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Anti-CD22 moxetumomab pasudotox achieved 46% complete remissions (CRs) in previously reported phase 1 testing in relapsed/refractory hairy cell leukemia (HCL; n = 28). The importance of minimal residual disease (MRD) after CR in HCL is unknown. A 21-patient extension cohort received 50 µg/kg every other day for 3 doses in 4-week cycles. These patients plus 12 previously reported at this upper dose level received 143 cycles without dose-limiting toxicity. The combined 33-patient cohort achieved 64% CR and 88% overall response rates, with median CR duration of 42.4 months. Of 32 50-µg/kg patients evaluable for MRD by bone marrow aspirate flow cytometry (most stringent assessment), median CR duration was 13.5 (4.9-42.4) months in 9 MRD-positive CRs vs 42.1 (24.0-69.2) months in 11 MRD-negative CRs (P < .001). Among MRD-negative CRs, 10 patients had ongoing CR, 9 without MRD, at end of study. To our knowledge, moxetumomab pasudotox is the only nonchemotherapy regimen that can eliminate MRD in a significant percentage of HCL patients, to enhance CR duration. Repeated dosing, despite early neutralizing antibodies, increased active drug levels without detectable toxicity from immunogenicity. The activity and safety profiles of moxetumomab pasudotox support ongoing phase 3 testing in HCL. This trial was registered at www.clinicaltrials.gov as #NCT00586924.

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.

Unconjugated monoclonal antibodies that target hematopoietic differentiation antigens have been developed to treat hematologic malignancies. Although some of these have activity against chronic lymphocytic leukemia and hairy cell leukemia, in general, monoclonal antibodies have limited efficacy as single agents in the treatment of leukemia. To increase their potency, the binding domains of monoclonal antibodies can be attached to protein toxins. Such compounds, termed immunotoxins, are delivered to the interior of leukemia cells based on antibody specificity for cell surface target antigens. Recombinant immunotoxins have been shown to be highly cytotoxic to leukemic blasts in vitro, in xenograft model systems, and in early-phase clinical trials in humans. These agents will likely play an increasing role in the treatment of leukemia.

