Development of Recombinant Toxins to Treat Hematologic Malignancies

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Overview. We focus on targeted therapy for hematologic malignancies, particularly hairy cell leukemia (HCL) and poor-prognosis variant HCLv. In patients, we tested combinations of chemotherapy and anti-CD20 Mab rituximab and small molecules inhibiting BRAF, MEK, BTK, and BCL2. Moxetumomab pasudotox (Moxe), a recombinant immunotoxin containing an anti-CD22 Fv fragment and truncated Pseudomonas exotoxin, was developed in our lab in collaboration with Ira Pastan and David FitzGerald. Based on clinical trials we led, Moxe was FDA approved for HCL/HCLv in 2018 but is currently unavailable, awaiting a pharmaceutical company to take over development for HCL/HCLv and more common lymphomas. A clinical trial of anti-CD22 CART in HCL/HCLv is underway with very exciting results. In the lab, we use clinical samples from patients to investigate treatment efficacy and toxicity, and to understand the biology and pathogenesis of HCL/HCLv. We created B-cell lines with mutations observed in HCL/HCLv patients. We perform whole exome sequencing (WES) and other next-generation sequencing (NGS) of HCL/HCLv samples which has elucidated information about the pathogenesis of HCL and its variants. Development of MAb-chemotherapy combinations for early and relapsed/refractory HCL. For 35 years, cladribine or pentostatin alone was standard 1st and 2nd line treatment of HCL, but without evidence of cure. In a randomized trial, we reported that 1st line concurrent cladribine-rituximab (CDAR) eradicates MRD in 97% vs 32% of patients with cladribine alone (CDA). Delayed rituximab, given when MRD is detected in blood, eradicates MRD in 2/3 of patients with most MRD-free CRs persisting at a median follow-up of 6.5 years. Of 68 patients treated with either approach, only 1 progressed to the point of needing next treatment, vs 28% of 90 historical patients treated with CDA alone and followed with blood counts until needing retreatment (p less than 0.0001). Thus, while CDAR has highest MRD-free CR rate, CDA alone with delayed rituximab is also a new 1st line standard of care. We recently completed a 25-patient validation cohort of the CDAR regimen and a randomized trial of CDAR vs CDA + delayed rituximab in patients with once-relapsed HCL. We also established CDAR as a new standard of care for early HCLv with 95% CRs and 80% MRD-free CRs. To study other purine analogs combined with rituximab, a randomized trial of pentostatin-rituximab vs bendamustine-rituximab was completed and showed both regimens as highly effective, particularly in eradicating MRD, albeit with chemotherapy toxicities. Targeted therapy for HCL. The BRAF V600E mutation is a driver for ~90% classic HCL and absent in patients with unmutated IGHV4-34 immunoglobulin rearrangement, the latter described by our group in 2009. We inhibited BRAF V600E+ with Dabrafenib and its downstream pathway MEK with Trametinib, as part of a multicenter trial in different BRAF V600E+ histologies. We treated several patients with anaplastic thyroid cancer (ATC), a rapidly fatal disease also expressing BRAF V600E, leading to FDA approval of Dabrafenib-Trametinib for ATC. We initiated our own trial of BRAF inhibitor Encorafenib and MEK inhibitor Binimetinib in HCL, which is achieving an unprecedented CR rate >90%. For HCL/HCLv patients lacking V600E, we began a trial of Binimetinib alone, which has resulted in major responses including CR. We treated 20 of the 37 patients enrolled on the multicenter BTK inhibitor Ibrutinib study and reported low response rates but excellent progression-free survival (PFS). While agents targeting BRAF, MEK and BTK usually do not eliminate MRD, our patients on Encorafenib-Binimetinib or Binimetinib may receive rituximab or other CD20 Mab once they achieve MRD+ CR for a year, to convert MRD+ to MRD-free CR. Finally, we are leading a multicenter CTEP-sponsored trial of BCL2 inhibitor Venetoclax in patients with relapsed/refractory HCL. Development of anti-CD22 approaches for B-cell malignancies. Based on our published phase 1-3 results for Moxe, the FDA approved Moxe for relapsed/refractory HCL in 2018. We completed a trial at NIH of Moxe with rituximab (MoxeR) to decrease immunogenicity, decrease HCL/HCLv tumor burden, and hasten MRD-free CR. Meeting these goals, MoxeR achieved an MRD-free CR rate of 72% in 18 patients, surpassing the MRD-free CR rate of 57% from Vemurafenib-Rituximab. Thus, Moxe is a lifesaving drug for HCL/HCLv, and in our experience, MoxeR is the most effective regimen for this disease. At this time further development of Moxe will require another company taking over development, using the FDA-approved master cell bank still at NIH which is available to be licensed. Once a new lot of Moxe is made, we propose 2 different trials to greatly increase its utility. The first is a phase 2 trial in 26 patients with non-Hodgkin's lymphomas (NHL), including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL), and marginal zone lymphoma (MZL) who are in MRD+ CR after chemotherapy, to convert MRD+ to MRD-free CR. The rationale for this trial is that leukemic cells from patients with NHL are highly sensitive to Moxe ex vivo like HCL cells. The 2nd trial would be a phase 2 trial of MoxeR in early HCL/HCLv, where 24 1st-line and 24 2nd-line patients would receive 1 dose of rituximab to decrease HCL burden and normal B-cells, and 2 weeks later begin MoxeR every 2 weeks until MRD-free CR. The 2nd trial alone should greatly increase the use of Moxe because of the easier and safer method of administration and expanding the eligible population from at least 3rd-line to all lines of treatment. Finally, in collaboration with Nirali Shah in the Pediatric Branch, we are also targeting CD22 using chimeric antigen receptor (CAR) T-cells, and have recently published a lifesaving MRD-free CR. Using samples from HCL/HCLv patients, we are sequencing immunoglobulin rearrangements (IgH) unique to each HCL patient, to study HCL biology and to test patients after treatment for MRD by RQ-PCR and next-generation sequencing (NGS). With the support of the HCL Foundation, whole exome sequencing (WES) and RNA transcriptome analysis were undertaken. We exomed several hundred HCL/HCLv samples. We recently reported for the first time 20 cases of non-V600E BRAF mutations, including a 5-amino acid deletion. This is a high-risk group, several of whom we have treated on our binimetinib trial and achieved CRs. We also found over 50 patients with MAP2K1 mutations with very high-risk disease who we are further studying and in some cases treating. We are studying MAP2K1 mutations in the lab by using the TALEN gene editing method to produce B-cell cell lines with these mutations. We are also using CRISPR to produce B-cell lines with BRAF V600E and non-V600E BRAF mutations. These new cell lines should be useful for screeding drugs for efficacy against HCL/HCLv and high-risk variants. In collaboration with the Cancer Data Science Laboratory, we have also studied single cell sequencing of HCL/HCLv samples to understand the many types of mutations leading to malignancy in patients with HCLv and HCL lacking BRAF V600E. We believe this work will be critical for the development of new inhibitors to target mutations present not only in HCL/HCLv but also in more common solid tumors that rely on the MAP kinase pathway. Finally, to better target HCL/HCLv, new potential drugs and drug combinations are also being tested in cytotoxicity assays against fresh patient cells, including BRAF, MEK, BTK, and BCL2 inhibitors. Cytotoxicity studies of BTK inhibitors combined with venetoclax have been tested as preparation for a combination trial to follow-up the venetoclax single-agent trial.