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 1×108, 3×108 or 1×109 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.

We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense s (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 thimidinecontaining 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 s ), and was dependent on ABL kinase. We conclude that thymidinecontaining PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.

Chronic lymphocytic leukemia (CLL) cells express high-levels of Bcl-2-family proteins that inhibit apoptosis, enhancing leukemia-cell survival and drug-resistance. AT-101 (Ascenta Therapeutics, Inc.) is an orally active BH3-mimetic that inhibits the anti-apoptotic activity of Bcl-2, Bcl-XL, and Mcl-1, and induces CLL cells to undergo apoptosis. (Prada et. al., ASH 2005). AT-101 also enhanced the cytotoxicity of rituximab for CLL cells in vitro (James et. al., ASH 2005). These in vitro effects of AT-101 were concentration and time-dependent. We are conducting a phase 2 trial to evaluate the safety and activity of AT-101 by two dosing schedules, used in combination with rituximab to treat patients with relapsed/refractory CLL. We previously reported (Castro et. al., ASH 2006) on 12 patients who received up to 12 weeks of AT-101, 30 mg daily for 3 out of every 4 weeks, with rituximab, 375 mg/m2 for 12 doses (total dose = 4,500 mg/m2) on days 1, 3, 5, 8, 15, 22, 29, 31, 33, 40, 57, 59, 61. Here we describe initial results from a second cohort (n=6) treated with intermittent, “pulse” AT-101, 80 mg/d on days 1–3 and 15–17 of each 28-day cycle, in combination with weekly rituximab, 375 mg/m2/week. To date, 6 patients have received “pulse” AT-101. Patient demographic characteristics and risk prognostic status (ZAP70, IgVH mutational status, and cytogenetics / FISH) are comparable between the two dose cohorts. Gastrointestinal (GI) toxicity, the most notable adverse effect of AT-101 with daily administration, appears reduced with intermittent AT-101; 2/6 patients have had NCI-CTCAE Grade 1–2 GI toxicity, and 0/6 have had Grade 3–4 ileus, compared with 11/12 and 2/12 patients, respectively, in the daily dose cohort. Apoptosis of CLL cells evaluated by Annexin V FACS at the time of maximum AT-101 concentration, was present in 18–45% of cells in 4 of the 6 patients after a single 80 mg dose of AT-101. By comparison, apoptosis after a 30 mg AT-101 dose appeared lower and was detected in approximately 1–15% of cells. After 80 mg of AT-101, plasma concentrations of up to 6.6 μM have been observed compared with concentrations of approximately 0.8–1.8 μM after a 30 mg dose in the daily dose cohort. In the “pulse” AT-101 cohort we have observed partial responses (PR) in 3 patients while the other 3 are still receiving treatment. Five (5) out of 12 patients had a PR in the previously reported AT-101 continuous administration group. Intermittent administration of AT-101 with a “pulse” dose regimen appears associated with an increased pro-apoptotic effect in vivo and higher plasma concentrations, as well as reduced toxicity, when compared with daily dosing. Enrollment continues to confirm these observations and assess whether clinical activity in combination with rituximab is increased with “pulse” dosing of AT-101.

