Development of Recombinant Immunotoxins for Hairy Cell Leukemia

Hairy cell leukemia (HCL) is an indolent B-cell malignancy with excellent initial response to purine analogs pentostatin or cladribine, but patients are rarely, if ever, cured. Younger patients will usually need repeat chemotherapy which has declining benefits and increasing toxicities with each course. Targeted therapies directed to the BRAF V600E mutation and Bruton’s tyrosine kinase may be helpful, but rarely eradicate the minimal residual disease (MRD) which will eventually lead to relapse. Moxetumomab pasudotox (Moxe) is an anti-CD22 recombinant immunotoxin, which binds to CD22 on HCL cells and leads to apoptotic cell death after internalization and trafficking of the toxin to the cytosol. Phase I testing achieved a complete remission (CR) rate of 57% in relapsed/refractory HCL. Most CRs were without MRD and eradication of MRD correlated with prolonged CR duration. Patients were often MRD-free after five years. Important mild-moderate toxicities included capillary leak and hemolytic uremic syndromes which could be prevented and managed conservatively. A phase 3 trial met its endpoint of durable CR with acceptable toxicity, leading to FDA approval of Moxe for relapsed/refractory HCL, under the name Lumoxiti. Moxe combined with rituximab is currently being evaluated in relapsed/refractory HCL to improve the rate of MRD-free CR.

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Moxetumomab pasudotox for hairy cell leukemia: preclinical development to FDA approval

Blood Advances ◽

10.1182/bloodadvances.2019000507 ◽

2019 ◽

Vol 3 (19) ◽

pp. 2905-2910 ◽

Cited By ~ 3

Author(s):

Adam Yuh Lin ◽

Shira Naomi Dinner

Keyword(s):

Hairy Cell Leukemia ◽

Residual Disease ◽

Remission Rate ◽

Lymphoblastic Leukemia ◽

Disease Free Survival ◽

Cell Surface Expression ◽

Hairy Cell ◽

Cell Leukemia ◽

Recombinant Immunotoxin ◽

Fda Approval

Abstract Moxetumomab pasudotox (MP) is an immunotoxin that recently received US Food and Drug Administration (FDA) approval for the treatment of hairy cell leukemia (HCL) that has failed at least 2 prior lines of therapy, including a purine analog. MP is a recombinant immunotoxin that consists of an anti-CD22 immunoglobulin variable domain genetically joined to Pseudomonas exotoxin (PE38). Unlike most antibody-drug conjugates, which use a chemical linker, recombinant DNA techniques are used to produce MP. MP and its predecessor, BL22, were initially developed to treat non-Hodgkin lymphoma, acute lymphoblastic leukemia, and HCL. However, MP was found to be particularly effective in HCL due to the high level of CD22 cell-surface expression. The recent pivotal phase 3 trial of MP in relapsed/refractory HCL demonstrated a durable complete remission rate of 30%, and 85% of complete responders achieved minimal residual disease negativity, which is associated with improved disease-free survival outcomes in HCL. In addition to an exceptional depth of response, MP appears to be less immunosuppressive than purine analogs. MP is generally well tolerated but has unique toxicities, including capillary leak syndrome and hemolytic uremic syndrome, which are poorly understood. This review will encompass the preclinical and clinical development of MP, with particular attention to its current indication in HCL.

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The Biology of Classic Hairy Cell Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms22157780 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7780

Author(s):

Jan-Paul Bohn ◽

Stefan Salcher ◽

Andreas Pircher ◽

Gerold Untergasser ◽

Dominik Wolf

Keyword(s):

Hairy Cell Leukemia ◽

Infectious Complications ◽

Braf V600e ◽

Hairy Cell ◽

Cell Leukemia ◽

Purine Analogues ◽

B Cell Malignancy ◽

Activating Mutation ◽

Open Issue ◽

Almost All

Classic hairy cell leukemia (HCL) is a rare mature B-cell malignancy associated with pancytopenia and infectious complications due to progressive infiltration of the bone marrow and spleen. Despite tremendous therapeutic advances achieved with the implementation of purine analogues such as cladribine into clinical practice, the culprit biologic alterations driving this fascinating hematologic disease have long stayed concealed. Nearly 10 years ago, BRAF V600E was finally identified as a key activating mutation detectable in almost all HCL patients and throughout the entire course of the disease. However, additional oncogenic biologic features seem mandatory to enable HCL transformation, an open issue still under active investigation. This review summarizes the current understanding of key pathogenic mechanisms implicated in HCL and discusses major hurdles to overcome in the context of other BRAF-mutated malignancies.

