ALBERT

Description: Background: Cirmtuzumab is a first-in-class humanized mAb specific for ROR1, an oncoembryonic antigen found on CLL and cancer stem cells of various cancers, but not on normal post-partum tissues. Work has defined that ROR1 serves as a receptor for Wnt5a, which induces non-canonical Wnt-signaling that promotes planar-cell-polarity, migration, stem-cell renewal, and proliferation. Recent studies demonstrated that Wnt5a binds to ROR1 and induces activation of RhoA and Rac1, promoting leukemia-cell migration and proliferation, respectively. By binding a functional epitope in the extracellular domain of ROR1, cirmtuzumab can block ROR1-dependent, non-canonical Wnt5a-signaling. Methods: We conducted a phase 1 trial of cirmtuzumab in patients (pts) with progressive, relapsed/refractory CLL who were not amenable to approved therapies. The primary aims of the study were to evaluate the safety and tolerability of cirmtuzumab and determine the maximum tolerated dose and/or recommended phase 2 dose. Secondary endpoints included evaluation of antibody pharmacokinetics (PK) and pharmacodynamics (PD). To address these endpoints, pts received only 4 biweekly infusions of antibody at doses ranging from 15 mcg/kg to 16 mg/kg in a standard 3+3 pt-per-cohort schema. Results: As of a planned analysis, 20 pts have received cirmtuzumab. Pts tolerated cirmtuzumab extremely well without any noted drug-related severe adverse events or dose-limiting toxicities. Anemia (7 pts), thrombocytopenia (4 pts), and neutropenia (3 pts) were the most common AEs, were primarily grade 1, and were likely due to late-stage-disease-related cytopenias. We developed an ELISA assay to measure serum levels of cirmtuzumab capable of binding to the targeted epitope of ROR1. Peak concentrations were detected within one hour after the completion of each infusion. The serum concentrations of active cirmtuzumab increased with each subsequent infusion. We detected significant levels of cirmtuzumab for at least eight weeks following the last infusion and determined that it has half-life of 〉24 days (d). Twenty-four hours following the 1st infusion, the leukemic cells of pts treated at doses 〉/= 2 mg/kg had inactivation of RhoA and Rac1, which were each observed to be activated in all cases prior to therapy. Loss of GTPase activation also was observed for CLL cells sampled at later time points. Clinically, we observed that most pts had an initial increase in the ALC without evidence of disease progression, similar to what is observed with targeted therapies that inhibit B-cell-receptor/chemokine signaling. Such a redistributive lymphocytosis (defined as an increase in the ALC of 10% or more) was observed in 5 of 9 pts who received doses /=2mg/kg. The lymphocytosis typically peaked 24 hours after dosing (median 36% increase, range 10-76%), with a subsequent reduction to at or below baseline levels. Most pts had stable disease when assessed at the completion of treatment. Of evaluable pts, 12 had stable disease and 2 had progressive disease, both of whom received

Description: Background: Chimeric Antigen Receptor (CAR) T cell therapy is a promising cancer immunotherapy that is growing exponentially. The doubling time of medical knowledge in 2010 was 3.5 years, and the projection for 2020 is just 73 days. In the last five years, the number of PubMed publications on cancer applications of CAR T cells has tripled. Therefore, to remain updated in the field represents a challenge for patients, care providers and researchers. In this review we provide a focused summary of the currently ongoing clinical trials, with a comprehensive overview of advances in CAR T cell therapy, beyond CD19, emphasizing on antigenic targets, development phases, and leading sponsor pharmaceutical companies. Methods: We retrieved the available data from the national registry of clinical trials (clinicaltrials.gov) using the following keywords: "CAR T cell", "CAR T cell and cancer", "chimeric antigen receptor", "CAR T AND tumor antigen", 'CAR T cell antigens", "Tumor antigens targeted by CAR T cells", "engineered T cells", "modified T cell", "CAR T cells in Cancer", "CAR T cell therapy", "CAR T cell therapy AND Cancer" until December 31, 2018 and manually excluded the trials unrelated to CAR T-cell therapies on cancer, by reviewing the detailed information provided on the website as well as preliminary data published. Results: The analysis included 271 clinical trials posted on the clinicaltrials.gov website from the United States by the cut-off date. For efficacy analysis, we retrieved information from 52 trials, by NCT number on a PubMed search. The majority of CAR T clinical research is focused on hematological cancer (57%), followed by CNS 8%, GI 6%, Skin 5%, Genitourinary 4%, Breast 4%, Gynecologic 4%, Respiratory 3%, Sarcoma 2%, Mesothelioma 2% and others 5%. The most used target in CAR T cell therapy and the leaders in phase 3 trials are CD19 (42%) and BCMA (12%), followed by CD20, NY-ESO-1, Mesothelin, HER2, GD2, MAGE-A3 and CD30. An essential step in CAR T cell therapy development is the selection of the right antigen/target. Here, we provide an overview of the clinically relevant targets that are actively being using by clinical trials in the United States. For example, CD19 appears to be a leading target regarding CAR T cell therapy on cancer with 116 trials (42% of total CAR T cells trials) on going just in the United States with a significant increment in the previous years. Similarly, with BCMA is one of the targets with more phase 3 trials (Figure 1) with promising results on patients with Multiple Myeloma with and the objective response of 85%, CR 45%, and PFS of 11.8 months. Second-generation CARs with either CD28 or 4-1BB as costimulatory signaling domain are preferred, with 4-1BB being the most commonly chosen. Conclusions: Our findings show growing trends in the development of CAR T cell-based therapies, combination and possible retargeting therapies in the future for solid tumor and hematologic malignances; taking into account the amount of important information and the complexity of the database, we have developed this analysis to understand how to generate in the future a friendly platform for researchers and patients to have an detailed overview of the clinical trials in cellular therapies Disclosures No relevant conflicts of interest to declare.

