Mary Crowley Medical Research Center/Texas Oncology PA, Dallas, TX (site 005); Hoag Cancer Center, Newport Beach, CA (site 002); Louisiana State University, New Orleans, LA (site 004); Baylor Sammons Cancer Center, Baylor University Medical Center, Dallas, TX; Murex Pharmaceuticals, Inc., Dallas, TX; Institute for Health Care Research and Improvement, Baylor Health Care System, Dallas, TX, NovaRx Corporation, San Diego, CA, Sponsor of the IND

Background Ionizing radiation induces lipid peroxidation and forms reactive oxygen species (ROS) within the cell. Due to their highly reactive state, ROS have short diffusion distances and quickly transfer unbalanced electrons to neighboring molecules. This transfer cycle continues until it finally culminates in the generation of reactive alkenals such as 4-hydroxynonenal (4HNE). Under normal conditions, intracellular 4HNE levels are controlled by conjugation to glutathione and are actively transported from the cell; RLIP76 protein has been determined to be the major transport protein involved in the efflux of 4HNE-conjugates. Radiation induces significant oxidative stress, and the increased levels of 4HNE conjugates overwhelm transport capacity. When this occurs, further conjugation is inhibited and free 4HNE levels rise, triggering apoptosis. It was hypothesized that adding exogenous RLIP76 protein would reduce 4HNE levels and correspondingly increase the recovery from acute radiation syndrome (ARS) or completely protect individuals exposed to lethal doses of radiation. Methods The National Institutes of Health has developed animal models of ARS so that medical countermeasures can be tested in accordance with the FDA Animal Rule, since testing in people is clearly not ethical. One of these models, the C57BL/6 mouse, has been successfully utilized by Terapio to evaluate the efficacy of recombinant human RLIP76 encapsulated in liposomes (RLIP76-PL). These studies were 30-day survival studies of mice exposed to total body irradiation of 7.45-8.75 Gy. Administration of RLIP76-PL was subcutaneous or intramuscular on a prophylactic (administered up to 20 h prior to irradiation) or therapeutic (administered up to 36 h after irradiation) schedule. Results As a prophylactic, there was 100% survival compared to 33% of the controls when mice were given three doses and exposed to 8.1 Gy. As a therapeutic, the drug is not administered until 24hrs after irradiation and remarkably, there is 92% survival compared to 8% of the controls. Conclusions Combined data of many studies show that when compared to controls, treated mice exposed to LD50s, LD70s, or LD90s resulted in survival ranging from 50-80% over the controls. RLIP76-PL is a strong candidate to protect the population from acute radiation exposure.

Background: Metastatic melanoma is an incurable disease with an average survival of less than one year. Talabostat is a novel dipeptidyl peptidase inhibitor with immunostimulatory properties.Methods: This phase II, open label, single arm study was conducted to evaluate the safety and efficacy of 75-100 mg/m(2) cisplatin combined with 300-400 mcg talabostat bid for 6, 21-day cycles. The primary endpoint was overall response. The rate of complete responses, duration of overall objective response, progression-free survival (PFS), and overall survival were the secondary endpoints.Results: Six objective partial responses were recorded in the 74 patients (8.1%) in the intention-to-treat population. Five of these responses involved the 40 evaluable patients (12.5%). Thirty-one percent of patients reported SAEs to the combination of talabostat and cisplatin.Conclusion: Acceptable tolerability was observed in the intention-to-treat population and antitumor activity was observed in 12.5% of evaluable patients, which is not greater than historical expectation with cisplatin alone.