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Responses in Refractory Hairy Cell Leukemia to a Recombinant Immunotoxin

Blood ◽

10.1182/blood.v94.10.3340.422k19_3340_3348 ◽

1999 ◽

Vol 94 (10) ◽

pp. 3340-3348 ◽

Cited By ~ 109

Author(s):

Robert J. Kreitman ◽

Wyndham H. Wilson ◽

David Robbins ◽

Inger Margulies ◽

Maryalice Stetler-Stevenson ◽

...

Keyword(s):

Hairy Cell Leukemia ◽

Bone Marrow Aspirate ◽

Residual Disease ◽

Hairy Cell ◽

Cell Leukemia ◽

Single Cycle ◽

Pseudomonas Exotoxin ◽

Pseudomonas Exotoxin A ◽

Circulating Cells ◽

Recombinant Immunotoxin

We report major responses in 4 of 4 patients with hairy cell leukemia (HCL) who have recently been treated on a phase I trial with the recombinant immunotoxin LMB-2. The immunotoxin, designed to target CD25+ malignancies, is composed of the Fv portion of the anti-Tac (anti-CD25) antibody, fused to a 38-kD truncated form of Pseudomonas exotoxin A, and has previously been called anti-Tac(Fv)-PE38. All 4 HCL patients were resistant to standard and salvage therapies for HCL, including 2-chlorodeoxyadenosine (CdA) and interferon , and all patients responded to LMB-2 after a single cycle. One patient treated with 2 cycles had a complete remission (CR), with regression of HCL cells from the blood and marrow and resolution of splenomegaly and pancytopenia. As is typical for patients in CR after treatment with CdA, minimal residual disease was detectable by flow cytometry of the bone marrow aspirate. This patient has not relapsed after 11 months. Three other patients had 98% to 99.8% reductions in malignant circulating cells. These results represent a proof of principal that targeted therapy with recombinant Fv-containing proteins can be clinically useful. LMB-2 may be an effective new therapy for patients with chemotherapy-resistant CD25+HCL.

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Immunoconjugates and new molecular targets in hairy cell leukemia

Hematology ◽

10.1182/asheducation.v2012.1.660.3798659 ◽

2012 ◽

Vol 2012 (1) ◽

pp. 660-666 ◽

Cited By ~ 19

Author(s):

Robert J. Kreitman

Keyword(s):

Hairy Cell Leukemia ◽

Poor Prognosis ◽

Residual Disease ◽

Single Agent ◽

Braf V600e ◽

Late Relapse ◽

Hairy Cell ◽

Cell Leukemia ◽

Purine Analogs ◽

Purine Analog

Abstract Hairy cell leukemia (HCL) is a B-cell malignancy that in its classic form is exquisitely sensitive to single-agent purine analog therapy, but that is associated in many patients with late relapse and eventual purine analog resistance. Minimal residual disease, which is present in most patients achieving complete remission with purine analogs, retains Ags that are ideal for targeted therapy. Rituximab, which targets CD20, is active as a single agent, particularly if combined with purine analogs. Recombinant immunotoxins targeting either CD25 or CD22 and containing truncated Pseudomonas exotoxin have achieved major responses in relapsed/refractory HCL. Moxetumomab pasudotox in phase 1 testing achieved responses in 86% of such patients (complete in 46%) without dose limiting toxicity and often without MRD. Soluble CD22 has been used for improved detection and monitoring of HCL, particularly the poor-prognosis variant that lacks CD25. Ig rearrangements unique for each HCL patient have been cloned, sequenced, and followed by real-time quantitative PCR using sequence-specific reagents. Analysis of these rearrangements has identified an unmutated IGVH4-34–expressing poor-prognosis variant with immunophenotypic characteristics of either classic or variant HCL. The BRAF V600E mutation, reported in 50% of melanomas, is present in > 85% of HCL cases that are both classic and express rearrangements other than IGVH4-34, making HCL a potential target for specific inhibitors of BRAF V600E. Additional targets are being defined in both classic and variant HCL, which should improve both detection and therapy.

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Resolution of Hairy Cell Leukemia Minimal Residual Disease by Both BRAF and Clone-Specific Real-Time Quantitative PCR (RQ-PCR) After Treatment with Moxetumomab Pasudotox.

