86 a highly compact epitope based marker suicide gene for safer and easier adoptive t cell gene therapy

86 A Highly Compact Epitope Based Marker Suicide Gene for Safer and Easier Adoptive T Cell Gene Therapy Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & C[.]

Molecular Therapy Volume 20, Supplement 1, May 2012

CANCER-TARGETED GENE & CELL THERAPY

for tumor-associated antigens (TAAs) preferentially expressed on

melanoma cells Recognition of cell-surface TAAs independent of

major histocompatibility complex can be achieved by introducing a

TAA-specifi c chimeric antigen receptor (CAR) on T cells using gene

therapy This approach is being used in clinical trials to adoptively

transfer CD19-specifi c CAR+ T cells in patients with B-lineage

malignancies To generate T-cell therapy for melanoma we targeted

a TAA derived from human endogenous retroviruse (HERV), whose

genome stably integrated into humans millions of years ago During

oncogenesis, biologically active variants of HERV, such as the

envelope (env) protein of HERV-K, are expressed on the surface of

melanoma, but not normal cells To target HERV-K, T cells were

engineered to express a CAR specifi c for this env protein, by replacing

the antigen-binding exodomain of CD19-specifi c CAR with the scFv

sequence of a HERV-K env specifi c monoclonal antibody This new

CAR was cloned as a transposon into our Sleeping Beauty (SB) system

which we have adapted for human application DNA plasmids coding

for the HERV-K env-specifi c CAR and SB transposase were

electro-transferred into primary human T cells and genetically modifi ed CAR+

T cells were selectively propagated on irradiated artifi cial antigen

presenting cells (aAPC) expressing HERV-K env and desired T-cell

co-stimulatory molecules After co-culture on g-irradiated aAPC, 95%

of CD3+ T cells expressed the CAR and these CAR+ T cells were able

to specifi cally kill HERV-Kenv+ melanoma targets in vitro in contrast

to CARneg T cells Specifi city of these CAR+ T cells was proven by

expressing the HERV-K env protein in antigen negative EL4 mouse

cells which were preferentially killed compared to HERV-Kenvneg

EL4 parental cells

In aggregate, these data demonstrate for the fi rst time that T cells

targeting an active ancient retrovirus can be used as an immunotherapy

for melanoma, using an approach that has translational appeal for

clinical trials

Nanoparticle Designed for the Treatment of

Multiple Myeloma, Has Anti-Tumoral Activity in

Lymphoma

Zhongda Liu,1 Terence Tang,1 Catherine A Taylor,1 Qifa Zheng,1

Sarah Francis,1 John E Thompson,1,2 Richard Dondero.2

1 Dept of Biology, University of Waterloo, ON, Canada; 2 Senesco

Technologies Inc., Bridgewater, NJ.

INTRODUCTION: The eukaryotic translation initiation factor

5A (eIF5A) is the only known protein to be regulated by

post-translational addition of a hypusine residue Hypusine-eIF5A has

been identifi ed as a marker of neoplastic growth and metastasis,

and suppression of hypusinated eIF5A sensitizes multiple myeloma

(MM) cells to apoptosis In vitro cell studies and in vivo xenograft

studies have demonstrated that simultaneous suppression of eIF5A expression and over-expression of a non-hypusinable pro-apoptotic mutant of eIF5A (eIF5AK50R) potently induces apoptosis in MM cells SNS01-T is a gene therapy nanoparticle targeted to the treatment

of MM and is comprised of: a B cell-specifi c plasmid expressing eIF5AK50R; an siRNA targeting the native eIF5A that promotes growth of cancer cells; and polyethylenimine A multiple ascending dose Phase 1b/2a open-label study has been initiated to evaluate the safety and tolerability of six weeks of twice-weekly administration

of SNS01-T by intravenous infusion in patients with relapsed or refractory MM Inasmuch as the B29 promoter driving expression

