anti-CD22 Recombinant Immunotoxin Research Articles

Effect of rituximab on remissions without minimal residual disease and immunogenicity in patients with relapsed/refractory hairy cell leukemia receiving moxetumomab pasudotox.

e19015 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) was FDA-approved for relapsed/refractory hairy cell leukemia (HCL) but is now unavailable due to vial expiration until a company resumes development. The phase 3 complete remission (CR) rate was 41%, higher in patients with lower anti-drug antibodies (ADA). Minimal residual disease (MRD) eradication led to longer CR duration. HCL and the poorer prognosis variant HCLv strongly express CD22 and CD20. A clinical trial was done to determine if Rituximab could decrease immunogenicity by killing normal B-cells in HCL patients receiving Moxe and hasten MRD-free CR by reducing HCL tumor burden. Methods: To permit Rituximab enough time to reduce ADA and tumor burden, it was administered 3 days before cycle 1 day 1 at 375 mg/m2, and Moxe was given by 30-minute infusion days 1, 3 and 5. On subsequent 28-day cycles, patients received Moxe days 1, 3 and 5, and Rituximab prior to Moxe on day 1. Patients received up to 4 cycles past MRD-free CR, or up to 8 total cycles if MRD-free CR was achieved after cycle 4. The ADA assay determined percent neutralization by serum of the cytotoxicity of Moxe on CD22+ Raji cells. Results: After 13 patients received Moxe-Rituximab (MoxeR) without dose-limiting toxicity (DLT), meeting the phase 1 endpoint, 5 additional patients received Moxe with the biosimilar Ruxience (MoxeR). The first 3 patients received Moxe at 30 mcg/Kg/dose and subsequent patients 40 mcg/Kg/dose. All 18 were evaluable for toxicity and response. Although no DLT, one patient had transient grade 3 hemolytic uremic syndrome during cycle 3 without significant symptoms or need for therapy. Of the 18 patients, 15 (83%) responded, 14 (78%) achieved CR and 13 (72%) MRD-free CR by blood and bone marrow (BM) aspirate flow cytometry and BM biopsy immunohistochemistry. MRD-free CRs included one with HCLv. Even though Moxe vial expiration prevented enrollment of the 26 planned patients needed for a 1-sided p-value <0.025 compared to Moxe alone where 30 (47%) of 64 phase 1-3 patients achieved MRD-free CR, the 53% relative improvement with MoxeR achieved a 1-sided p-value of 0.05. Compared to 29 (48%) of 61 evaluable phase 1-3 patients who received Moxe alone with high ADA levels, 4 (29%) of 18 patients had high ADA levels to MoxeR (p=0.048). At 14-51 (median 35.3) months of follow-up, all but 2 of 13 MRD-free CRs are continuing, with relapse-free survival 14-45 (median 34.5) months. Conclusions: Despite enrolling slightly fewer patients than planned, MoxeR was safe and more effective than Moxe alone at achieving MRD-free CR, probably due to lower immunogenicity and faster reduction of HCL/HCLv tumor burden. Since non-Hodgkin’s lymphoma (NHL) cells from patients are sensitive to Moxe like HCL, MoxeR could be tested after NHL treatment to convert MRD+ to MRD-free CRs. Clinical trial information: NCT03805932 .

Moxetumomab Pasudotox in Hairy Cell Leukaemia: A Profile of Its Use.

Moxetumomab pasudotox (Lumoxiti®), an anti-CD22 recombinant immunotoxin, is an important treatment option that is approved in adults with relapsed or refractory hairy cell leukaemia (HCL) who have received at least two prior lines of treatment with systemic therapies including purine nucleoside analogues. In a pivotal phase III trial, treatment with moxetumomab pasudotox resulted in approximately one third of patients achieving durable complete response lasting more than 6 months, as well as improvements in other haematological parameters and disease-related symptoms. Moxetumomab pasudotox had a generally manageable tolerability profile; the most common treatment-related adverse events (AEs) included nausea, peripheral oedema, headache and pyrexia. AEs of special interest (including haemolytic uraemic syndrome and capillary leak syndrome) were generally manageable and reversible with monitoring and supportive care.

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

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.

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.

Population pharmacokinetics, efficacy, and safety of moxetumomab pasudotox in patients with relapsed or refractory hairy cell leukaemia.

