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. 2015 Jan 23;4(3):e994446.
doi: 10.4161/2162402X.2014.994446. eCollection 2015 Mar.

IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo

Mythili Koneru  1 , Terence J Purdon  1 , David Spriggs  1 , Susmith Koneru  2 , Renier J Brentjens  1
Affiliations

IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo

Mythili Koneru et al. Oncoimmunology. .

Abstract

A novel approach for the treatment of ovarian cancer includes immunotherapy with genetically engineered T cells targeted to ovarian cancer cell antigens. Using retroviral transduction, T cells can be created that express an artificial T cell receptor (TCR) termed a chimeric antigen receptor (CAR). We have generated a CAR, 4H11-28z, specific to MUC-16ecto antigen, which is the over-expressed on a majority of ovarian tumor cells and is the retained portion of MUC-16 after cleavage of CA-125. We previously demonstrated that T cells modified to express the 4H11-28z CAR eradicate orthotopic human ovarian cancer xenografts in SCID-Beige mice. However, despite the ability of CAR T cells to localize to tumors, their activation in the clinical setting can be inhibited by the tumor microenvironment, as is commonly seen for endogenous antitumor immune response. To potentially overcome this limitation, we have recently developed a construct that co-expresses both MUC16ecto CAR and IL-12 (4H11-28z/IL-12). In vitro, 4H11-28z/IL-12 CAR T cells show enhanced proliferation and robust IFNγ secretion compared to 4H11-28z CAR T cells. In SCID-Beige mice with human ovarian cancer xenografts, IL-12 secreting CAR T cells exhibit enhanced antitumor efficacy as determined by increased survival, prolonged persistence of T cells, and higher systemic IFNγ. Furthermore, in anticipation of translating these results into a phase I clinical trial which will be the first to study IL-12 secreting CAR T cells in ovarian cancer, an elimination gene has been included to allow for deletion of CAR T cells in the context of unforeseen or off-tumor on-target toxicity.

Keywords: AAPCs, artificial antigen presenting cells; ADCC, antibody-dependent cellular cytotoxicity; ALL, acute lymphocytic leukemia; CAR, chimeric antigen receptor; EGFRt, truncated epidermal growth factor; EOC, epithelial ovarian cancer; i.p., intraperitoneal; IL-12, interleukin-12; i.v., intravenous; MDSC, myeloid-derived suppressor cells; PBL, peripheral blood leukocytes; PBMCs, peripheral blood mononuclear cells; scFv, single-chain fragment antibody; TAA, tumor-associated antigen; TCR, T cell receptor; TIL, tumor-infiltrating lymphocytes; Tregs, regulatory T cells.; IL-12; MUC16; chimeric antigen receptors; human ovarian cancer; tumor microenvironment.