Moxetumomab pasudotox-tdfk is a first-in-class recombinant CD22-directed cytotoxin approved in the US for the treatment of adult patients with relapsed/refractory hairy cell leukemia (HCL). The pivotal, multicenter, open-label, single-arm trial (Study 1053; NCT01829711) evaluated the efficacy, safety, immunogenicity, and pharmacokinetics of moxetumomab pasudotox-tdfk monotherapy in patients with relapsed/refractory HCL. Here we present the final analysis, describing the long-term follow-up of patients in Study 1053, with a median follow-up of 24.6 months (range 1.2-71.7). Eligible patients had received ≥ 2 prior systemic therapies, including ≥ 2 courses of a purine nucleoside analog (PNA) or 1 course of a PNA followed by ≥ 1 course of rituximab or a BRAF inhibitor. Patients received moxetumomab pasudotox-tdfk 40 µg/kg intravenously on days 1, 3, and 5 of each 28-day cycle for 6 cycles, or until minimal residual disease (MRD)-negative complete response (CR), disease progression, initiation of alternate therapy, or unacceptable toxicity. Disease response and immunohistochemistry MRD status were determined by blinded independent central review. The primary endpoint was durable CR (achieving CR and maintaining hematologic remission [HR] for > 180 days). Durable CR with HR ≥ 360 days was measured in this final analysis. HR was defined as hemoglobin ≥ 11.0 g/dL, absolute neutrophil count ≥ 1.5 × 103/µL, and platelet count ≥ 100 × 103/µL without receiving transfusions or growth factors within the 4 preceding weeks of assessment. Eighty patients (63 males; median age 60 years [range 34-84]) were enrolled and treated with moxetumomab pasudotox-tdfk. The median number of prior systemic therapies was 3 (range 2-11); 48.8% of patients were PNA-refractory and 37.5% were unfit for PNA retreatment. The median number of treatment cycles administered was 6 (range 1-7). The durable CR rate was 36.3% (29/80 patients; 95% confidence interval [CI]: 25.8-47.8%), durable CR rate with HR ≥ 360 days was 32.5% (26/80 patients; 95% CI: 22.4-43.9%), and the overall CR rate was 41.3% (33/80 patients; 95% CI: 30.4-52.8%) (Table). Overall, 27/33 (81.6%) patients who achieved a CR also achieved MRD-negative status (33.8% of all patients). HR was achieved by 64/80 patients (80.0%). The median duration of HR from CR was 62.8 months (Figure) and median progression-free survival was 41.5 months (range 0.0+ to 71.7). Per the primary analysis, the most frequent treatment-related adverse events (AEs) were nausea (28%), peripheral edema (26%), headache (21%), and pyrexia (20%). Treatment-related Grade 3/4 AEs were reported in 24 patients (30.0%) and serious AEs in 28 (35.0%). Treatment-related AEs led to study drug discontinuation in 8 patients (10.0%): hemolytic uremic syndrome (HUS), n = 4 (5.0%); capillary leak syndrome (CLS), n = 2 (2.5%); and increased blood creatinine, n = 2 (2.5%). Grade ≥ 3 CLS events occurred in 2 patients (2.5%) and Grade ≥ 3 HUS occurred in 5 patients (6.3%). In general, CLS and HUS events were manageable and reversible with appropriate supportive care and monitoring. Based on primary and follow-up analyses of serum creatinine, there was no decline in renal function over time; 12 months post-treatment all mean (SD) serum creatinine laboratory values stayed within normal limits, which suggests current management strategies for HUS (oral hydration, i.v. fluid supplementation on the day of infusion, and the use of dexamethasone) are adequate. With 24.6 months of follow-up, 4 deaths were reported: 2 due to study disease progression and 2 due to an AE (1 each of pneumonia and septic shock). Moxetumomab pasudotox-tdfk treatment was associated with a manageable safety profile. Moxetumomab pasudotox-tdfk achieved a high rate of durable responses, demonstrating the ability to achieve MRD negativity in heavily pretreated patients with HCL. Disclosures Kreitman: Genentech: Research Funding. Dearden:Abbvie: Honoraria; Genentech: Honoraria; Janssen: Honoraria; Sanofi: Honoraria. Zinzani:MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VERASTEM: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELLTRION: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GILEAD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; IMMUNE DESIGN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSAPHARMA: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ROCHE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANDOZ: Membership on an entity's Board of Directors or advisory committees; KYOWA KIRIN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANOFI: Consultancy; PORTOLA: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Robak:Amgen: Consultancy, Other: Travel grant; UCB: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grant, Research Funding; Roche: Consultancy, Other: Travel grant, Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel grant, Research Funding; Gilead: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Acerta: Research Funding; Morphosys AG: Research Funding; Takeda: Consultancy, Research Funding. le Coutre:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Gjertsen:Research Council of Norway: Research Funding; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BerGenBio: Consultancy; KinN Therapeutics AS: Equity Ownership; Seattle Genetics: Consultancy; ERA PerMed: Research Funding; Haukeland University Hospital / University of Bergen: Employment; ACTII AS: Equity Ownership; Astellas: Consultancy; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees; The Norwegian Cancer Society: Research Funding; Helse Vest Health Trust: Research Funding; EU Horizon 2020: Research Funding. Troussard:Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Other: Research Support; Sysmex: Other: Research Support. Roboz:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Karlin:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support. Kuptsova-Clarkson:AstraZeneca: Employment, Equity Ownership. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership.

BACKGROUND: Hairy 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. METHODS: A 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. RESULTS: We 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 CONCLUSIONS: RQPCR 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. Figure. Figure. Disclosures Yao: 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.

Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.

A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC50) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.

Background: Moxetumomab pasudotox (Moxe) is a recombinant immunotoxin that targets CD22. Moxe showed high clinical activity in a phase II trial against hairy cell leukemia (Kreitman et al, 2012) and a phase III trial has recently completed enrollment. Moxe also demonstrated objective responses in 30% of children with multiply relapsed acute lymphoblastic leukemia (ALL) (Wayne et al AACR 2014). In a xenograft model with the ALL cell line KOPN-8, Moxe as monotherapy clears the murine bone marrow (BM) from leukemia blasts (Mueller et al, CCR 2016). However, mice eventually relapse and die of progressive disease. Goal: To characterize the ALL cells responsible for relapse and to devise a relapse-preventing treatment. Methods/Results: The ALL cell lines KOPN-8 and REH were transduced with luciferase. The systemic bioluminescence signal in KOPN-8 bearing mice was abrogated after three i.v. bolus-doses of Moxe. Relapse was subsequently detected at various, apparently randomly distributed BM sites which possibly were the origin of systemic relapse. The systemic relapse remained Moxe-sensitive for several treatment cycles, but the bioluminescence signal at the discrete BM sites persisted. After several treatment cycles a Moxe-resistant systemic relapse emerged and mice died of progressive disease as determined by bioluminescence and flow cytometry. The Moxe-resistant cells showed a loss of CD10, and a 2.5-fold decrease in surface CD19 and CD22. CD22 mRNA levels and total CD22 protein by Western Blot were reduced up to 7-fold, indicating a profound reduction of intracellular CD22 in resistant cells. RNA deep sequencing revealed a global transcriptional change including down-regulation of B-cell specific genes suggesting a less mature state of KOPN-8 in the Moxe resistant blasts. We had previsouly demonstrated an epigenetic mechanism of Moxe resistance in vitro (Hu et al, Leuk Res 2013). Because most of the genetically homogeneous KOPN-8 cells growing in murine BM were Moxe sensitive but cells at discrete sites persisted, we hypothesized that locally induced epigenetic changes might partially explain the resistance. We therefore combined Moxe with the DNA-methyltransferase inhibitor 5-Azacytidine (5-AZA). KOPN-8 bearing mice treated with Moxe or 5-AZA alone survived longer than untreated mice, but eventually died from leukemia. However, mice that received the combination of 5-AZA and Moxe were disease free at 4 months after treatment by flow cytometry and histopathology. The combination of 5-AZA and Moxe also improved responses over each agent alone in a second ALL xenograft model with the ALL cell line REH. Conclusion: The relapse of KOPN-8 after treatment with Moxe, possibly from discrete BM sites is consistent with BM-niche induced resistance. This niche-induced resistance is further supported by locally persisting cells after repeated treatment cycles, while blasts that repopulate other BM sites remain rIT-sensitive. That relapse at discrete BM-sites is prevented by 5-AZA suggests that epigenetic modifications contribute to induction of localized resistance in vivo. rIT-resistant KOPN-8 relapse shows a profound reduction in total CD22 which may contribute to resistance to CD22-directed rIT. The marked increase of Moxeefficacy in vivo by the addition of 5-AZA makes this combination a promising approach for further clinical testing in children and adults with ALL. Disclosures Wayne: Kite Pharma: Honoraria, Other: Travel support, Research Funding; Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Pastan:NIH: Patents & Royalties: Co-Inventor on NIH Patent.