PurposeGeneration of broad cytotoxic T-lymphocyte responses against multiple epitopes and tumor-associated antigens (TAAs) may provide effective immunotherapy in patients with cancer. We evaluated a single-vial peptide vaccine consisting of nine HLA-A2 supertype-binding epitopes (two native and seven analog epitopes modified for optimal HLA binding or T-cell receptor stimulation) covering five TAAs and the universal helper pan-DR epitope, formulated as a stable emulsion with incomplete Freund's adjuvant (Montanide ISA 51; Seppic SA, Paris, France). The clinical efficacy, safety, and multiepitope immunogenicity of IDM-2101 was evaluated in patients with stage IIIB or IV non-small-cell lung cancer (NSCLC).Patients and MethodsA total of 63 patients were enrolled who were positive for HLA-A2. End points included survival, safety, and immune response. IDM-2101 (previously EP-2101) was administered every 3 weeks for the first 15 weeks, then every 2 months through year 1, then quarterly through year 2, for a total of 13 doses. Epitope-specific cytotoxic and helper T-lymphocyte immunogenic responses were measured by the interferon gamma enzyme-linked immunosorbent spot assay.ResultsNo significant adverse events were noted. Low-grade erythema and pain at the injection site were the most common adverse effects. One-year survival in the treated patients was 60%, and median survival was 17.3 months. One complete and one partial response were identified. Survival was longer in patients demonstrating an immune response to epitope peptides ( P < .001).ConclusionIDM-2101 was well tolerated, and evidence of efficacy was suggested.

RLIP76 is a multifunctional membrane protein that transports glutathione conjugates of electrophilic compounds and other xenobiotics including chemotherapy agents out of cells. The protein is overexpressed in lung carcinomas, ovarian carcinomas, and melanomas. The protein also binds Ral and participates in mitotic spindle function, clathrin-dependent endocytosis, and triggers GTPase-activating protein activity. It is found throughout the cell, in membrane, cytosol, and the nucleus, and is known to shift between these compartments in response to stress. Loss of RLIP76 by antibody or antisense therapy is associated with increased sensitivity to radiation and chemotherapy. Conversely, liposomally delivered RLIP may treat poisoning and wounds.

21134 Background: Clofarabine (CLOLAR®) is a next-generation nucleoside analog designed as a hybrid molecule to improve the efficacy and minimize the extramedullary toxicity of other nucleoside analogs. Such analogs are known to have a variety of effects on both humoral and T-cell immune function. Fludarabine, the drug most closely related to clofarabine, causes major alterations in the CD4:CD8 lymphocyte ratios, associated with marked increases in the rate of opportunistic infections. In contrast, gemcitabine has a selective detrimental effect on the B-lymphocyte subset. Therefore, while conducting a phase I dose-finding trial of clofarabine in patients with solid tumors, we investigated its effects on lymphocyte sub-populations. Methods: A phase I dose-finding study is ongoing in patients with solid tumors who receive clofarabine once a week for 3 weeks every 28 days. Blood samples were collected from patients in the highest dose levels before, during, and after clofarabine administration for lymphocyte quantitation and phenotyping using flow cytometry. Results: Of the 11 patients who had samples collected, 5 currently have both baseline and postbaseline values available (1 at the 129 mg/m2 dose level and 4 at the 103 mg/m2 dose level). Overall, the percentage of T cells and CD8 suppressor cells did not decrease significantly with clofarabine administration. In 1 patient where data from 2 cycles were available, CD4 cells decreased slightly after the first cycle, suggesting a possible cumulative effect. However, in all 5 patients CD19 cells decreased significantly after the first infusion of clofarabine and remained suppressed for the duration of the study period. Conclusions: Although clofarabine is a purine analog (as is fludarabine), its effects on lymphocyte subsets more closely resemble gemcitabine, a pyrimidine analog. If substantiated, these findings should have bearing on future decisions about appropriate drug combinations involving clofarabine. No significant financial relationships to disclose.

OncoGel is a novel depot formulation of paclitaxel designed for intralesional injection with a sustained paclitaxel delivery over approximately 6 weeks from a single administration. This phase 1 study was designed to characterize the toxicity, pharmacokinetics and preliminary antitumor activity associated with OncoGel administered directly into solid tumors. OncoGel was injected into 18 superficially accessible advanced solid cancerous lesions among 16 adult patients for whom no curative therapy was available. Four dose cohorts were evaluated, ranging from 0.06 to 2.0 mg paclitaxel/cm3 tumor volume. OncoGel injections were generally well tolerated. There was one report of grade 3 injection site pain for a patient in the 0.25 mg paclitaxel/cm3 tumor volume dose cohort. Other adverse events considered related to the study drug included mild to moderate local responses to the injection itself. Systemic levels of paclitaxel were detectable only in 3.3% of the samples analyzed (range: 0.53-0.71 ng/ml). For the 14 patients evaluable for disease progression, stable disease was noted among six patients and progressive disease among eight patients. Although the maximum tolerated dose was not identified, the planned maximum dose was administered in the study. OncoGel delivered intralesionally at doses up to 2.0 mg paclitaxel/cm3 tumor volume was well tolerated and paclitaxel remained localized at the injection site, confirming design principles to minimize systemic exposure. Therefore, localized paclitaxel administration using OncoGel merits continued clinical development.

