Courier and U.S. Mail Address: UCSD Moores Cancer Center Blood & Marrow Transplantation Division 3855 Health Sciences Drive, #0960 La Jolla, CA 92093-0960 USA
Abstract: Ad-ISF35 is an adenovirus (Ad) vector that encodes a mouse-human chimeric CD154. Ad-ISF35 induces activation of chronic lymphocytic leukemia (CLL) cells converting them into CLL cells capable of promoting immune recognition and anti-leukemia T-cell activation. Clinical trials in humans treated with Ad-ISF35-transduced leukemia cells or intranodal injection of Ad-ISF35 have shown objective clinical responses. To better understand the biology of Ad-ISF35 and to contribute to its clinical development, we preformed studies to evaluate biodistribution, persistence and toxicity of repeat dose intratumoral administration of Ad-ISF35 in a mouse model. Ad-ISF35 intratumoral administration induced tumor regression in more than 80% of mice bearing A20 tumors. There were no abnormalities in the serum chemistry. Mice receiving Ad-ISF35 presented severe extramedullary hematopoiesis and follicular hyperplasia in the spleen and extramedullary hematopoiesis with lymphoid hyperplasia in lymph nodes. After Ad-ISF35 injection, the vector was found primarily in the injected tumors with a biodistribution pattern that showed a rapid clearance with no evidence of Ad-ISF35 accumulation or persistence in the injected tumor or peripheral organs. Furthermore, pre-existing antibodies against Ad-5 did not abrogate Ad-ISF35 anti-tumor activity. In conclusion, intratumoral administration of Ad-ISF35 induced tumor regression in A20 tumor bearing mice without toxicities and with no evidence of vector accumulation or persistence.Cancer Gene Therapy advance online publication, 9 March 2012; doi:10.1038/cgt.2012.6.
Abstract: New therapies for chronic lymphocytic leukemia (CLL) are needed, particularly those that can eradicate residual disease and elicit anti-CLL immune responses. CD40 ligation on CLL cells, which can be achieved using adenovirus encoding chimeric CD154 (Ad-ISF35), enhances their ability to function as antigen presenting cells and increases their sensitivity to clearance by immune-effector mechanisms. In this study, we report the results of a first-in-man Phase I trial of intranodal direct injection (IDI) of Ad-ISF35 in patients with CLL to evaluate toxicity, safety, and tolerability. Fifteen patients received a single IDI of 1-33 x 1010 Ad-ISF35 viral particles (vp), with a defined maximum tolerated dose as 1x1011 vp. Although the most common adverse events were transient grade 1-2 pain at the injection site and flu-like symptoms following IDI, some patients receiving the highest dose had transient, asymptomatic grade 3-4 hypophosphatemia, neutropenia, or transaminitis. Increased expression of death receptor, immune co-stimulatory molecules, and Ad-ISF35 vector DNA was detected in circulating CLL-cells. Notably, we also observed preliminary clinical responses, including reductions in leukemia cell counts, lymphadenopathy, and splenomegaly. Six patients did not require additional therapy for more than 6 months, and 3 achieved a partial remission. In conclusion, Ad-ISF35 IDI was safely delivered in patients with CLL and induced systemic biological and clinical responses. These results provide the rationale for phase II studies in CLL, lymphomas, and CD40-expressing solid tumors.
Abstract: Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.
Abstract: The treatment of low- and intermediate-grade subtypes of malignant lymphoma continues to evolve. Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma (NHL) and is generally considered incurable. Although high response rates can be achieved with initial chemotherapy, median survival is only 3-4Â years. Intensified consolidation with high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) has been reported to improve progression-free survival (PFS), but most patients eventually relapse. Indolent lymphoma accounts for 35% of all NHL and is associated with a median survival of 9 years. Similar to MCL, it is also generally considered incurable, and the PFS also appears to be improved following HDT/ASCT. We initiated a pilot study to evaluate idiotype (Id) vaccination following HDT and ASCT for patients with MCL, indolent, and transformed NHL to evaluate the ability of Id-keyhole limpet hemocyanin (KLH) to induce immune responses, and to evaluate overall survival (OS) and PFS. We treated 15 patients: 8 with MCL, 4Â with follicular lymphoma, 1 with small lymphocytic lymphoma, and 2 with transformed lymphoma. After a median follow-up of approximately 6.3 years (range: 1-9), PFS and OS at 9.05 years from time of ASCT are 59% and 52%, respectively.