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Figures

Figure 1.
Figure 1.
Design and production of IL-12 secreting 4H11-28z T cells and in vitro studies of 4H11-28z/IL-12 T cells compared to second-generation 4H11-28z CAR T cells. (A) Schema of 4H11-28z and 4H11-28z/IL-12 retroviral vectors. 4H11 scFv: MUC16-specific scFv derived from heavy (VH) and light (VL) chain variable regions of the mAb 4H11; CD28: human CD28 transmembrane and cytoplasmic signaling domains; ζ chain: human TCRζ chain cytoplasmic signaling domain; flexi human IL-12 (hIL-12f); LTR: 5′ and 3′ long terminal repeat; black box: CD8 leader sequence; gray box: (Gly4Ser)3 linker. (B) Flow cytometric analysis of transduction efficiency using a 4H11-CAR specific antibody. Transduction efficiencies achieved were routinely >50%. Data shown is representative of >7 independent experiments. (C) The ability of CAR T cells to lyse SKOV3 (MUC-16ecto) ovarian cancer cells was assessed using a 4-h 51Cr release assay. CAR T cells targeting tumor cells demonstrated increased killing by 4H11-specific CARs compared to irrelevant non-specific CAR T cells (1928z and 19-28z/IL-12). Data shown is representative of four independent experiments. (D) Peripheral mononuclear cells (PBMCs) were exposed to conditioned media overnight from viral producers and the amount of IFNγ in each sample was assessed using a luminex assay. Elevations in IFNγ was seen in 4H11-28z/IL-12 compared to 4H11-28z conditioned media. Data shown is representative of two independent experiments.
Figure 2
Figure 2
(See previous page). (G) CD16+ NK-92 cells were used in an ADCC assay to determine the ability of cetuximab or rituximab to mediate clearance of EGFRt/4H11-28z/IL-12 CAR T cells. The samples were analyzed by flow cytometry for the decrease in percentage of CAR T cells with the antibody compared to CAR T cells in the absence of antibody. EGFRt/4H11-28z/IL-12 CAR T cells showed increased ADCC when treated with cetuximab compared to rituximab (p < 0.05). Cetuximab did not induce ADCC on 4H11-28z/IL-12 CAR T cells. Data shown is mean percentage of ADCC (±SEM) from three independent experiments.
Figure 3.
Figure 3.
In vivo antitumor efficacy of EGFRt/4H11-28z/IL-12 CAR T cells. (A) Mice inoculated with 1 × 10 SKOV3 (MUC-16ecto+) tumor cells i.p. were treated with i.p. infusion of 2.5 × 10 CAR T cells 2 weeks later. Survival of SKOV3 tumor bearing mice was significantly enhanced with 4H11-28z/IL-12 and EGFRt/4H11-28z/IL-12 CAR T cells compared to 4H11-28z CAR T cells (**p < 0.05). Data shown is from two independent experiments. (B) Mice inoculated with 1 × 10 SKOV3 (MUC-16ecto+) tumor cells i.p. were treated with i.v. or i.p. infusion of 2.5 × 10 CAR T cells 2 weeks later. Survival of SKOV3 tumor bearing mice was significantly enhanced with i.p. infusion of 4H11-28z/IL-12 CAR T cells compared to i.v. infusion of 4H11-28z/IL-12 CAR T cells (**p < 0.05). Data shown is from one experiment. (C) Representative bioluminescent imaging (BLI) of tumor progression following i.p administration of tumor alone compared to mice treated with 19-28z and 4H11-28z/IL-12 CAR T cells (i.p.) 2 week post-tumor administration. Day 13 was imaged one day prior to CAR T cell treatment. Data shown is representative of five mice from two independent experiments. (D) Mean radiance of tumor progression imaged with BLI in mice bearing SKOV3(MUC-16ecto+) tumors and treated 2 weeks later. Mice treated with 4H11-28z/IL-12 and EGFRt/4H11-28z/IL-12 CAR T cells have less tumor compared to untreated control mice at day 27. Data shown is mean radiance from five mice and representative of two experiments. (E) H&E staining of SKOV3(MUC-16ecto+) tumor in the liver of untreated mice (i and ii) at day 35 and normal liver at day 93 in SKOV3(MUC-16ecto+) tumor bearing mice treated with EGFRt/4H11-28z/IL-12 (iii). Data shown is representative of three mice from two independent experiments. (F) Mice inoculated with 1 × 10 SKOV3 (MUC-16ecto+) tumor cells i.p. were treated with i.p. infusion of 2.5 × 10 CAR T cells 4 weeks later. Survival of SKOV3 tumor bearing mice was significantly enhanced with 4H11-28z/IL-12 and EGFRt/4H11-28z/IL-12 CAR T cells compared to 4H11-28z CAR T cells (**p < 0.05). Data shown is from two independent experiments.
Figure 4.
Figure 4.
Increased CAR T cell persistence and serum IL-12 levels in mice treated with EGFRt/4H11-28z/IL-12 CAR T cells. (A) Mice were inoculated with 1 × 10 SKOV3 (MUC-16ecto+) tumor cells i.p. followed 2 weeks later by i.p. infusion of 2.5 × 10 CAR T cells. Peripheral blood (PB) of mice 6 d post-CAR treatment with 4H11-28z/IL-12 T or 4H11-28z CAR T cells was collected and analyzed by flow cytometry for presence of CAR T cells by staining with Ab specific for hCD3 and 4H11-CAR. PB from mice treated with 4H11-28z/IL-12 has increased amounts of CAR T cells compared to mice treated with 4H11-28z CAR T cells. Data shown is representative of four mice from two independent experiments. (B) Serum levels of IFNγ in mice treated with CAR T cells 6 d post-T cell infusion were analyzed by luminex assay. Increased serum IFNγ was shown in mice treated with 4H11-28z/IL-12 and EGFRt/4H11-28z/IL-12 compared to mice treated with 4H11-28z (**p < 0.05). Mice treated with EGFRt/4H11-28z/IL-12+cetuixmab shows no difference in serum IL-12 compared to mice treated with 4H11-28z (*p > 0.05). Data shown is mean (±SEM) from four mice, representative of two independent experiments. (C) Serum levels of IFNγ in mice treated with CAR T cells 35 d post-T cell infusion were analyzed by luminex assay. No statistically significant differences were identified between any of the groups (p > 0.05). Data shown is mean (±SEM) from four mice, representative of two independent experiments. (D) Serum levels of IL-12(p70) in mice treated with CAR T cells 4, 11, 20, and 35 d post-T cell infusion were analyzed by luminex assay. Mice treated with 4H11-28z/IL-12 showed increased IL-12(p70) on days 11 and 20 compared to day 35 post-CAR T cell injection (**p < 0.05). Mice treated with 4H11-28z showed no increase in IL-12 (p70) on any of the days post-CAR T cell injection (*p > 0.05). Data shown is mean (±SEM) from four mice, representative of two independent experiments.
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