3525 Background: ARQ 197 is a selective inhibitor of the c-Met receptor tyrosine kinase, an oncogene implicated in tumor invasiveness, metastasis, cancer cell proliferation, resistance to apoptosis, chemoresistance and angiogenesis. The c-Met RTK is a high-affinity receptor for hepatocyte growth factor (HGF). c-Met and HGF are dysregulated in a broad spectrum of cancers, thus inhibitors of c-Met could be promising targeted agents deserving clinical investigation. Methods: A phase 1 dose escalation study in metastatic pts who failed standard therapy was initiated to determine safety, tolerability, RP2D, pharmacokinetics, pharmacodynamics and preliminary antitumor activity of ARQ 197. Cycles consisted of twice-daily oral dosing of ARQ197 for two out of 3 weeks. Results: Thirty-eight pts were enrolled with data available for 36 pts (21M/15F; median age 61). Ten cohorts were assessed ranging from 10 to 360 mg/day. ARQ 197 was well tolerated and no DLT was observed. All treated pts achieved plasma drug concentrations significantly above the in vitro IC50. Doses through 70 mg b.i.d. revealed Cmax and AUC(0–12hr) increased linearly. There was no further increase in systemic exposure in pts treated with 90 through 180 mg b.i.d. There was notable inter-patient variability in Cmax and AUC(0–12hr), typical of oral dosing. The effects of cytochrome P450 polymorphism will be discussed. Adverse events (N=29) were generally mild with the most common being: fatigue (24 %), diarrhea (21%), and constipation (21%). Grade 3 or greater events possibly or probably related to ARQ 197 include: elevated ALP (3%), ALT (3%) and AST (3%). Of the 38 pts enrolled, 36 received at least one complete cycle of ARQ 197 with 33 evaluable for efficacy. Two pts achieved a PR (1 confirmed) and 19 had stable disease (SD) 21 10+ to 34+ weeks. Conclusions: Based on pharmacokinetic data, a RP2D for ARQ 197 was determined to be 120 mg b.i.d. Higher oral doses did not result in increased systemic systemic exposure to the drug. Eleven pts remain on study and continue to receive ARQ 197 treatment. A favorable adverse event profile and encouraging signs of anti-tumor activity was observed and continuous daily dosing of ARQ 197 is now being explored to better define clinical toxicity and efficacy. [Table: see text]

Lucanix™ is a non-viral gene based allogeneic tumor cell vaccine. Preclinical studies demonstrate enhancement of tumor antigen recognition as a result of TGF-β2 inhibition. We performed a randomized dose variable phase II trial involving stage II, IIIA, IIIB and IV NSCLC. Each patient received one of 3 doses (1.25, 2.5, 5.0x107 cells/injection) of Lucanix™ on a monthly or every other month schedule to a maximum of 16 injections. Immune function, safety and anticancer activity were monitored. Seventy five patients (2 stage II, 12 IIIA, 15 IIIB, 46 IV), received a total of 550 vaccinations. No significant (≥ grade 3) adverse events probably or definitely associated with administration of the vaccine were observed. A dose-related survival difference was demonstrated in patients who received ≥ 2.5 × 107 cells/injection versus those who received <2.5 × 107 cells/injection (p=0.0069). Focusing on the sixty- one late stage (IIIb/IV) evaluable patients, a 15% partial response rate was achieved. The estimated probabilities of surviving 1 and 2 years were 68% (95% CI: 55%, 80%) and 52% (95% CI: 35%, 68%) for the two higher-dose groups combined and was 39% (95% CI: 22%, 66%) and 20% (95% CI: 4%, 36%) for the low dose group. Immune function was explored in the 61 advanced stage (IIIB/IV) patients. Cytokine production (IFN-γ, p=0.006; IL-6, p=0.004; IL4, p=0.007) was induced, antibody mediated response to vaccine HLA antigen was observed (p=0.014) and cell mediated response showed a correlation trend (p=0.086) in patients achieving stable disease or partial response compared to those with progressive disease. In conclusion, Lucanix™ is safe and well tolerated. A survival advantage is suggested in patients who receive ≥ 2.5x107 cells/ injection thereby supporting the justification for further phase III evaluation.

