396 BARF1 specific t cells for the adoptive immunotherapy of EBV positive nasopharyngeal carcinoma

396 BARF1 Specific T Cells for the Adoptive Immunotherapy of EBV Positive Nasopharyngeal Carcinoma Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell T[.]

Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

S152

CANCER - IMMUNOTHERAPY II

(59%±6%) than control T cells (11%±8%) even at the 5:1

effector-to-target ratio in 51Cr release assays Furthermore, in long-term

coculture assays, CAR.MCSP+ T cells effi ciently and consistently

eliminated several MCSP+ targets including melanoma (SEMNA and

CLB, residual tumors: 0.1%±0.06% and 0.1%±0.1, respectively),

mesothelioma (PH1 and MILL: 3.8%±3.1%; 3.2%±5%), head and

neck carcinoma (PCI-30, 0.5%±0.5%), and basal breast carcinoma

(UACC-812 and MDA-MB-231: 5.7% ±6.1%; 3.1% ±2.5%,

respectively) while having no effect on a MCSP– targets (38% ±10%)

As expected all tumor cells expanded in coculture with control T

cells The antitumor activity of CAR.MCSP+ T cells was paralleled

by release of Th1 cytokines, such as IL2 (from 6±10 pg/μL to 190±98

pg/μL) and IFNg (from 105±48 pg/μL to 3710±975 pg/μL) upon

coculture with different MCSP+ tumors Both CAR.MCSP transgenic

CD4+ and CD8+ cells proliferated in response to SEMNA tumor cells

as compared to control T cells, as assessed by CFSE dilution assays

We also generated a third generation CAR encoding CD28 and 4-1BB

endodomains However, since this construct did not show superior

function in vitro as compared to the CD28 endodomain we selected

the latter for the following in vivo experiments Using NSG mice

(n=10/group) either melanoma (SEMNA) or head and neck carcinoma

(PCI-30) or basal breast carcinoma (UACC-812) cells were engrafted

s.c Across all tumor models, mice treated with CAR.MCSP+ T

lymphocytes consistently showed tumor control (753mm3±350mm3;

18.5mm3±10mm3; 28mm3±13mm3) as compared to mice receiving

control T lymphocytes (7126mm3±2500mm3; 190mm3±75mm3; 166

mm3±64mm3) by days 40-50 post tumor engraftment In summary,

CAR.MCSP-redirected T cells can be used for the treatment of a

variety of solid tumors

395 Tumor Recurrences Share Immunogenic

Antigens across Both Tumor Types and Primary

Treatments Which Can Be Therapeutically

Targeted with VSV-cDNA Libraries

Nicolas Boisgerault,1 Jose Pulido,1 Timothy Kottke,1 Oliver

Donnelly,2 Esteban Celis,3 Jill Thompson,1 Rosa Diaz,1 Kevin

Harrington,4 Hardev Pandha,5 Peter Selby,2 Alan Melcher,2 Richard

Vile.1

1 Mayo Clinic, Rochester; 2 University of Leeds, Leeds, United

Kingdom; 3 H Lee Moffi tt Cancer Center, Tampa; 4 The Institute of

Cancer Research, London, United Kingdom; 5 University of Surrey,

Guildford, United Kingdom.

cDNA libraries expressed from the highly immunogenic Vesicular

Stomatitis Virus (VSV) platform can treat established tumors in

both prostate and melanoma models Viral stocks can be delivered

systemically, do not have to target tumor and generate potent T cell

responses against a variety of different tumor associated antigens

(TAA) which, cumulatively, lead to rejection of well established

subcutaneous tumors Following different suboptimal primary

treatments of tumor bearing mice in which initial tumor regression is

followed by aggressive recurrence, VSV-cDNA libraries constructed

from these recurrent tumors can, if administered early enough,

prevent tumor recurrence However, the VSV-cDNA libraries

which are active against primary tumors are ineffective against

recurrences, showing that the antigenic profi le of recurrent tumors

is substantially different from that of the primary tumors Using an

in vitro splenocyte-based assay, we cloned TAA from VSV-cDNA

libraries of recurrent melanoma and prostate tumors, generated by

different primary treatments, which fall into three clearly defi ned

classes In the fi rst, (treatment-specifi c TAA) proteins are lost/

gained in recurrences, relative to primary tumors, which are highly

specifi c to the nature of the primary treatment (such as loss of

the HSVtk protein from recurrences derived from initial HSVtk/

Ganciclovir treatment) The second class (tumor type-specifi c TAA)

