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Development of Recombinant Toxins to Treat Hematologic Malignancies

Robert Kreitman

4 Collaborator(s)

Funding source

National Cancer Institute (NIH)
We focus on targeted therapy using recombinant immunotoxins for hematologic malignancies, particularly hairy cell leukemia (HCL), and other new therapies for HCL. Recombinant immunotoxins contain truncated forms of Pseudomonas exotoxin (PE) fused to anti-CD25 or anti-CD22 Fv fragments. Anti-CD25 LMB-2 is currently undergoing phase II testing with chemotherapy in adult T-cell leukemia (ATL). Anti-CD22 moxetumomab pasudotox, previously called HA22 or CAT-8015), is an affinity-matured form of BL22 for targeting hematologic malignancies, particularly HCL and pediatric acute lymphoblastic leukemia (ALL). We test 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 use clinical samples from patients to investigate treatment efficacy and toxicity, and to better understand the biology and pathogenesis of HCL. Development of anti-CD22 recombinant immunotoxins for CD22+ B-cell malignancies. Moxetumomab pasudotox, previously called HA22 or CAT-8015, is an affinity-matured mutant of a parent recombinant immunotoxin BL22, made and tested preclinically in collaboration with Ira Pastan's lab. Moxetumomab pasudotox contains the Fv fragment of the anti-CD22 MAb RFB4 fused to a truncated form of PE called PE38. In Phase I testing of Moxetumomab pasudotox in 49 patients at 5-50 ug/Kg every other day for 3 doses (QOD x3), complete remission (CRs) were observed in 28 (57%), with overall response rate (ORR) 88%. At the highest dose level, the CR rate was 64%, with 62% ongoing at a median of 37 months. 90% of 21 CRs continue with resolved blood counts. No dose limiting toxicity (DLT) was observed on the phase I trial. Two patients had grade 2 HUS, milder than HUS seen with BL22. Based on this efficacy and toxicity profile, the FDA recommended a pivotal trial using a single-arm non-randomized design, with a primary endpoint of 28% CRs in 77 patients. Of 9 patients enrolled, and 6 evaluable for response, 5 (83%) achieved CR, all without minimal residual disease (MRD). This is important because the phase 1 trial showed lower relapse without compared to with MRD. New sites will be joining this multicenter trial soon. In pediatric ALL, 25% CRs have been achieved, with the trials are being directed in collaboration with Dr. Alan Wayne, now at USC, and also Dr. Nirali Shah at NIH. Development of anti-CD25 recombinant immunotoxin LMB-2 for CD25+ leukemias. We enroll HCL patients ineligible to receive HA22 on a phase II trial of LMB-2, with 1 CR and 3 PRs for an ORR of 36% out of 11 patients. The trial will meet its primary endpoint with 2 more responses, after which it will close. To investigate immunotoxin combinations with chemotherapy, we target ATL where rapid disease progression and immunogenicity limit efficacy of LMB-2 as a single agent. We developed a mouse model showing synergistic antitumor activity combined with LMB-2. In a phase II trial, ATL patients receive fludarabine and cyclophosphamide prior to LMB-2, with ORR 62% including 46% CRs in 13 evaluable patients. Our goals are to complete this trial with limited LMB-2 drug remaining. We collaborate with Dr. Raffit Hassan who found that pentostatin and cyclophosphamide can prevent immunogenicity in mesothelioma patients receiving PE38 fused to anti-mesothelin Fv (SS1P), despite the higher immunogenicity in solid compared to hematologic tumors. Development of MAb-chemotherapy combinations for early and relapsed/refractory HCL. For the past 25 years, cladribine alone, or less commonly pentostatin alone, has been the standard 1st and 2nd line treatment of HCL, but is non-curative in most patients. To determine the value of rituximab added to cladribine, newly diagnosed or once-relapsed HCL patients are randomized to cladribine with either immediate or 6-month delayed rituximab, and MRD at 6 months and other time points measured. For newly diagnosed HCL patients, randomization of the 68-patient cohort has completed. Simultaneous cladribine + rituximab is highly effective in eradicating MRD, and delayed rituximab, when needed, usually eradicates MRD. A new 25 patient cohort has begun accrual to determine the feasibility of combined cladribine + rituximab as an outpatient. Long-term follow-up will determine if there is a difference between immediate and delayed rituximab with first-line cladribine, but this trial establishes the efficacy and safety of either approach. Once-relapsed HCL patients continue randomization in a separate stratification of the trial. In HCLv, combined rituximab + cladribine is highly effective, with our published report supporting this combination as a new standard of care for early HCLv. To study pentostatin-rituximab and bendamustine-rituximab (BR) combinations in HCL for the first time prospectively, a randomized trial is underway in multiply relapsed HCL. Our recent report of the toxicity portion of the trial using 2 dose levels of bendamustine establish BR as a highly effective combination in multiply relapsed HCL, particularly in eradicating MRD. Targeted therapy for HCL. Although the BRAF V600E mutation is thought to be present in 100% of classic HCL, we showed that up to 20% lack V600E, particularly those with the poor prognosis IGHV4-34 immunoglobulin rearrangement, described by our group in 2009. Nevertheless, most HCL patients remain potentially treatable using BRAF inhibition. For the first time ever in HCL, we began treating V600E+ HCL patients by inhibiting both BRAF and its downstream pathway MEK, particularly those ineligible for immunotoxin therapy. This trial is being performed as part of a Glaxosmithkline-sponsored multicenter registration trial in many different BRAF V600E+ histologies. Ibrutinib is an inhibitor of Bruton's tyrosine kinase (BTK), approved in CLL and mantle cell lymphoma, and BTK is also a target expressed by HCL cells. We have joined a multicenter CTEP/Pharmacyclics trial of ibrutinib in HCL and HCLv and have already enrolled patients with both diagnoses. Laboratory research with moxetumomab pasudotox and other therapies for HCL. One goal is to improve the efficacy of CD22 targeting by further engineering immunotoxin structure, both to decrease immunogenicity and increase efficacy. In collaboration with Ira Pastan's lab, engineered mutants of moxetumomab pasudotox are tested in cytotoxicity assays of primary HCL, HCLv, and CLL cells obtained from patients on approved LMB and other NIH protocols. New potential drugs are also being tested in cytotoxicity assays, including BRAF, MEK, BTK and PI3K inhibitors. A 2nd goal is to sequence immunoglobulin rearrangements (IgH) unique to each HCL patient, to study HCL biology and to design patient-specific PCR assays for MRD. This RQ-PCR test is able to detect 1 HCL in 1 million normal cells. This work reached a breakthrough recently when cloning of IgH, previously possible only when the pre-treatment HCL count was high enough to identify the IgH, was successfully accomplished by deep sequencing. Deep sequencing (MiSeq) provides millions of 'reads' where malignant clones can be identified statistically. We are also using RNA/DNA from these samples to better characterize the 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. We are also discovering new genes, including HLA types, which are characteristic of patients with HCL and/or HCLv, and may shed light on the pathogenesis and possible new treatments of the disease.

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