OverviewThe Clinical Immunotherapy Section of the Laboratory of Molecular Biology is focused on targeted therapy using both recombinant immunotoxins for hematologic malignancies, particularly hairy cell leukemia (HCL), and rituximab plus chemotherapy for HCL. The recombinant immunotoxins contain truncated forms of Pseudomonas exotoxin (PE) fused to anti-CD25 or anti-CD22 Fv fragments. The anti-CD22 recombinant immunotoxin HA22, a high-affinity version of BL22, is being developed for the treatment of HCL, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and, in collaboration with Dr. Wayne in NCI's Pediatric Oncology Branch, pediatric acute lymphoblastic leukemia (ALL). The anti-CD25 recombinant immunotoxin LMB-2 is currently undergoing phase II testing in conjunction with chemotherapy in patients with adult T-cell leukemia (ATL), where complete remissions (CRs) have been observed. Meanwhile, we have taken advantage of our large HCL population to perform clinical trials with combinations of chemotherapy and rituximab to answer questions relevant to the optimal therapy of newly diagnosed and multiply relapsed HCL, and also to better understand the behavior of HCL in immunotoxin-treated patients. In the laboratory, we are using clinical samples from patients to answer questions about treatment efficacy and toxicity, to better understand the biology and origin of HCL, and to model in vivo how to combine recombinant immunotoxins with chemotherapy to effectively treat a more aggressive disease like ATL.Development of anti-CD22 recombinant immunotoxins for CD22+ B-cell malignancies. To effectively target CD22+ drug-resistant B-cell malignancies without chemotherapy, BL22 was originally created using the Fv fragment of the anti-CD22 MAb RFB4 fused to a truncated form of PE called PE38. BL22 in phase I-II testing achieved CR rates of 47-61% in chemoresistant HCL, with overall response rates (ORR) of 72-81%. A completely reversible hemolytic syndrome (HUS) was observed in 8 (12%) of 69 HCL patients. BL22 was less effective in chronic lymphocytic leukemia (CLL), probably due to low CD22 expression. We used hot spot mutagenesis to increase the affinity of BL22. The resulting protein, called HA22, CAT-8015 or moxetumomab pasudotox, contains THW instead of SSY in the antigen binding site of VH. This resulted in 14-fold increased binding affinity for CD22, due to lower off-rate, and up to >100-fold increased cytotoxicity. Moxetumomab pasudotox had antitumor activity in murine xenograft studies, and an acceptable safety profile in cynomolgus monkeys. In Phase I testing of 28 patients at 5-50 ug/Kg every other day for 3 doses (QOD x3), CRs were observed in 13 (46%), with an ORR of 86%. All but 1 CR lasted >1 year. A multicenter phase I trial in CLL and NHL was also initiated with 23 patients so far enrolled. In pediatric ALL, a much more aggressive disease, 4 (24%) of 17 achieved CR with a more dose-intense administration of HA22. A pivotal trial is currently being planned, with the goal of registration for relapsed/refractory HCL. Development of anti-CD25 recombinant immunotoxin LMB-2 for CD25+ leukemias. We have continued to enroll HCL patients ineligible to receive HA22 on a phase II trial of LMB-2, and have achieved major responses including an ongoing CR. To investigate immunotoxin combinations with chemotherapy, we decided to target ATL where rapid disease progression and immunogenicity limit efficacy of LMB-2 as a single agent. We developed a mouse model showing that gemcitabine reduced tumor interstitial soluble CD25 (sCD25) and showed synergistic antitumor activity combined with LMB-2. In an ongoing phase II trial, ATL patients receive fludarabine and cyclophosphamide prior to LMB-2, with ORR 58% including 33% CRs in 12 evaluable patients. Our goals are to complete this trial with limited LMB-2 drug remaining, and if activity is promising, propose to have additional LMB-2 made for additional studies or develop a new agent for CD25 targeting.Development of MAb-chemotherapy combinations for early and relapsed/refractory HCL. For the past >20 years, cladribine alone (or less commonly pentostatin alone) has been the standard 1st and 2nd line treatment of HCL, but appears non-curative in most patients. To determine if rituximab should be added to cladribine, newly diagnosed or once-relapsed HCL patients are randomized to cladribine with either immediate or >6-month delayed rituximab, and minimal residual disease (MRD) at 6 months and other time points measured. So far 76 patients have been enrolled to this 150-patient trial, with the goal to compare both MRD rates at 6 months and long-term blood MRD-free survival. To study pentostatin-rituximab and bendamustine-rituximab combinations in HCL for the first time prospectively, a randomized trial is underway in multiply relapsed HCL with 2-way crossover between the 2 arms. Our clinical goal is to determine the best regimen from the standpoint of toxicity and efficacy, and determine the capacity of either regimen for eradicating HCL MRD.Laboratory research connected with moxetumomab pasudotox and other therapies for HCL. A major goal is to improve the efficacy of CD22 targeting by further engineering of the immunotoxin structure. In collaboration with Ira Pastan's lab, the toxin was further truncated to little more than the ADP-ribosylating domain III, producing a liposomal protease resistant mutant of HA22 called HA22-LR which we found to have cytotoxicity >10-fold improved for primary CLL cells and equal toward HCL compared to moxetumomab pasudotox. Identification and mutation of 8 immunogenic epitopes in domain III led to HA22-LR-8M, which is also more cytotoxic than moxetumomab pasudotox toward CLL. This molecule could improve the CR rate of moxetumomab pasudotox toward HCL by preventing immunogenicity, and also improve activity toward CLL/NHL. A 2nd goal is to sequence immunoglobulin rearrangements unique to each HCL patient, to study HCL biology and to design patient-specific PCR assays for MRD. We have previously found this test (RQ-PCR) able to detect 1 HCL in 1 million normal cells. We now have 6 HCL patients treated with BL22 or moxetumomab pasudotox who are RQ-PCR-negative up to 9 years after achieving CR, and additional patients negative after receiving rituximab combined with chemotherapy. We plan to test additional patients to determine correlations between MRD and clinical outcome. We are also using RNA/DNA from these samples to better characterize B- and T- cell repertoire in patients treated with immunotoxins compared to chemotherapy and/or rituximab, and to study fundamental questions related to HCL biology, including those related to the V600E mutation recently discovered in HCL. BRAF as a target in HCL. BRAF testing using pyrosequencing has shown that up to 20% of classic HCL patients lack the V600E mutation published by others to be present in 100% of HCL cases. All classic HCL patients with the poor prognosis IGHV4-34 immunoglobulin rearrangement were wild-type for BRAF. Nevertheless, most HCL patients remain potentially treatable using BRAF inhibition. Plans are underway to treat V600E+ HCL patients with BRAF inhibition, particularly those ineligible for immunotoxin therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010301-15
Application #
8552658
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$991,402
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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