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: The NFκB transcription factors p50/p65 regulate the expression of genes encoding various growth-promoting factors and anti-apoptotic proteins, such as the cellular inhibitors of apoptosis (c-IAPs), Caspase-8/Flice-inhibitory protein (FLIP), A1 (also known as Bfl1), tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) and 2 (TRAF2). Furthermore, constitutive activation of NFκB has been observed in many tumor types, supporting the notion that activation of NFκB can play a causal role in tumor development and/or progression. Studies have shown chronic lymphocytic leukemia (CLL) cells experience activation of NFκB in vitro upon ligation of their surface immunoglobulin (Ig), which commonly possesses polyreactive-binding activity for many self-antigens. Other studies also have found that CLL cells from different patients vary in their capacity to undergo B-cell-receptor signaling following ligation of their surface Ig receptors, a capacity that appears associated with leukemia-B-cell expression of the zeta-associated protein of 70 kD (ZAP-70). We examined whether CLL B cell expression of ZAP-70 also was associated with the capacity to activate NFκB upon surface Ig ligation. For this we used CLL B cells of 8 different patients that expressed ZAP-70 and CLL B cells from 8 other patients that had negligible expression of this tyrosine kinase (as assessed by immunoblot and flow cytometric analysis). The CLL B cells of these two groups of patients had similar expression levels of surface, allowing us to use a F(ab’)2 anti-human IgM (anti-μ) to effect comparable surface Ig receptor ligation. Following treatment with anti-μ, we observed early and sustained degradation of IκB-α, thereby releasing cytoplasmic p50/p65 to the nucleus - the hallmark of NFκB activation. Moreover, this was associated with subsequent increased expression of NFκB target genes. In contrast, similar events were not observed following treatment with anti-μ in the cases lacking expression of ZAP-70. Also, activation of NFκB in ZAP-70+ cases was associated with a greater release of intracellular calcium and calcium flux following treatment with anti-μ than observed in ZAP-70-negative cases. Both calcium flux and activation of NFκB induced by anti-μ in these leukemia cells could be inhibited by Cyclosporine-A, indicating that these responses were mediated via a calmodulin-calcineurin-dependent pathway. These studies reveal that expression of ZAP-70 in B cell CLL is associated with a greater capacity to induce activation of NFκB following ligation of surface Ig, a characteristic that might account for the more aggressive clinical behavior of patients with leukemia B cells that express this tyrosine kinase. Moreover, if constitutive activation via ligation of surface Ig with self-antigen in vivo leads to activation of NFκB, then targeting the calmodulin-calcineurin-dependent pathway might have therapeutic potential for this subset of patients with this disease.

Description: 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. (Blood. 2005;106:2506-2512)

