A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer

Abstract

Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity.

Figures

FIG. 1.

PSCA expression in tumor and normal tissues. (a) Quantitative RT-PCR analysis of PSCA expression in 437 tumor samples of 18 different histologies. Results shown for each individual sample (dots) grouped by cancer histology. Horizontal lines in each dataset represent median expression value for a given histology. Dotted line marks expression level of thousand copies of PSCA per million copies of ACTB. (b) Quantitative RT-PCR analysis of PSCA expression in 40 cell lines derived from pancreatic cancer. PSCA expression higher than a thousand copies per million copies of ACTB (dotted line) was found in 18 cell lines. “>” means higher than the upper limit of the standard curve. (c) Quantitative RT-PCR results for a panel of cDNA derived from human normal tissues. ACTB, beta-actin; PSCA, prostate stem cell antigen; RT-PCR, reverse transcription polymerase chain reaction.

FIG. 2.

Human monoclonal antibody-based anti-PSCA CARs demonstrate superior expression and reactivity. (a) Schematic representation of three different anti-PSCA CAR designs. bm2B3-28BBZ is a third-generation CAR containing an antigen-recognition domain derived from a humanized mouse monoclonal antibody (bm2B3) and a signaling domain containing CD28, 4-1BB (CD137), and CD3-zeta moieties. Ha1-4.117-28Z and Ha1-4.117-28BBZ are second- and third-generation CARs, respectively, containing an antigen-binding domain derived from fully human Ha1-4.117 antibody. They differ in their signaling domain, composed of a CD28 plus CD3-zeta moiety in Ha1-4.117-28Z or CD28 plus 4-1BB (CD137) plus CD3-zeta in Ha1-4.117-28BBZ. (b) FACS analysis of CAR expression in primary human T cells. OKT3-stimulated peripheral blood mononuclear cells were transduced twice with retroviral vectors encoding the indicated CAR constructs. A Herceptin-based anti-Her2/neu CAR was used as a positive control. SA-PE: cells incubated with secondary staining (streptavidin-PE) alone as isotype control. (c) Anti-PSCA CARs endow human T lymphocytes with specific reactivity against PSCA-expressing targets. In vitro-transduced T cells were cocultured O/N with the indicated targets. IFNg release was measured in culture supernatants by enzyme-linked immunosorbent assay. Bar charts show results from representative experiments (values represent the average of duplicates) for different donors. Target cells: LNCaP (prostate cancer), LNCaP-PSCA and DU145-PSCA (prostate cancer lines engineered to express PSCA), HPAC and PANC-1 (pancreatic cancer), 624mel (melanoma), U251-vIII (glioma), NHDF-A2 (normal fibroblasts), SKOV3 (ovarian cancer), and H1299-A2 (lung cancer). White bars, bm2B3-CD28BBZ CAR; gray bars, Ha1-4.117-28Z CAR; black bars, Ha1-4.117-28BBZ CAR; dotted bars, GFP control; horizontal-line bars, mock transduced control. Ab, antibody; CAR, chimeric antigen receptor; FACS, fluorescence-activated cell sorting; IFNg, interferon-gamma.

FIG. 3.

PSCA and MSLN as targets for pancreatic cancer immunotherapy. (a) Quantitative RT-PCR analysis of PSCA and MSLN expression in samples of human PDA and normal tissues. Quantitation was performed in parallel for both genes in order to compare their expression in malignant and healthy tissue. (b) Surface expression of anti-PSCA and anti-MSLN CARs. Histograms showing results of protein-L staining, analyzed by FACS, of primary human T lymphocytes engineered to express the two versions of Ha1-4.117-based anti-PSCA CARs or the anti-MSLN CAR. GFP was used as a control of transduction. (c) IFNg release upon coculture of human primary T cells transduced with the indicated CARs against PSCA or MSLN. Horizontal-line bars, GFP-transduced control T cells; black bars, Ha1-4.117-28Z CAR; white bars, Ha1-4.117-28BBZ CAR; gray bars, anti-MSLN CAR; dotted bars, mock transduced control. Expression of PSCA and MSLN shown as copies of mRNA per million copies of ACTB mRNA. MSLN, mesothelin; PDA, pancreatic ductal adenocarcinoma.

FIG. 4.

PSCA CARs effectively treat established tumors in NSG mice. (a) Growth curves after treatment with CAR-expressing human T cells or controls, in two independent experiments. Tumor area expressed as mean of five tumor bearing mice±SEM. Filled circles, PSCA-28Z CAR; filled squares, PSCA-28BBZ CAR; open squares, GFP; open triangles, untransduced CD4 alone; open diamonds, vehicle alone. Arrows indicate day of adoptive cell transfer; arrowheads indicate administration of rhIL-2 intraperitoneal. (b) Number of CAR transgene in genomic DNA of splenocytes, obtained at the end of the treatment, analyzed by quantitative PCR. Undetectable expression results were assigned a value of 0.1. NSG, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ.

FIG. 5.

Immunohistochemistry staining of PSCA CAR-treated tumors. (a) PSCA expression in remaining tumors after treatment. Immunohistochemistry of tumor sections showing strong PSCA staining in control groups and greatly reduced staining in PSCA CAR-treated mice. Scale bars represent 2000 μm in the top row and 200 μm in the bottom row. (b) Immunohistochemical analysis of FAP in PSCA-28Z CAR-treated tumor and control GFP-treated tumor. Scale bars represent 1000 μm in the top row and 200 μm in the bottom row. FAP, fibroblast activation protein.