ALBERT

Description: Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.

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: Key Points USP22 deficiency in Ras-driven myeloproliferative neoplasm blocks myeloid differentiation promoting acute myeloid leukemia. USP22 is a PU.1 deubiquitylase that positively regulates PU.1 stability and the expression of myeloid-differentiation genes.

Description: Mutations in Ras pathway are among the most common gene abnormalities in several types of cancer including hematological malignances. Ras gene (NRAS or KRAS) mutations are found in 20-40% of myeloid malignances and in 15-30% of acute lymphoblastic leukemia. Mutations in KRAS are the second most frequently mutated. Expression of the most common KRAS mutation (G12D) in mouse hematopoietic cells causes a fatal myeloproliferative neoplasm (MPN). The disease observed in these mice resembles the human MPNs juvenileand chronic myelomonocytic leukemias characterized by leukocytosis, splenomegaly and anemia. Patients with these MPNs typically survive for 10 months to 4 years and are still considered incurable. Previous studies comparing mouse and human samples led to the identification of USP22 (Ubiquitin Specific Peptidase 22) as part of a self-renewal gene expression signature in different types of cancer including hematological malignances. We analyzed the publicly available database, Oncomine, and showed that USP22 is highly expressed in acute myeloid and lymphoid leukemias. Moreover, USP22 was found as part of an oncogenic network in acute myeloid leukemia with mutations in an upstream activator of Ras signaling. Thus, to study a possible role of Usp22 in Ras-induced myeloproliferative disease, we generated KrasG12D/+ Mx1Cre/+ (hereby named KM), KrasG12D/+ Mx1Cre/+ Usp22F/F (KMU) and Mx1Cre/+ Usp22F/F (MU) mice. Analysis of peripheral blood cells showed no changes in blood cell counts in MU mice compared to those of control mice, indicating that Usp22 deletion does not affect steady state hematopoiesis. As expected, KM mice had an increase in neutrophils and monocytes as previously reported. In contrast, KMU mice showed a dramatic decrease in neutrophil counts, suggesting that USP22 is possibly involved in KRAS-induced myeloproliferative disease. Analysis of bone marrow populations showed no significant changes in the frequency of HSCs, GMP, CMP or MEP populations between KM and KMU mice. However, there was a statistically significant decrease in the Gr1+ CD11b+ population in KMU compared to KM mice. This decrease was mostly associated with a decrease in Gr1 expression and accumulation of CD11b+ cells. We also performed studies using colony-forming assays in methylcellulose media. Bone marrow cells from KM mice were able to form colonies in the absence of cytokines as previously reported. However, Usp22 deletion (KMU mice) decreased cytokine-independent growth by more than 50%. Previous reports have shown that transplantation of KM bone marrow cells lead to the generation of myeloproliferative disease and thymic lymphomas in recipient mice. To investigate whether Usp22 deletion could limit the generation of these malignances, we transplanted bone marrow cells from KM and KMU mice into lethally irradiated mice. Although KMU recipient mice died earlier than KM recipient mice for unknown reasons, myeloproliferative disease was observed in only 25% of KMU recipient mice compared to 75% of KM recipient mice. Moreover, the thymus size of KMU recipient mice was statistically significant smaller than those of KM recipient mice. In summary, we found that Usp22 may play an important role in Kras-driven myeloid and lymphoid malignances. Usp22 deletion in KM mice induced a dramatic decrease in neutrophil counts, decreased numbers of Gr1+ CD11b+ cells in the bone marrow and decreased cytokine-independent growth of KM bone marrow cells. Moreover, transplantation of KMU cells into lethally irradiated recipients resulted in decreased generation of myeloid and lymphoid malignances. We are currently performing additional studies to dissect the molecular mechanism by which Usp22 affects Kras-driven hematological malignances. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

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 4638 High-dose methylprednisolone (HDMP) and the fully human monoclonal anti-CD 20 Ofatumumab each can produce partial responses in CLL, even though complete remissions are not often observed following treatment with either agent alone. Previously we have reported that the combination of Rituximab and HDMP is an effective non-myelosuppressive treatment combination for previously untreated CLL patients as well as in patients that are fludarabine refractory. We report here our single institution experience of treatment using the combination of HDMP and Ofatumumab in CLL patients that were not considered to be good candidates for chemotherapy treatment due to comorbidities, poor performance status, profound cytopenias or because refractory status to fludarabine and/or alemtuzumab. Eight patients with progressive, symptomatic CLL were treated with HDMP 1 g/m2 IV daily × 3 every 28 days for three consecutive cycles, and ofatumumab administered based on package insert instructions (300 mg dose # 1 followed by 11 doses of 2,000 mg over a 6 month period) along with prophylactic antimicrobial therapy. The main objectives were to determine the safety, toxicity and clinical efficacy of this regimen. All of the patients were males with a median age of 69 years (range 49–78). The median of prior treatments was 4.5 including 4 patients that have received previously HDMP and rituximab and two patients that underwent matched unrelated donor stem cell transplant. All patients have been previously treated with rituximab, 75% of the patients failed or were intolerant to fludarabine and/or aletuzumab and 88% had high-risk disease by the modified Rai classification. Most of the patients had bulky disease with median lymph node product of 42 cms2 and median splenomegaly of 6. 5 cm (range 0–17) below the left costal margin. The median lymphocyte count was 28,000 cells/mm3. 75% of the patients had high-risk prognostic markers including unfavorable cytogenetics, unmutated IgVH region genes or high expression levels of ZAP-70. All patients completed the planned therapy with no major side effects or toxicities. There was no evidence of marrow suppression and even patients with pancytopenia improved their peripheral blood counts with this salvage regimen. During treatment patients experience significant decrease in peripheral white blood cell counts, increase in hemoglobin, elevation of platelets and a dramatic reduction in lymphadenopathy and splenomegaly. Response assessment based on the IW-CLL 2008 criteria showed that the overall response rate was 50% (4 partial remissions), 25% of patients had stable disease and the remainder showed progressive disease. Overall, these data suggest that the combination of HDMP and Ofatumumab is a safe and effective salvage regimen for high-risk CLL patients that otherwise were not candidates for additional treatment. The response rates observed for the combination of HDMP and Ofatumumab in this group of patients appear to be favorable with the majority of patients achieving a response to therapy or experiencing disease stabilization. Moreover, HDMP and Ofatumumab treatment was not associated with bone marrow suppression, which is a major limiting factor for treatment administration, making this regimen a potential valid alternative for high-risk CLL patients. Additional clinical studies of this combination are warranted. Disclosures: No relevant conflicts of interest to declare.

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.