To characterize the pharmacokinetics (PK) of moxetumomab pasudotox, an anti-CD22 recombinant immunotoxin, in adults with relapsed or refractory hairy cell leukaemia, we examined data from a phase 1 study (Study 1001; n = 49) and from the pivotal clinical study (Study 1053; n = 74). Data from both studies were pooled (n = 123) to develop a population PK model. Covariates included demographics, disease state, liver and kidney function, prior treatment, and antidrug antibodies (ADAs). Exposure-response and exposure-safety were analysed separately by study. A 1-compartment model with linear elimination from the central compartment and 2 clearance (CL) rates was developed. Moxetumomab pasudotox was cleared more rapidly after cycle 1, day 1 (CL1 = 24.7 L/h) than subsequently (CL2 = 3.76 L/h), with high interindividual variability (116 and 109%, respectively). In Study 1053, patients with ADA titres >10 240 showed ~4-fold increase in CL. Higher exposures (≥median) were related to higher response rates, capillary leak syndrome and increased creatinine (Study 1053 only), or grade ≥3 adverse events (Study 1001 only). Clinical benefits were still observed in patients with lower exposure or high ADA titres. Despite a high incidence of immunogenicity with increased clearance, moxetumomab pasudotox demonstrated efficacy in hairy cell leukaemia.

Abstract CN07-03: Recombinant immunotoxins for hematologic malignancies

Abstract Recombinant immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p<0.0001) compared to those remaining blood RQ-PCR+. Both BMA flow cytometry and RQ-PCR were associated with prolonged CR duration if negative, but blood flow cytometry was not helpful since only one patient with CR remained flow-positive. Molecular remissions assessed by blood analysis may be useful to determine the number of cycles of Moxe to administer to achieve long-term CR or possibly cure. To achieve molecular remissions with fewer cycles, Moxe is being tested with rituximab to facilitate tumor reduction and decrease anti-drug antibody formation. Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03

A novel approach to assess domain specificity of anti-drug antibodies to moxetumomab pasudotox, an immunotoxin with two functional domains

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.

Moxetumomab pasudotox for the treatment of hairy cell leukemia

ABSTRACTIntroduction: Cladribine and pentostatin are the drugs of choice in the treatment of hairy cell leukemia (HCL). Recently, immunotoxin moxetumomab pasudotox has been introduced to improve the prognosis in relapsed and refractory HCL.Areas covered: This review discusses the mechanism of action, safety, and efficacy of moxetumomab pasudotox in HCL patients. A literature review of the MEDLINE database for articles in English concerning immunotoxins, moxetumomab pasudotox, and hairy cell leukemia was conducted via PubMed. Publications from 2000 through December 2018 were scrutinized. The search terms used were immunotoxins and moxetumomab pasudotox in conjunction with hairy cell leukemia. Conference proceedings from the previous five years of the American Society of Hematology, European Hematology Association and American Society of Clinical Oncology were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles.Results/conclusion: Moxetumomab pasudotox, a novel recombinant anti-CD22 immunotoxin, was well tolerated and active in the previous phase 1 and 3 studies in patients with HCL. The drug has been approved in 2018 by the FDA for the treatment of patients with relapsed/refractory HCL who had at least two prior systemic therapies including at least one purine nucleoside analog.Expert opinion: The use of moxetumomab pasudotox is a promising new strategy for the treatment of HCL.

Hairy cell leukemia: present and future directions

Hairy cell leukemia (HCL) is an indolent B-cell malignancy, with long-term responses to purine analogs, but with decreasing efficacy and increasing toxicity with repeated courses. Leukemic cells express CD22, CD20, CD25, tartrate-resistant acid phosphatase (TRAP), annexin 1A (Anxa1), and BRAF V600E mutation. HCLv, lacking CD25, Anxa1, TRAP, and BRAF V600E, is more aggressive and less purine analog-sensitive. A molecularly defined IGHV4-34+ variant is also resistant whether HCL or HCLv immunophenotypically. Traces of HCL cells, termed minimal residual disease (MRD), accompany most with complete remission (CR) and may cause relapse. Rituximab has limited single-agent activity, but frequent CR without MRD when combined with purine analog, albeit with chemotherapy toxicities. The anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox can achieve MRD-negative CR in multiply relapsed HCL without chemotherapy toxicities and was FDA approved in 2018 as Lumoxiti. Investigational oral non-chemotherapy options also include Vemurafenib or Dabrafenib/Trametinib targeting BRAF V600E ± MEK, and Ibrutinib targeting Bruton’s tyrosine kinase.

Molecular Remissions with Anti-CD22 Recombinant Immunotoxin Moxetumomab Pasudotox Are Associated with Improved Complete Remission Durations during Phase I and III Testing

