521 multiplex genome editing of TCR°CD52 genes as a platform for “off the shelf� adoptive t cell immunotherapies

521 Multiplex Genome Editing of TCR°/CD52 Genes as a Platform for “Off the Shelfâ€? Adoptive T Cell Immunotherapies Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Soci[.]

Molecular Therapy Volume 22, Supplement 1, May 2014

CANCER-IMMUNOTHERAPY I

IL-12 and Decorin (hDCN) (RdB/IL12/hDCN) as suitable therapeutic

adjuvant to overcome immune suppression by down-regulation of

TGF-β through hDCN Intratumoral administration of RdB/IL12/

hDCN promoted enhanced antitumor effects compared with an

oncolytic adenovirus expressing IL-12 or hDCN alone (RdB/IL12

or RdB/hDCN, respectively) We showed markedly elevated levels

of interferon-γ (IFN-γ), tumor necrosis factor-a (TNF-a) and IFN-γ

secreting cells in RdB/IL12/hDCN-treated tumors Moreover, Treg

in draining lymph nodes (DLNs) dramatically decreased in mice

treated with RdB/IL12/hDCN, suggesting an association with the

down-regulation of TGF-β in tumor treated with RdB/IL12/hDCN

Consistent with these data, tumor tissue injected with RdB/IL12/

hDCN showed increased infi ltration of CD4+ T and CD8+ T cells

infi ltration Furthermore, hDCN enhanced viral distribution and tumor

penetration, leading to improved virus-mediated cancer gene therapy

by overcoming the extracellular matrix barrier within tumor masses

Together, these results provide new insight into the ability of hDCN

to eliminate immune suppression and demonstrate that adenovirus

co-expressing IL-12 and hDCN is a promising therapeutic tool for

cancer treatment than adenovirus expressing IL-12 alone

519 In Vivo Boosting of CAR-Redirected Virus

Specifi c CTLs by Artifi cial Antigen Presenting

Cells

Ignazio Caruana,1 Gerrit Weber,1 Barbara Savoldo,1 Gianpietro

Dotti.1

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

Houston, TX.

Virus-specifi c cytotoxic T lymphocytes (CTLs) redirected with a

GD2-specifi c chimeric antigen receptor (GD2.CAR) induces objective

clinical responses in patients with relapsed/refractory neuroblastoma

(NB) [Pule et al Nat Med 2006] However the in vivo persistence of

these cells remains limited Using Cytomegalovirus-(CMV)-specifi c

CTLs as a model, we sought to boost GD2.CAR-CMV-CTLs in

vivo by using artifi cial antigen presenting cells (aAPC) based on the

K562 cell line, encoding the pp65-CMV antigen and CD40L/OX40L

costimulatory molecules The rational of this vaccine design relies on

the in vivo pp65-antigen-cross presentation to professional dendritic

cells (DC) by apoptotic/necrotic bodies originating from irradiated

pp65-modifi ed/K562 CD40L induces DC maturation, while OX40L

is mediating CD4+ T-cell activation As shown in Table 1, we found

that gene modifi ed aAPC reproducibly reactivated CMV-CTLs from

note the co-expression of both OX40L and CD40L (aAPC/CD40L/

OX40L/pp65) showed the highest activation of CMV-CTLs in vivo

(*p=0.048)

(SFU/10 5 cells) In vitro IFNγ In vivo IFNγ

aAPC/CD40L/OX40L/pp65 477±91 101±21*

Using the same NSG model, we also found that when CMV-CTLs

were genetically modifi ed to express the GD2.CAR, vaccinations in

vivo with aAPC/CD40L/OX40L/pp65 also increases the frequency

of IFNg+ T cells responding to GD2 (71±24 IFNg+SFU/105 cells) and

pp65 antigen (85±16), compared to mice vaccinated with the control

aAPC/CD40L/OX40L (23±8, p=0.048, and 41±11, p=0.035, for GD2

and pp65, respectively) This boosting ultimately promoted better

aAPC/CD40L/OX40L/pp65 had better tumor reduction compared

to mice boosted with control aAPC/CD40L/OX40L (p=0.002) Forty-eight percent were tumor free, while only 8% of mice boosted with control aAPC/CD40L/OX40L cleared the tumor (p≤0.037) Although K562-derived vaccines have been extensively used in clinical trials, to further increase the safety profi le of this approach

we also incorporated the inducible caspase9 safety switch We found that its activation effectively eliminates aAPC which had purposely been engrafted in NSG mice to mimic an unforeseen circumstance of aAPC accidentally inoculated without prior irradiation In conclusion,

we have identifi ed a strategy based on an “off the shelf” vaccine

that can be used to stimulate CAR-redirected virus-specifi c CTLs in

vivo ensuring their longer persistence, which results in an improved

antitumor activity

520 A PD1-CD28 “Switch Receptor” Is Able to Augment Mesothelin-Directed Chimeric Antigen Receptor T Cell Therapy in a Resistant In Vivo Model of Human Tumor

Edmund K Moon,1 Raghuveer Ranganathan,1 Jing Sun,1 Xiaojun Liu,2 Carl H June,2 Steven M Albelda,1 Yangbing Zhao.2

1 Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 2 Department of Pathology and Laboratory Medicine, Perelman School of Medicine

at the University of Pennsylvania, Philadelphia, PA.