Description: We have previously reported that intermediate dose cyclophosphamide followed by sequential GM-CSF and G-CSF (iCy/GM/G) provides efficient mobilization for patients undergoing autografting. Furthermore, the predictable time course of mobilization with this regimen obviates the need for weekend leukaphereses (Blood 2003: 957a). Recently, the addition of rituximab to mobilization regimens for B-cell NHL has been shown to be effective at depleting contaminating B-cells from the leukapheresis product. However, the effect of rituximab administered for in-vivo purging, on mobilization and stem cell collection parameters is unclear. We compared leukapheresis (LP) yield parameters, and the time course of stem cell mobilization in 23 consecutive B-cell NHL patients mobilized with iCy/GM/G plus rituximab (group 1) with 27 consecutive B-cell NHL patients mobilized with the same regimen without rituximab (group 2). The iCy/GM/G regimen consisted of cyclophosphamide 1.5g/m2 (d1), GM-CSF 500 mcg/d (d 3–7), G-CSF (d 8 until completion of LP) 600mcg/d for weight ≤80kg, 960 mcg for weight 〉 80 kg. Rituxan was administered at 375mg/m2 as a single dose on d8. LP was begun on d 11 irrespective of WBC. D1 was usually a Friday in order to avoid weekend LP. Patients underwent up to 20 liter LP for ≤ 5 days (median =3, range 1–5 for both groups) with a target collection of 〉 5 x 10e6 CD34+ cells/kg. The groups were well matched for median age, gender, number of prior chemotherapy regimens (median=2 for both groups), prior pelvic XRT and histological subtype of B-NHL (p=NS in all cases). The estimated (Kaplan-Meier) cumulative probability of achieving a target collection of 2 x 10e6 CD34+ cells/kg on d 1–5 was 0.43, 0.70, 0.78, 0.84, 0.84 respectively for group 1 and 0.22, 0.69, 0.77, 0.84, 0.84 respectively for group 2. The corresponding probabilites of achieving 5 x 10e6 CD34+ cells/kg on d 1–5 were 0.22, 0.39, 0.57, 0.57, 0.57 (group 1) and 0.11, 0.30, 0.46, 0.59, 0.59 (group 2) (p=NS Log-rank test). Percentage of CD34+ cells in the LP product (LP CD34%) was measured daily. Maximums LP CD34% was seen on LP d1 for both groups with a fall on subsequent days (p=NS between groups 1 and 2). Toxicities experienced were generally mild consisting mostly of bone pain and fevers and were similar in both gropups. No patient required admission for febrile neutropenia. The number of CD34+ cells infused were similar for both groups (median 5.9 vs.5.7 x10e6 CD 34+ cells/kg). Median time to reach ANC 〉 500/mm3 and platelets 〉 20,000/mm3 were identical between groups 1 and 2 (d11 and d 10 respectively). These data show that the addition of rituximab administered on d 8 to the iCy/GM/G regimen in patients with B-NHL does not impair the yield of CD34+ cells, or the tolerability of the regimen. Furthermore, the time course of the mobilization and therefore the predictbility of the collection is not compromised. Maximum cumulative yield of CD34+ cells is achieved within 4 days of LP with no patient benefitting from a fifth day of collection. The additional cost and inconvenience of weekend leukapheresis can be avoided in all cases using this regimen.

Description: 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.

Description: High-dose gluocorticoids and the anti-CD20 mAb Rituximab each can effect partial responses in patients with chronic lymphocytic leukemia (CLL), although complete and durable responses to treatment with either of these agents have not been reported. We examined whether patients with relapsed and/or refractory CLL could respond to these agents when used in combination in a pilot clinical trial. Fourteen patients with progressive, symptomatic, and relapsed/refractory disease were treated with three four-week cycles of high-dose Methylprednisolone (HDMP) at 1gr/m2 daily for 5 days and weekly Rituximab® at 375mg/m2 for four weeks. The median age of the patients was 60 years, the male to female ratio was 4:1, the ECOG performance status was 〈 2, and the average number of prior treatments was 2. All patients failed or were intolerant to fludarabine and 86% had high-risk disease by the modified Rai classification. Sixty-five percent of the patients had CLL cells that expressed ZAP-70 and unmutated immunoglobulin variable region genes. Response assessment was performed at the end of each cycle, two months after completion of treatment, and each 3–6 months thereafter until the patients experienced disease progression and/or required further treatment. Objective responses were observed in all 14 patients, with 6 patients achieving a complete response (CR) and the remainder a partial response (PR) as per the NCI-working group criteria. We observed a significant decrease in peripheral white blood cell (WBC) counts, increase in hemoglobin, elevation of platelet counts and a dramatic decrease in lymphadenopathy and splenomegaly. Five of the treated patients have not required further treatment with a median follow up of 26 months. Of these 5 patients 3 have maintain a CR. The median time to progression (TTP) was 12 months. Overall, the treatment was well tolerated and all the patients, except one, completed 3 cycles of therapy. The majority of adverse events were Grade I-II (fluid retention, cough, transient hyperglycemia, fatigue). In addition, we observed 7 episodes of grade III-IV toxicity secondary to (anemia, CMV esophagitis and GI bleeding with one case each and two cases each of thrombocytopenia and neutropenia). These data suggest that treatment with the combination of Rituximab® and HDMP has increased activity over treatment with either agent alone and may produce durable complete responses in patients with refractory and / or relapsed CLL.