169 combination oncolytic herpes simplex virus and dendritic cell immunotherapy for the treatment of established murine neuroblastomas

169 Combination Oncolytic Herpes Simplex Virus and Dendritic Cell Immunotherapy for the Treatment of Established Murine Neuroblastomas ties in various cancer cell lines, including prostate cancer We a[.]

ties in various cancer cell lines, including prostate cancer We also

reported that intratumoral administration of adcnoviral vector

mediatedGliprl (AdGliprl) significantly reduced primary tumor

and spontaneous lung metastasis in a preclinical mouse model of

metastatic prostate cancer (Hum Gene Ther 14, 91-101, 2003)

These preclinical studies led to an ongoing neoadjuvant gene therapy

Phase 1/11 clinical trial in which AdGLIPR I is being tested by direct

intratumoral injection prior to radical prostatectomy (IND# 13033)

Based on our ongoing studies ofGLlPR I function, we hypothesized

that GLiPRI may promote macrophage mediated anti-tumoral

ac-tivities In the current study,we analyzed the anti-tumoral activities

ofGliprlgene modified Mfll METHODS: Peritoneal exudates Mfll

were infected withAdGliprl or control Adv/CMV/Bgal24hr before

use I-IBSS,uninfected Mfll, Bgal gene-modified Mfll (Bgal/Mfll),

or Gliprl gene-modified Mfll (Gliprl/Mdi) were injected directly

into orthotopic mouse prostate cancer (metastatic 178-2 BMA) on

day 7 after tumor cell inoculation.At day 21, primary tumors and

spontaneous lung metastasis were evaluated.For survival analysis,

animals were monitored daily and euthanized when moribund

RESULTS: There were no significant differences in macrophage

viability after transduction of AdGliprl compared with contro1

FACS analysis showed an increase in the number of cells positive

for MI-ICc1assll antigen, CD40, and CD80 in Gllpr l/Mdi compared

to uninfected Mfll or Bgal/Mfll IL-12 secretion from Gliprl/Msb in

vitro was significantly increased compared to uninfected Mfllor Bgal/

Mfll GliprllMfll induced significant suppression of primary tumor

growth (I 029mg) compared with Bgal/Mfll (2414mg) or uninfected

Mfll (2691mg) (P<O.OOO I and P=0.0002, respectively) GliprllMfll

also demonstrated significant suppression of spontaneous lung

metastasis (mean 2.0) compared with Bgal/Mfll (5.9) or uninfected

Mfll (5.9) (P=0.0204 and P=0.0204, respectively) A significant

survival advantage was demonstrated for Glipr l/Mrb compared with

control Bgal/Mfll injected animals (27.8 vs, 23.0 days,respectively,

P=0.0008) Serum IL-12 levels were significantly elevated on day

5 in Gliprl/Moinjected animals compared with control Bgal/Mfll

injected animals Splenocyte-derived cytotoxic natural killer cell

activity was enhanced on day 2,and on day 7 tumor-specific

T-lymphocyte activities were significantly increased after Gliprl/Mfll

injection, compared with control BgalfMfll CONCLUSIONS:We

have demonstrated potentially anticancer therapeutic activities of

Gliprl gene-modified macrophage in a preclinical mouse model of

metastatic prostate cancer This approach may be useful for prostate

cancer therapy

168 Allograft Vaccine for Epithelial Cancers

Yucheng Tang,IHakan Akubulut,IJonathan Maynard,ILine

Ped-ersen,IAlbert Deisseroth.'

'Gene Therapy Program, Sidney Kimmel Cancer Center, San

Diego , CA.