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.

To determine whether the multidrug resistance gene MDR1could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that theMDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Many B-cell malignancies express the CD22 antigen on their cell surface. To kill cells expressing this antigen, the RFB4 monoclonal antibody (MoAb) has been linked chemically with either deglycosylated ricin A chain or truncated versions of Pseudomonas exotoxin. These immunotoxins exhibited selective cytotoxic activity for CD22+ cells and antitumor activity in nude mouse models bearing human B-cell lymphomas. To construct a recombinant immunotoxin targeted to CD22, we first cloned the variable portions of the heavy and light chains of RFB4. The cloned Fv fragments were joined by a newly created disulfide bond to form a disulfide stabilized (ds) construct. The RFB4 construct was combined by gene fusion with PE38, a truncated version of PE. The recombinant immunotoxin was then expressed in Escherichia coli, purified by column chromatography and tested for cytotoxicity activity. RFB4(dsFv)PE38 retained its binding activity for CD22, was very stable at 37°C and exhibited selective cytotoxic activity for CD22+-cultured cell lines. Because of its favorable binding characteristics and potency for CD22-positive cell lines, RFB4(dsFv)PE38 was tested for antitumor activity in a nude mouse model of human lymphoma. CA46 cells were injected subcutaneously and then treated with the RFB4(dsFv)PE38 immunotoxin. Antitumor activity was dose responsive and was not evident when an irrelevant immunotoxin was administered on the same schedule.

The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is a potential target for toxin-directed therapy, because it is overexpressed on many leukemias and solid tumors and apparently not on stem cells. To investigate the potential therapeutic use of GM-CSF toxins, we fused human GM-CSF to truncated forms of either Pseudomonas exotoxin (PE) or diphtheria toxin (DT) and tested the cytotoxicity of the resulting GM-CSF–PE38KDEL and DT388–GM-CSF on human gastrointestinal (GI) carcinomas and leukemias. Toward gastric and colon cancer cell lines, GM-CSF–PE38KDEL was much more cytotoxic than DT388–GM-CSF, with IC50s (concentration resulting in 50% inhibition of protein synthesis) of 0.5 to 10 ng/mL compared with 4 to 400 ng/mL, respectively. In contrast, toward leukemia lines and fresh bone marrow cells DT388–GM-CSF was more cytotoxic than GM-CSF–PE38KDEL. The cytotoxicity of both GM-CSF–PE38KDEL and DT388–GM-CSF toward the human cells was specific, because it could be competed by an excess of GM-CSF. Binding studies indicated that human GM-CSF receptors were present on all of the human GI and leukemic cell lines tested, at levels of 540 to 3,700 sites per cell (kd = 0.2 to 2 nmol/L), and the number of sites per cell did not correlate with the cell type. A similar pattern of cytotoxicity was found with recombinant immunotoxins binding to the transferrin receptor, in that anti-TFR(Fv)–PE38KDEL was much more cytotoxic than DT388–anti-TFR(Fv) toward GI cells, but both were similar in their cytotoxic activity toward leukemia cells. The fact that PE is more effective than DT in killing GI but not leukemic tumor cells targeted by GM-CSF indicates a fundamental difference in the way PE or DT gains access to the cytosol in these cells. GM-CSF–PE38KDEL and DT388–GM-CSF deserve further evaluation as possible treatments for selected tumors.