Adoptive Cell therapy (ACT) using genetically-modifi ed T cells has shown great promise in diseases such as melanoma and hematologic malignancies Our group has recently applied this paradigm-changing technology to malignant pleural mesothelioma (MPM) by creating

T cells that express a chimeric antigen receptor (CAR) that targets the highly expressed tumor antigen, mesothelin (mesoCAR T cells) Previously published work showed the mesoCAR T cells could reduce the size of human MPM cell line (M108) fl ank tumors in mice Based on this data, a Phase I trial in MPM patients using mesoCAR

T cells has begun One limitation of ACT using tumor-infi ltrating lymphocytes (TILs) using their native T cell receptors has been functional inhibition This important phenomenon has not yet been fully explored in CAR signaling Employing a novel resistant in-vivo model of tumor-induced mesoCAR T cell hypofunction, we have demonstrated upregulation of PD1 on tumor-infi ltrating mesoCAR T cells and of PDL1 on tumors infi ltrated by those cells To block this inhibition, we have created a PD1-CD28 “switch receptor” in which the extracellular domain of PD1 was linked to the cytoplasmic domain

of CD28 and expressed along with a mesoCAR receptor Genetically modifying mesoCAR T cells with a PD1-CD28 switch receptor led

to increased pro-infl ammatory cytokine secretion, infi ltrating T cell proliferation, tumor control, and resistance to tumor-induced hypofunction

521 Multiplex Genome Editing of TCRα/CD52 Genes as a Platform for “Off the Shelf” Adoptive T-Cell Immunotherapies

Laurent Poirot,1 Brian Philip,2 Cécile Schiffer-Mannioui,1 Sophie Derniame,1 Agnes Gouble,1 Sylvain Arnould,1 Julianne Smith,1 Martin Pule,2 Andrew Scharenberg.1

1 Cellectis Therapeutics, Paris, France; 2 Department of Hematology, UCL Cancer Institute - University College of London, London, United Kingdom.

Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a powerful approach to treating cancer However, a limitation of this approach

is that CAR T-cells must be generated on a bespoke basis To permit adoptive T cell therapy using allogeneic T cells, we have developed

a platform for the large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donor T-cells We overcome the

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

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CANCER-IMMUNOTHERAPY I

key barriers to the adoptive transfer of 3rd party CAR T-cells via

application of Transcription Activator-Like Effector Nucleases

(TALEN) gene editing technology – we eliminate the potential for

T-cells bearing alloreactive TCR’s to mediate GvHD by disrupting

the TCRalpha constant (TRAC) gene, and we exploit the requirement

for preparative lymphodepletion for engraftment of allogeneic CAR

T-cells through disruption of the CD52 gene Simultaneous editing

of the TRAC and CD52 genes results in TCR/CD52-defi cient T-cells,

which can be administered with concomitant alemtuzumab-mediated

lymphodepletion/ immunosuppression to both promote engraftment

and suppress rejection In order to achieve simultaneous inactivation

of the TRAC and CD52 genes in primary human T-cells, we developed

and optimized a method for electroporation of TALEN mRNAs into

T-cells that consistently achieves 40% double knock out rates

This TALEN electroporation step was incorporated following

transduction of a CAR gene cassette into a two-week, large scale,

process for GMP manufacturing of TCR/CD52-defi cient CAR T-cells

We provide proof of concept for the general applicability of this

approach by manufacturing a TCR/CD52-defi cient universal CD19

CAR T-cell (UCART19), and demonstrating that this product does not

mediate alloreactivity, and can be selectively engrafted in the presence

of alemtuzumab while maintaining potency equivalent to standard

CD19 CAR T-cells in an orthotopic CD19+ lymphoma model

We propose that this approach may be used as a platform for large

scale manufacturing of allogeneic CAR T-cell products directed

against arbitrary targets for administration as “off the shelf”

immunopharmaceuticals

522 Single Edited T Cells Redirected Towards

NY-ESO-1 Ensure Tumor Rejection Without

Inducing Xenogeneic GvHD

Sara Mastaglio,1 Pietro Genovese,1 Zulma Magnani,1 Barbara

Camisa,1 Elisa Landoni,1 Elena Provasi,1 Angelo Lombardo,1

Andreas Reik,2 Nicoletta Cieri,1 Maurilio Ponzoni,1 Fabio Ciceri,1

Claudio Bordignon,3 Michael C Holmes,2 Philip D Gregory,2

Luigi Naldini,1 Chiara Bonini.1

1 San Raffaele Scientifi c Institute, Milan, Italy; 2 Sangamo

BioSciences Inc, Richmond; 3 MolMed SpA, Milan, Italy.