Blood ◽

10.1182/blood.v120.21.2896.2896 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2896-2896 ◽

Cited By ~ 1

Author(s):

Robert J. Kreitman ◽

Evgeny Arons ◽

Sapolsky Jeffrey ◽

Laura Roth ◽

Hong Zhou ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Hairy Cell Leukemia ◽

Residual Disease ◽

Braf V600e Mutation ◽

Braf V600e ◽

Hairy Cell ◽

Cell Leukemia ◽

Specific Primer ◽

Minimal Residual

Abstract Abstract 2896 Background: Moxetumomab pasudotox is an anti-CD22 recombinant immunotoxin containing truncated Pseudomonas exotoxin which was recently reported to achieve a complete remission rate of 46% in 28 patients with relapsed/refractory hairy cell leukemia (HCL). An additional 20 patients were treated at the highest dose level and are now fully evaluable for response and minimal residual disease (MRD) determinations. RQ-PCR using clone-specific primers and a clone-specific TaqMan probe is capable of detecting one HCL cell in 106normal cells. Recently reported methods to detect the HCL-associated BRAF V600E mutation include pyrosequencing (5–10% sensitivity) and PCR (0.1–0.23% sensitivity). Methods: Moxetumomab pasudotox was administered to 16 patients at 5–40 ug/Kg every other day for 3 doses (QODx3) and to 32 patients at 50 ug/Kg QODx3, via 1–16 (median 4) cycles per patient at 4-week intervals. Complete remission (CR) required resolution of cytopenias and elimination of HCL in the blood and marrow by standard microscopy, but MRD could be present by flow cytometry of blood or bone marrow aspirate (BMA) or immunohistochemistry (IHC) of the bone marrow biopsy (BMBx). Blood and marrow from patients were also tested by PCR using consensus primers. When immunoglobulin (Ig) rearrangements could be cloned, RQ-PCR using clone-specific primer and probe was performed. To detect MRD by the BRAF V600E mutation, BRAF quantitative PCR (BRAF-qPCR) was performed on cDNA samples, using mutant-specific primer, and SYBR-Green detection followed by melting point analysis. MRD testing for BRAF-qPCR, unlike clone-specific RQ-PCR, did not require prior cloning of the Ig rearrangement. Results: All 198 cycles of moxetumomab administered to 48 patients were evaluable for toxicity and response. No dose limiting toxicity was observed, although 2 patients as previously reported had a grade 2 hemolytic uremic syndrome with transient grade 1 platelet and creatinine abnormalities. Of the 48 HCL patients at all dose levels, there were 26 (54%) CRs, with an overall response rate (ORR) of 88%. Of 32 at 50 ug/Kg QODx3, there were 19 (59%) CRs with an ORR of 91%. Of these 19 CRs, 11 (58%) achieved MRD negativity by repeated flow cytometry of both BMA and blood and IHC of BMBx. Flow cytometry of the BMA was the most sensitive conventional test of MRD. Of the 9 CRs at 50 ug/Kg QODx3 evaluable by clone-specific RQ-PCR of blood, 5 negative were also flow-negative, and 4 positive were also flow-positive (p=0.008). BRAF-qPCR on cDNA from limiting dilutions of BRAF V600E+ Colo-205 cells into BRAF wild-type cells achieved consistent detection at 1:105dilution (0.001%). Of 10 flow-negative CRs at 50 ug/Kg QODx3 evaluated by BRAF-qPCR, all 10 (100%) were BRAF-qPCR negative, including 4 which were nonevaluable by RQ-PCR due to inability to clone the Ig rearrangements prior to treatment. Currently 12 (63%) of the 19 CRs at 50 ug/Kg QODx3 are ongoing at 6–47 (median 21) months, including 10 (91%) of 11 MRD-negative vs 2 (25%) of 8 MRD+ CRs (p=0.006). Conclusions: Moxetumomab pasudotox is active in relapsed and refractory HCL and has a safety profile supporting further development for this disease. Retreatment on this trial could not necessarily be extended to achieve MRD-negative BMAs or molecular remission by RQ-PCR using sequence-specific or BRAF primers. However, these tests might be useful in the future to guide retreatment, optimize CR durability and possibly eradicate the HCL clone in selected patients. This summary contains investigator reported data. This study was sponsored by MedImmune, LLC, and supported by NCI's Intramural Research Program and the Hairy Cell Leukemia Research Foundation. Disclosures: Kreitman: NIH: Co-inventor on the NIH patent for Moxetumomab Pasudotox, Co-inventor on the NIH patent for Moxetumomab Pasudotox Patents & Royalties. Off Label Use: Moxetumomab Pasudotox is an experimental agent for CD22+ hematologic malignancies. FitzGerald:NIH: Coinventor on the NIH patent for Moxetumomab Pasudotox, Coinventor on the NIH patent for Moxetumomab Pasudotox Patents & Royalties. Fei:AstraZeneca: Stock, Stock Other; MedImmune, LLC: Employment. Ibrahim:AstraZeneca: Stocks, Stocks Other; MedImmune: Employment. Pastan:NIH: Coinventor on NIH patent for moxetumomab pasudotox, Coinventor on NIH patent for moxetumomab pasudotox Patents & Royalties.