contains proteins which are lost/gained in recurrences only of certain

histological types (such as a variant of CD44 which is acquired by relapsed TC2 prostate tumors but not B16 melanoma recurrences) Finally, the VSV-cDNA technology has identifi ed a class of proteins (recurrence-specific TAA) which are present in recurrences of different tumor types and independently of the nature of the primary treatment These recurrence-specifi c TAA are associated with control

of DNA replication and progression through the cell cycle, such as Topoisomerase IIa, cdc7 kinase and YB-1 Signifi cantly, we have shown that these proteins can serve as genuine recurrent-specifi c TAA, in that mice cured by recurrent-specifi c VSV-cDNA libraries contain T cells which recognize epitopes from these proteins Finally,

we have used these data to show that it is possible to treat recurrences both of different tumor types, and generated from different primary treatments, with a cocktail of recurrence-specifi c TAA expressed from VSV These data suggest that 1) early recurrences across tumor and treatment types share common molecular pathways by which they

evolve in vivo and 2) that by identifying these common,

recurrence-specifi c antigens it may be possible to devise both immunotherapeutic, and chemotherapeutic, strategies to treat recurrent tumors across a variety of histological types and primary treatments

396 BARF1-Specifi c T Cells for the Adoptive Immunotherapy of EBV-Positive Nasopharyngeal Carcinoma

Mamta Kalra,1 Minhtran C Ngo,1 Ulrike Gerdemann,1 Ann Leen,1

Chrystal Louis,1 Cliona M Rooney,1 Stephen Gottschalk.1

1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston.

Background: The majority of nasopharyngeal carcinomas (NPC)

are positive for Epstein-Barr virus (EBV), and the outcome for patients with recurrent/refractory disease remains poor Treatment

of NPC patients with EBV-specifi c cytotoxic T cells (EBV-CTLs) has been promising, resulting in clinical responses However, the antitumor activity of EBV-CTLs in patients with bulky disease was limited This lack of effi cacy may simply be quantitative in that current methods of CTL generation induce limited T-cell responses to the EBV antigens selectively expressed in NPC (EBNA1, LMP1, LMP2, BARF1) While T-cell responses to EBNA1, LMP1, and LMP2 have been studied in detail and clinical studies with EBNA1-, LMP1-, and LMP2-specifi c T cells are in progress, little is known about BARF1-specifi c T-cell responses The goal of the present study was to (i) characterize BARF1-specifi c T-cell responses in EBV-positive healthy donors and NPC patients, (ii) map immunogenic T-cell epitopes, and (iii) expand BARF1-specifi c T cells for the adoptive immunotherapy

of EBV-positive NPC Methods: Peripheral blood mononuclear cells

(PBMCs) from EBV-positive healthy donors (n=16) and NPC patients (n=7) were stimulated with a BARF1 overlapping peptide library consisting of 53 15-mer peptides After 8-10 days of culture, T-cell responses were determined by IFN- Elispot assay using either the entire library or 5 BARF1 peptide mini-pools, each containing 10 to

13 peptides Subsequently, in responding donors, BARF1-specifi c T-cell lines were generated by consecutive rounds of stimulation with BARF1-loaded autologous dendritic cells (DCs) for epitope mapping

and functional analysis Results: Twelve (68%) of 16 healthy donors

and 5 (71%) of 7 NPC patients showed T-cell reactivity towards BARF1 Of these BARF1-positive subjects, 9/12 healthy donors and 4/5 patients recognized distinct peptide pools The majority of CD8-restricted epitopes were mapped to the N- (pool 1) and C-termini (pool 4), while CD4-restricted epitopes were located in the middle of BARF1 (pools 2 and 3) Detailed screening of reactive pools in four healthy donors resulted in the identifi cation of 17 immunodominant peptides For one of the CD8-restricted epitopes we have so far mapped the minimal peptide length and determined that it is restricted

