Boosts Immunotherapy Response in Gastroesophageal Cancer

Peritoneal carcinomatosis, which is observed in one-third of patients who present with metastatic gastroesophageal adenocarcinoma, is resistant to chemotherapy and immunotherapy and is associated with a median survival of 3–5 months.1–4

The immune microenvironment in gastroesophageal adenocarcinoma peritoneal metastases—which is characterized by M2 macrophages, inhibitory cytokines, and low levels of tumor-infiltrating lymphocytes—hinders the response to immunotherapy.5–7 To tilt the immune environment in favor of effector responses, chimeric antigen receptor (CAR) T-cell therapy has been administered to boost the number of tumor-infiltrating lymphocytes in these resistant tumors.8 CARs are synthetic receptors that have been engineered to direct T cells to target a cancer-associated antigen, such as mesothelin (MSLN), which is overexpressed on gastroesophageal adenocarcinoma cells and is associated with tumor aggressiveness.9 To overcome the challenges of poor infiltration of adoptively transferred immune effector cells into solid tumors, we investigated intrapleural (regional) delivery of CAR T cells, which, in preclinical studies, achieved superior efficacy, compared with intravenous (systemic) administration, even at 30-fold lower doses, and augmented the CD4 CAR T-cell helper function to sustain a higher number of CD8 CAR T cells.10 Intrapleural delivery of MSLN-targeted CAR T cells was translated to a phase I clinical trial (n=27 patients) and was shown to be safe and effective; 41 patients have been treated to date in phase I/II studies, with systemic circulating CAR T cells detected >100 days after a single intrapleural infusion.11

Patients with peritoneal carcinomatosis from gastroesophageal adenocarcinoma often present with extraperitoneal sites of metastases;2 4 we therefore investigated the efficacy of intraperitoneal CAR T-cell therapy in mouse models of combined peritoneal and distant sites of disease.

KYAE-1 and SK-GT-4 gastroesophageal adenocarcinoma cells were obtained from Sigma-Aldrich. Because of the variable nature of the MSLN expression in cancer cells in culture, cells were transduced with human MSLN variant 1 (isolated from human ovarian cancer cells OVCAR-3) subcloned into an SFG retroviral vector to generate MSLN-overexpressing tumor cells (KYAE1-M and SKGT4-M). Cells were retrovirally transduced to express green fluorescent protein (GFP)–firefly luciferase fusion protein (KYAE1-GM, SKGT4-GM) to facilitate the performance of in vivo bioluminescence imaging (BLI). All cell lines used were periodically authenticated at the Antibody and BioResource Core Facility at Memorial Sloan Kettering Cancer Center using short tandem repeat profiling. In addition, all lines were routinely tested for mycoplasma contamination to ensure cell line identity and culture integrity throughout the study.

γ-retroviral vector construction and viral production

An MSLN-specific CAR was generated by linking a single-chain variable fragment specific for human MSLN to the CD28/CD3ζ signaling domains. A programmed cell death protein-1 (PD-1) dominant negative receptor (DNR) was generated by linking the extracellular portion of the PD-1 receptor to the M28z vector sequence.8 The CD3ζ domain was modified to contain a single functional immunoreceptor tyrosine–based activation motif, termed 1XX, resulting in a M28z1XXPD1DNR CAR construct. Each CAR sequence was inserted into the SFG γ-retroviral vector (provided by I Riviere, Memorial Sloan Kettering Cancer Center) and linked to a Myc-tag sequence. The CAR-encoding plasmids were then transfected into 293T H29 and 293VecRD114 packaging cells to produce the retrovirus. The sequences for the 28z MSLN CAR, PD-1 DNR, and 1XX CAR constructs used in this study are available in the following patent applications: WO2015188141A9,12 WO2017040945A1,13 and EP3732191A2.14

T-cell isolation and gene transfer

Peripheral blood leukocytes were isolated from the blood of healthy volunteer donors under a Memorial Sloan Kettering Cancer Center Institutional Review Board–approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated by low-density centrifugation on Lymphoprep (STEMCELL Technologies) and activated for 2 days with phytohemagglutinin (2 ug/mL; Remel). Two days after isolation, PBMCs were transduced with 293VecRD114-produced retroviral particles encoding for CARs and PD-1 DNR and spinoculated for 1 hour at 1800 g on plates coated with retronectin (15 µg/mL; r-Fibronectin, Takara Bio). Transduced PBMCs were maintained in interleukin-2 (IL-2) (20 UI/mL; Novartis).8 Both untransduced and prostate-specific membrane antigen (PSMA)–targeted CAR T cells were used as controls. We have previously reported that a 28z CAR targeting PSMA, an irrelevant antigen in this context, showed no efficacy in MSLN tumor models, comparable with untransduced T cells,15 with minimal background activity.