BACKGROUNDHairy cell leukemia (HCL) is an indolent B-cell malignancy strongly expressing CD22, with high sensitivity to purine analog chemotherapy, but relapses frequently occur, associated with minimal residual disease (MRD). Targeted non-chemotherapeutic approaches have shown efficacy but leave MRD. Moxetumomab pasudotox is an anti-CD22 recombinant immunotoxin recently reported in relapsed/refractory HCL to achieve 57% and 51% complete remission (CR) rates in phase 1 and 3 testing, respectively. Eradication of MRD by bone marrow flow cytometry or immunohistochemistry was associated with improved CR duration. We have previously reported that Taqman real-time quantitative PCR (RQPCR), which amplifies patient- and HCL-specific B-cell immunoglobulin rearrangements (IGHV), can detect one HCL cell in 106 normal cells. In the present study we determined whether MRD eradication measured by RQPCR is associated with improved CR duration. METHODSA total of 28 patients achieving CR to moxetumomab pasudotox in phase 1 (n=18) and phase 3 (n=10) trials were studied at the National Institutes of Health by multicolor flow cytometry of the bone marrow aspirate (BMA) and blood. Patients received moxetumomab pasudotox at 40-50 mcg/Kg on days 1, 3 and 5 of 2-7 cycles (median 5) at 4-week intervals. Blood and bone marrow samples were obtained to determine CR. In addition to end of treatment, bone marrow samples for MRD were generally obtained every 6-12 months for 2.5 years, then every other year. Blood flow cytometry was obtained every 6 months for 2.5 years, then yearly. Follow-up (time since treatment initiation) was 26.9-121.8 (median 84) months. For this analysis, CR for each bone marrow time point was determined by standard criteria for all patients independent of specific data-reporting requirements and data cut-off time points. RQPCR testing for each patient required cloning the immunoglobulin heavy chain variable regions and designing sequence-specific primers and probes. RNA samples were made from PAXgene tubes of blood and sodium-heparin tubes of BMA. RESULTSWe found that BMA RQPCR was most sensitive, followed by BMA flow cytometry, blood RQPCR, and blood flow cytometry. Of the 28 patients studied, 12 (43%) achieved MRD-free CR by all 4 tests and 2 of 12 patients (17%) relapsed. Seven (25%) were MRD+ by BMA RQPCR but negative by the other 3 studies, and none relapsed. One patient RQPCR+ and flow-negative in both blood and BMA relapsed. Seven patients flow-negative but RQPCR+ in blood, flow+ in BMA, and either BMA RQPCR+ or RQPCR not done, all relapsed. One patient with BMA RQPCR not done and the other 3 studies positive relapsed. With respect to RQPCR of blood as a single test, 3 of 20 negative vs 8 of 8 positive patients relapsed (p<0.001). Median CR duration was 13.0 months in blood RQPCR+ patients, vs not reached in blood RQPCR-negative patients, 17 (85%) of whom remain in CR at 11.6-112.9 (median 53) months of follow-up. With respect to BMA status, 2 of 12 patients negative vs 9 of 16 patients positive by either flow or RQPCR relapsed (p=0.054), and the 12 BMA MRD-negative patients had longer CR duration than the other 16 patients (p=0.018). CONCLUSIONSRQPCR negativity of blood is strongly associated with prolonged CR duration after moxetumomab pasudotox and may be a useful measure of MRD when bone marrow studies cannot be done. Additional testing will be needed to determine if blood RQPCR can be used to determine the optimal number of cycles of immunotoxin to administer. BMA RQPCR, though more sensitive than blood RQPCR, was not as useful due to fewer patients being evaluable and more time needed for BMA-RQPCR+ patients to relapse. Additional testing and follow-up may determine if BMA RQPCR negativity will correlate significantly with CR duration. [Display omitted] DisclosuresYao:MedImmune: Employment. Marshall:MedImmume: Employment. Pastan:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox. Kreitman:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox.

Moxetumomab Pasudotox: First Global Approval.

Moxetumomab pasudotox-tdfk (LUMOXITI™), an anti CD22 recombinant immunotoxin, has been developed by MedImmune and its parent company AstraZeneca for the treatment of hairy cell leukaemia. The product, discovered at the National Cancer Institute, is an optimised version of immunotoxin CAT-3888. Moxetumomab pasudotox is composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A, PE38. The Fv portion of moxetumomab pasudotox binds to CD22, a cell surface receptor expressed on a variety of malignant B-cells, thereby delivering the toxin moiety PE38 directly to tumour cells. Once internalised, PE38 catalyses the ADP ribosylation of the diphthamide residue in elongation factor-2 (EF-2), resulting in the rapid fall in levels of the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1), leading to apoptotic cell death. This article summarizes the milestones in the development of moxetumomab pasudotox leading to this first approval for the treatment of adults with relapsed or refractory hairy cell leukaemia who received at least two prior systemic therapies, including treatment with a purine nucleoside analogue. Development of moxetumomab pasudotox for non-Hodgkin’s lymphoma, chronic lymphocytic leukaemia and precursor cell lymphoblastic leukaemia/lymphoma was discontinued.