through HLA*B1562 Conclusion: T-cells specifi c for CD4- and

CD8-restricted BARF1 epitopes are present in PBMCs of 70% of

Molecular Therapy Volume 21, Supplement 1, May 2013

Copyright © The American Society of Gene & Cell Therapy S153

CANCER - IMMUNOTHERAPY II

EBV-seropositive healthy donors and EBV-positive NPC patients,

and can be expanded ex vivo Targeting BARF1 in addition to the

other EBV antigens expressed in NPC may improve the effi cacy of

adoptive immunotherapy approaches

397 IL-12 Increases Immune Responses

Induced by a Novel pDNA Prostate Cancer

Immunotherapy Approach in Non-Human Primates

Bernadette Ferraro,1 Amritha Balakrishnan,1 Jewell N Walters,1

Devin J Myles,1 Jian Yan,2 Amir S Khan,2 Niranjan Y Sardesai,2

David B Weiner.1

1 University of Pennsylvania School of Medicine, Philadelphia, PA;

2 Inovio Pharmaceuticals, Inc., Blue Bell, PA.

Prostate cancer (PCa) remains a significant public health

problem Current treatment modalities for PCa can be useful, but

may be accompanied by deleterious side effects and often do not

confer long-term control Accordingly, additional modalities, such

as immunotherapy, may represent an important approach for PCa

treatment Delivery of DNA vaccines with electroporation (EP) has

shown promising results for prophylactic and therapeutic targets in

a variety of species, and recently in humans (Bagarazzi et al., Sci

Transl Med 2012 Oct 10;4(155)) Application of this technology

for PCa immunotherapy strategies has been limited to single antigen

and epitope targets We sought to test the hypothesis that a broader

collection of antigens would improve the breadth and effectiveness

of a PCa immune therapy approach We concurrently explored if the

molecular adjuvant IL-12 would increase the magnitude of these

responses To this end, we developed highly optimized DNA vectors

encoding prostate-specifi c antigen (PSA), and prostate-specifi c

membrane antigen (PSMA), and six-transmembrane epithelial antigen

of the prostate (STEAP) as a three-pronged approach to immune

therapy of PCa Vaccine immunogenicity was evaluated in rhesus

macaques (NHPs) NHPs (n=4/group) received 4 immunizations of

DNA alone (DNA) or DNA with IL-12 (DNA+12) delivered with

EP at weeks 0, 6, 12, and 24 Immune responses were evaluated at

weeks 0, 14, 20 and 26 DNA alone elicited modest IFN production

by ELISpot at weeks 14 (153 SFU), 20 (55 SFU) and 26 (79 SFU)

IL-12 signifi cantly increased IFN production at weeks 14 (612

SFU), 20 (293 SFU) and 26 (394 SFU) Further characterization of

immune responses by fl ow cytometry demonstrated that at week 26

DNA+12 increased the level antigen-specifi c CD8+ T cells (0.43%)

compared to DNA (0.19%) Importantly, DNA+12 promoted CD8+

T cells with the potential for cytotoxic and effector function, as

evidenced by antigen-specifi c CD107a (0.34%) and T-bet (0.28%),

respectively There was also a strong humoral response as determined

by PSA- and PSMA-specifi c seroconversion These data support

further study of these immunogens as part of a novel approach to

immune therapy of PCa

398 Transgenic Expression of a Novel

Immunosuppressive Signal Converter on T Cells

Norihiro Watanabe,1 Usanarat Anurathapan,1 Malcolm K Brenner,1

Helen E Heslop,1 Ann M Leen,1 Cliona M Rooney,1 Juan F

Vera.1

1 Center for Cell and Gene Therapy, Baylor College of Medicine,

Houston, TX.

