SlideShare a Scribd company logo
1 of 2
Download to read offline
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
ABSTRACT
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
constructs.
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
(Fig.10, 11).
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).
EXPERIMENTAL APPROACH
CONCLUSIONS
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,
respectively.
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.
AACR poster VP_2014

More Related Content

What's hot

Medullary thyroid carcinoma
Medullary thyroid carcinomaMedullary thyroid carcinoma
Medullary thyroid carcinomaSoM
 
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...Karolina Megiel
 
Cancer Res-2014-Chakraborty-3489-500
Cancer Res-2014-Chakraborty-3489-500Cancer Res-2014-Chakraborty-3489-500
Cancer Res-2014-Chakraborty-3489-500Rachel Stupay
 
Aleta Bio CAR-TCR Summit 11 Sept 2019
Aleta Bio CAR-TCR Summit 11 Sept 2019Aleta Bio CAR-TCR Summit 11 Sept 2019
Aleta Bio CAR-TCR Summit 11 Sept 2019Paul D. Rennert
 
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...Sima Lev
 
ALETA CNS metastasis program 8 July 2020
ALETA CNS metastasis program 8 July 2020ALETA CNS metastasis program 8 July 2020
ALETA CNS metastasis program 8 July 2020Paul D. Rennert
 
Sima Lev The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBC
Sima Lev  The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBCSima Lev  The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBC
Sima Lev The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBCSima Lev
 
The CDKN2A
The CDKN2AThe CDKN2A
The CDKN2ARpat8312
 

What's hot (20)

Car T-cell therapy
Car T-cell therapyCar T-cell therapy
Car T-cell therapy
 
3768 rennert
3768 rennert3768 rennert
3768 rennert
 
Medullary thyroid carcinoma
Medullary thyroid carcinomaMedullary thyroid carcinoma
Medullary thyroid carcinoma
 
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...
JTM-Functional characterization of human Cd33+ And Cd11b+ myeloid-derived sup...
 
Cancer Res-2014-Chakraborty-3489-500
Cancer Res-2014-Chakraborty-3489-500Cancer Res-2014-Chakraborty-3489-500
Cancer Res-2014-Chakraborty-3489-500
 
Poster021808
Poster021808Poster021808
Poster021808
 
Aleta Bio CAR-TCR Summit 11 Sept 2019
Aleta Bio CAR-TCR Summit 11 Sept 2019Aleta Bio CAR-TCR Summit 11 Sept 2019
Aleta Bio CAR-TCR Summit 11 Sept 2019
 
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...
Animal Lectin Galectin-8 by Hadas Shatz-Azoulay, Yaron Vinik, Roi Isaac, Ulri...
 
Car t cell tumor board
Car  t cell tumor boardCar  t cell tumor board
Car t cell tumor board
 
ALETA CNS metastasis program 8 July 2020
ALETA CNS metastasis program 8 July 2020ALETA CNS metastasis program 8 July 2020
ALETA CNS metastasis program 8 July 2020
 
pc DNA3
pc DNA3pc DNA3
pc DNA3
 
Gpcr genetic variation
Gpcr genetic variationGpcr genetic variation
Gpcr genetic variation
 
JBC2
JBC2JBC2
JBC2
 
Sima Lev The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBC
Sima Lev  The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBCSima Lev  The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBC
Sima Lev The AXL PYK2 PKC axis as a nexus of stemness circuits in TNBC
 
Trecs
TrecsTrecs
Trecs
 
The CDKN2A
The CDKN2AThe CDKN2A
The CDKN2A
 
onc2013588a
onc2013588aonc2013588a
onc2013588a
 
Lepow Day Poster
Lepow Day PosterLepow Day Poster
Lepow Day Poster
 
journal.pbio.2000998
journal.pbio.2000998journal.pbio.2000998
journal.pbio.2000998
 
StntShwcs2015Abstract
StntShwcs2015AbstractStntShwcs2015Abstract
StntShwcs2015Abstract
 

Viewers also liked

Cancer hospital in india
Cancer hospital in indiaCancer hospital in india
Cancer hospital in indiaHCG Oncology
 
AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...
AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...
AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...Architects for Health
 
Cancer hospital litrature casestudy
Cancer hospital litrature casestudyCancer hospital litrature casestudy
Cancer hospital litrature casestudyAr. Sahid Akhtar
 
Energy Efficient Design Education Through Architectural Design Studio Projects
Energy Efficient Design Education Through Architectural Design Studio ProjectsEnergy Efficient Design Education Through Architectural Design Studio Projects
Energy Efficient Design Education Through Architectural Design Studio ProjectsKhaled Ali
 

Viewers also liked (6)

Cancer hospital in india
Cancer hospital in indiaCancer hospital in india
Cancer hospital in india
 
AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...
AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...
AfH Design Awards Winner 2015 Best Idea Award - New Cancer Centre at Guy’s Ho...
 