Phase 1 study of the anti-CD22 immunotoxin moxetumomab pasudotox for childhood acute lymphoblastic leukemia

Novel therapies are needed to overcome chemotherapy resistance for children with relapsed/refractory acute lymphoblastic leukemia (ALL). Moxetumomab pasudotox is a recombinant anti-CD22 immunotoxin. A multicenter phase 1 study was conducted to determine the maximum-tolerated cumulative dose (MTCD) and evaluate safety, activity, pharmacokinetics, and immunogenicity of moxetumomab pasudotox in children, adolescents, and young adults with ALL (N = 55). Moxetumomab pasudotox was administered as a 30-minute IV infusion at doses of 5 to 50 µg/kg every other day for 6 (cohorts A and B) or 10 (cohort C) doses in 21-day cycles. Cohorts B and C received dexamethasone prophylaxis against capillary leak syndrome (CLS). The most common treatment-related adverse events were reversible weight gain, hepatic transaminase elevation, and hypoalbuminemia. Dose-limiting CLS occurred in 2 of 4 patients receiving 30 µg/kg of moxetumomab pasudotox every other day for 6 doses. Incorporation of dexamethasone prevented further dose-limiting CLS. Six of 14 patients receiving 50 µg/kg of moxetumomab pasudotox for 10 doses developed hemolytic uremic syndrome (HUS), thrombotic microangiopathy (TMA), or HUS-like events, exceeding the MTCD. Treatment expansion at 40 µg/kg for 10 doses (n = 11) exceeded the MTCD because of 2 HUS/TMA/HUS-like events. Dose level 6B (ie, 50 µg/kg × 6 doses) was the MTCD, selected as the recommended phase 2 dose. Among 47 evaluable patients, an objective response rate of 32% was observed, including 11 (23%) composite complete responses, 5 of which were minimal residual disease negative by flow cytometry. Moxetumomab pasudotox showed a manageable safety profile and evidence of activity in relapsed or refractory childhood ALL. This trial was registered at www.clinicaltrials.gov as #NCT00659425.

Abstract 4595: Moxetumomab pasudotox spares CD4 and CD8 lymphocytes in multiply relapsed hairy cell leukemia patients, while prospective trials of first, second, and later lines of purine analogs show increasing toxicity

Abstract The purine nucleoside analogs cladribine and pentostatin damage DNA and cause long-term suppression of CD4 and CD8 lymphocytes in patients with hairy cell leukemia (HCL). There is increasing concern that these reductions, particularly in CD4+ T-cells, may be associated with opportunistic infections and secondary malignancies. The anti-CD22 recombinant immunotoxin moxetumomab pasudotox targets HCL without damaging DNA, and spares T-cells. In a phase 1 trial, 20 patients treated with 50 ug/Kg every other day for 3 doses for 2-8 (median 4) cycles were evaluated for T-cell subsets. CD4 cells increased -56% to 152% (median 46%) after end of treatment, decreasing in only 1 of the 20 patients. Because there is very little prospective data on the effect of purine analogs on T-cells, we analyzed data from 80 consecutively enrolled patients receiving cladribine in first line, 40 receiving cladribine in second line, and 52 receiving either pentostatin or bendamustine in third or later lines. The latter group was most appropriate to compare with moxetumomab pasudotox, which was also used in third or later-line treatment of HCL. CD4 counts after moxetumomab pasudotox were 85-600 (median 424), compared to 22-618 (median 73) after third or later line purine analog (p&amp;lt;0.0001). These CD4 counts after moxetumomab pasudotox were even higher than those after first line (86-954, median 259, p=0.0037) or second line (53-724, median 185, p&amp;lt;0.0001) cladribine. CD4 counts after first line cladribine were higher than those after second line cladribine (p=0004), which in turn were higher than those after third or later line purine analogs (p&amp;lt;0.0001). Even after 2 years, CD4 counts after third or later line purine analogs, 41-1004 (median 264), n=43, remained suppressed compared to 1st line cladribine, 191-1044 (median 392, n=68, p=0.0013). The percent decreases in CD4 counts after first line cladribine were significantly greater compared to those after moxetumomab pasudotox (median 63% decrease vs 46% increase, p&amp;lt;0.0001). CD8 counts were also preserved after moxetumomab pasudotox, increasing by -32% to 198% (median 46%). Similar differences between moxetumomab pasudotox and purine analog groups were observed with respect to CD8-cell suppression. Opportunistic infections, neutropenic fevers, and other infectious complications were much more common after purine analogs than after moxetumomab pasudotox, particularly in later lines of therapy. These data provide a rationale for avoiding repeated cycles of purine analog for relapsed HCL, and support the use of non-chemotherapy approaches like moxetumomab pasudotox before repeated courses of purine analog are tried. (Supported in part by MedImmune and NCI, Intramural Program). Citation Format: Robert J. Kreitman, Maryalice Stetler-Stevenson, Evgeny Arons, Ira Pastan. Moxetumomab pasudotox spares CD4 and CD8 lymphocytes in multiply relapsed hairy cell leukemia patients, while prospective trials of first, second, and later lines of purine analogs show increasing toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2017-4595

An immunoinhibition approach to overcome the impact of pre-existing antibodies on cut point establishment for immunogenicity assessment of moxetumomab pasudotox