The success ofvaccination is reduced in the aged immune system

and in the cancer host The Deisseroth laboratory has reported that

the sc injection of the Ad-sig-TAA/ecdCD40L adenoviral vector

prime followed by two TAAleedCD40L protein boost se injections

(hereafter designated TAAfecdCD40L VPP) can overcome anergy

in TAA.Tg mice,and can induce immunological memory for over

a year to tumor associated antigens (TAA).CD40L is a potent

im-munological activating signal required for induction ofboth cellular

and humoral immune responses, which is not expressed normally

in activated CD4 helper cells in the aged test subject (both mice

and humans) thereby reducing the response to vaccination among

older individuals The E7/ecdCD40L VPP vaccine has been shown

to increase the level of E7 specific antibodies and CD8 T cells in

old (18 months) as well as young (2 months) test mice We decided

to study the effect of adding the E7fecdCD40L VPP vaccine to total

body irradiation and allografting, The allodonor for donor

lyrnpho-S64

cyte infusions (DLI) was first vaccinated with the E7/ecdCD40L VPP vaccine starting on day -35 The recipient C57BLl6J mice were then injected sc with 100,000E7 TC-I cells on day -7 1,200 cGy of total body irradiation (TBI) were administered on day 0, 7 days following injection of the tumor cells I hour following the total body irradiation, the mice were transplanted with a single intravenous injection of 10 million T cell depleted bone marrow cells from an allodonor Spleen cells (50 million) collected from

an allodonor immunized one month earlier against the E7 protein with the E7fecdCD40L VPP vaccine, were given to the allorecipi-ent mice on day 3 (3 days following TBI and allotransplant) Thc recipient also received one sc injection of the E7fCD40L protein boost vaccination one week after allogeneic stem cell transplanta-tion We showed that the administration ofthe TBI and an allogeneic stem cell transplant 7 days post injectionof the E7 positive TC-I cancer cells, DLI from a E7/ecdCD40L VPP vaccinated donor 3 days following transplant (10 days after the E7 positive TC-I tumor injection), and a single E7fccdCD40L protein boost sc vaccination one week thereafter, resulted in a growth rate of the E7 positive tumor cells which was less than the control (injection oftumor cells followed in 7 days by TBI), or the animals in which the sc injection

of the E7 positive tumor cells was followed in 7 days by TBI and allograft, and E7fecdCD40L vaccination of the recipient This data shows that the TBI/DLI from an E7 VPP vaccinated donor/ allograft followed by E7 VPP vaccination is much more powerful than the same therapy without the DLI In addition,the addition of the DLI from an E7/ccdCD40L VPP vaccinated donor to TBI,allograft and post allograft E7fecdCD40L vaccination improves overall survival dramatically Finally, this approach makes possible the use ofyoung donors for old recipients,which may be a major advantage in re-storing a vigorous immune response to the vaccine in the elderly cancer recipient This would be feasible for elderly cancer patients

if one used the TAA/ecdCD40L vaccine with the non-mycloablativc allograft which is safe even in the older cancer patient

Virus and Dendritic Cell Immunotherapy for the Treatment of Established Murine Neuroblastomas

Christopher 1 Farrell,' Cecile M Zaupa,'Robert L.Martuza,' Samuel D Rabkin,' William T Curry.'

'Neurosurgery; Massachusetts Gen eral Hospital, Bo ston, MA.

Genetically-engineered, conditionally-replicating oncolytic viruses are capable of killing tumor cells by direct lysis Our laboratory and others have previously demonstrated that treatment with oncolytic HSV vectors also elicits induction ofa cell specific anti-tumor immune response as demonstrated by protection against tumor rechallenge and abrogation of this response in immunode-ficient mice In this study, we examined whether this immuno-therapeutic effect could be enhanced by combining oncolytic HSV with intratumoral administration of immature, ex-vivo generated dendritic cells (iDC).Subcutaneous N 18 neuroblastoma tumors were established in immunocompetentAfJmice and when tumors reached approximately 5mm in maximal diameter,the tumors were inoculated with G47A, an oncolytic I-ISV vector with engineered inactivation of ICP6 (ribonucleotide reductase) and deletions in the ICP34.5 and ICP47 genes Two days following viral infec-tion,iDC were directly administered into the tumor followed by

a second G47A inoculation three days later Significant reductions

in tumor volumes were observed at day 12 following treatment initiation in micereceivingcombinationtherapywithG47A+iDC

in comparison to those receiving G47A alone (4.7-fold reduction) and mock-treated mice (l5-foldreduction).Treatment with iDC in the absence of oncolytic virus had no demonstrable effect on tumor volume compared to the mock-treated group Additionally,survival was significantly prolonged in the combination therapy group as