L523S is an immunogenic lung cancer antigen that has demonstrated preclinical safety when the gene is injected intramuscularly as an expressive plasmid (pVAX/L5235) and when delivered following incorporation into an El B-deleted adenovirus (Ad/L523S). We performed a phase I clinical trial in 13 stage IB, IIA, and IIB non-small-cell lung cancer patients. pVAX/L523S (8 mg on days 0 and 14 in all cohorts) and Ad/L523S (1, 20, 400 x 10(9) vp on days 28 and 56, cohorts 1, 2, and 3, respectively) were administered to 3 patients in each of three cohorts. No significant toxic effect was identified. All but 1 patient demonstrated greater than or equal to twofold elevation in anti-adenovirus antibodies. One of 10 evaluable patients demonstrated L523S-specific antibody by direct IgG ELISA. Two patients developed disease recurrence and all remain alive after a median of 290 days follow-up. Results suggest a high level of safety but evidence of L523S-directed immune activation was limited, suggesting a need for modification of dose, schedule, and site of vaccination (i.e., intradermal) with further clinical testing.

We report the first long-term follow-up of adenoviral vector based therapeutics patients who previously received anticancer therapy. Over the period of 10/26/95 to 8/15/05 146 patients with stage IIIB/ IV NSCLC were treated as out patients. Patients participated in 1 of 8 adenoviral based therapeutic trials involving 6 different agents. All were evaluable for long term unexpected toxic effect and survival. The study agent and number of patients is as follows: Bystander GVAX, 35; GVAX, 53; Adenoviral p53, 51; Onyx 015, 5; TNFeride, 1; MDA7, 1. Early safety and activity results have previously been reported in the literature(see below). Male/female ratio was 55%/45%, and median age was 60 years (19–85 years). No long term unexpected toxicity was reported. Overall mean survival was 334 days (95% CI: 269 to 400), median survival was 199 (95% CI: 168 to 320) days. No difference in survival was demonstrated by gender (Log Rank, p = 0.187) or age (Log Rank, p = 0.392). Survival at 1 year and at 2 years for patients <60 years and > 60 years respectively was 32% and 16% vs. 27% and 5%. These results are consistent with expected survival described in the literature of similar advanced stage NSCLC patients over the same time period and suggest no evidence of long term adverse effect related to adenoviral vector therapy. Four patients remain alive more than 3 years after treatment. 1.Nemunaitis J, Jahan T, Ross H, et al. Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX® vaccine in advanced-stage non-small-cell lung cancer. Cancer Gene Therapy 2006 Jan 6; [epub ahead of print]. 2.Cunningham CC, Chada S, Merritt J, et al: Clinical and Local Biological Effects of an Intratumoral Injection of mda-7 (INGN 241) in Patients with Advanced Carcinoma; a Phase I Study. Molecular Therapy 2005 Jan; 11(1):149–59. 3.Nemunaitis J, Sternman D, Juhn K, et al. Phase I/II Study of GMCSF Gene- Modified Autologous Tumor Vaccine (GVAX) in Early and Advanced Stage Non Small Cell Lung Cancer. Journal of the National Cancer Institute 2004; 96: 326–331. 4.Senzer N, Mani S, Rosemurgy A, et al. TNFerade Biologic, an Adenovector with a radiation- inducible promoter, carrying the Human Tumor Necrosis Factor Alpha Gene: A phase I study in Patients with Solid Tumors. Journal of Clinical Oncology 2004 Feb; 22(4):592–601. 5.Nemunaitis J, Cunningham C, Buchanan A, et al: Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: Safety, feasibility and biological activity. Gene Therapy 8(10):746–59, 2001 May. 6.Nemunaitis J, Swisher G, Timmons T, et al: Adenovirus-mediated p53 gene transfer in sequence with Cisplatin to tumors of patients with non-small cell lung cancer. Journal of Clinical Oncology 18(3):609–622, 2000. 7.Swisher SG, Roth JA, Nemunaitis J, et al. Adenoviral-mediated p53 gene transfer in advanced non-small cell lung cancer. J Natl Cancer Inst 91(9):763–771, 1999. May.

PurposeBelagenpumatucel-L is a nonviral gene-based allogeneic tumor cell vaccine that demonstrates enhancement of tumor antigen recognition as a result of transforming growth factor beta-2 inhibition.Patients and MethodsWe performed a randomized, dose-variable, phase II trial involving stages II, IIIA, IIIB, and IV non-small-cell lung cancer patients. Each patient received one of three doses (1.25, 2.5, or 5.0 x 10(7) cells/injection) of belagenpumatucel-L on a monthly or every other month schedule to a maximum of 16 injections. Immune function, safety, and anticancer activity were monitored.ResultsSeventy-five patients ( two stage II, 12 stage IIIA, 15 stage IIIB, and 46 stage IV patients) received a total of 550 vaccinations. No significant adverse events were observed. A dose-related survival difference was demonstrated in patients who received >= 2.5 x 10(7) cells/injection ( P =.0069). Focusing on the 61 late-stage ( IIIB and IV) assessable patients, a 15% partial response rate was achieved. The estimated probabilities of surviving 1 and 2 years were 68% and 52%, respectively for the higher dose groups combined and 39% and 20%, respectively, for the low-dose group. Immune function was explored in the 61 advanced-stage ( IIIB and IV) patients. Increased cytokine production ( at week 12 compared with patients with progressive disease) was observed among clinical responders ( interferon gamma, P =.006; interleukin [IL] - 6, P =.004; IL-4, P =.007), who also displayed an elevated antibody-mediated response to vaccine HLAs ( P =.014). Furthermore, positive enzyme-linked immunospot reactions to belagenpumatucel-L showed a correlation trend ( P =.086) with clinical responsiveness in patients achieving stable disease or better.ConclusionBelagenpumatucel-L is well tolerated, and the survival advantage justifies further phase III evaluation.

PT-100 upregulates cytokine expression competitively inhibiting the dipeptidyl peptidase activity of fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). This dose-escalation study was conducted to evaluate the safety of PT-100 in patients receiving myelo-suppressive chemotherapy and to assess its effects on neutrophil recovery.PT-100 was administered orally for 7 days as a 200 mu g, 400 mu g, 800 mu g, or 1,200 mu g total daily dose ( divided twice daily) to 6, 6, 17, and 5 patients, respectively. Patients received 2 cycles of chemotherapy: The first cycle served as each individual patient's control. Patients had to develop Grade 3+ neutropenia in Cycle 1 in order to receive PT-100 in Cycle 2. Most patients received PT-100 on Days 2-8 of chemotherapy in Cycle 2, except at 800 mu g where an additional cohort (n=8) was treated on a Days 5-11 schedule. Five of 7 patients receiving 800 mu g on Days 2-8 experienced a >= 1-day improvement in Grade 3+ neutropenia in Cycle 2 versus Cycle 1. Overall, PT-100 was well tolerated. A reduction in chemotherapy-related nausea, vomiting, fatigue, alopecia, and diarrhea was noted in patients receiving PT-100. Edema/peripheral swelling, hypotension, hypovolemia, and dizziness were the most common nonhematologic adverse events considered related to PT-100. Two Grade 3 adverse events were considered related to PT-100: syncope (1,200 mu g) and orthostatic hypotension (800 mu g). A maximum tolerated dose was not reached. Given the accelerated neutrophil recovery, preclinical evidence of antitumor activity, and tolerable toxicities of PT-100, additional studies to optimize the PT-100 dosing schedule in patients receiving myelosuppressive chemotherapy are needed.

Onyx 015 is an E1B deleted conditional replicating adenovirus which has demonstrated control of local regional disease in prior studies [E. Lin and J. Nemunaitis, Cancer Gene Ther 11, 643 (2004)] but demonstrates limited effect when administered as an intravenous therapeutic. We hypothesized that systemic activity may be potentiated by reduction of viral clearance related to tumor necrosis factor a (TNF|[alpha]|).