Immunogenicity can impact PK, PD, efficacy and safety of biopharmaceuticals, and is often evaluated as a secondary objective in clinical studies. Methods to detect anti-drug antibodies (ADA) and neutralizing ADA (NAb) are semi-quantitative and utilize cut points to determine positive or negative samples. Assay cut points are established by the statistical analysis of treatment-naïve subject specimens that are assumed ADA and NAb-negative. Pre-existing antibodies to various biopharmaceuticals have been observed in treatment-naïve subjects and may artificially elevate the cut point, resulting in compromised assay sensitivities, inaccuracy in immunogenicity reporting and ultimately misleading assessment of the impact of immunogenicity on clinical outcomes. Although several approaches such as removal of pre-existing antibody samples or increasing the sample dilution could be used for cut point establishment to mitigate impact of pre-existing antibodies, they each have limitations, especially when a high prevalence of pre-existing antibodies is observed. Here we describe an innovative approach used to establish cut points for ADA and NAb assays of moxetumomab pasudotox (moxetumomab), a recombinant anti-CD22 immunotoxin, to which a high prevalence of pre-existing antibodies was observed. In order to overcome the challenges associated with this high prevalence and prevent establishment of an artificially elevated cut point, we developed an immunoinhibition approach that allowed generation of pseudo ADA and NAb-negative populations for cut point determination. Immunoinhibition was performed by adding excess moxetumomab (for ADA) or a non-CD22 binding PE38-containing immunotoxin, CAT-5001 (for NAb), to treatment-naive samples prior to evaluating samples for cut point establishment. This approach successfully eliminated pre-existing antibody activity in treatment-naive samples, enabling establishment of more accurate ADA and NAb assay cut points. A comparative analysis of the clinical immunogenicity results using cut points derived with immunoinhibition and without immunoinhibition (conventional method) demonstrated that the immunoinhibition approach markedly improved detection sensitivity and accuracy of immunogenicity characterization in patient samples. This innovative approach provides an alternative, practical solution for immunogenicity assay cut point establishment when biopharmaceuticals have a high prevalence of pre-existing antibodies.

Immunotoxin Activity Against B-Lineage Leukemia and Lymphoma Is Exposure Time Dependent: Implications for Trial Design

Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT) Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (HCL) (Kreitman et al 2012), but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR 2014). In contrast, HA22 is active vs primary blasts from 24/35 (68%) children with ALL in vitro (Mussai et al 2010). The apparent discrepancy between clinical response rates and in vitro cytotoxicity against ALL might be explained by the short serum half-life observed in clinical trials (approximately 1 hour). We hypothesized that blasts need to be exposed to RIT for longer in order to die. We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11, which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These modifications have been associated with pre-clinical improvements including reduced side effects, prolonged half-life, and diminished immunogenicity. LMB-11 achieved sustained CR in a Burkitt lymphoma animal model (Bera et al 2014). In the current study, we used LMB-11 and HA22 to evaluate activity against ALL and to try to identify methods to maximize in vitro cytotoxicity with the goal to increase clinical response rates in future clinical trials.Methods: Cytotoxicity was assessed with growth inhibition assays (WST-8). ALL cells were incubated with RIT for various times, washed, re-plated for a total of 72 h, stained for apoptosis by Annexin V-PE/7-AAD, and cell death analyzed by flow cytometry. NSG mice were inoculated with 5 million ALL cells IV on day 1 and treated with RIT from day 8 either IV or by continuous infusion with ALZET osmotic pumps implanted into the peritoneal cavity. Bone marrow (BM) samples were analyzed by flow cytometry for ALL infiltration and apoptosis.Results:In vitro cytotoxicity (IC50) of cell lines varied 210-fold (0.02 to 4.6 ng/ml). The time that cells had to be exposed to RIT to kill >90% of the cells varied greatly from <30 minutes to >4 days. We observed similar exposure time variability on 8 primary ALL samples. Despite this variability, for 57% (8/14) of all samples tested, the two toxins HA22 and LMB-11 were similar in the time to induce cell death. For 38% (5/14) of samples, HA22 was up to 10-fold more active and killed more rapidly than LMB-11. One example was KOPN-8, which was killed after less than 30 minutes exposure to HA22, but needed more than 9 hours exposure to LMB-11 for similar effects. The difference in exposure time dependent killing between the two toxins was related to intracellular RIT-processing. Using two newly developed ALL xenograft models with KOPN-8 or HAL-01, we found that response in vivo correlated directly with the time needed to kill the cells in vitro. Because RITs have a short serum half-life in mice (<30 minutes) but killing of KOPN-8 or HAL-01 cells needed 9 h or 24 h exposure to LMB-11, we replaced bolus dosing with small doses given at frequent intervals or with continuous infusion. The change in dosing strongly improved response of both KOPN-8 and HAL-01 xenografts (table 1).Conclusion: The previously unrecognized high variability of the time a CD22-targeting immunotoxin requires to kill ALL blasts provides a potential explanation for differences between in vitro cytotoxicity and the response rates observed in clinical trials. Our data indicate that ALL patients might show improved responses to anti-CD22 immunotoxins if treated with continuous infusion rather than the currently used bolus injection.Table 1In vivo response to immunotoxin improved with change in dosing.ALL Xenograft Cell lineRITChange in dosingChange in responseKOPN-8LMB-11QOD → frequent injectionsSD → CRHAL-01HA22QOD → continuous infusionPD → PRRIT = recombinant immunotoxin, QOD = every other day, IP = intraperitoneal, SD = stable disease, CR = complete response, PD = progressive disease, PR = partial response. DisclosuresWayne:Medimmune: Honoraria, Other: travel support, Research Funding; NIH: Patents & Royalties; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding. Pastan:NIH: Patents & Royalties: Coinventor on NIH Patent.

