209 TCR gene editing in a single step of t cell activation to redirect t cell specificity and prevent GvHD

209 TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene[.]

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

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in vitro Furthermore, to assess the anti-tumor effect of anti-FSHR

T-cells, we established human OvCa xenografts in NSG mice by

subcutaneous flank injection of CaOV3 cancer cell line followed

by two intravenous redirected T-cell administrations As shown by

caliper measurements, FSHR-redirected T-cells elicited anti-tumor

potential with suppressed outgrowth of established tumor Tumor

growth was statistically reduced in comparison to control, GFP

engineered T-cell treated group or PBS treated animals These data

suggest that systemic delivery of anti-FSHR T-cells to mice bearing

FSHR-expressing tumors may result in tumor trafficking, activation,

and lysis at location of tumor

In summary, we have successfully exploited for the first time a

novel therapeutic antigen, the FSHR protein, expressed on tumor

cell surface, to deliver T-cell based immune-therapy of cancer Given

its highly restricted expression in cancer, cancer-associated vessels

and gonadal tissues, the rationale for FSHR targeting is strong and

supports further exploration of FSHR-targeted immune therapy

approaches for cancer patients

207 Optimal Activation of Both CD28 and 4-1BB

in CAR-Targeted T Cells Results in Enhanced

Tumor Eradication

Zeguo Zhao,1 Sjoukje J.C van der Stegen,1 Maud Condomines,1

Fabiana Perna,1 Gertrude Gunset,1 Jason Plotkin,1 Michel

Sadelain.1,2

1 Center for Cell Engineering, Memorial Sloan Kettering Cancer

Center, New York, NY; 2 Immunology Program, Sloan Kettering

Institute, New York, NY.

Chimeric Antigen Receptors (CARs) are a novel drug class,

consisting of engineered receptors for antigen that redirect and

reprogram T-cell function We have shown in both xenogeneic tumor

models as well as in leukemia patients, that CD19-targeted T-cells

are capable of inducing complete remission of relapsed or refractory

B cell malignancies The inclusion of appropriate co-stimulation is

essential for the potency, quality and durability of the CAR T cell

response Here we show that the co-stimulatory domain incorporated

within the CAR dictates the kinetics of in vivo anti-tumor response

and T-cell accumulation Additionally, expression of co-stimulatory

ligands complementary to the second-generation CAR provides an

optimal balance of cytolytic activity and T-cell accumulation resulting

in superior anti-tumor function The combination of 19-28z CAR with

4-1BBL was associated with activation of the endogenous Interferon-β

(IFNβ) pathway in an IRF7-dependent manner This enabled very

small T-cell doses to eradicate systemic established leukemia in a

xenogeneic model The unexpected role of the IRF7/IFNβ pathway

provides insight into the synergy behind co-stimulatory molecules in

T cell activation and a novel mechanism through which engineered

T cells can increase tumor control

208 Single-Cell Gene Expression Profiles

of Genetically Modified T Cells for Adoptive

Immunotherapy

Sonny O Ang,1 Laurence J.N Cooper.1

1 Pediatrics, University of Texas MD Anderson Cancer Center,

Houston, TX.

System-level characterization of cell states requires detailed

portrayal of cellular components such as gene transcripts or

proteins Here, we exploit a robust single-cell platform technology

to characterize T cells expanded ex vivo intended for human

application Coupled with advanced custom analytics, we identify

“drivers” (relatively few, but vital) genes which orchestrate T-cell

activation and clonal expansion programming, versus “passengers”

(relatively many, less critical) or resultant/downstream transcripts

Detailed analyses reveal that there is considerable variability between individual activated T cells, implying that cell state may undergo multiple stepwise transitions in a stochastic manner Compared to interrogation of bulk T-cell populations (e.g., analyzed using RNA-Seq, microarrays, and Northern blotting), where heterogeneity in expression signals is attenuated by temporal and cell-cycle averaging, the genetic heterogeneity between cells in isogenic populations may

be a reflection of fundamental phenomena such as transcriptional bursting Our model describes cell state oscillation in tandem with transcriptional repressive (closed chromatin) and permissive conformations (open chromatin), versus simpler probabilistic models of transcription T-cell variability can significantly impact upon individual cell behavior within seemly isogenic populations, and may be essential in molding survival and cytotoxicity within certain contexts, such as the rapidly changing and stressful tumor microenvironments We find that lactate dehydrogenase A (LDHA)

is a leading indicator for the metabolic adaptation during T-cell activation and together with other energetics related genes such pyruvate kinase isoform 2 (PKM2), can be harnessed to adjust our culture conditions during ex vivo expansion These cellular fingerprints suggest useful ways for tuning the balance between oxidative and non-oxidative glucose metabolism to enhance the killing and thus therapeutic potential of our T cells Our framework

to harness catalogs of gene expression data into clinically-applicable insights can be adapted for meaningful characterization of other therapeutically-relevant source populations such as hematopoietic stem cells and reprogrammed NK cells This is being reduced to practice by understanding the heterogeneity within and between genetically modified T-cell products generated for clinical use

