Mice were injected i.p. to be made to the second generation CAR to overcome the harsh solid tumor microenvironment and enhance antitumor function. We have developed a second generation CAR, 4H11-28z, containing a single-chain fragment antibody (scFv) that recognizes MUC16ecto, with promising preclinical results.11 MUC16ecto is the retained extracellular portion of the glycosylated mucin, MUC16. MUC-16 consists of a cytoplasmic tail, a transmembrane domain, and an extracellular portion. The extracellular region contains a cleavage site distal to which includes 16C20 tandem repeats of 156 amino acids, each with potential glycosylation sites.12 Of note, MUC16ecto remains on the cell surface after the distal N-terminus of MUC-16 called CA-125 (a well-known ovarian tumor antigen routinely used for monitoring disease) is cleaved and released.13 About 80% of women with epithelial ovarian cancer (EOC) have elevated levels ( ?65U/mL) of serum CA-125, and a majority of these patients have been found to express MUC16ecto on their tumor cells.12,14 MUC16ecto is also expressed on normal tissues including the uterus, fallopian tubes, and ovaries, which are removed initially during surgery as part of treatment, and also at low levels in the trachea.15 Therefore, expression of MUC16ecto in patients with advanced ovarian cancer is likely to be highly specific to the tumor cells, so this antigen offers an excellent target for immunotherapy of EOC.12 We have recently shown that 4H11-28z T cells eradicate orthotopic human ovarian cancer xenografts in SCID-Beige mice in a subset of treated mice.11 Herein, we have further modified CAR T cells to secrete IL-12, a stimulatory cytokine which acts through multiple mechanisms to enhance CD8+ T cell function and potentially overcome the inhibitory microenvironment.16 Previous clinical trials in ovarian cancer have used intraperitoneal (i.p.) injection of recombinant IL-12 with some efficacy, but these studies resulted in various treatment-related toxicities.17 In this report we show that MUC16ecto-specific CAR T cells that secrete IL-12 can enhance antitumor efficacy and as a safety measure, incorporated an elimination gene. Results Generation of Pyridoxamine 2HCl 4H11-28z/IL-12 T cells We have previously constructed a SFG retroviral vector encoding a second-generation CAR targeted to MUC-16ecto (4H11-28z) using a monoclonal antibody specific for MUC-16ecto antigen (4H11 hybridoma) (Fig. 1A -top panel).11 The second-generation CAR contains a scFv domain that allows for antibody recognition along with CD28 and CD3 signaling domains. We further modified the 4H11-28z construct with an IRES element followed by a gene encoding human IL-12 p35 and p40 subunit fusion connected via a flexi linker (flexi-IL-12 (hIL-12f); Fig. 1A -bottom panel). To assess the function of the 4H11-28z/IL-12 CAR (also referred to as armored CAR), healthy donor T cells isolated from peripheral blood were retrovirally transduced to express either the 2nd generation CAR, armored Pyridoxamine 2HCl CAR, or an irrelevant CAR targeted to CD19 (19-28z and 19-28z/IL-12) as controls. Transduction efficiency was determined using flow cytometry and was comparable for all constructs (Figs. 1B and 2B-right panel, 0.05). Open in a JV15-2 separate window Figure 1. Design and production of IL-12 secreting 4H11-28z T cells and 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; chain: human TCR chain cytoplasmic signaling domain; flexi human IL-12 (hIL-12f); LTR: 5 and 3 long terminal repeat; black box: CD8 leader sequence; gray Pyridoxamine 2HCl 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.