Introduction to oncology
Introduction to oncologyIntroduction to oncology
Introduction to oncology
 
Cancer hospital litrature casestudy
Cancer hospital litrature casestudyCancer hospital litrature casestudy
Cancer hospital litrature casestudy
 
Energy Efficient Design Education Through Architectural Design Studio Projects
Energy Efficient Design Education Through Architectural Design Studio ProjectsEnergy Efficient Design Education Through Architectural Design Studio Projects
Energy Efficient Design Education Through Architectural Design Studio Projects
 
Principles of oncology
Principles of oncology   Principles of oncology
Principles of oncology
 

Similar to AACR poster VP_2014

Sequencing the Human TCRβ Repertoire on the Ion S5TM
Sequencing the Human TCRβ Repertoire on the Ion S5TMSequencing the Human TCRβ Repertoire on the Ion S5TM
Sequencing the Human TCRβ Repertoire on the Ion S5TMThermo Fisher Scientific
 
Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...
Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...
Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...S KANDAKUMAR CUDDALORE
 
CAR T-cell Therapy_A New Era in Cancer Immunotherapy
CAR T-cell Therapy_A New Era in Cancer ImmunotherapyCAR T-cell Therapy_A New Era in Cancer Immunotherapy
CAR T-cell Therapy_A New Era in Cancer ImmunotherapyTuhin Samanta
 
Sezary syndrome part 2
Sezary syndrome   part 2Sezary syndrome   part 2
Sezary syndrome part 2Bob Marcus
 
Ph D Swati Dhar
Ph D Swati DharPh D Swati Dhar
Ph D Swati DharSwati Dhar
 
20614 ftp
20614 ftp20614 ftp
20614 ftpsofiles
 
Multiplex energy metabolism
Multiplex energy metabolismMultiplex energy metabolism
Multiplex energy metabolismMatt Sanderson
 
Car t-cell-based-immunotherapy-basic-principles
Car t-cell-based-immunotherapy-basic-principlesCar t-cell-based-immunotherapy-basic-principles
Car t-cell-based-immunotherapy-basic-principlesCreative Biolabs
 
Recent advances in paediatric oncology
Recent advances in paediatric oncologyRecent advances in paediatric oncology
Recent advances in paediatric oncologymadurai
 
Immunotherapy: Focus on CAR-T cells
Immunotherapy: Focus on CAR-T cellsImmunotherapy: Focus on CAR-T cells
Immunotherapy: Focus on CAR-T cellsZeena Nackerdien
 
CAR-T cell immunotherapy for solid tumors
CAR-T cell immunotherapy for solid tumorsCAR-T cell immunotherapy for solid tumors
CAR-T cell immunotherapy for solid tumorsSushma Ahirwar
 
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...JohnJulie1
 
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...daranisaha
 
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...semualkaira
 

Similar to AACR poster VP_2014 (20)

Sequencing the Human TCRβ Repertoire on the Ion S5TM
Sequencing the Human TCRβ Repertoire on the Ion S5TMSequencing the Human TCRβ Repertoire on the Ion S5TM
Sequencing the Human TCRβ Repertoire on the Ion S5TM
 
Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...
Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...
Tumor Microenvironment-Mediated Construction and Deconstruction of Extracellu...
 
CAR T-cell Therapy_A New Era in Cancer Immunotherapy
CAR T-cell Therapy_A New Era in Cancer ImmunotherapyCAR T-cell Therapy_A New Era in Cancer Immunotherapy
CAR T-cell Therapy_A New Era in Cancer Immunotherapy
 
EACR-P0ster-17
EACR-P0ster-17EACR-P0ster-17
EACR-P0ster-17
 
CAR- T Cell
CAR- T CellCAR- T Cell
CAR- T Cell
 
Sezary syndrome part 2
Sezary syndrome   part 2Sezary syndrome   part 2
Sezary syndrome part 2
 
JCI9908476
JCI9908476JCI9908476
JCI9908476
 
Ph D Swati Dhar
Ph D Swati DharPh D Swati Dhar
Ph D Swati Dhar
 
20614 ftp
20614 ftp20614 ftp
20614 ftp
 
Multiplex energy metabolism
Multiplex energy metabolismMultiplex energy metabolism
Multiplex energy metabolism
 
Car t-cell-based-immunotherapy-basic-principles
Car t-cell-based-immunotherapy-basic-principlesCar t-cell-based-immunotherapy-basic-principles
Car t-cell-based-immunotherapy-basic-principles
 
Recent advances in paediatric oncology
Recent advances in paediatric oncologyRecent advances in paediatric oncology
Recent advances in paediatric oncology
 
PlOSone paper
PlOSone paperPlOSone paper
PlOSone paper
 
Immunotherapy: Focus on CAR-T cells
Immunotherapy: Focus on CAR-T cellsImmunotherapy: Focus on CAR-T cells
Immunotherapy: Focus on CAR-T cells
 
publi Toulouse
publi Toulousepubli Toulouse
publi Toulouse
 
CAR-T cell immunotherapy for solid tumors
CAR-T cell immunotherapy for solid tumorsCAR-T cell immunotherapy for solid tumors
CAR-T cell immunotherapy for solid tumors
 
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
 
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
 
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
Non-Synonymous Variants of Methylenetetrahydrofolate Reductase C677T Gene Pol...
 
Car tcell therapy
Car tcell therapyCar tcell therapy
Car tcell therapy
 

AACR poster VP_2014

  • 1. 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 ABSTRACT 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 constructs. 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 (Fig.10, 11). 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). EXPERIMENTAL APPROACH CONCLUSIONS 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, respectively. 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.