We have previously shown that human renal cell carcinoma (RCC) cells express large numbers of interleukin-13 receptors (IL-13R), a newly described hemopoietic growth factor receptor. To target tumor cells that express IL-13R, we have produced a chimeric protein composed of human IL-13 and a derivative of Pseudomonas exotoxin A, termed PE38QQR. We report here that IL13-PE38QQR is highly cytotoxic to many human RCC cell lines. IL-13R-negative cell lines or cell lines expressing low numbers of IL-13R ( < 300 sites/cell) that include human bone marrow- derived cells were not susceptible to the cytotoxic effect of IL 13- PE38QQR. The sensitivity of RCC cells to IL13-PE38QQR correlated positively with the density of IL-13R. The cytotoxic activity of IL13- PE38QQR was competed by an excess of IL-13 in a protein synthesis inhibition assay and confirmed by a clonogenic assay. Even though IL-13 and IL-4 are homologues and IL-4R and IL-13R have been proposed to share a receptor subunit, IL-4 did not compete for the cytotoxicity mediated by IL13-toxin on RCC. IL13-PE38QQR competes for [125I]-IL-13 binding sites on RCC cells, although at a lower affinity than the wild- type recombinant cytokine. Human T-cell, B-cell, and monocytic cell lines are unresponsive to the cytotoxic action of IL13-PE38QQR. Thus, our results indicate that IL13-PE38QQR is highly cytotoxic to human RCC cells, although it is not cytotoxic to a variety of normal hematopoietic cells. IL13-PE38QQR should be further investigated preclinically for the treatment of human RCCs.

Background: In patients with hairy cell leukemia (HCL), the clinical importance of minimal residual disease is unknown; particularly by bone marrow aspirate (BMA) multicolor flow cytometry. It is often assumed that agents failing to eradicate MRD may prove equally effective as those that do. Moxetumomab pasudotox is a recombinant immunotoxin containing truncated Pseudomonas exotoxin fused to an anti-CD22 Fv. After the dose-escalation phase of a phase I trial in 28 patients with relapsed/refractory HCL treated with 5-50 ug/Kg every other day for 3 doses (QOD x3), a complete remission (CR) rate of 46% and overall response rate (ORR) of 86% were reported. Two cases of reversible grade-2 hemolytic uremic syndrome (HUS) were also reported, with transient grade I platelet and creatinine changes. Methods: To determine response and toxicity at a fixed dose, Twenty-one additional patients were enrolled at the highest dose level, totaling 33 patients at 50 ug/Kg QOD x3 and 49 patients overall. Patients required at least 2 prior courses of purine analog, or 1 course each of purine analog and rituximab if response duration to the first purine analog was Results: No dose limiting toxicity or additional HUS cases were observed in any of the 202 cycles administered to 49 patients. Of 33 patients receiving 143 cycles of 50 ug/Kg QOD x3, the overall response rate was 88% and 21 (64%) achieved CR. Of 20 CRs followed for response duration, the median duration of CR (disease-free survival, DFS) was 42 mo (range 5 - 72), with 12 (60%) of 20 remaining in CR at a median of 42 mo. In addition to the 21 CRs achieved at 50 ug/Kg, there were 2 CRs out of 4 patients treated at 40 ug/Kg, and 5 CRs out of 12 patients treated at lower dose levels. Of the 28 CRs, MRD by BMA flow cytometry was evaluated in all except in 1 patient each at 50 and 40 ug/Kg, and 2 patients at lower doses. By BMA flow cytometry, MRD-negative CR was achieved in 11 (34%) of 32 at 50 ug/Kg, 1 of 3 at 40 ug/Kg, and 0 of 10 at lower doses (Fisher's Exact comparison above vs below 40 ug/Kg, p=0.041). MRD+ CRs included 9 at 50 ug/Kg, 0 at 40 ug/Kg, and 3 at lower doses. For the 12 MRD-negative CRs at 40-50 ug/Kg, DFS has not been reached, compared to a DFS of 17 mo in the 9 remaining MRD+ CRs at 40-50 ug/Kg (p Conclusion: Moxetumomab pasudotox can eliminate HCL MRD, and MRD eradication significantly correlated with CR durability. In HCL, particularly after multiple relapses, CRs should optimally be achieved without MRD. To our knowledge, moxetumomab pasudotox is the only known agent capable of eliminating MRD in HCL in a high percentage of patients without causing myelosuppressive toxicities common to chemotherapy. Its activity and safety profile support multicenter pivotal phase III testing, which is currently ongoing. 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 Lanasa: MedImmune: Employment. FitzGerald:NIH: Patents & Royalties: Coinventor on NIH Patent. Pastan:NIH: Patents & Royalties: Coinventor on NIH Patent.