Chimeric antigen receptor (CAR)-transduced T cells are promising

tools for the treatment of cancers To extend this therapeutic modality

to prostate cancer we have constructed a 2nd generation CAR targeting

the tumor antigen PSCA (2G.CAR-PSCA), which provides cells with

the ability to kill PSCA+ prostate tumor cells (51.7±1.2% specifi c lysis

of Du145 cells at 20:1 E:T) However, many tumors including prostate

cancer secreting TGF, which inhibits in vivo T cell proliferation,

activation and function Our group has previously demonstrated that

adoptively-transferred T cells can be protected from the inhibitory effects of TGF through the transgenic expression of a truncated, dominant-negative receptor (DNRII), which blocks transmission of TGF signal We have now extended this strategy by converting the inhibitory signal into an activation stimulus for T cells We generated

a chimeric cytokine receptor expressing the exodomain of TGFRII linked to the endodomain of toll-like receptor (TLR) 4 and GFP (RIID4) We transduced primary T cells with RIID4 and obtained 69.3±6.0% transduction which was stable for >60 days of culture

To address whether transgenic expression of RIID4 protected against TGF, we modifi ed 2G.CAR-PSCA T cells to co-express either the dominant negative DNRII or RIID4 receptors These T cells were then stimulated weekly with PSCA+ tumor cells (K562-PSCA) with

or without exogenous TGF1 (5ng/mL) In the absence of TGF1, 2G.CAR-PSCA, 2G.CAR-PSCA(DNRII) or 2G.CAR-PSCA(RIID4)

T cells proliferated at similar levels for 30 days (6.4x102, 2.5x103, 5.9x103 fold, respectively) But in the presence of TGF1, 2G.CAR-PSCA T cells did not expand, and cultures failed within 2 weeks

In contrast, transgenic expression of DNRII or RIID4 protected the cells from the inhibitory impact of this cytokine (7.4 and 21 fold at

2 weeks of culture, respectively) To determine whether there were long term differences between DNRII- and RIID4-modifi ed cells we monitored cell expansion and found that only RIID4-modifi ed T cells were able to expand for >60 days in the presence of TGF1 (3.0x105

fold) while DNRII cells began to contract after 30 days in culture (0.72 fold) Administration of TGF1 also selected 2G.CAR-PSCA(RIID4)

T cells, leading to an enrichment in this transgenic cell population over time (from 63.6% to 93.3%) This modifi cation appears to be safe since the administration of TGF1 alone was insuffi cient to drive transgenic T cell proliferation (0.04 fold) and the withdrawal

of antigenic stimulation resulted in T cell contraction (0.02 fold) Finally, to address whether this modifi cation could improve the anti-tumor activity of CAR-T cells, we co-cultured 1x106 fi refl y-luciferase-Du145 cells, which express PSCA and produce TGF1,

2G.CAR-PSCA(RIID4) T cells After 6 days we observed superior control of tumor growth by RIID4-expressing T cells compared with DNRII or CAR alone conditions (Total Flux; 9±0.1x109, 10±1x109, 20±1x109

p/s, respectively) Thus, suggest that RIID4 not only protects cells from the inhibitory effects TGF but converts this cytokine signal into one which is stimulant

399 Hypomethylating Agent 5-aza Cytidine Exposure To Improve AP1903 Treatment for Apoptosis Induction of iCasp9/∆CD19 Gene Modifi ed T Cells

Elodie Bole-Richard,1 Jean-Marie Certoux,1 Idir Idirene,1 Laurent Jossot,1 Eric Deconinck,1,2 Fabrice Larosa,2 Etienne Daguindau,2

Christophe Borg,1,2 Pierre Tiberghien,1 Christophe Ferrand,1

Marina Deschamps.1

1 UMR1098-EFSBFC-SFR FED 4234, Besancon, France;

2 Hematology, CHU Jean Minjoz, Besancon, France.

Haematopoietic transplantation may result in serious complications, notably graft versus host disease (GvHD) T-lymphocyte depletion

of the bone marrow graft is able to prevent GvHD, while increasing the risk of rejection and reducing the anti-leukemic effect An alternative issue is to use a suicide gene system that allows in-vivo conditional T cell depletion Based on our experience reporting drawbacks of the suicide gene HSV-tk expressing donor T cells, we investigate the use of human derived inducible caspase 9 (iCasp9/ iC9) and truncated CD19 (CD19) expressing T-cell This allows rapid apoptosis after exposure to a Chemical Inducer of Dimerization (CID; AP1903, Bellicum Pharmaceuticals) As reported in-vivo by others, we found a drug responsiveness of some iC9/CD19+ gene modifi ed cells (GMC) after CID in-vitro exposure Such fi ndings