Molecul ar Therapy V olume 15 S upplement I• \by 2<)07

C opyright ©The Americ m Society {I t Gene Therapy

compared to theG47t.alone and mock-treated groups ELISPOT

analysis demonstrated that combined therapy was accompanied by

a nearly four-fold increase in N18-specific IFNy producing cells

compared to virus treatmentalone In conclusion,

intratumorally-administered immature dendritic cells significantly enhanced the

anti-tumorefficacyofoncolytic HSV in an established tumor model

of neuroblastoma This increased activity is associated with an

in-crease in the percentage of IFNy producing cells, likelyreflecting

enhancement of the systemic anti-tumor immune response elicited

by oncolytic HSV

Using Multiple IV Administrations of IL-12 Plasmid

Nanopolyplexes

Jason G Fewell, Jennifer S Rice,Miranda Hickman, Majed M

Malar, Elaine Brunhoeber, Casey Pence, Gregory Slobodkin,

Danny H Lewis, Khursheed Anwer

'Biology and Pharmacology, Expression Genetics, Inc,

Hunts-ville, AL.

Renal cell cancer accounts for approximately 3% of adult

ma-lignancies and -12,000deaths per year The most common site of

metastatic involvement for this cancer is the lung where it appears

in -75% of the cases of metastatic disease Treating primary lung

tumors or tumors resulting from metastasis is exceptionally difficult

as evidenced by poor patient outcome While hormone therapy and

chemotherapy are known to have little effect on metastatic renal

cell carcinoma (RCC), high dose interterleukin-2 and other

im-munotherapieshave been shown to have some benefit The strong

anti-cancer properties of interleukin-12 ([L-12) make it a

sought-after target for therapeutic development that could be particularly

useful for treating RCC metastases The effects of IL-12 are

medi-ated through induction of local and systemic immune responses

and strong anti-angiogenic characteristics and are associated with

T-Iymphoeyteand natural killer (NK) cell proliferation,activationof

cytotoxic' l-lymphocytesand secretionofinterferon-gamma (IFN-y)

The anti-angiogenic effects result from IFN-y induced stimulation

of MIG,IP-IO and direct inhibitory effects on VEGF mRNA For

studies presentedhere,a novel polymericdeliverysystem(BD15-12)

has beendeveloped.This PEl-based co-blockpolymerutilizes

biode-gradable linkages and is designed to produce high expression levels

in-vitro and in vivo Results show that BD15-12 formulated with

a plasmid encoding for murine IL-12 and delivered intravenously

(IV)is an effective inhibitor of experimentally induced metastatic

lung tumors For these studies BALB/C mice were injected (IV)

withlxtO' Rencacells.At days 4,9 and 16 after Rencacell

admin-istration mice were treated IV with formulated plasmid The lungs

were harvested 1 day after the last injection for eytokine analysis

and tumor nodule quantification Treatment produced significant

levels of m1L-12 in lungs that plateau at DNA dose levels above

9/lg,while IFN-ylevels were increasingly higher up to a 30/lg dose

A significant85% dose-dependent reduction inthe number of tumor

nodules occurred treated mice.Additional modificationswere made

to the polymer to reduce the moderate systemic toxicity associated

with high treatment doses Through a series ofoptimization studies

it was shown that the additionof'aspecific amount

of'methoxypoly-ethyleneglyeol (PEG) functionalgroups prevents an increase in liver

enzymes following multiple IV administrations while preservingthe

overallefficacy levelof treatment The advantage of this approach

is the ability to administer IL-12 repeatedly with minimal toxicity

Additionalwork is being performed to evaluate BD15-12 for use

in administeringinhibitoryRNAs Preliminaryresults have shown

the ability to produce 70% knockdown of reporter gene following

co-administration (IV) of formulated luciferase reportergene

plas-mid with a plasplas-mid encoding for a luciferase short-hairpin.Future

experiments wiII be designed to examine combination approaches

Molecular Therapy Volum e 15.Supplcm mt 1 \1 ",· 2007

Cop yrighf © The: American$OOI.1'}' of Gene Therapy

utilizing both therapeutic protein ovcrexpression with RNAi for treating metastatic RCC

171 Preclinical Evaluation of an iCD40-Dendritic Cell Vaccine for Prostate Cancer

Natal ia Lapteva,1MamathaR Seethamrnagari ,' BrentA Hanks,1

KevinM.Slawin,' David M.Spencer.'

