Prostate Cancer Immunotherapy Development
Using Tumor-specific CAR Design
Victor Prima*, Sergei Kusmartsev* and Johannes Vieweg
Department of Urology, College of Medicine, University of Florida, Gainesville, FL
Prostate cancer that has progressed to metastatic disease remains largely untreatable. One of the most
promising modalities for treatment of advanced cancer is immunotherapy based on adoptive transfer of T
lymphocytes. It was demonstrated that T lymphocytes expressing tumor-specific chimeric artificial receptor
(CAR) exert potent anti-tumor activity upon adoptive transfer into tumor-bearing host. Recent studies
identified Prostate-specific membrane antigen (PSMA) and ligands for NK cell –activating receptors (e.g.
NKG2D ligands) as the targets primarily expressed on tumor cells but absent on most normal tissues.
We generated two sets of chimeric DNA constructs: CAR-PSMA and CAR-NKG2D, that can be used for
transduction of human T cells. Both of them consist of an antigen-recognizing domain bound to the
transmembrane and intracellular domains of various signal-transducing and/or cell-activating molecules
and a signaling molecule. Extracellular antigen-recognizing domain of CAR-PSMA consists of single chain
anti-PSMA antibody (scFv) while in another construct set it is human NKG2D receptor. Intracellular CD3 ξ-
chain functions as a signaling molecule triggering T-cell activation. Co-stimulatory proteins CD28, CD40L or
OX40L which can compensate for the lack of co-stimulation from the target tumor cell are either fused to
CD3 ξ-chain as intracellular domains or are expressed on cell surface from bi-cistronic constructs.
Upon transduction of human T cells with these constructs, transduced cells presented surface expression
of the chimeric scFv anti-PSMA or NKG2D, respectively. In vitro experiments demonstrated specific
binding activity and cytotoxicity of human T lymphocytes expressing anti-PSMA or NKG2D CARs against
prostate cancer cells which creates the basis for a viable prostate cancer immunotherapy development.
*VP and SK contributed equally to the project.
The authors would like to thank Galante Foundation for the generous support.
Generation of chimeric DNA constructs encoding human TCR signaling domains. The specific cDNA
sequences employed in our constructs incorporate various domains of human T and NK cell receptor
signaling fused to the CD3ξ chain at the 3’-end (Fig.2). Original full-lengths human cDNA clones were
obtained from NIH Mammalian Gene Collection (MGC). Following functional domain analysis and
oligonucleotide design the In-Fusion system of DNA cloning (Clontech) was used to assemble the CAR
Generation of CD3-CD28-anti-PSMA DNA construct. In order to engineer the T cells that express
receptor specific for human prostate-specific membrane antigen (PSMA) we used the strategy of
consecutive CAR constructs assembling (Fig.3). cDNA sequences encoding the human CD28 and CD3ξ
chains were linked to variable domains of anti-PSMA antibody (scFv) derived from the J415 hybridoma
(ATCC) (Fig.4). In this chimeric anti-PSMA receptor the scFv serves as an extracellular antigen-binding
domain, CD28 domain stimulates cell proliferation and cytokine expression, and CD3ξ chain is the
intracellular signaling module activating T cell upon antigen engagement. Transgene expression here is
driven by a composite promoter consisting of CMV enhancer/modified beta-actin promoter – CBA (Fig.5).
Generation of CD3-NKG2D DNA construct. To generate T cells with ability to kill NKG2D-ligand–bearing
tumor cells, cDNA sequences encoding the human NKG2D receptor and CD3ξ chain were assembled to
make the chimeric NKG2D receptor. NKG2D is a type II protein, in which the N-terminus is located
intracellularly whereas the CD3ξ chain is a type I protein with the C-terminus in the cytoplasm. To create an
NKG2D-CD3ξ fusion protein, the initiation codon ATG was placed ahead of the cDNA encoding the
cytoplasmic region of the CD3ξ chain followed by wild-type NKG2D cDNA. Upon expression, the orientation
of the CD3ξ domain is reversed inside the cells. The extracellular domain derived from NKG2D serves as
antigen-binding part of the receptor, and CD3ξ chain is the intracellular signaling module activating T cell
upon antigen engagement. The schematic maps of the expression constructs generated for assessment of
functional roles of NKG2D and PSMA CARs are shown in Fig.5. The CAR-expressing retroviruses were
produced using standard molecular techniques.
Functionality of cell transduction techniques and transgene expression was assessed with HEK-293
and Jurkat cells as modeling cell lines. Our data show successful retroviral delivery of the chimeric
constructs with high level of ectopic expression both in the modeling cell lines (Fig.6) and in the T cells
Specific binding activity of anti-tumor CARs. Assays for the ability of CAR- transduced T cells to bind the
target cells expressing PSMA or/and NKG2D ligands on their surface confirmed specificity of this interaction
(Fig.7 as an example).
Cytotoxic T lymphocytes (CTL) activity
On day 7-8 after infection with CAR RV or control RV (mock) cultured T cells were collected, washed with
PBS and mixed with tumor cells at cell ratios 10:1 or 25:1 in complete RPMI-1640 medium in 96 well plates.
Cell mixtures were cultured at 370 C in humidified CO2 incubator for twenty hours. CTL activity was
measured using LDH cytotoxicity assay (Sigma-Aldrich) according to the manufacturer’s protocol. The
preliminary data of in vitro tests indicate cytotoxicity of human T lymphocytes expressing anti-PSMA or
NKG2D CARs against the cells expressing cancer-related ligands (Fig.8).
We generated two sets of chimeric DNA constructs: CAR-PSMA and CAR-NKG2D,
consisting of an antigen-recognizing domain and various domains of T and NK cell receptor
signaling fused to the CD3ξ.
Upon viral transduction of human T cells with these constructs, transduced cells
presented significant surface expression of the chimeric scFv anti-PSMA or NKG2D,
Binding specificity and cytotoxicity of human T lymphocytes expressing recombinant
anti-PSMA or NKG2D CARs make feasible the development of effective immunotherapy
against prostate cancer.