Chronic lymphocytic leukemia (CLL) is an ideal disease for therapeutic vaccine strategies. While the leukemia cells are usually stealth-like, avoiding T cell recognition, they can be manipulated through ligation of CD40 on their surface to become very effective antigen presenting cells (APCs). Ligation of CD40 leads to expression of CD80, CD86 and upregulation of CD54. Other biochemical changes occur upon ligation of CD40, including upregulation of CD95, DR5, and expression of Bid, predisposing the leukemia cells to death-receptor-induced apoptosis. CLL cells can be made to express CD154 (CD40-ligand) using a replication-defective adenovirus. A phase I clinical trial with autologous CLL cells transduced to express murine CD154 previously demonstrated tolerability and clinical activity with this strategy (Blood96:2917, 2000). More recently, a recombinant CD154 (ISF35) was produced, based on the human CD154 backbone, incorporating murine sequences needed for expression on CLL cells and with the proteinase cleavage site removed. We evaluated this new transgene in a phase I clinical trial, expecting to have similar tolerability to the murine CD154. Transduction of CLL cells results in expression of ISF35, ligation of CD40 on transduced and bystander cells, and the resultant downstream changes needed for antigen presentation and sensitivity to death receptor-induced apoptosis. We conducted a phase I study of a single infusion of autologous CLL cells transduced to express ISF35. Three dose levels were evaluated with 3 patients (pts) each: 1×108, 3×108, & 1×109 transduced cells. Infusions were well tolerated, no acute infusion-related toxicities were observed. ISF35-related toxicities consisted of grade 1–2 flu-like symptoms that occurred several hrs after the infusion and consisted of fever, arthralgia, myalgia, nausea, vomiting, and fatigue lasting 2–4 days and resolving in all cases. There were no dose-limiting toxicities at any dose level. Biologic responses were seen at all doses, there was no dose-response relationship. There were consistent decreases in absolute lymphocyte counts at all dose levels, indicating a therapeutic effect. This was not dose-related, and ALC returned to pre-treatment level after 1–2 months post-infusion. There was consistent induction of CD95 and DR5 expression on bystander cells in vivo by 3 days following infusion, which lasted 2–3 weeks. Furthermore, consistent induction of Bid expression in bystander cells was seen by wk 1, also lasting 2–3 wks. Finally, consistent increases in absolute T cell counts (both CD4+ & CD8+) were seen, peaking 1–4 wks post infusion. These results demonstrate that ISF34-transduced autologous leukemia cells can be given safely at up to 1×109 transduced cells, without dose-limiting toxicities, and resulting in phenotypic and biochemical changes in bystander leukemia cells in vivo that render the cells able to present antigen and priming them for death-receptor-induced apoptosis. Furthermore, clinical responses were seen with reduction in leukemia counts and increases in absolute T cell counts. We expect that multiple, sequential doses will be needed for maximal therapeutic effect with this strategy. Given these results, we have developed a phase II trial of repeated doses of autologous ISF35-transduced leukemia cells for patients with CLL.

6605 Background: CLL pts with early stage disease are often observed under a “watch and wait” approach. Unmutated immunoglobulin variable-region gene (IgVH); expression of zeta chain associated kinase (ZAP-70); CD38 expression; and cytogenetics abnormalities identify pts with a tendency for early disease progression. Whether pts with these high-risk features may benefit from early intervention warrants investigation in clinical trials. Gossypol, a naturally occurring compound in cotton seeds has been extensively studied in clinical trials and is well tolerated with a favorable safety profile. AT-101, a derivative of R-(-)-gossypol binds to Bcl-2 family proteins and induces apoptosis of CLL cells in vitro. Dysregulated expression of Bcl-2 proteins is critical for CLL leukemogenesis and is an attractive therapeutic target. Methods: We conducted a phase I study to evaluate the safety and tolerability of single agent AT-101 in treatment naïve CLL pts with high-risk features. Clinical activity, pharmacokinetics, and pharmacodynamics were assessed. Results: 7 pts were treated with AT-101 at doses of 20–40mg daily. Pt characteristics: median age 55, median Rai stage II, elevated ZAP-70 (57%), high CD38 (71%), unmutated IgVH (57%), trisomy 12 (43%), and loss of 17p (43%). AT-101 was well tolerated with no grade 4 toxicities, hospitalizations, deaths. Maximum toxicity in 6 evaluable pts was grade III transaminase elevation at week 7, with complete resolution following discontinuation of AT-101. Other most common adverse events, all Grade 1–2 include: elevated transaminases; nausea; fatigue; diarrhea; and hypokalemia. No hematologic toxicity was observed. 5/6 pts had decrease in lymphocyte count, 6/6 had reduction in lymphadenopathy, and 5/5 with palpable spleens had reduction in spleen size. Cmax for 30mg (n=3) and 40mg (n=2) dose of AT-101 was 570ng/ml and 660ng/ml at a Tmax of 4.3 and 4.4 hrs. Tmax correlated with in vivo apoptosis studies revealing maximum leukemic cell apoptosis occurring at 4hrs and poly(ADP-ribose) polymerase cleavage. Conclusions: AT-101 is safe and well tolerated, induces in vivo leukemia cell apoptosis, and may have clinical activity in previously untreated pts with CLL and high risk features. No significant financial relationships to disclose.

Ad-ISF35, an adenovirus vector encoding a membrane-bound engineered CD154 chimeric protein (ISF35), induces complete A20 lymphoma tumor regression in mice after intratumoral direct injection (IDI). Ad-ISF35 induced durable local and systemic antitumor responses associated with a rapid tumor infiltration of macrophages and neutrophils as well as increased levels of proinflammatory cytokines in the tumor microenvironment. Ad-ISF35 IDI transduced preferentially fibroblasts and macrophages present in the tumor microenvironment, and ISF35 protein expression was observed in only 0.25% of cells present in the tumor. Moreover, Ad-ISF35 IDI induced upregulation of CD40 in tumor and immune regulatory cells, including those that did not express ISF35, suggesting the presence of a strong bystander effect. These responses resulted in the generation of IFN-γ-secreting cytotoxic lymphocytes and the production of specific cytotoxic antibodies against lymphoma cells. Overall, cellular immune therapy based on ISF35 induced phenotypic changes in the tumor cells and tumor microenvironment that were associated with a break in tumor immune tolerance and a curative antitumor effect in this lymphoma mouse model. Our data highlight the potential activity that modulation of costimulatory signaling has in cancer therapy.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the monoclonal accumulation of well-differentiated CD5 B cells. Previously, we reported that CLL cells that use unmutated immunoglobulin VH (IgVH) genes and/or express ZAP-70 are more responsive to signaling induced by ligation of surface IgM compared to cells that use mutated IgVH and lack expression of ZAP-70 (Chen et al. Blood 2005) Because signaling through the Ig receptor appears to play a role in the pathogenesis or progression of CLL, targeting the Ig signal transduction pathway in CLL might have therapeutic utility, particularly for those patients with high-risk disease. Dasatinib (Sprycel) is a tyrosine kinase inhibitor (TKI) with antiproliferative activity against hematological and solid tumor cell lines and it is FDA approved for the treatment of patients with CML and Ph+ ALL. Contrary to Imatinib (Gleevec), Dasatinib is a potent TKI not only of the Abl family of kinases but also of Src kinases, which regulate Ig-receptor signal transduction and govern the early events following Ig receptor ligation. Because Dasatinib TKI profile and its potential role inhibiting BCR signaling we studied its in vitro activity in CLL. Primary leukemia cells from 40 different CLL patients were evaluated. We found that Dasatinib, but not Imatinib, induced apoptosis in CLL cells at doses that were pharmacologically achievable. The IC50 for Dasatinib was in the 30–100 nM range. Interestingly, the pro-apoptotic activity of Dasatinib in CLL cells was not observed in normal B, T cells or blood mononuclear cells of healthy donors, suggesting that Dasatinib has a specific effect on CLL cells. Dasatinib induced apoptosis in CLL cells in a time and concentration dependent manner. Peak apoptosis occurred after 2 hours of in vitro exposure. Leukemia cells that expressed ZAP-70 were significantly more sensitive to Dasatinib-induced apoptosis than CLL cells lacking expression of ZAP-70. In addition, Dasatinib enhanced in vitro the pro-apoptotic activity of Rituximab and Fludarabine in CLL cells. Dasatinib treatment of CLL cells induced changes in the expression profile of apoptosis related genes as well as changes in apoptosis related proteins such as cleavage of PARP-1 and Caspases. Moreover, CLL cells treated in vitro with Dasatinib showed reduced tyrosine kinase activity measured by ELISA and also by immunoblots of CLL-cell lysates using specific phospho-TK antibodies. In addition, Ig receptor signaling following surface IgM ligation was decreased in CLL cells that were pre-treated with Dasatinib relative to that of untreated CLL cells. In conclusion, Dasatinib induces apoptosis of CLL cells at low nanomolar concentrations that do not appear to affect the viability of B cells, T cells, or blood mononuclear cells of healthy adults. CLL cells that expressed ZAP-70 were significantly more sensitive to Dasatinib than CLL cells that lacked expression of ZAP-70. This process was associated with impairment of BCR signaling, decrease TK activity and regulation of genes and proteins related to apoptosis. In addition, treatment of CLL cells with Dasatinib enhanced the in vitro activity of Rituximab and Fludarabine. These results reveal that Dasatinib is potentially active in CLL, providing a rationale for clinical trials evaluating its clinical activity in the treatment of patients with this disease.

No abstract