'Scott Dept of Urology, Baylor College ofMedicine, Houston, TX llmll/unology, Baylor Coflege ofMed icine, Houston, TX.

Clinical trialsutilizing dendritic cell (DC)-based cancer vaccines have been performed in patients with a variety of cancers including prostate, melanoma, renal,and colorectal cancer Despite encour-aging observations in these trials, vaccine-induced antigen-specific

T cell responses have been largely insufficienttoeffectivelyreduce tumor burden or prevent tumor progression in most patients.This suggests that furtherenhancements in the potency of DC-based can-cer vaccines are required to achieve a meaningful clinical response

To enhance DC-based vaccines, we used the combination of an inducible CD40 receptor (iCD40) and TLR-4 ligands.The iCD40 receptor permits targeted, reversible activation ofCD40 in vivo and has been previously shown to enhance tumor regression in mouse models more efficiently than DCs activated in vitro with LPS and CD40L Using iCD40 in combination with LPS or elinieal analog MPL, we assessed multiple parameters associated with monocyte-derived DC (MoDC) activation, including levels of maturation markers, lL-6, IL-12p70, and HLA class I and IIepitope-speciflcT cell responses, as well as MoDC migration in vitro and in vivo Fur-thermore, use of iCD40-modified and LPS-stimulated MoDCs led

to targeted expansion of autologous antigen-specific T cells against both melanocyte antigen (MAGE3) and the prostate tumor-associ-ated antigen,prostate-specific membrane antigen (PSMA) These observationsformthe technological foundation for a planned phase

1111 trial in patients with advanced,hormone-refractory prostate cancer (planned launch: 4th quarter 2007).Men with progressive, metastatic, androgen-independent prostate cancer will be recruited for the study In this Phase 1111 dose escalation trial, 3 patients will

be treated at each of three escalating doses (lxlO"7, 2x10"7 and 4xlO"7) of iCD40-modified MoDCs pulsed with PSMA either weekly or bi-weekly (total of6 doses) Ifno dose-limiting toxicity (DLT) is experienced at the maximum dose, additional patientswill

be treated at this dose and schedule For patients who remain free from disease progression,vaccinations will continue every 8 weeks for up to 48 weeks The primary endpoints of the trial arc to deter-mine the maximum tolerated dose (MTD) and the optimal biologic dose and schedule (OBD) The secondary goals arc to determine survival,freedom from objective progression, PSA-doubling time, FACT-P quality-or-life and pain scores Finally, ongoing improve-ments to iCD40 that incorporate synergistic eleimprove-ments from both CD40 and TLRs will also bediscussed These enhanced DCs(eDCs) promise to greatly improve the utility of DC-based vaccines This work was supported by the NIH (ROI-CA120411)

(MCCRC Results)

John J Nemunaitis.l-v'Phillip B Maples,1 ,2Neil Senzer,I.2.3.4 Rosemarie Arzaga,? Kristen Hege..l

'Cancer Resea rch, Mary Crowley Cancer Research Centers

(,HC-CRC), Dallas, TX ; 'Cancer Research, Murex Pharmaceuticals, Inc , Dallas, rx.·'Cancer Research, Baylor Sammons Cancer Center, Dallas, TX ; 'Cancel' Research, Texas Oncology, P.A ,

Dallas ,r x;.sCancer Research, Cell Genesys, Inc, South San Francisco, CA

Previously we reportedsafety and activity ofGVAX, a granulo-cyte macrophage gene transduced autologous malignantcell vaccine

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