Abstract A157: Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia

Abstract Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT) Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (Kreitman et al 2012), but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR 2014). We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11, which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These modifications have been associated with reduced side effects, prolonged half-life, diminished immunogenicity, and better responses in in a Burkitt lymphoma animal model (Bera et al 2014). Activity of HA22 and LMB-11 on ALL cells varies significantly in vitro and in vivo (Müller et al AACR 2015). With the goal to improve activity against ALL, we combined CD22-targeting RITs with the multi-kinase inhibitor H89. H89 inhibits PKA (135 nM) and other kinases such as S6K1 (80 nM), MSK1 (120 nM), and ROCK2 (270 nM) (Davies et al 2000). Methods: In vitro cytotoxicity (IC50) was determined by WST-8 assay, and cell death by Annexin V-PE/7-AAD flow cytometry. An in vitro screen for H89 targets was performed by Reaction Biology Corp. Results: 10 uM H89 specifically enhanced activity of LMB-11 or HA22 10- fold on KOPN-8; activity of controls were unchanged. H89 also increased RIT activity by 5 to 10-fold on the ALL cell lines REH, SEM, HAL-01, and Nalm-6 and on 4 primary ALL samples. H89 did not change the rates of RIT-internalization or of furin-cleavage in KOPN-8. However, the time to ADP-ribosylate EF2 and arrest protein synthesis was shortened from 10 to 4 h with H89. We tested phosphorylation states of downstream targets in H89 treated KOPN-8 cells. Phosphorylation level of the PKA targets p-CREB or p-ATF2 were not reduced by H89. A high baseline phosphorylation of rpS6 or GSK-3β at the S6K1 target site was decreased to undetectable levels 15 minutes after addition of H89 to KOPN-8 and MCL1 fell by 50% within 15 minutes. In KOPN-8, a low dose of 5 ng/ml LMB-11 or 10 uM H89, respectively lowered MCL1 but did not induce apoptosis; MCL1 was undetectable and PARP-cleavage induced when H89 and LMB-11 were combined. The more specific S6K1 inhibitor PF-4708671 showed only 2-fold enhancement of RIT-induced cytotoxicity. The ROCK2 inhibitor Stemolecule similarly enhanced cytotoxicity 2-fold. MSK-1 inhibition or PKA-inhibition as well as PKA activation did not alter RIT activity. We performed a kinase screen on 359 kinases of which 21 were inhibited more than 90% by H89. None of the additionally identified kinases showed enhancement of RIT activity once blocked with relevant inhibitors. Despite combinations of small molecule inhibitors (e.g. ROCK2 and S6K1; GSK-3β and ROCK2), we were not able to mimic the 10-fold enhancement of RIT-induced cytotoxicity observed with H89. Conclusion: H89 strongly enhances CD22-targeting RITs against ALL. The increase in activity was in part due to inhibition of S6K1 and ROCK2 and was reflected by a faster onset of ADP-ribosylation of EF2 indicating improved trafficking of RITs through the various intracellular compartments. H89 and RIT lowered MCL1 levels by a distinct mechanism and synergistically induced intrinsic apoptosis. Further pre-clinical testing to assess in vivo efficacy of this combination is ongoing. Citation Format: Fabian Mueller, Xiu Fen Liu, Alan S. Wayne, Ira Pastan. Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A157.

Class II human leucocyte antigen DRB1*11 in hairy cell leukaemia patients with and without haemolytic uraemic syndrome.

