Successful eradication of established peritoneal ovarian tumors in SCID-Beige mice following adoptive transfer of T cells genetically targeted to the MUC16 antigen

Abstract

Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For this reason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient's own T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigen receptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovarian cancer.

Experimental design: We have generated several CARs targeted to the retained extracellular domain of MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitro biology of human T cells retrovirally transduced to express these CARs by coculture assays on artificial antigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD(+) ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacy of MUC-CD-targeted T cells in SCID-Beige mice bearing peritoneal human MUC-CD(+) tumor cell lines.

Results: CAR-modified, MUC-CD-targeted T cells exhibited efficient MUC-CD-specific cytolytic activity against both human ovarian cell and primary ovarian carcinoma cells in vitro. Furthermore, expanded MUC-CD-targeted T cells infused through either i.p. injection or i.v. infusion into SCID-Beige mice bearing orthotopic human MUC-CD(+) ovarian carcinoma tumors either delayed progression or fully eradicated disease.

Conclusion: These promising preclinical studies justify further investigation of MUC-CD-targeted T cells as a potential therapeutic approach for patients with high-risk MUC16(+) ovarian carcinomas.

Figure 1

Design and in vitro analysis of MUC-CD targeted CARs. (A) Schematic diagram of the first generation 4H11z and second generation 4H11-28z retroviral vectors. 4H11scFv: MUC16 specific scFv derived from the heavy (V_H) and light (V_L) chain variable regions of the monoclonal antibody 4H11; CD8: CD8 hinge and transmembrane domains; CD28: CD28 transmembrane and cytoplasmic signaling domains; ζ chain: T cell receptor ζ chain cytoplasmic signaling domain; LTR: long terminal repeat; black box: CD8 leader sequence; grey box: (Gly₄Ser)₃ linker; arrows indicate start of transcription. (B) FACS analysis of human T cells retrovirally transduced to express either the 4H11z or 19z1 CAR. (C) 4H11z⁺ but not 19z1⁺ T cells expand on 3T3(MUC-CD/B7.1) AAPC. CAR⁺ were co-cultured on 3T3(MUC-CD/B7.1) AAPC monolayers at 3 × 10⁶ CAR⁺ T cells/well of a 6 well plate. Proliferation of CAR⁺ T cells was assessed by FACS in combination with viable T cell counts as assessed by trypan blue exclusion assays.

Figure 2

In vitro comparison of T cells modified to express the first generation 4H11z CAR to T cells modified to express the second generation co-stimulatory 4H11-28z CAR. (A) CAR⁺ T cells were co-cultured on MUC-CD monolayers with (right panel) or without B7.1 (left panel). 1 × 10⁶ CAR⁺ T cells per well were co-cultured on AAPC monolayers in 12 well tissue culture plates in cytokine-free medium. Total viable T cell counts were assessed on days 2, 4 and 7, by trypan blue exclusion assays. (B) Antigen specific cytokine’s secretion by MUC-CD⁺ targeted T cells. (C) Expansion of CAR⁺ T cells following 3 cycles of stimulation on 3T3(MUC-CD/B7.1). Arrows indicate 1st and 2nd cycles of restimulation on AAPCs. (D) FACS analysis of the CAR⁺ T cell fraction of 4H11-28z⁺ T cells increased following each weekly cycle of stimulation. (I) FACS following initial transduction, (II) FACS at 7 days following first stimulation on AAPCs, (III) FACS at 7 days following second stimulation on AAPCs.

Figure 3

Upon co-culture, 4H11-28z⁺ T cells specifically expand and lyse MUC-CD⁺ tumor cells. (A) Cytotoxicity assays of 4H11-28z⁺ T cells targeting OV-CAR3(MUC-CD) and SK-OV3(MUC-CD) tumor cells demonstrate efficient cytotoxicity mediated by 4H11-28z⁺ T cells T cells from healthy donors compared to control 19-28z⁺ T cells. (B) Antigen specific proliferation of MUC-CD targeted T cells after co-culture with OV-CAR3(MUC-CD) or SK-OV3(MUC-CD) tumor cells. CAR⁺ T cells were co-cultured with wild type or MUC-CD expressing tumor cells at 1:1 ratio for 7 days. Absolute T cell numbers assessed on days 2, 4 and 7 following co-culture with ovarian tumor cells. The T cell seeding density for T cell proliferation assay was 1 × 10⁶/mL. (C) Antigen-specific IL-2 and IFN-γ secretion by 4H11-28z transduced T cells after 2 days of co-culture on ovarian cancer cells. (D) Healthy donor T cells or autologous T cells isolated from ascites, modified to express the 4H11-28z CAR lyse primary patient ascites-derived MUC-CD⁺ tumor cells when compared to T cells modified to express the control 19-28z CAR.

Figure 4

Eradication of OV-CAR3(MUC-CD) tumors after i.p. treatment with first and second generation of MUC-CD targeted T cells. (A) Intraperitoneal injection of OV-CAR3(MUC-CD) tumors in untreated SCID-Beige mice results in abdominal distension and nodular peritoneal tumors. At 5 weeks post intraperitoneal injection of OV-CAR3(MUC-CD) tumor cells mice developed ascites as evidenced by a distended abdomen (center panel) when compared to a tumor free mouse (left panel). Post mortem visualization of the peritoneum demonstrates nodular tumor masses (arrows) within the abdominal cavity (right panel). (B) Intraperitoneal injection of 4H11z⁺ and 4H11-28z⁺ T cells either delay tumor progression or fully eradicate disease. Kaplan-Meier survival curve of SCID-Beige mice treated with first or second generation of MUC-CD targeted T cells.

Figure 5

MUC-CD targeted 4H11-28z⁺ T cells traffic to peritoneal OV-CAR3(MUC-CD/GFP-FFLuc) tumors following systemic intravenous infusion resulting in efficient anti-tumor efficacy. (A) BLI of tumor progression of representative i.p. and i.v. 4H11-28z⁺ T cell treated mice with ultimately progressive disease following treatment compared to BLI of tumor progression in a representative control 19-28z⁺ T cell treated mouse. (B) Kaplan-Meier survival curve of SCID-Beige mice treated i.p. or i.v. with 4H11-28z⁺ T cells. Tumor eradication is enhanced after either ip or iv infusion of 4H11-28z⁺ T cells when compared to control treated mice. Both ip and iv 4H11-28z⁺ T cell treated mice exhibited statistically enhanced survival when compared to 19-28z⁺ T cell treated control cohorts while survival between the i.p. and i.v. treated 4H11-28z⁺ T cell cohorts was not statistically significant (p=0.22). (C) Systemically injected CFSE stained 4H11-28z⁺ T cells traffic to advanced i.p. OV-CAR(MUC-CD) tumors. Presence of i.v. injected CFSE labeled 19-28z⁺ control T cells (left panel) and 4H11-28z⁺ T cells (right panel) 1 day following infusion into SCID-Beige mice with OV-CAR(MUC-CD) tumors injected 7 days earlier as assessed by FACS analysis of single cell OV-CAR3(MUC-CD) tumor suspensions.

Figure 6

Eradication of BLI evident OV-CAR3(MUC-CD) tumors in SCID-Beige mice by ip infusion of 4H11-28z⁺ T cells. (A) BLI of 4H11-28z⁺ T cell treated mice with either relapsed disease (middle row) or eradicated disease (bottom row) compared to a representative 19-28z⁺ T cell treated control mouse. (B) Kaplan-Meier survival curve of SCID-Beige mice with advanced OV-CAR3(MUC-CD/GFP-FFLuc) tumors treated i.p. with 4H11-28z⁺ T cells.