209 TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD

Pietro Genovese,1 Sara Mastaglio,2 Zulma Magnani,2 Elisa Landoni,2,3 Barbara Camisa,2 Giulia Schiroli,1,3 Elena Provasi,1 Angelo Lombardo,1,3 Andreas Reik,4 Nicoletta Cieri,1 Maurilio Ponzoni,2 Fabio Ciceri,2 Claudio Bordignon,2 Michael C Holmes,4 Philip D Gregory,4 Luigi Naldini,1,3 Chiara Bonini.2

1 TIGET, Milan, Italy; 2 San Raffaele Scientific Institute, Milan, Italy; 3 San Raffaele University, Milan, Italy; 4 Sangamo BioSciences, Richmond.

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer adoptive immunotherapy Yet, TCR gene transfer into mature T cells results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities Thus, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction

of tumor-specific TCR genes (Provasi et al, Nat Med 2012) While successful, the complete editing requires multiple manipulation steps involving repeated cell activation cycles and transductions To reduce the duration and complexity of cell product generation, we recently developed a ‘single TCR editing’ (SE) procedure, based on the disruption of the endogenous TCR α chain only followed by the transfer of the tumor specific TCR genes This SE method generates redirected T cells fully devoid of their natural TCR repertoire in a single round of cell activation We validated the SE protocol exploiting

an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by

a considerable proportion of high risk multiple myeloma) The SE strategy allowed rapid production of high numbers of tumor specific

T cells enriched for an early differentiation phenotype When fucntionaly tested (co-culture, γ-IFN and 51Cr release) against the

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CanCer – Immunotherapy, CanCer VaCCInes I

U266 myeloma cell line (NY-ESO-1+HLA-A2+), all NY-ESO-1

redirected T cells showed a strikingly high killing activity However,

when we assess the alloreactive potential of the different redirected T

cells in mixed lymphocyte reactions, we observed that the allogeneic

lysis by SE T cells was markedly lower (p=0.05) than that of

conventional TCR transfer cells (TR) These results were validated

in NSG mice, where the genetically modified T cells were infused

after the engraftment of the U266 myeloma All animals treated

with tumor specific T cells were completely myeloma-free at the

time of sacrifice, demonstrating the powerful anti-tumor potential

of the NY-ESO-1 redirected T cells However, the overall survival

of mice treated with TR vs SE cells was 26% vs 100% respectively

(p<0,001) corresponding with a significant difference in acute and

chronic GvHD occurrence (71% vs 0%, p<0,001) Consistently,

histopathological analysis of human T cell infiltration in the organs

revealed a significantly higher score in mice treated with TR cells

(p<0.001) The relative simplicity of the SE protocol enables rapid

generation of highly performing tumor specific T cells, fully devoid of

their endogenous TCR repertoire, and thus incapable of participating

in GvHD Such single TCR edited cells thus potentially represent a

further advance in adoptive immunotherapy for cancer

210 Complete Control of Tumor Growth In Vivo

By a Synthetic Consensus Multi-Antigen DNA

Immune Therapy for Prostate Cancer

Bernadette Ferraro,1 Jewell N Walters,2 Emma L Reuschel,2

Amrthia Balakrishnan,2 Matthew P Morrow,1 Jian Yan,1 Amir

S Khan,1 Niranjan Y Sardesai,1 Laurent M Humeau,1 David B

Weiner.2

1 Inovio Pharmaceuticals, Inc, Plymouth Meeting, PA; 2 University

of Pennsylvania School of Medicine, Philadelphia, PA.