of eIF5AK50R is active in all B cell lineages, the activity of SNS01-T

as an anti-cancer agent in non-Hodgkin’s Lymphomas (NHL) of B cell origin was assessed using both mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL) xenograft models METHODS: In vitro biological activity of SNS01-T was assessed using RT-qPCR, and uptake of nanoparticles was assessed by FACS analysis of cells treated with fl uorescently-labeled SNS01-T Anti-tumoral activity was evaluated using MCL and DLBCL xenograft models with twice-weekly intravenous injections of SNS01-T at doses ranging from 0.093 mg/kg to 0.75 mg/kg RESULTS: SNS01-T transfected MM, MCL, and DLBCL cell lines and resulted in suppression of endogenous eIF5A mRNA and accumulation of the eIF5AK50R transgene Twice-weekly dosing of SNS01-T at doses > 0.18 mg/kg resulted in statistically signifi cant inhibition of tumor growth in both MCL and DLBCL xenograft models In the MCL xenograft model, tumor growth inhibition of 52.7 %, 79.1 %, and 89.0 % was observed for SNS01-T doses of 0.18 mg/kg, 0.375 mg/

kg, and 0.75 mg/kg, respectively Similar results were obtained in the DLBCL model Median survival in the MCL xenograft model was 21 days for control mice and up to 42 days (p = 0.002; logrank test) for mice treated with SNS01-T (0.75 mg/kg) SNS01-T also increased survival in the DLBCL model from 20 days for control mice to 45 days (p = 0.002; logrank test) for mice treated with 0.375 mg/kg SNS01-T CONCLUSION: SNS01-T is a gene therapy nanoparticle that modulates expression of eIF5A to kill cancers of

B cell origin and is being investigated in the clinic in patients with multiple myeloma SNS01-T is biologically active in other B cell cancers, including MCL and DLBCL MCL and DLBCL xenograft tumor growth was signifi cantly inhibited following intravenous administration of SNS01-T, and treatment provided a signifi cant survival advantage The results support the clinical investigation of SNS01-T in NHL patients

Suicide Gene for Safer and Easier Adoptive T-Cell Gene Therapy

Brian Philip,1 Barry Flutter,1 Ronjon Chakraverty,1 Amit Jathoul,1

Martin Pule.1

1 Haematology, UCL Cancer Institute, London, United Kingdom.

Introduction: A compact marker-suicide gene reliant on off-the-shelf reagents would offer considerable utility for adoptive T-cell therapy Marker genes enable measurement of transduction effi ciency and allow sorting of transduced cells while suicide genes allow deletion of administered T-cells in case of toxicity Previous marker genes include the Neomycin resistance gene, truncated nerve growth factor receptor and CD34; however all of these possess limitation to application including immunogenicity, unexpected biological activity and long coding regions respectively Similarly, several suicide genes have been described with Herpes Simplex Virus Thymidine Kinase and inducible Caspase 9 in clinical use Here limitations include either immunogenicity or limited availability of the inducing drug Aim: We sought to generate a compact marker-suicide gene that enables both clinical grade sorting and effective deletion using CD34 cliniMACS and Rituximab respectively By using minimal epitopes required for

Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy

S36

CANCER-TARGETED GENE & CELL THERAPY

binding, we hoped to greatly reduce the size of the construct as well

as rendering it biologically inert Results: We fi rst sought to locate the

epitope from CD34 which binds QBEnd10, the monoclonal antibody

used in Miltenyi cliniMACS CD34 selection system By epitope

mapping, we determined that this antibody binds a linear fragment

of CD34 16 amino acids long which demonstrates equal binding of

QBEnd10 as the native CD34 antigen Next we sought to incorporate

Rituximab binding epitopes to engender Rituximab triggered cellular

killing After much optimisation we determined that a construct

designated (RQR8), composed of two Rituximab binding epitopes

fl anking a single QBEnd10 epitope on the CD8 stalk demonstrates

effective sorting using cliniMACS CD34 system and effi cient deletion

by both CDC and ADCC with Rituximab

We have demonstrated functional co-expression of RQR8 with both

chimeric antigen receptors and transgenic T-cell receptors Finally,

we tested RQR8 in vivo using a C57BL/6 to Balb/c murine model

of GvHD We demonstrated almost complete in vivo depletion of

RQR8 expressing T-cells within 7 days of antibody administration

and subsequent resolution of GvHD

Conclusions: RQR8 is a 136 amino acid combined marker-suicide

gene This enables clinical grade selection and cellular deletion

with off-the shelf GMP reagents and pharmaceutical respectively

Its minimal size allows co-expression of multiple additional genes

We hope RQR8 will make therapy with engineered T-cells simpler,

cheaper and safer

87 Preclinical and Clinical Evaluation of Oncolytic Immunotherapy with

Ad5/3-E2F1-Δ24-GMCSF (CGTG-602), a GM-CSF Producing

Adenovirus Targeted to Tumors on Four Levels

Tuuli Ranki,1 Lotta Kangasniemi,1 Petra Ahokas,1 Minna Oksanen,2

Kaarina Partanen,3 Kalevi Kairemo,3 Leena Laasonen,3 Anna Kanerva,2 Vincenzo Cerullo,2 Akseli Hemminki.2

1 R &D, Oncos Therapeutics, Helsinki, Finland; 2 Cancer Gene Therapy Group, University of Helsinki, Finland; 3 International Comprehensive Cancer Center Docrates, Helsinki, Finland.

Oncolytic adenoviruses that express granulocyte-macrophage colony-stimulating factor (GM-CSF) induce anti-cancer immunity while acting directly on cancer cells by oncolysis Viral oncolysis releases tumor epitopes for antigen presenting cells (APC), GMCSF stimulates APC proliferation, maturation and migration APCs induce tumor-specifi c cytotoxic T-cells and, thus, an oncolytic virus coding for GM-CSF can induce potent antitumor immunity Systemically elevated cytokine levels can result to toxic side effects, however, and therefore GM-CSF expression has to be effectively restricted to tumor tissue To this end, we engineered a serotype 5-based GMCSF producing oncolytic adenovirus, CGTG-602 (Ad5/3-E2F1-Δ24-GMCSF), with E2F-1 promoter controlled expression of constant region 2 mutant E1A, resulting in replication dependent production

of GMCSF from E3 CGTG-602 has dual mechanisms for p16-Rb pathway selectivity and features a chimeric 5/3 fi ber that allows CAR-independent entry to tumor cells CGTG-602 was found to be

a specifi c and potent anti-cancer agent when tested preclinically in

vitro and in an immunocompetent Syrian hamster model 13 patients

with solid tumors refractory to standard treatments were treated with CGTG-602 Treatments were well tolerated and tumor-specifi c and adenovirus-specifi c T-cell immunity was seen Frequent changes in serum antibody levels against tumor associated antigens were seen after treatment with CGTG-602 and these changes seemed to correlate with other signs of effi cacy Overall, with regard to tumor marker or radiological responses, signs of antitumor effi cacy were seen in 9/12 evaluable patients (75%) Median survival was 135 days which is unusually high for a chemotherapy refractory population CGTG-602

is an attractive candidate for further clinical development

Human Endostatin Adenovirus (E10A) in Combination with Paclitaxel and Cisplatin in Patients with Head and Neck Carcinomas

Wen Ye, Ranyi Liu, Jiangxue Wu, Lixia Li, Zhongzhen Guan, Wenlin Huang

State Key Laboratory of Oncology, Cancer Center, Sun Yat-Sen University, Guangzhou, Guangdong, China.

Background Recombinant adenovirus encoding human Endostatin

gene (E10A) has been proved to be an effi cient and low-toxic drug candidate against solid tumors by inhibiting VEGF-induced tumor angiogenesis in preclinical and phase I clinical trials Here we investigated the effi cacy and safety of E10A in combination with paclitaxel and cisplatin for the treatment of patients with advanced head and neck squamous cell carcinoma (HNSCC) or nasopharyngeal carcinoma (NPC) in an open-label, phase II, multicenter clinical trial

with a randomize manner Methods We randomly assigned 70 of

140 eligible patients with recurrent or metastatic HNSCC or NPC into test group to receive E10A (at a dose of E10A of 1.0×1012 VP (virus particles) intratumoral injection on day1 and 8) plus cisplatin (at a dose of 25 mg/m2 per day on day 3 to day 5) and paclitaxel (at

a dose of 160 mg/m2 on day 3) every 3 weeks for a maximum of 4 cycles, while other 70 eligible patients in the control group to receive chemotherapy (cisplatin and paclitaxel) only Tumors were evaluated every 2 cycles during treatment and every 3 months in the follow-up