaged once a week. Two days after the first injection, the continuous injection was administered daily for a total of three injections. The non-treatment group was the control group. (A) Imaging was obtained once weekly for 4 weeks, until D27. (B) Bar graph of luminescence intensity over a period 27 days. Student's t-tests were used to compare each individual group to the non-treatment group. (C) The survival of the mice was measured.
<b>Figure 5.</b>
Figure 5.
Repeated i.p. injection of CARMA-hMeso cells showed better activity in a mouse hMeso-expressing ovarian tumor model compared to a single injection. Mouse ovarian tumors expressing hMeso were initiated by i.p. injection of 1 × 104 Defb29 Vegf-luc/Hmeso cells into nude mice (N = 10/group) on D0. (A) Tumor-bearing mice were treated by i.p. injection (once weekly, three doses) with PBS (control) or CARMA-hMeso cells at 1 × 107 on D4 (1 × 1e7); 5 × 107 on D4 (1 × 5e7); 1 × 107 on D4, D11, and D18 (3 × 1e7), or 5 × 107 on D4, D11, and D18 (3 × 5e7). Survival of tumor-bearing mice is shown through D90. (B) Tumor-bearing mice were treated by i.p. injection with phosphate PBS; 5 × 107 control cells (Control T); or CARMA-hMeso cells at 5 × 107 on D4 (1 × CAR T), 5 × 107 on D4, D11, and D18 (3 × CAR T), or 5 × 107 on D4, D11, D18, D25, D32, and D39 (6 × CAR T). Survival of tumor-bearing mice is shown through D130.

Similar articles

See all similar articles

Cited by 6 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback