ALBERT

Description: ISF35 is a novel CD40-binding protein designed to maximize stable, high-level surface-expression of this potent immuno-stimulatory molecule on cells transduced to express this protein. In a recent clinical study, patients with chronic lymphocytic leukemia (CLL) received intravenous infusions of autologous leukemia cells transduced ex vivo to express ISF35 using a replication-defective adenovirus vector (Ad-ISF35). This treatment was well tolerated, did not have dose-limiting toxicity, and had apparent clinical activity. Moreover, injection of autologous, ISF35-expressing CLL cells induced upregulation of death receptors and pro-apoptotic proteins on bystander, non-infected CLL cells, resulting in acute cytoreductions in leukemia cell counts and reductions in the size of lymph nodes and spleen of the treated patients (Wierda et al. Blood. 2007; 110: a-2040). Because of this and preclinical studies demonstrating specific anti-lymphoma activity of Ad-ISF35 when directly injected into growing tumor nodules of experimental animals, we conducted a first-in-man phase I clinical study, evaluating the safety of direct intra-nodal injections of Ad-ISF35 in patients with CLL. Fifteen patients, ranging in age from 45 to 71 years (median age 55, 10 male and 5 female), with progressive CLL (Rai stage III and IV) and leukemia-cell doubling times of 0.9 to 22.5 months (median 3.3 months) participated in a dose-escalation study involving successive cohorts of 3 patients each. The patients in each cohort received a single injection of 1x1010, 3 x1010, 1x1011 or 3x1011 Ad-ISF35 viral particles into a pathologically enlarged axillary lymph node under ultrasound guidance. Intranodal injection with Ad-ISF35 was well tolerated. Adverse events included erythema, swelling and/or pain at the site of injection and “flu like symptoms”, which occurred primarily during the first 24 hours of treatment. At the two highest dosing cohorts, three of three patients in the 3x1011 cohort and two of six patients in the 1x1011 cohort developed Grade 3 and 4 asymptomatic and transient neutropenia and hypophosphatemia one to three weeks after the injection of Ad-ISF35. In all patients, the neutropenia and hypophosphatemia resolved following administration of filgastrim and oral phosphate. Although Ad-ISF35 was injected into only one axillary lymph node, we observed significant reductions in absolute numbers of CLL cells in the blood and reductions in the size of all lymph nodes and the spleen in 14 of 15 evaluable patients. The reduction was durable in 9 patients and the leukemia cell counts have remained below pre-treatment levels for up to four months after a single intra-nodal injection of Ad-ISF35, even in patients who had rapid CLL doubling times prior to therapy. Although we have no evidence for dissemination of Ad-ISF35 beyond the injected lymph node, we observed up-regulation of death receptors, immune co-stimulatory molecules, and pro-apoptotic proteins in the circulating, non-infected CLL cells of the treated patients, suggesting a bystander effect. In addition, we observed increased blood levels of interferon-gamma and interleukin-12 beginning 8 hours after injection. In summary, we found that direct intranodal injection of Ad-ISF35 into patients with CLL was well tolerated and had systemic biologic and clinical activity, suggesting that this approach might be effective in the treatment of patients with this disease and other B-cell lymphomas.

Description: Abstract 1476 Ad-ISF35 is a replication-defective adenovirus (Ad) vector that encodes ISF35, which is a potent immuno-stimulatory CD40 binding protein designed to maximize stable, high-level cell-surface expression of this molecule. ISF35 can induce expression of costimulatory and death receptor molecules on chronic lymphocytic leukemia (CLL) cells in vitro and in vivo. Ad-ISF35 has shown to be safe and to have potential activity in two clinical studies in CLL patients, one using infusions of autologous CLL cells trasduced ex-vivo with Ad-ISF35 and the second one using a single intranodal injection of Ad-ISF35. From this second study we have observed that the maximum tolerated intranodal dose (MTD) was 3.3 × 1010 viral particles (vp) due to development of asymptomatic and transient hypophosphatemia, SGOT/AST elevation and neutropenia in some patients treated with higher doses. Using this MTD dose (3.3 × 1010 vp), we designed a clinical study to evaluate the toxicity and efficacy of repeat doses of intranodal Ad-ISF35 administration in CLL patients. Ten patients with median age of 66 years (range 56–74, 80% male and 20% female), with progressive CLL (Rai stage III and IV) and median leukemia-cell doubling times of 6 months participated in this repeat dose intranodal administration study involving up to six biweekly injections with a fixed dose of 3.3 ×1010 vp. Intranodal Ad-ISF35 injections were done into pathologically enlarged lymph nodes accessible by palpation using ultrasound guidance. Repeat dose intranodal administration of Ad-ISF35 was well tolerated by the majority of patients. The median number of injections was 5. One patient developed transient and asymptomatic Grade III hyponatremia after injection #4 considered to be a dose-limiting toxicity (DLT). No dose adjustments were required in any of the patients and we have not observed cumulative dose effect or long-term toxicities after a median follow up of 14 months. Other adverse events included erythema, swelling and/or pain at the site of injection and “flu-like symptoms”, which occurred primarily during the first 24 hours after each injection and were self-limited. The majority of patients experienced 〉50% reduction in leukemia cell counts (70% of patients), lymphadenopathy (80% of patients) and/or splenomegaly (70% of patients) during the course of treatment. Response assessment based on IW-CLL-08 criteria showed and overall response rate of 30% with 3 patients achieving a partial response, 4 patients with stable disease and 3 patients with disease progression. Although we have no evidence of Ad-ISF35 vector expression beyond the injected lymph node, we observed upregulation of death receptors, immune costimulatory molecules, and pro-apoptotic proteins in the circulating, non-transduced CLL cells of the treated patients suggesting a bystander effect. Cytokine expression analysis in the sera of treated patients showed increased levels of interferon-gamma and interleukin-6 beginning 8 hours after injection. In addition, we observed evidence of antibody production against Ad-ISF35 with the ability in some cases to neutralize vector transduction in vitro. However, we did not see antibody formation against human CD154. In summary, we found that repeat dose Ad-ISF35 intranodal administration via direct injection in patients with CLL was well tolerated and had systemic biologic and clinical activity that appears to be mediated by the effect of non-transduced bystander cells. These data suggest that intranodal administration of Ad-ISF35 may be safe and potentially effective in the treatment of patients with CLL or other B-cell lymphomas. Disclosures: Kipps: Memgen, LLC: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 376 Chronic lymphocytic leukemia (CLL) cells that have del(17p) typically have loss of functional P53, rendering these cells refractory to standard chemotherapeutic agents, which require activation of P53 for their cytotoxic activity. However, del(17p) CLL cells co-cultured in vitro with cells transduced to express the CD40L (CD154) activate another member of the P53 family of proteins, namely P73, which, like P53, can induce transcription of death receptors and pro-apoptotic proteins and sensitize cells to the cytotoxic activity of “P53-dependent” drugs, such as Fludarabine (F-ara-A). Moreover, transduction of del(17p) CLL cells with a replication-defective adenovirus (Ad) encoding recombinant CD154 (Ad-ISF35) can induce such changes in both transduced and bystander CLL cells in vitro. To examine whether a similar activity could be obtained in vivo, we are conducting a phase Ib clinical study in subjects with high-risk CLL who are refractory to fludarabine or have evidence of del(17p). In this study, subjects receive three IV doses of 3×108 autologous CLL cells that had been transduced ex vivo with Ad-ISF35 and two weeks latter they are treated with a truncated chemoimmunotherapy regimen involving 3 monthly courses of fludarabine, cyclophosphamide and rituximab (FCR). P53-defective CLL cells from treated patients were initially resistant to F-ara-A induced apoptosis with IC50 〉 10μM prior to treatment. CLL cells collected from patients ≥ 24 hours after the first infusion of autologous Ad ISF35-trasduced CLL cells became sensitive to the cytotoxic effects of F-ara-A, with IC50 0.3-1 μM. Enhanced sensitivity to F-ara-A was associated with induced expression of Bid, DR5, CD95, and P73 by circulating non-transduced “bystander” CLL cells, an effect lasting ≥ 2 weeks following IV infusion. To date, two subjects have completed treatment and this has been well tolerated without serious adverse events. The most common adverse events have been transient fever, malaise and fatigue associated to infusion of Ad-ISF35 transduced cells and cytopenias after treatment with FCR. Both subjects have achieved a compete response, one of them without detectable minimal residual disease (MRD) by sensitive multiparameter flow cytometry of marrow mononuclear cells obtained 3 months following completion of treatment. Moreover, these patients have complete resolution of lymphadenopathy and organomegaly by exam and by whole body computed tomography. These results indicate that Ad-ISF35-cell-gene therapy can sensitize P53-deficient CLL to “P53-dependent” cytotoxic agents in vivo, allowing for effective treatment of patients who otherwise would be resistant to standard forms of therapy. Disclosures: Prussak: Memgen: Employment.

Description: Abstract 1470 Ad-ISF35 is a replication-defective adenovirus (Ad) vector encoding a membrane-stable chimeric CD154 (ISF35) that can induce expression of costimulatory and death receptor molecules in chronic lymphocytic leukemia (CLL) cells in vitro and in vivo. Ad-ISF35 has shown to be safe and to have potential activity in two clinical studies in CLL patients (Wierda et al, Leukemia, 2010 in press; Castro et al. Blood 2008 112: Abstract 2100). However, the activity, immunogenicity, safety and biodistribution of Ad-ISF35 intratumoral administration have not been evaluated in a solid tumor model. To study these questions, we use BALB/c mice bearing subcutaneous A20 Non-Hodgkin Lymphoma (NHL) tumors as an in vivo cancer vaccine model. A20 tumor bearing mice were injected intratumoraly with single or repeat dose administration of Ad-ISF35 [3×109 or 3×1010 viral particles (vp)], Ad-Empty or control vehicle on days 0, 7 and 14. Treated animals were sacrificed on days 2, 5, 19 and 39. Clinical observations, comprehensive necropsy, hematology, serum chemistry, histopathology, cytokine and protein expression as well as Ad-ISF35 vector biodistribution and ISF35 transgene expression were analyzed. Ad-ISF35 injection in A20 tumor-bearing mice was well tolerated with mild toxicities that included mild and transient elevation in RBC, hemoglobin and hematocrit treated males. In both sexes we observed a transient increase in neutrophils on day 2 after Ad-ISF35 injection, and there were no abnormalities in serum chemistry suggestive of major organ dysfunction. Histopathology assessment of ten different organs did not show differences between the controls and Ad-ISF35 injected mice. Ad-ISF35 biodistribution showed that the viral vector was primarily found in the injected tumors and underwent a rapid clearance with no evidence of accumulation or persistence in the injected tumor or peripheral organs. Half of the Ad-ISF35 positive samples showed evidence of ISF35 transgene expression measured by real time q-PCR. Immunohistochemistry analysis of A20 tumors 48 hours after Ad-ISF35 intratumoral injection showed a massive infiltration of monocytes, neutrophils and T-cells as well as evidence of tumor cell apoptosis that correlated with tumor shrinkage followed by complete regression in 80% of Ad-ISF35 injected mice but not in the control animals. Following Ad-ISF35 injection, we observed CD154 upregulation in 0.25% of the tumor cells while there was a 100-fold increase in the number of cells expressing CD40 suggesting a potent bystander effect in non-transduced tumor cells. Analysis of the cytokine expression profile in tumor cells from mice treated with Ad-ISF35 intratumoral injection showed increased expression of IL-6, IFN-γ and MCP-1 as early as 48 hours after injection; this was not observed in the control mice. Mice that were cured from A20 tumors after intratumoral Ad-ISF35 injection developed antibodies and cytotoxic T cells that reacted specifically with A20 cells and prevented the development of A20 tumors after rechallenge. In summary, we show here that Ad-ISF35 intratumoral injection in a solid tumor NHL mouse model can function as an in vivo cancer vaccine breaking tumor tolerance and inducing complete tumor regression and immunity to tumor rechallenge. Ad-ISF35 intratumoral administration was well tolerated and showed a favorable biodistribution viral vector profile with rapid clearance and no evidence of vector persistence. Ad-ISF35 intratumoral injection induced expression of immunomodulatory and chemotactic cytokines as well as humoral and cellular anti-tumoral responses. Moreover, the effect of intratumoral Ad-ISF35 administration appears to be associated with a potent bystander effect in non-transduced cells. These data provide the rationale for development of Ad-ISF35 intratumoral administration as an in vivo cancer vaccine strategy in patients with NHL and other solid tumors. Disclosures: Kipps: Memgen LLC: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 1472 Background: Chronic lymphocytic leukemia (CLL) cells with del(17p) typically have loss of functional P53, rendering them refractory to chemotherapeutic agents. However, del(17p) CLL cells activated by CD40L (CD154) are induced to express pro-apoptotic factors that re-sensitize cells to the cytotoxic activity of P53-dependent drugs, such as fludarabine (F-ara-A). Chemotherapy re-sensitization is mediated in part by induction of p73, a p53-related transcription factor. To examine whether a CD154-based therapeutic strategy can be developed in vivo for del(17p) and/or fludarabine refractory CLL, a phase 1b clinical study evaluating an autologous cellular gene immunotherapy is being conducted. Autologous CLL cells transduced ex vivo with a replication defective adenovirus vector encoding a membrane-stable, re-engineered form of CD154 (Ad-ISF35) are administered, followed by standard courses of FCR in subjects with high-risk fludarabine refractory and/or del(17p) CLL. Methods: Subjects with fludarabine refractory and/or del(17p) receive three IV doses (one dose every two weeks) of 3×108 autologous CLL cells that have been transduced ex vivo with Ad-ISF35. Two weeks following the third dose of Ad-ISF35-transduced cells, subjects receive standard monthly cycles of fludarabine, cyclophosphamide and rituximab (FCR). Study endpoints include analysis of safety and efficacy. Correlative analyses are conducted for evidence of drug re-sensitization, regulation of apoptotic pathways, cytokine analysis, and humoral immune responses to the adenovirus vector and ISF35 transgene. Results: To date, four patients have completed treatment. Two patients have achieved a compete response, one of them without detectable minimal residual disease (MRD) by sensitive multiparameter flow cytometry of marrow mononuclear cells after completion of treatment. These responses have been durable after a median follow up of 18 months. One patient achieved a partial response with complete resolution of lymphocytosis, lymphadenopathy and splenomegaly, but residual CLL in the bone marrow. The remaining patient had progressive disease despite an initial response to both infusion of Ad-ISF35-transduced cells and FCR chemoimmunotherapy. Infusion of Ad-ISF35 transduced cells has been well tolerated. Overall, the most common adverse events have been transient fever, malaise and fatigue associated with infusion of Ad-ISF35-transduced cells and cytopenias after treatment with FCR. Prior to ISF35 treatment, CLL cells from patients were resistant to F-ara-A induced apoptosis (IC50 〉 10μM). However, one day following the first infusion of Ad-ISF35-transduced CLL cells, patient cells became sensitive to F-ara-A (IC50 0.3–1 μM). In addition, pro-apoptotic factors, including Bid, DR5, CD95, and P73 were induced in the non-transduced “bystander” CLL population following ISF35 infusion. These pro-apoptotic effects persisted ≥ 2 weeks following IV infusion. The sera from treated patients showed increase in IL-6 and IFN-γ after infusion of Ad-ISF35 transduced CLL cells. Despite evidence of anti-adenovirus antibody responses in the treated patients, there was no detectable anti-human CD154 production before or after ISF35 treatment. Conclusions: The results indicate that Ad-ISF35-cell-gene therapy can sensitize P53-deficient CLL to “P53-dependent” cytotoxic agents in vivo, allowing for effective and durable clinical responses. These data are very encouraging and suggest that this unique chemoimmunotherapy re-sensitization strategy could offer a valuable treatment option for patients who otherwise would be resistant to standard forms of therapy. Disclosures: Cantwell: Memgen: Employment. Kipps:Memgen, LLC: Membership on an entity's Board of Directors or advisory committees.

Description: Antimicrobial peptides constitute an excellent alternative against conventional antibiotics because of their potent antimicrobial spectrum, unspecific action mechanism and low capacity to produce antibiotic resistance. However, a potential use of these biological molecules as therapeutic agents is threatened by their low stability and susceptibility to proteases. In order to overcome these limitations, encapsulation in biocompatible polymers as poly-lactic-glycolic-acid (PLGA) is a promising alternative for increasing their stability and bioavailability. In this work, the effect of new synthetic antimicrobial peptides GIBIM-P5S9K (G17) and GAM019 (G19) encapsulated on PLGA and acting against methicillin resistant Staphylococus aureus (MRSA) and Escherichia coli O157:H7 was studied. PLGA encapsulation allowed us to load around 7 µg AMPs/mg PLGA with an efficiency of 90.5%, capsule sizes around 290 nm and positive charges. Encapsulation improved antimicrobial activity, decreasing MIC50 from 1.5 to 0.2 (G17NP) and 0.7 (G19NP) µM against MRSA, and from 12.5 to 3.13 µM for E. coli O157:H7. Peptide loaded nanoparticles could be a bacteriostatic drug with potential application to treat these bacterial E. coli O157:H7 and MRSA infections, with a slow and gradual release.

Description: Abstract 2544 The clinical presentation of chronic lymphocytic leukemia (CLL) is variable and includes at least two distinct clinical subtypes: indolent or progressive disease. Several prognostic markers such as the mutational status of the IgVH genes or ZAP-70 expression identify patients that will have more aggressive clinical courses. We have previously reported that ZAP-70 expression in CLL requires the support and stability provided by the Hsp90 activated complex (Hsp90-AC) and that Hsp90 inhibitors induce apoptosis in CLL cells preferentially in patients with high–risk/rapid progressive disease. Hsp90 requires the conformation of a multimeric complex with other co-chaperones (Hop, p23) to become active and function as a chaperone for mutated, over-expressed, misfolded or constitutively activated proteins. However, to measure Hsp90-AC activity is challenging and requires protein assays that are semi-quantitative and not always can provide a functional read out. To address this problem we designed a test to measure Hsp90-AC by taking advantage of the higher avidity of this complex to bind Hsp90 inhibitors such as 17-AAG. We hypothesize that Hsp90-AC activity is an independent prognostic marker of disease progression in CLL and to test this we measured the ability of 17-AAG to inhibit Hsp90-AC and promote apoptosis in vitro in a cohort of previously untreated CLL patients. This evaluation was performed in samples collected at the time of diagnosis and the results were correlated with the time from diagnosis to first treatment (t-Tx1) as well as the presence of other known prognostic markers. Ninety-five previously untreated CLL patients were included in this cohort. In vitro sensitivity/apoptosis to Hsp90-AC inhibition using 17-AAG (1μg/ml) was measured by flow cytometry using DiOC6 and PI after 48 hours of incubation. Only samples with more than 60% viability were included and we selected a cut-point of ≥ 53% induction of apoptosis to determine if a sample was sensitive or not to Hsp90-AC inhibition (This cut-point was selected using a ROC analysis for optimal diagnostic performance). We found that 37 patients (39%) were sensitive to 17-AAG. These patient had a median time from diagnosis to first treatment (t-Tx1) of 7 years compared to 4.2 years in patients that were resistant to 17-AAG (p=0.04). In addition, 37 patients (39%) had unmutated IgVH genes with a median t-Tx1 of 8.7 years compared to 4.2 years in patients with mutated IgVH genes (p=0.02) and 41 patients (43%) had high levels of ZAP-70 expression (〉20% by flow cytometry) and this group of patients had a median t-Tx1 of 7 years vs. 4.2 years in ZAP-70 negative patients (p=0.03). We evaluated the association between the degree of Hsp90-AC inhibition (17-AGG sensitivity) and IgVH mutational status and found a strong correlation between these two variables with shorter t-Tx1 in patients that were IgVH unmutated and sensitive to Hsp90 inhibitor (p=0.0003). We found a similar strong correlation between ZAP-70 expression and sensitivity to Hsp90-AC inhibitor (p=0.003). In our patient cohort, we saw that IgVH, ZAP-70 and Hsp90 inhibitor sensitivity are independent prognostic markers in CLL (Cox regression, p=0.002). We found that the sensibility and specificity of ZAP-70 test for high-risk CLL (sensitivity 81.1% [95% IC 67–95%] and specificity 77.6%[95% IC 65–90%]) was increased when the Hsp90-AC inhibitor sensitivity test was added (sensitivity 87% [95% IC 71–100%] and specificity 91%[95% IC 79–100%]). In conclusion, in vitro inhibition of Hsp90-AC in CLL using 17-AAG is an independent prognostic maker and strong predictor of the need for treatment. Hsp90-AC inhibition showed a high correlation with IgVH mutational status and ZAP-70 expression. Additionally, patients sensitive to 17-AAG whose leukemic cells also highly express ZAP-70 appear to have the worst prognosis with the shortest t-Tx1. Our data suggest that in vitro sensitivity to 17-AAG at the time of diagnosis serves as a quantitative surrogate marker for Hsp90-AC activity and correlates with disease progression in CLL. This is the first time that such correlation has been established in a cohort of cancer patients and shows the relevance of the Hsp90 chaperone system in cancer biology and disease progression. Disclosures: No relevant conflicts of interest to declare.