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 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 ( 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 1. In vivo response to immunotoxin improved with change in dosing. ALL Xenograft Cell line RIT Change in dosing Change in response KOPN-8 LMB-11 QOD → frequent injections SD → CR HAL-01 HA22 QOD → continuous infusion PD → PR RIT = recombinant immunotoxin, QOD = every other day, IP = intraperitoneal, SD = stable disease, CR = complete response, PD = progressive disease, PR = partial response. Disclosures Wayne: 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.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989). Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown. To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells. ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice. In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40. IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365–380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively. All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50. The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50.

We have previously shown that the variable domains of the monoclonal antibody anti-Tac [anti-Tac(Fv)] can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor- bearing cells. We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL). In anti-Tac(Fv)- PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253–608 of PE and the -KDEL mutation at the carboxyl terminus. In DT388-anti- Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT. PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and [3H]-leucine incorporation was measured. Anti-Tac(Fv)-PE40KDEL was cytotoxic to 7 of the 14 patient samples, with half-maximal inhibition of protein synthesis (IC50) achieved at 1.2 to 9 ng/mL (1.8 to 13 x 10(-11) mol/L). DT388-anti- Tac(Fv) was cytotoxic to 11 of the 14 samples, with IC50s ranging from less than 1 to 250 ng/mL. DT388-IL-2, in which the first 388 amino acids of DT are attached to IL-2, was marginally cytotoxic toward only 4 of 13 CLL samples tested with IC50s ranging from 100 to 550 ng/mL. Trypan blue staining of cells from several patients indicated that inhibition of protein synthesis correlated with cell death. Binding assays using [3H]-anti-Tac indicated that the CLL cells from nine of the patients contained between 400 and 2,500 sites per cell. Cells from another patient, which were resistant to both anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), had less than 100 sites per cell. We conclude that anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv) can kill CLL cells which have low numbers of IL-2 receptors, and should be investigated further for therapy of this disease.

IL-6-PE4E is a recombinant protein consisting of interleukin-6 (IL-6) fused to a mutant form of Pseudomonas exotoxin in which four basic amino acids are changed to glutamate (PE4E). The chimeric toxin has been previously shown to specifically kill malignant hepatic, prostatic, epidermoid, and myeloma cell lines in vitro. To explore the possible clinical utility of IL-6-PE4E, particularly as an agent for ex vivo purging of marrow for autologous bone marrow transplantation (ABMT), we tested malignant cells from patients with multiple myeloma for sensitivity to this chimeric toxin. Ficoll-purified bone marrow cells were incubated with and without IL-6-toxin for 2 to 3 days. Eight of the 15 myeloma patients had cells that were sensitive to IL-6-toxin as measured by a decrease in the level of protein synthesis. Cells from five patients were very sensitive to IL-6-PE4E, with 50% inhibition of protein synthesis (ID50) achieved at or below 6 ng/mL (7 x 10(-11) mol/L). Cells from three additional patients showed moderate sensitivity, with ID50s between 30 and 140 ng/mL. The remaining seven samples showed little or no sensitivity, with ID50s greater than or equal to 400 ng/mL. Normal bone marrow cells or normal BFU-E and CFU-GM were resistant to the IL-6-toxin even at 1,000 ng/mL. Neither IL-6, IL- 2-PE4E, nor an enzymatically deficient mutant of IL-6-PE4E was cytotoxic toward the myeloma cells, indicating that the cytotoxic effect of IL-6-PE4E required the adenosine diphosphate-ribosylation function as well as the specific ligand. Our data suggest that IL-6- toxin could be effective in ex vivo marrow purging in selected multiple myeloma patients who are candidates for ABMT, and that this toxin should also be investigated further for in vivo therapy.

Many human cancers that are initially responsive to chemotherapy eventually fail to respond to treatment. For some drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow (BM) limit cytotoxic therapy. Approaches to prevent or circumvent BM toxicity are therefore a high priority of research on dose escalation protocols. In this study, we have transplanted BM cells from transgenic mice that constitutively express physiologic amounts of a functional human multidrug resistance (MDR1) cDNA to lethally irradiated C57BL/6 x SJL F1 mice (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and BM specimens as indicated by Southern blot analysis, and expressed MDR1 messenger RNA in BM samples as detected by slot blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal BM were drug sensitive. Finally, the chemoprotection afforded by the MDR1 gene could readily be reversed by adding chemosensitizers such as cyclosporin A and R-verapamil to chemotherapy. Hence, it appears that BM cells expressing the human MDR1 gene maintain this function after transplantation to host animals for a minimum of 10 months, and confer multidrug resistance to these BM recipients. This selective advantage conferred by expression of the MDR1 cDNA suggests a strategy for the use of MDR1 gene therapy in cancer chemotherapy and for the introduction of otherwise nonselectable genes into BM.

Background Moxetumomab 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. Methods Moxetumomab 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. Results There 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 Conclusions Moxetumomab 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.

Abstract 2488 Hairy cell leukemia (HCL) is a B-cell malignancy which is thought to originate in most patients after the B-cell contacts antigen. To determine whether certain HLA types are preferentially expressed in HCL, HLA class I and class II allele frequencies at low resolution were collected from 247 HCL patients including 233 Caucasian, 5 Black, 5 Hispanic, 2 Asian and 2 Hawaiian/Pacific Islanders. Out of 494 total alleles from the 247 patients, the most frequent were HLA-A*02 (145, 29%), HLA-B*07 (58, 12%), HLA-C*07 (141, 29%), and HLA-DRB1*11 (76, 15%). In comparison with normal donors, only HLA-DRB1*11 was preferentially expressed in HCL, with a population frequency among the 233 Caucasians of 70 (30%), compared to 17% of a database of USA Caucasians (n=61655, p Because it was recently reported that HLA-DRB1*11 is a strong risk factor for acquired ADAMTS13 deficiency-related thrombotic microangiopathy, HCL patients who had hemolytic uremic syndrome (HUS) after anti-CD22 recombinant immunotoxin BL22 were examined for HLA expression at the DRB1 locus. BL22 was previously reported to be associated in HCL with a 12% risk of completely reversible grade 3–4 HUS mainly during the 2nd or 3rd retreatment cycle. We found that of 49 HCL patients treated with at least 2 cycles of BL22, most (71%) of 7 HCL patients with HUS expressed HLA-DRB1*11, compared to only 21% of 42 without HUS (p=0.015). Even when considering all 66 evaluable HCL patients who received BL22, including the 17 who received just 1 cycle, expression of HLA-DRB1*11 was more frequent in those with compared to without HUS (63% of 8 vs 24% of 58 patients, p=0.038). In the poor-prognosis variant of HCL, termed HCLv, we found that the most common HLA-DRB1 gene expressed was HLA-DRB1*04, found in 19 (56%) of 34 patients, compared to 30% of the normal USA Caucasian database (n=61655, p=0.002) and 27–37% in the 8 smaller databases mentioned above (p values 0.016 and 0.001–0.01). HLA-DRB1*04 expression was recently reported to be significantly reduced in patients with idiopathic thrombotic microangiopathy. In BL22-associated HUS in HCL, DRB1*04 expression was not increased and none had HCLv. Moxetumomab pasudotox, an affinity-matured version of BL22, binds with 14-fold higher affinity to CD22 due to mutations in the CDR3 domain of the heavy chain, and was recently reported in 28 HCL patients to be associated with only 2 cases of transient grade 2 HUS, defined as grade 1 creatinine and platelet abnormalities. No HUS was observed in 20 additional HCL patients treated. Despite the similarity in the population incidences of HLA-DRB1*11 in those receiving moxetumomab pasudotox and BL22 (35% vs 29%), the grade 3–4 HUS risk was significantly lower with moxetumomab pasudotox than with BL22 (p=0.02). We conclude that 1) HLA-DRB1*11 is preferentially expressed in HCL, 2) HLA-DRB1*04 is preferentially expressed in HCLv, and 3) HLA-DRB1*11 constitutes a potential marker in HCL for susceptibility to HUS from BL22, but not from moxetumomab pasudotox, possibly because the latter molecule binds more specifically to CD22 and avoids HUS. Disclosures: Off Label Use: BL22 and Moxetumomab Pasudotox are experimental agents for CD22+ malignancies. Pastan:NIH: Coinventor on NIH patents for BL22 and Moxetumomab, Coinventor on NIH patents for BL22 and Moxetumomab Patents & Royalties. Kreitman:NIH: Coinventor on NIH patents for BL22 and Moxetumomab, Coinventor on NIH patents for BL22 and Moxetumomab Patents & Royalties.

Abstract 2611 CD22 is expressed on the surface of B cell hematologic malignancies such as acute lymphoblastic leukemia (ALL). CD22 is a Siglec family lectin present on B cells, starting at the pre-B cell stage of development, but is not expressed on plasma cells. CD22 consists of 7 extracellular Ig domains and is found in 2 isoforms, one of which is missing the second and third N-terminal Ig domains. We generated CAR modified T cells containing anti-CD22 extracellular binding motifs fused to intracellular signaling domains for T cells activation (CD3 zeta) or costimulation (CD28 or 4-1BB). The binding motifs were derived from scFvs that targeted a membrane distal epitope of CD22, Ig domain 3, (BL22 and a higher affinity HA22 motif) or that bound a more membrane proximal, Ig domains 5–7, of CD22 (m971). The CAR constructs we generated were second-generation (CD28 and CD3 zeta; or, 4-1BB and CD3 zeta) or third generation (CD28, 4-1BB and CD3 zeta signaling domains). A CH2CH3 spacer domain from IgG1 was added in some constructs to examine the impact of extending the scFv-derived binding domain away from the transduced T cell membrane. In vitro cellular cytotoxicity and cytokine release experiments with 4 B cell-ALL cell lines (REH, SEM, NALM6, KOPN8) as well as the CD22 (+)ve Daudi and Raji cell lines were performed. Our results demonstrate that addition of the CH2CH3 domain did not improve tumor lysis and that standard affinity BL22 and higher affinity HA22-derived scFv epitopes were equivalent. With regard to signaling domains, second generation constructs were better than third generation constructs both in vitro and in vivo. In comparison between second generation constructs, CD28 containing domains outperformed 4-1BB with regard to lytic activity and cytokine release. Most surprising was the activity of the m971-derived scFv binding epitope. m971-CAR had significantly higher killing activity, a far more robust cytokine release profile, and superior in vivo activity. NSG mice were injected i.v. with 0.5× 106 NALM6-GL cells (pre-B cell ALL line engineered to express luciferase). Three days later, when disease was evident, mice were treated with 1×107 CAR+ T cells, and then followed by bioluminescent imaging to measure disease burden. The m971 CAR was significantly more potent at tumor clearance than our previously developed most active construct expressing the HA22-derived scFv domain (Figure 1). Disease progressed rapidly when non-transduced T cells were used (mock). We are currently examining the activity of different signaling domains on m971 CAR efficacy in vivo and directly comparing the anti-CD22 m971 CAR to the CD19 CAR currently being evaluated in clinical trials. These studies will guide future anti-CD22 CAR-based anti-leukemia immunotherapy trials. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 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. This 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.

Abstract 4313 CD22 is expressed on a number of hematologic malignancies. A recombinant immunotoxin composed of an anti-CD22 Fv fused to a 38 kDa fragment of Pseudomonas exotoxin A (Moxetumomab pasudodtox, HA22) is currently being evaluated in the treatment of B cell malignancies. We used the Fv sequence to construct a series of chimeric antigen receptors (CARs) to determine the optimal affinity, domain structure, and signaling required for optimal anti-leukemic activity. The original anti-CD22 binding domain, BL22, or a newer generation high-affinity domain, HA22, were fused to transmembrane and signaling sequences derived from the TCR zeta-chain, CD28, and CD137. In some constructs we extended the binding domain away from the membrane using constant domains from IgG (CH2CH3). Retroviral gene vectors were used to transduce activated primary T cells with CAR constructs. We found: HA22 Fv, 2 as opposed to 3 signaling motifs, and non-CH2CH3 containing CARs were superior in short-term in vitro tumor cell cytolysis assays, indicating that Fv affinity, signaling, and 3-D structure of the CAR all impact the anti-leukemic effectiveness of CARs. Finally, direct comparison to CD19-specific CAR showed an equivalent or superior killing activity in 2 out of 3 ALL lines tested. Using Scatchard analysis to define the number of CD22 moleules on the surface, the ALL line with the lowest number of CD22 molecules is also the least able to be killed by CAR-transduced T cells. The supeior lytic activity of CD19-CAR for this single cell line was due to preservation of high levels of CD19 at the cell surface. We are currently testing the in vivo activity of anti-CD22 CAR in an immunodeficient mouse model bearing human cell lines and primary patient-derived ALL. Our results indicate that CD22-CAR should be developed for the immunotherapy of CD22+ hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2575 Background: Adult T-cell leukemia (ATL) is a highly aggressive CD25+ HTLV-1-associated malignancy with median survivals of 4 and 9 months for leukemic and lymphomatous forms, respectively. There is no standard therapy capable of altering median survival by more than a few months. The anti-CD25 recombinant immunotoxin LMB-2 as a single agent was previously reported in phase I testing to have activity, but limited by rapid tumor growth between cycles and immunogenicity. Human CD25+ xenografts in mice contained soluble CD25 (sCD25) levels >100-fold higher than in serum, which were rapidly depleted by chemotherapy. Treatment with fludarabine was previously reported to be associated with lower immunogenicity to murine antibodies (HAMA), and the fludarabine-cyclophosphamide (FC) combination was reported associated with reductions in normal T- and B-cells. Methods: To prevent the formation of neutralizing antibodies and enhance anti-tumor activity, leukemic or lymphomatous ATL patients received FC chemotherapy with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 1, 2, and 3, and 2 weeks later began cycles every 3 weeks in which FC was administered on days 1, 2 and 3, followed by LMB-2 30–40 ug/Kg on days 3, 5 and 7. Formation of neutralizing antibodies and clinical response were determined with each cycle. Results: Interim results with 8 evaluable patients receiving 28 cycles (2–5 each) include an ORR of 50% with 2 CRs and 2 PRs. In 6 patients with circulating ATL cells detectable in the blood, reductions of ATL cells varied from 90.8 to 100% compared to baseline. CRs were associated with complete resolution of chest wall and pelvic masses, circulating ATL cells, and bone marrow involvement as assessed by immunohistochemistry and flow cytometry. One of 2 PRs is ongoing for nearly 1 year after complete resolution of skin involvement and partial decrease in circulating ATL cells. One of 2 CRs recurred after 6 months only in a sanctuary site (testis). The toxicity of LMB-2 was not increased by FC and 40 ug/Kg of LMB-2 days 3, 5 and 7 was not associated with dose-limiting toxicity (DLT). While the original doses of FC used were also without DLT, escalation to 30 and 300 mg/m2/day x3 resulted in severe thrombocytopenia in 1 of 2 patients. The most common toxicities, based on percent of cycles observed, were neutropenia (69%), lymphopenia (52%), leukopenia and nausea (48%), and anemia, thrombocytopenia and fever (41%). With FC, normal T-cells were reduced 0–95% (median 70%), but normal B-cells were reduced 91–100% (median 96%). Two (25%) patients made high levels of neutralizing antibodies after 3–4 cycles of LMB-2. Conclusions: LMB-2 is effective in ATL when preceded by FC, and can achieve major responses including CR. Additional patients will be needed to determine if FC can delay immunogenicity significantly, and allow enough cycles to result in long-term remission from this disease (Supported in part by NCI, intramural research program, NIH). Disclosures: Kreitman: NIH: Patents & Royalties. Off Label Use: Use of investigational agent and FC chemotherapy for treatment of ATL. Pastan:NIH: Patents & Royalties.