T cell receptor (TCR) gene transfer has yielded promising clinical

results in cancer patients To permanently remove the expression of

the endogenous TCR and the risk of TCR chain mispairing, our group

developed a TCR gene editing approach, based on the combination

of somatic knockout of the endogenous TCR genes (by transient exposure to alpha and/or beta chain specifi c Zinc Finger Nucleases

- ZFNs) and introduction of tumor-specifi c TCR genes by lentiviral vectors (Provasi, Genovese et al Nat Med 2012) While the complete editing (CE) procedure requires multiple manipulation steps, ‘single TCR editing’ (SE), based on the disruption of a single endogenous TCR chain, followed by gene transfer of a tumor specifi c TCR, enables the generation of redirected T cells devoid of their natural TCR repertoire with a single round of T cell activation, improving the feasibility of its clinical translation By completely abrogating the endogenous repertoire, single TCR editing overcomes the risk of Graft versus Host Disease (GvHD) after allogeneic hematopoietic stem cell transplantation We exploited a HLA-A2 restricted TCR specifi c for NY-ESO-1, expressed by several solid tumors and hematological malignancies, to compare safety and effi cacy of unedited TCR transferred (TR), SE and CE T cells Genetically modifi ed T cells were tested in vitro for TCR expression, differentiation phenotype and killing ability, and in vivo in NSG mice engrafted with the U266 (HLA-A2+ and NY-ESO-1+) myeloma cell line Gene editing ensured the preservation of an early differentiated T cell phenotype, enriched

in central memory and stem memory T cells Upon lentiviral transfer

of the NY-ESO-1-specifi c TCR, we observed signifi cantly higher levels of the tumor-specifi c TCR expression, measured as dextramer binding capacity, in edited versus transferred T cells Edited T cells were more effi cient than transferred T cells in killing NY-ESO-1+ myeloma cell lines Importantly, SE and CE T cells displayed no reactivity against NY-ESO-1- targets In NSG mice, NY-ESO-1 redirected SE and CE T cells completely eliminated the NY-ESO1+ HLA-A2+, WT1- U266 myeloma line (19 out of 19 mice), that accumulated in the bone marrow in the presence of WT1-redirected

CE T cells (5 out of 5 mice) Importantly, mice infused with CE or SE

T cells did not develop xenogeneic GvHD In conclusion, the TCR single editing approach allows the rapid generation of large numbers

of tumor specifi c T cells with high anti-tumor effi cacy and, being devoid of off-target reactivity, unable to cause GvHD

523 Clinical Application of TCR Gene-Transduced Lymphocytes for Patients With Epitherial Cancer and Other Types of Malignancy:

In Vivo Persistence of Adoptively Transferred TCR Gene-Transduced Lymphocytes With Anti-Tumor Reactivity in Patients

Hiroaki Ikeda,1 Shinichi Kageyama,1 Naoko Imai,1 Yoshihiro Miyahara,1 Mikiya Ishihara,1 Naoyuki Katayama,2 Hirofumi Yoshioka,3 Daisuke Tomura,3 Ikuei Nukaya,3 Junichi Mineno,3 Kazuto Takesako,3 Hiroshi Shiku.3

1 Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Japan; 2 Hematology and Oncology, Mie University Graduate School of Medicine, Tsu, Japan; 3 Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Japan.

Engineering the antigen receptor gene in patients’ lymphocytes

is one promising strategy to create antigen-specifi c lymphocytes without senescent phenotypes The strategy provides an opportunity

to extend the application of adoptive T cell therapy for cancer patients However, this concept has not been well verifi ed in patients with epithelial cancer or hematological malignancy

We completed a phase I clinical trial of TCR gene therapy targeting MAGE-A4 to treat esophageal cancer patients without lympho-depleting pre-conditioning The trial was designed as a cell-dose escalation consisting of three cohorts, 2x108, 1x109 and 5x109 cells/ patient The treatment was tolerable with no adverse events associated with transferred cells In all patients, the transferred lymphocytes were detected in their peripheral blood in a dose-dependent manner during the fi rst 14 days The infused cells persisted more than 5 months after the transfer and sustained the reactivity to the antigen-expressing