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Phase 1 trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox combined with rituximab for relapsed/refractory hairy cell leukemia.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.7036 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 7036-7036

Author(s):

Robert J. Kreitman ◽

Maryalice Stetler-Stevenson ◽

Constance Yuan ◽

Hao-Wei Wang ◽

Hong Zhou ◽

...

Keyword(s):

Bone Marrow ◽

Hairy Cell Leukemia ◽

Phase 1 ◽

Tumor Burden ◽

Braf V600e ◽

Hairy Cell ◽

Cell Leukemia ◽

Phase 3 ◽

Phase 1 Trial ◽

Recombinant Immunotoxin

7036 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) is FDA-approved for hairy cell leukemia (HCL) patients who have received at least two prior systemic therapies including a purine nucleoside analog. In phase 3 testing the complete remission (CR) rate was 41%, and response was higher in patients with lower tumor burden and lower titers of antidrug antibodies (ADA). Phase 1 testing indicated that most CRs were without minimal residual disease (MRD) and eradication of MRD was associated with prolonged CR duration. Monoclonal antibody (Mab) rituximab binds to CD20 on HCL cells and induces apoptosis or immune-mediated killing, but as a single-agent achieved only 13% CRs in relapsed HCL requiring therapy. In a phase 1 trial to determine safety, rituximab was combined with Moxe, with the goal to help reduce tumor burden and to prevent or delay ADA by killing normal B-cells. Methods: To allow rituximab sufficient time to accomplish both goals, it was infused 3 days before day 1 of cycle 1 at 375 mg/m2, and Moxe was given by 30-minute infusion on days 1, 3 and 5. On repeat cycles of Moxe days 1, 3 and 5, rituximab was given on day 1. Cycles were generally spaced 4 weeks apart. Moxe was begun at a lower dose, 30 rather than the 40 mcg/kg dose used in phase 3 in case the rituximab would increase its toxicity. Bone marrow aspirate flow cytometry, which can detect 0.002% HCL cells, was the most sensitive test used for MRD detection, much more sensitive than BRAF V600E digital droplet PCR (ddPCR) or bone marrow biopsy immunohistochemistry (IHC). Patients could receive 4 cycles past MRD-free CR, but not more than 8 cycles. Results: Three patients received Moxe at 30 mcg/Kg/dose and 6 received 40 mcg/Kg/dose, all without dose limiting toxicity (DLT). There was no evidence of hemolytic uremic syndrome or capillary leak syndrome. To prevent intravascular hypovolemia due to expected third spacing, patients were encouraged to drink one cup per hour of water or other fluid from days 1 to 8 and take dexamethasone 4 mg orally if headache or nausea prevented good oral hydration. Of the 9 patients, 7 (78%) achieved CR after 2 (n = 6) or 3 (n = 1) cycles, and achieved MRD-free CR after 2 (n = 3), 4 (n = 3) or 6 (n = 1) cycles. No patients became infected with COVID-19. Conclusions: This phase 1 trial met its primary endpoint of determining whether rituximab could be safely combined with Moxe and will enroll 4 additional patients to further access clinical activity. Further testing will determine whether addition of a CD20 Mab to Moxe significantly improves clinical outcome compared to Moxe alone, particularly long-term MRD-free CR rate. Clinical trial information: NCT03805932.