Prostate cancer (PCa) is one of the leading causes of cancer

deaths among men with limited treatment options Accordingly,

new approaches, such as immunotherapy, may represent important

approaches for PCa treatment Electroporation (EP) delivered DNA

vaccines has recently shown promising results for therapeutic

treatment of HPV early disease in humans Application of this

technology for PCa immunotherapy strategies has been limited

to single antigen and epitope targets with limited success We

hypothesized that a broader collection of antigens adjuvanted by

plasmid encoded IL-12 would bypass immune tolerance and improve

the breadth and effectiveness of a PCa immunotherapy approach We

tested this hypothesis in NHP for immune tolerance effects as well as

in the highly relevant TRAMP-C2 challenge model We developed

highly optimized DNA vectors encoding consensus antigens for

important PCa targets prostate-specific antigen (SynCon PSA),

prostate-specific membrane antigen (SynCon PSMA), and human

six-transmembrane epithelial antigen of the prostate (STEAP) In mice

the vaccines demonstrated potent IFNγ production by ELISpot (2740

SFU) and robust immune responses in the CD4+ (0.53%) and CD8+

(3.0%) T cell compartments Further, sera from immunized mice

reacted in ELISA with relevant targets and specifically stained LNCaP

cells, a human PCa cell line, as well as human PCa tumor sections,

supporting that the vaccine antigens induced relevant antibody

responses Vaccination of Rhesus Macaques, which share greater

than 98% identity with humans, showed robust anti-PSA, PSMA and

STEAP IFNγ production (612 SFU), potential for cytotoxic T cell

function, and antigen specific seroconversion supporting the ability

of these constructs to break tolerance The therapeutic potential of

PSMA, STEAP, and the combination of PSMA and STEAP, alone

or with the molecular adjuvant IL-12, was evaluated in mice in the

TRAMP-C2 tumor model Alone, PSMA, STEAP or PSMA+STEAP

demonstrated prolonged survival and a modest impact on tumor

growth However, the combination of synthetic vaccine antigens

with IL-12 resulted in 100% efficacy in treatment and clearance of tumors resulting in 100% survival These data support further study

of this novel immune therapy of PCa

211 CD2, the First Identified T cell Co-Stimulator, Demonstrates More Effective Chimeric Antigen Receptor Activity Over CD28 and 4-1BB

Avery D Posey, Jr.,1 Carl H June.1

1 Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA.

CD2, first identified as T11 sheep erythrocyte receptor protein, was originally classified as the trigger for an alternative T cell activation pathway and later as a costimulatory molecule that synergized with CD3 activation Co-stimulation of T cells with CD2 augments CD3-mediated signaling cascades, IL-2 production, and proliferation The field dedicated to the development of novel second- and third-generation chimeric antigen receptors (CARs) has focused on the inclusion endodomains from CD28 superfamily and TNFRSF members as costimulation, but the use of CD2/SLAM family of costimulatory molecules has not been explored Here, we report the development of an anti-mesothelin (SS1) chimeric antigen receptor co-stimulated with the endodomain of CD2 juxtaposed to the CD3z activation domain (SS1CD2z) Human T cells modified with SS1CD2z demonstrate comparable cytotoxicity of tumor cell lines in vitro as SS1z, SS1BBz, and SS128z SS1CD2z T cells proliferation similar to SS128z and better than SS1BBz in vitro SS1CD2z T cells exhibit a calcium flux in response to antigen that is half the amplitude

of the response seen for SS128z, while no calcium flux is observed for SS1BBz Similarly, all three CARs exhibit differential signaling profiles, consistent with the signaling of CD28 superfamily, TNFRSF, and CD2/SLAM superfamily pathways Luminex analysis revealed that IL-2 production is superior among SS128z and SS1CD2z, while SS1BBz T cells produce less IFNγ production is comparable between all CAR groups Importantly, SS1CD2z cells produce minimal quantities TNFα similar to SS1BBz cells; contrary to SS128z cells, which produce large quantities of the neurotoxic cytokine SS1CD2z

T cells exhibited a fast and durable anti-tumor response against a subcutaneous mesothelioma xenograft model, while both SS128z and SS1BBz T cells lagged in terms of response rate These results suggest that CAR co-stimulation via CD2 can produce potent anti-tumor activity, T cell proliferation and favorable cytokine profiles Exploration of other CD2/SLAM superfamily members for CAR co-stimulation could be beneficial to cancer patients and the cellular therapy community

212 Of States and Fates: Predicting T-Cell Immunity By the Numbers

Colleen M O’Connor,1 Sonny O Ang,1 Jianrong Dong,2 Sourindra

N Maiti,1 Luay Nakhleh,2 Laurence J.N Cooper.1

1 Pediatrics, MD Anderson Cancer Center, Houston, TX; 2 Computer Science, Rice University, Houston, TX.

The immune system is a complex network of checks and balances

in constant flux Quantitative characterization of the system dynamics, sampled via constituent components such as T cells, combined with mathematical modeling enabled us to obtain “statistical pattern recognition” of immune states and transitions over time Here we show a statistical framework to characterize immune states for adoptive immunotherapy using serial infusions of activated polyclonal

T cells into companion canines diagnosed with B-cell non-Hodgkin Lymphoma (NHL) post CHOP chemotherapy regimen as a model for human disease We applied multiplexed gene profiling techniques to assess changes in gene expression data from 10 companion canine patient clinical samples and gene regulatory networks (GRN)