Frequencies of human leucocyte antigens (HLA) were determined in 287 classic hairy cell leukaemia (HCL) patients. With respect to both population (n = 287) and allele (2n = 574) frequency respectively, the most common HLA class I and II antigens expressed were HLA-A*02 (49·1% and 28·6%), HLA-B*07 (21·3% and 11·1%), HLA-C*07 (46·7 and 28·2%), HLA-DQB1*03 (62·7% and 37·3%), HLA-DRB1*11 (30·0% and 16·0%) and HLA-DRB4*01 (45·3% and 29·6%). In comparing 6-14 databases of control Caucasians to 267 Caucasian HCL patients, only HLA-DRB1*11 was consistently over-represented in HCL, 31·1% of patients vs. 17-19·9% of controls (P = 0·0055 to <0·0001) and 16·5% of alleles vs. 6·5-12·3% of control alleles (P = 0·022 to <0·0001). HLA-DRB1*11 is a known risk factor for acquired thrombotic microangiopathy. Anti-CD22 recombinant immunotoxin BL22 in HCL was associated with a 12% incidence of completely reversible grade 3-4 haemolytic uraemic syndrome (HUS), mainly during the second or third retreatment cycle. Of 49 HCL patients receiving ≥2 cycles of BL22, 7 (14%) had HUS and HLA-DRB1*11 was expressed in 71% of 7 with HUS compared with only 21% of 42 without (P = 0·015). These data suggest that DBR1*11 may be a marker for increased susceptibility to HCL and, among HCL patients, could be a risk factor for BL22-induced HUS.

Pharmacokinetic Analysis Of Response In Hairy Cell Leukemia Treated By Anti-CD22 Recombinant Immunotoxin Moxetumomab Pasudotox

BackgroundMoxetumomab pasudotox, an anti-CD22 recombinant immunotoxin, contains truncated Pseudomonas exotoxin and is currently undergoing pivotal phase III testing for relapsed and refractory hairy cell leukemia (HCL). The dose-escalation portion of the phase I trial was reported to achieve an overall response rate (ORR) of 86% including 46% complete remissions (CRs) in 28 HCL patients. A completely reversible form of grade 2 hemolytic uremic syndrome (HUS), based on laboratory changes but lacking clinical signs, was observed in 2 patients, and no dose-limiting toxicity (DLT) was observed. To determine the relationship between response and CD22 antigen ‘sink' from high CD22 density on HCL cells, plasma levels were studied. Those analyzed included the original 28 in the dose-escalation arm, an additional 20 at the highest dose level enrolled prior to 2012, and one additional patient enrolled prior to initiation of the pivotal trial. MethodsMoxetumomab pasudotox was administered to 49 patients at 5, 10, 20, and 30 µg/kg every other day for 3 doses (QODx3, n=3 each dose level), and then 4 and 33 patients received 40 and 50 µg/kg QODx3, respectively. Patients received retreatment cycles at 4 week intervals, with retreatment halted after progressive disease, high levels of neutralizing antibodies, HUS, or 2 cycles after achievement of MRD-free CR. MRD was studied using bone marrow biopsy (BMBx) immunohistochemistry (IHC), and flow cytometry of blood or bone marrow aspirate (BMA). Pharmacokinetics was determined from cytotoxicity assays to quantify active moxetumomab pasudotox in the plasma. ResultsThere were 28 (57%) CRs and 15 (31%) PRs for an ORR of 88% out of 49 patients. While CRs were insignificantly more frequent at 50 µg/kg QODx3 vs lower doses (64% of 33 vs 44% of 16, p=0.23), the difference in MRD-free CR rate was significant (39% vs 6%, p=0.02). Of the 21 CRs at 50 µg/kg QODx3, 13 (62%) remain in CR at a median relapse-free follow-up time of 32 months, and relapse occurred in 7 (88%) of 8 with MRD vs 1 (8%) of 13 without MRD (p<0.001). With respect to pharmacokinetics assessed on day 5 (3rd dose) of the 1st cycle (C1D5), dose level correlated with both peak level (r=0.58, p<0.0001) and area under the curve (AUC, r=0.45, p=0.003), but these varied within each dose level. Focusing on the 50 µg/kg QODx3 dose level, CR was more likely with lower volume of distribution (Vd, p=0.014) and clearance (p=0.02). With respect to PK parameters as a result of tumor burden, circulating HCL count correlated with C1D5 clearance (r=0.86, p<0.0001), and spleen diameter correlated with clearance on both C1D1 (r=0.50, p=0.01) and C1D5 (r=0.78, p<0.0001). An inverse correlation was observed between spleen diameter and peak level on C1D1 (p=0.027), and trends (p=0.06) were observed between spleen diameter and peak level on C1D5, and AUC on both C1D1 and C1D5. ConclusionsMoxetumomab pasudotox can achieve durable CR without MRD in relapsed and refractory HCL, with a safety profile supporting its current pivotal testing, including a dose level consistent with the activity of the 50 µg/kg QODx3 phase I dose. Our results also indicate that lower HCL tumor burden minimizes the CD22 ‘sink' effect allowing higher plasma levels, and suggest that patients treated while tumor burden is relatively low may have improved chance of durable CR. Moreover, strategies for improving the efficacy of moxetumomab pasudotox might include combination or sequential use of other therapies to decrease HCL burden.This summary contains investigator reported data. This study was sponsored by MedImmune and supported by NCI's Intramural Research Program and the Hairy Cell Leukemia Research Foundation. Disclosures:Kreitman:NIH: Co-inventor on the patent for moxetumomab pasudotox, Co-inventor on the patent for moxetumomab pasudotox Patents & Royalties, Employment, Research funding via CRADA with MedImmune Other. Arons:NIH: Employment. Stetler-Stevenson:NIH: Employment. Wilson:NIH: Employment. FitzGerald:National Cancer Institute, NIH: Employment, Moxetumomab Pasudotox, Moxetumomab Pasudotox Patents & Royalties. Pastan:NIH: Employment, Inventor on immunotoxin patents, which are all assigned to NIH, Inventor on immunotoxin patents, which are all assigned to NIH Patents & Royalties.

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.

AbstractAbstract 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.

Decreased Expression of Diphthamide Genes Is Responsible for the Resistance to the Anti-CD22 Immunotoxin Moxetumomab Pasudotox (m. pasudotox) in Acute Lymphoblastic Leukemia (ALL)/Non-Hodgkin Lymphoma (NHL) Cell Lines.

AbstractAbstract 2443 Background:Complete remissions have been observed in some but not all children in an ongoing Phase 1 trial of the anti-CD22 recombinant immunotoxin moxetumomab pasudotox (m. pasudotox, HA22, CAT-8015) for relapsed and refractory ALL and NHL. Objectives:To develop ALL/NHL cell lines resistant to m. pasudotox in attempt to uncover possible mechanisms of immunotoxin resistance and to use this information to try to improve clinical outcomes. Methods:Using the ALL cell line HAL-01 and the Burkitt NHL cell line CA46, we generated two cell lines (HAL-01-R and CA46-R, respectively) that were resistant to killing by m. pasudotox by repeated exposure to sub-lethal doses of m. pasudotox. We studied the basis of their resistance. Results:Cytotoxicity was markedly reduced in the resistant cell lines (Table) and m. pasudotox was unable to ADP-ribosylate and inactivate elongation factor-2 (EF2). In HAL-01-R this was due to a low level of DPH4 mRNA and protein, which prevented diphthamide biosynthesis and rendered EF2 refractory to m. pasudotox. Analysis of the promoter region of the DPH4 gene showed that the CpG island was heavily methylated. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells. A second resistance mechanism in the diphthamide pathway was identified in the CA46-R line. There was a small decrease in CD22 site density and internalization rate in CA46-R cells, which were insufficient to account for m. pasudotox resistance. In contrast, protein synthesis measured by 3H-leucine incorporation was not reduced in CA46-R cells suggesting a defect in diphthamide modification of EF2. Consistent with this hypothesis was the finding that m. pasudotox catalyzed the incorporation of ADP-ribose into EF2 in extracts of sensitive CA46 cells, but not in CA46-R cells, although EF2 protein levels were similar in the two cell lines. We examined the expression of the six genes required for diphthamide synthesis (DPH1–5 and WDR85). Only the levels of WDR85 mRNA and protein were substantially decreased in CA46-R cells. We used shRNA to knock down WDR85 mRNA and protein levels and tested the cells for sensitivity to m. pasudotox. The IC50 increased 10-fold from 0.6 ng/ml to 6.0 ng/ml. Conversely, we restored WDR85 protein expression into CA46-R cells by transduction of resistant cells with a WDR85 expressing vector, which partially restored sensitivity to m. pasudotox. Altogether, these findings show that a reduction in WDR85 protein is sufficient to cause m. pasudotox resistance. To study why WDR85 expression is reduced in CA46-R cells, a high resolution SNP array analysis of the WDR85 gene was performed. A homozygous deletion at Chr9:140,468,104–140,610,581 (Build: GRCh37/19) was detected, which accounts for the inability of the cells to make WDR85 protein. Conclusion:We isolated two immunotoxin-resistant ALL/NHL cell lines and found that resistance was associated with reduced expression of diphthamide synthesis genes required to correctly post-translationally modify EF2. As a consequence, m. pasudotox was unable to ADP-ribosylate and inactivate EF2. Reduced expression of DPH4 in HAL-01-R was related to hypermethylation and could be prevented by 5-azacytidine. Reduced expression of WDR85 in CA46-R was due to a deletion of the gene. These represent novel mechanisms of immunotoxin resistance that will be of interest to explore in future studies.HAL-01HAL-01-RCA46CA46-RIC50 (ng/mL)1.1>1000.6>100This study was supported by the Intramural Research Program of the NIH, National Cancer Institute (NCI), Center for Cancer Research and by a Cooperative Research and Development Agreement between MedImmune, LLC and the NCI. Disclosures:No relevant conflicts of interest to declare.