Abstract: Preclinical data demonstrate enhanced antitumor effect when lumiliximab, an anti-CD23 monoclonal antibody, is combined with fludarabine or rituximab. Clinical data from a phase 1 trial with lumiliximab demonstrated an acceptable toxicity profile in patients with relapsed or refractory chronic lymphocytic leukemia (CLL). We therefore pursued a phase 1/2 dose-escalation study of lumiliximab added to fludarabine, cyclophosphamide, and rituximab (FCR) in previously treated CLL patients. Thirty-one patients received either 375 mg/m(2) (n = 3) or 500 mg/m(2) (n = 28) of lumiliximab in combination with FCR for 6 cycles. The toxicity profile was similar to that previously reported for FCR in treatment of relapsed CLL. The overall response rate was 65%, with 52% of patients achieving a complete response (CR), which compares favorably with the CR rate previously reported for the FCR regimen alone in relapsed CLL. The estimated median progression-free survival for all responders was 28.7 months. The addition of lumiliximab to FCR therapy is feasible, achieves a high CR rate, and does not appear to enhance toxicity in previously treated patients with CLL. A randomized trial comparing lumiliximab plus FCR with FCR alone is underway to define the benefit of this combination in relapsed CLL. This trial was registered at clinicaltrials.gov as NCT00103558.
Abstract: Ligation of CD40 on chronic lymphocytic leukemia (CLL) cells induces phenotypic and biochemical changes that facilitate CLL cell-T cell interactions and enhances the sensitivity of CLL cells to clearance by adaptive and innate immune-effector mechanisms. CLL cells can be transduced to express CD40 ligand (CD154) using a replication-defective adenovirus vector, thereby cross-linking CD40 on transduced and non-transduced, bystander CLL cells. In a previous study, patients received infusions of autologous CLL cells, transduced to express murine CD154 (mCD154), which induced anti-leukemic immune responses, but also anti-mCD154 antibodies. In this study, we report a phase I study, in which patients were infused with 1 × 10(8), 3 × 10(8) or 1 × 10(9) autologous CLL cells transduced ex vivo to express ISF35, a humanized, membrane-stable CD154. Infusions were well tolerated and consistently followed by reductions in blood lymphocyte counts and lymphadenopathy. After infusion, circulating CLL cells had enhanced or de novo expression of CD95, DR5, p73 and Bid, which enhanced their susceptibility to death-receptor-mediated or drug-induced apoptosis, including CLL cells with deletions at 17p13.1 (del(17p)). Two patients who had CLL with del(17p) had subsequent chemoimmunotherapy and responded well to treatment. In summary, infusions of autologous, ISF35-transduced CLL cells were well tolerated, had biological and clinical activity, and might enhance the susceptibility of CLL cells with del(17p) to chemoimmunotherapy.
Abstract: We observed that high-dose methylprednisolone (HDMP) and rituximab was well tolerated and had promising activity when used in combination to treat patients with fludarabine-refractory chronic lymphocytic leukemia (CLL). This prompted us to evaluate the use of these agents in frontline therapy. A total of 28 patients with a median age of 65 years enrolled in this study. Patients received HDMP at 1 g/m(2) each day for 3 days during each of the three 4-week cycles together with rituximab and prophylactic antimicrobial therapy. The treatment was well tolerated with few adverse events of grade III or higher. The overall response rate was 96% (N=27). Nine patients (32%) achieved a complete remission (CR), two of which were without detectable minimal residual disease (MRD). Six patients with MRD received consolidation with alemtuzumab; five of these patients achieved an MRD-negative CR. With over 3 years of follow-up median progression-free survival was 30.3 months with only 39% of patients requiring additional therapy, and an overall survival was 96%. This study demonstrates that HDMP and rituximab is an effective nonmyelosuppressive treatment combination for patients with CLL that warrants consideration particularly for patients with limited myeloid reserve that might not tolerate standard treatment regimens.
Abstract: Autologous peripheral blood stem/progenitor cell transplantation (APBSCT) has been investigated as a potential therapeutic option to improve outcome in patients with acute myelogenous leukemia (AML). However, its optimal role in treatment for adults in remission has not been clearly established. We performed a retrospective analysis on 45 patients aged 21 to 73 years (median 51 years) with de novo AML who underwent APBSCT stratified by age, complete remission status, and cytogenetic risk. The 5-year disease-free survival (DFS) for all patients was 33.9% (95% confidence interval [CI], 20.1%-53.7%) and overall survival (OS) was 43.6% (CI, 29.2%-62.8%). For patients under the age of 60 years, the 5-year DFS for intermediate and high cytogenetic risk was 53.3% (CI, 23.5%-85.6%) and 50.0% (CI, 16.1%-100.0%); the 5-year OS for patients under the age of 60 years with low, intermediate, and high cytogenetic risk was 80.0% (CI, 40.0%-100.0%), 60.0% (CI, 31.2%-90.7%), and 75.0% (CI, 39.0%-100.0%), respectively. For patients over the age of 60 years, the 5-year DFS and OS for intermediate cytogenetic risk was 21.4% (CI, 7.9%-58.4%) and 21.4% (CI, 7.9%-58.4%). The DFS and OS of these patients are comparable to the historic survival of those who underwent allogeneic stem cell transplantation when adjusted by age. In addition, there was no treatment-related mortality (TRM). We conclude that APBSCT is a reasonable and safe intensive consolidation for patients with AML who do not have a suitable HLA-matched donor.
Abstract: We examined the clinical response of fludarabine-refractory CLL patients treated with high-dose methylprednisolone (HDMP) and rituximab. Fourteen patients were treated with three cycles of rituximab (375 mg/m(2) weekly for 4 weeks) in combination with HDMP (1 gm/m(2) daily for 5 days). All patients were refractory to fludarabine and 86% had high-risk disease by the modified Rai classification. In all, 79% of the patients had CLL cells that expressed ZAP-70 and three patients had poor prognostic cytogenetics. The overall response rate was 93% and the complete remission rate was 36%. The median time-to-progression was 15 months and the median time-to-next treatment was 22 months. Median survival has not been reached after a median follow up of 40 months. Four patients have died of progressive disease. Patients tolerated the treatment well and serious adverse events were rare. This allowed patients to receive all planned treatments on schedule with no dose modifications. All but one patient responded to treatment and the overall survival and time-to-progression were superior to those of other published salvage regimens.
Abstract: We examined the sera of six patients before and after i.v. infusions of autologous chronic lymphocytic leukemia (CLL) cells transduced ex vivo with an adenovirus encoding CD154 (Ad-CD154). Five patients made high-titer antibodies against adenovirus and three made IgG reactive with a leukemia-associated surface antigen, which we identified as ROR1. Anti-ROR1 antibodies were not detected in the sera of untreated patients. We generated anti-ROR1 mAbs and found they reacted specifically with the CLL cells of all patients, but not with nonleukemic leukocytes, a wide variety of normal adult tissues, or blood mononuclear cells, including CD5(+) B cells of healthy adults. ROR1 could bind Wnt5a, which induced activation of NF-kappaB when coexpressed with ROR1 in HEK293 cells and enhanced the survival of CLL cells in vitro, an effect that could be neutralized by posttreatment anti-ROR1 antisera. We conclude that patients with CLL can break immune tolerance to ROR1, which is an oncofetal surface antigen and survival-signaling receptor in this neoplastic disease.
Abstract: Interpatient variability in the kinetics of peripheral blood progenitor cell (PBPC) mobilization is commonly seen with conventional chemotherapy-based mobilization regimens. This necessitates the availability of leukapheresis (LP) facilities 7 days a week.
Abstract: Intravenous infusion of autologous chronic lymphocytic leukemia (CLL) cells transduced with an adenovirus encoding CD40-ligand (CD154) caused rapid reductions in leukemia-cell counts and lymphnode size. We hypothesized that CD40-ligation via CD154 sensitized CLL cells to death-receptor-mediated apoptosis. We found that CD154-expressing cells induced expression of CD95 and the BH3-interacting-domain death agonist (Bid) in CLL, regardless of whether the leukemia cells had functional p53. Such treatment also induced p73, a p53-related transcription factor regulated by c-Abl kinase, and enhanced the sensitivity to fludarabine (F-ara-A) of CLL cells lacking functional p53. Transduction of CLL cells with an adenovirus encoding p73 also induced Bid and CD95 and enhanced the sensitivity to F-ara-A of p53-deficient CLL cells. However, inhibition of c-Abl with imatinib suppressed CD154-induced expression of p73, p73-induced expression of Bid and CD95, and blocked the sensitization of p53-deficient CLL cells to CD95-mediated or F-ara-A-induced apoptosis. Conversely, CLL cells transduced with an imatinib-resistant c-Abl mutant could be induced by CD154 to express p73 and Bid even when treated with imatinib. These results indicate that CD154 can sensitize leukemia cells to apoptosis via the c-Abl-dependent activation of p73 and mitigate the resistance of p53-deficient CLL cells to anticancer drug therapy.
Abstract: We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense (G3139-PS), with other PS-ODN or phosphodiester-ODN (PO-ODN) in their relative capacity to induce apoptosis of chronic lymphocytic leukemia (CLL) B cells in vitro. Surprisingly, we found that thymidine-containing PS-ODN, but not PO-ODN, induced activation and apoptosis of CLL cells independent of BCL-2 antisense sequence or CpG motifs. All tested thimidine-containing PS-ODN, irrespective of their primary sequences, reduced the expression of Bcl-2 protein and increased the levels of the proapoptotic molecules p53, Bid, Bax in CLL cells. Apoptosis induced by thymidine-containing PS-ODN was preceded by cellular activation, could be blocked by the tyrosine-kinase inhibitor imatinib mesylate (Gleevec), and was dependent on ABL kinase. We conclude that thymidine-containing PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.
Abstract: We assessed remission rates and toxicity in 24 consecutive elderly (age>or=60) patients with untreated Acute myeloid leukemia (AML) who received the anthracycline-free combination of fludarabine, cytosine arabinoside and G-CSF (FLAG) as initial induction chemotherapy at our center. CR was achieved following one cycle of FLAG in 14 patients (58%). Another four patients cleared blasts from their bone marrow by day 30 without complete platelet recovery. Three patients died from infections prior to neutrophil recovery (12%). No other grade 3/4 toxicities and no clinically significant mucositis were seen. No significant association was found between age, WBC and cytogenetic risk group with likelihood of achieving CR. Fifteen patients proceeded to consolidation therapy and seven patients received a stem cell transplant (six autologous, one allogeneic). Primary induction with FLAG in elderly AML patients achieves a high remission rate without prohibitive mucosal or cardiac toxicity and may thus be considered as an alternative to standard anthracycline-based regimens in this setting.
Abstract: Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 microM) in the presence of 0.1 mM H2O2 demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.
Abstract: The zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL.
Abstract: We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgV(H) mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.
Abstract: The clinical use of oligonucleotide (ODN) therapeutics has been hampered by their limited ability to penetrate intact cells. To identify ODN properties that would facilitate cellular uptake, we developed a repetitive selection procedure using an ODN library containing at least 10(14) different molecules and human B lymphoma cells as a target. Natural phosphodiester single-stranded DNA ODNs (R-aptamers) were obtained after 10 rounds of selection. A common feature in the R-aptamers was guanine-rich 3' terminal sequences, and many also contained potential immunostimulatory (ISS) CpG sequence motifs. Two R-aptamers (R10-60 and D-R15-8) with the predominant shared characteristics were selected for further study on primary human chronic lymphocytic leukemia (CLL) B cells, which are well known to be difficult to transfect and activate. Flow cytometry analysis of the CLL cells demonstrated that the fluorochrome-labeled R-aptamers were internalized much more efficiently than nonselected random sequence ODN. Studies on sequence modifications indicated that efficient uptake required ODN multimerization, that was promoted by guanine-rich sequences at the 3' terminus. In addition, CLL cells that were exposed to the aggregating R-aptamers containing CpG motifs were strongly activated, as indicated by upregulation of CD40 levels as compared to cells treated with nonaggregating CpG R-aptamers. Together, these findings suggest that the sequence compositions in R-aptamers that promote multimerization and contain optimal ISS CpG motifs facilitate the delivery of ISS-ODN to CLL cells and enhance the activation of these cells.
Abstract: The field of BMT has entered a new phase based on insights into basic molecular and cellular biology. The mechanism of cure mediated by allogeneic HSCT is now believed to be at least partially mediated by cellular attack against tumor-associated antigens. Better understanding of these cellular targets and the means of targeting them specifically will allow a more precise application of cellular therapy than is currently possible. This in turn may make it possible to design therapies that are increasingly selective without the collateral toxicities of GVHD, infection, and direct organ damage that currently are the limiting features of allogeneic HSCT as practiced until recently. However, it remains a challenge to demonstrate that disease-control using reduced intensity preparative regimens is at least comparable to that achieved by traditional myeloablative HSCT. This will require carefully controlled studies in each the major diseases that have shown sensitivity to the immune-mediated effects of HSCT. The chapters below will highlight progress towards the goals of elucidating the role of NST.
Abstract: The flow cytometric analysis of apoptosis in lymphocytes from in vivo samples has been difficult because of the low frequency of apoptotic events. To overcome this obstacle, many investigators have relied on in vitro incubations to increase the number of apoptotic cells before analysis. In this report, we show that an adaptation of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay for use in flow cytometry can be used to detect rare apoptotic lymphocytes from freshly harvested LN suspensions. This approach is both specific and extremely sensitive. This method also is amenable to multiparameter analyses and allows a phenotypic analysis of these rare apoptotic cells. However, we observed that some monoclonal antibodies can stain apoptotic-but not viable-cells nonspecifically. Therefore, the specificity of all antibodies to stain apoptotic cells was confirmed in competition assays.
Abstract: A major mechanism maintaining immune tolerance is the deletion of potentially autoreactive thymocytes by apoptosis during development in the thymus. Previous reports suggest that apoptosis is induced by high avidity signals transduced via the T cell receptor; however, the role of signals transduced by other cell surface receptors during thymic selection remains poorly understood. Fas, a member of the TNF receptor family, has been shown to induce apoptosis in mature peripheral T cells; however, the effects of Fas on negative selection of thymocytes have not been previously detected. Using a sensitive terminal deoxynucleotidyl transferase method to detect apoptotic cells, we found that mutant Fas molecules in lpr mice decrease the sensitivity of thymocytes to T cell receptor-mediated apoptosis and that blockade of Fas-Fas ligand interactions in vivo can inhibit antigen-induced apoptosis of thymocytes in non-lpr mice. Thus, we have shown that Fas, in conjunction with antigen-specific signals, can modulate apoptosis during negative selection of thymocytes.