CAR T-cell cytotoxicity assays

The cytotoxicity of CAR T cells was determined using a standard 51Cr-release assay. M28z1XXPD1DNR CAR T cells and control untransduced T cells were cultured with MSLN-overexpressing target cells (KYAE1-M, SKGT4-M) at effector to target (E:T) ratios ranging from 1:4 to 64:1. Cytotoxicity was determined after 18 hours of incubation.15 Data are reported as the mean of triplicate measurements±SE and were analyzed using Microsoft Excel (Microsoft) or GraphPad Prism (GraphPad Software).

In vitro CAR T-cell proliferation and accumulation assays

CAR T cells were stained with CellTrace Violet cell proliferation stain using a 1 µM working solution, in accordance with the manufacturer’s instructions (Thermo Fisher Scientific). M28z1XXPD1DNR CAR T cells (180e3 CAR T cells/24-well plate) and MSLN-overexpressing tumor cells (60e3 tumor cells/24-well plate; KYAE1-M, SKGT4-M) were plated at an E:T ratio of 3:1 in triplicate. After incubation for 4 and 7 days, T cells were collected, T-cell numbers were counted using a hemacytometer, and CAR T cells were assessed for CAR+ percentage and CellTrace Violet fluorescence intensity using flow cytometry. Plotted CAR T-cell accumulation numbers were adjusted for CAR+percentage.

In vitro CAR T-cell repeat-antigen-stimulation assay

To assess accumulation after repeat antigen stimulation, MSLN-overexpressing tumor cells (KYAE1-M, SKGT4-M) were irradiated and plated at a density of 30e3 tumor cells per cm2 in 6-well plates in triplicate. At 24 hours after tumor cell plating, M28z1XXPD1DNR CAR T cells were plated at an E:T ratio of 2:1. After 2 days of incubation, T cells were collected and counted using a hemacytometer and were assessed for CAR+percentage. Plotted CAR T-cell accumulation numbers were adjusted for CAR+ percentage. T cells were then restimulated under the same conditions, for a total of three antigen stimulations.

In vitro cytokine-release assay

M28z1XXPD1DNR CAR T cells (180e3 CAR T cells/24-well plate) and MSLN-overexpressing tumor cells (60e3 tumor cells/24-well plate; KYAE1-M, SKGT4-M) were plated at an E:T ratio of 3:1 in triplicate. After 20 hours of incubation, cell-culture supernatant was collected. Human interferon-gamma, IL-2, and tumor necrosis factor-alpha were quantified using the Luminex Assay (Thermo Fisher Scientific).

Flow cytometry and T-cell proliferation labeling

Fluorochrome-conjugated antibodies to MSLN (anti-MSLN PE-conjugated Rat IgG2A FAB32652P; R&D Systems), CD45, CD3 (PE/Cy7 anti-human CD3 Clone HI3A, 300316; BioLegend), CD4 (APC Mouse Anti-Human CD4, 55349; BD Pharmingen), CD8 (FITC Mouse Anti-Human CD8, 561948; BD Pharmingen), programmed death-ligand 1 (PD-L1), PD-1, and Myc-tag (PE Mouse Anti-Human Myc, 9B11; Cell Signaling Technology) were used. All flow cytometric analyses were performed on either a BD FACSCalibur or an LSR II (BD Biosciences) flow cytometer; data were analyzed using FCS Express software (V.7). Flow cytometry of in vivo xenograft tumor samples was performed after filtration in a 40 µm filter and blocking with an antimouse Fc block.

Gastroesophageal cancer peritoneal carcinomatosis mouse models

The experimental procedures used for the animal studies were approved by the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center. NSG mice (The Jackson Laboratory) were injected intraperitoneally with 5e6 KYAE1-GM or SKGT4-GM gastroesophageal adenocarcinoma cells expressing MSLN and GFP-luciferase in 200 µL of serum-free media. M28z1XXPD1DNR CAR T cells or control (untransduced) T cells were injected in 200 µL of serum-free media by intraperitoneal or intravenous injection in doses of 1e5, 2e5, or 5e5 cells per mouse. Flank tumor was established via injection of 5e6 KYAE1-M or KYAE tumor cells in serum-free media. Flank-tumor volumes were calculated as tumor volume (mm3) = [width (mm)]2 × [length (mm)]/2. Ascites fluid was obtained from the mouse peritoneum after euthanasia, and the TGF-β1 concentration was determined using the Luminex assay (Thermo Fisher Scientific).