Lucanix|[trade]| is a non-viral gene based allogeneic tumor cell vaccine. Preclinical studies demonstrate enhancement of tumor antigen recognition as a result of TGF-|[beta]|2 inhibition.

7120 Background: Talabostat (T) is an oral inhibitor of dipeptidyl peptidases such as fibroblast activation protein found on the stroma of tumors. Administration of T stimulates cytokine and chemokine production in humans and animals. In a mouse xenograft model, T significantly enhanced the activity of docetaxel in the A549 model of NSCLC. Methods: Open-label, single-arm, Phase 2 study in up to 41 evaluable patients with Stage IIIB/IV NSCLC using the Simon 2-stage design. Two responses (PR or CR) by WHO criteria required in the first 21 evaluable patients (defined as completion of 2 cycles with a response assessment by the investigator) to expand enrollment to 41. Up to six 3-week cycles of docetaxel (D) 75mg/m2 (Day1) with appropriate pre-medication and T-200μg administered orally BID Days 2–15. Dose-escalation of T to 300μg BID allowed in subsequent cycles depending on tolerability. Single-agent T may be continued past 6 cycles at the discretion of the investigator. Prior failure of a platinum-based therapy required with no more than 2 prior regimens allowed. ECOG PS 0–1. Primary endpoint is disease response; secondary endpoints include survival, progression-free survival, and incidence of clinically-significant events of neutropenia or anemia. Results: In the first 21 evaluable patients (10 men, 11 women), the median age is 58 (range 34–80); 14 patients had 1 prior treatment and 7 had 2. Partial responses have been seen in 2 patients. The median PFS is 19 weeks. 8 patients have continued on single-agent T beyond 6 cycles, 4 of whom had SD for at least an additional 6 weeks. The most frequently reported adverse events are edema NOS/peripheral (71%), fatigue (57%), neutropenia and leukopenia (each 43%), cough, hypotension, LDH increase, and arthralgia (each 29%). Grade 3/4 events included neutropenia, leukopenia (each 25%), and febrile neutropenia, edema, and dyspnea (all 10%). 6 patients have died during the study; 5 due to PD and one to an unspecified cardiovascular event. Conclusions: In the initial cohort of 21 patients, 2 PRs were reported. The median PFS is 19 weeks. The most common AEs are edema, fatigue, and neutropenia/leukopenia. Enrollment continues. Updated study results for all 41 evaluable patients will be presented at the annual meeting. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Point Therapeutics Point Therapeutics Point Therapeutics Point Therapeutics

Over the last several years, attempts have been made to use the tumoricidal effects of tumor necrosis factor (TNF)-α to treat cancer. Many of these studies demonstrated dose-limiting systemic side effects from high concentrations of TNF-α. The recent focus has been on developing a local delivery system for TNF-α to minimize the systemic response.

The mda-7/IL-24 cDNA was isolated almost a decade ago in a screen for genes differentially upregulated following growth arrest and terminal differentiation of a human melanoma cell line employed as an in vitro cell differentiation model. The underlying rationale for the screen was that oncogenesis arises from a cellular dedifferentiation process culminating in uncontrolled proliferation and acquisition of invasive and metastatic potential. Identification of genes upregulated during the process of reactivation of faulty or inoperational differentiation maintenance programs was postulated to have cancer gene therapeutic potential. In this context, it is heartening to note that mda-7/IL-24 has made a methodical and progressive journey, from an unidentified novel sequence with little homology to known genes at its time of isolation to currently having the status of a molecule belonging to the IL-10-related family of cytokines, with considerable cancer gene therapeutic potential. Extensive in vitro and in vivo human tumor xenograft studies have established its transformed cell apoptosis-inducing capacity in various model systems. It has recently taken an important step for a candidate cancer gene therapeutic molecule, in the ultimate goal of benchtop to clinic, by being currently utilized in human Phase I/II clinical trials. This review provides a current perspective of our understanding of mda-7/IL-24, including established and more recent information about the molecular properties, specificity of anti-tumor-cell apoptosis-inducing activity, and underlying mechanisms of this action relative to its cancer gene therapeutic potential.