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Faculty Opinions recommendation of The utility of BRAF V600E mutation-specific antibody VE1 for the diagnosis of hairy cell leukemia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725292083.793544947 ◽

2018 ◽

Author(s):

Sascha Dietrich

Keyword(s):

Specific Antibody ◽

Hairy Cell Leukemia ◽

Braf V600e Mutation ◽

Braf V600e ◽

Hairy Cell ◽

Cell Leukemia

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Moxetumomab pasudotox in heavily pre-treated patients with relapsed/refractory hairy cell leukemia (HCL): long-term follow-up from the pivotal trial

Journal of Hematology & Oncology ◽

10.1186/s13045-020-01004-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Robert J. Kreitman ◽

◽

Claire Dearden ◽

Pier Luigi Zinzani ◽

Julio Delgado ◽

...

Keyword(s):

Hairy Cell Leukemia ◽

Residual Disease ◽

Braf Inhibitor ◽

Nucleoside Analogs ◽

High Rate ◽

Hairy Cell ◽

Cell Leukemia ◽

Long Term Follow Up

Abstract Background Moxetumomab pasudotox is a recombinant CD22-targeting immunotoxin. Here, we present the long-term follow-up analysis of the pivotal, multicenter, open-label trial (NCT01829711) of moxetumomab pasudotox in patients with relapsed/refractory (R/R) hairy cell leukemia (HCL). Methods Eligible patients had received ≥ 2 prior systemic therapies, including ≥ 2 purine nucleoside analogs (PNAs), or ≥ 1 PNA followed by rituximab or a BRAF inhibitor. Patients received 40 µg/kg moxetumomab pasudotox intravenously on Days 1, 3, and 5 of each 28-day cycle for up to six cycles. Disease response and minimal residual disease (MRD) status were determined by blinded independent central review. The primary endpoint was durable complete response (CR), defined as achieving CR with hematologic remission (HR, blood counts for CR) lasting > 180 days. Results Eighty adult patients were treated with moxetumomab pasudotox and 63% completed six cycles. Patients had received a median of three lines of prior systemic therapy; 49% were PNA-refractory, and 38% were unfit for PNA retreatment. At a median follow-up of 24.6 months, the durable CR rate (CR with HR > 180 days) was 36% (29 patients; 95% confidence interval: 26–48%); CR with HR ≥ 360 days was 33%, and overall CR was 41%. Twenty-seven complete responders (82%) were MRD-negative (34% of all patients). CR lasting ≥ 60 months was 61%, and the median progression-free survival without the loss of HR was 71.7 months. Hemolytic uremic and capillary leak syndromes were each reported in ≤ 10% of patients, and ≤ 5% had grade 3–4 events; these events were generally reversible. No treatment-related deaths were reported. Conclusions Moxetumomab pasudotox resulted in a high rate of durable responses and MRD negativity in heavily pre-treated patients with HCL, with a manageable safety profile. Thus, it represents a new and viable treatment option for patients with R/R HCL, who currently lack adequate therapy. Trial registration ClinicalTrials.gov identifier: NCT01829711; first submitted: April 9, 2013. https://clinicaltrials.gov/ct2/show/NCT01829711

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BRAF mutation in hairy cell leukemia

Oncology Reviews ◽

10.4081/oncol.2014.253 ◽

2014 ◽

Cited By ~ 6

Author(s):

Ahmad Ahmadzadeh ◽

Saeid Shahrabi ◽

Kaveh Jaseb ◽

Fatemeh Norozi ◽

Mohammad Shahjahani ◽

...

Keyword(s):

B Cell ◽

Hairy Cell Leukemia ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Braf V600e ◽

Common Mutation ◽

Hairy Cell ◽

Cell Leukemia ◽

Mitogen Activated Protein ◽

Almost All

BRAF is a serine/threonine kinase with a regulatory role in the mitogen-activated protein kinase (MAPK) signaling pathway. A mutation in the RAF gene, especially in BRAF protein, leads to an increased stimulation of this cascade, causing uncontrolled cell division and development of malignancy. Several mutations have been observed in the gene coding for this protein in a variety of human malignancies, including hairy cell leukemia (HCL). BRAF V600E is the most common mutation reported in exon15 of BRAF, which is observed in almost all cases of classic HCL, but it is negative in other B-cell malignancies, including the HCL variant. Therefore it can be used as a marker to differentiate between these B-cell disorders. We also discuss the interaction between miRNAs and signaling pathways, including MAPK, in HCL. When this mutation is present, the use of BRAF protein inhibitors may represent an effective treatment. In this review we have evaluated the role of the mutation of the BRAF gene in the pathogenesis and progression of HCL.

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