Abstract 248 Background: Most pediatric patients with acute lymphoblastic leukemia (ALL) are cured. For the 10 to 20% of patients with relapsed or refractory ALL, novel therapies are needed to overcome chemotherapy resistance. CD22 is an antigen commonly expressed on B-lineage ALL blasts. Moxetumomab pasudotox (previously known as CAT-8015 or HA22) is a recombinant immunotoxin composed of the variable domain of an anti-CD22 monoclonal antibody fused to a 38 Kda truncated form of Pseudomonas exotoxin A. Methods: We are conducting a multicenter, open-label, Phase I, dose-escalation study to determine the maximum tolerated dose and to assess the safety profile, activity, and immunogenicity of moxetumomab pasudotox in pediatric patients with relapsed/refractory hematologic malignancies. Eligibility criteria include age 6 months to 24 years, CD22+ B-lineage ALL or non-Hodgkin lymphoma, ≥1 prior salvage therapy, and no isolated testicular or central nervous system (CNS) disease. Moxetumomab pasudotox is administered as a 30-minute intravenous (IV) infusion at doses of 5, 10, 20, 30, or 40 mcg/kg, every other day for 6 doses, every 21 days. Patients receive concomitant IV hydration, pre-medication with acetaminophen, ranitidine, and diphenhydramine, and CNS prophylaxis with intrathecal hydrocortisone, cytarabine, and methotrexate. An initial cohort (A) consisting of an accelerated dose-escalation phase (one patient each treated at 5, 10, and 20 mcg/kg dose levels) followed by standard 3+3 dose-escalation at 30 mcg/kg dose has been completed. To reduce the incidence of capillary leak syndrome (CLS), a second ongoing cohort (B) was added to the study in which dexamethasone (2.5 mg/m2 every 12 hours) is coadministered during treatment cycle 1 around moxetumomab pasudotox doses. Standard 3+3 dose-escalation is being followed starting at the 20 mcg/kg dose level. Results: 21 patients 4 – 21 years of age (median, 11) with ALL have been treated; 7 in Cohort A and 14 in Cohort B (Table). They had received 2 – 8 prior regimens (median 4) and 17 were refractory to chemotherapy and 10 had undergone stem cell transplant. 1 – 4 treatment cycles (median, 1) of moxetumomab pasudotox were administered. The most common adverse events (regardless of attribution) reported to date are decreased hemoglobin, decreased platelets, increased aspartate aminotransferase (AST), pyrexia, decreased neutrophils, tachycardia, increased alanine aminotransferase (ALT), hypokalemia, increased weight, and decreased white blood cells. The most common treatment-related adverse events were increased weight, increased AST, increased ALT, and hypoalbuminemia. Most (70%) of the treatment-related toxicities have been mild and reversible. CLS that was dose-limiting was observed in 2 of 7 patients in Cohort A (30 mcg/kg dose level) and in none of 14 patients in Cohort B. One patient treated at 40 mcg/kg developed refractory hypercalcemia and died of a cardiac arrhythmia during attempted central venous catheter placement. That dose level (5B) was expanded and is currently accruing. Of the 17 (81%) patients evaluable for response (Table), objective responses were achieved in 5 (29%), including 4 (24%) complete responses (CR) and 1 (6%) partial response (PR). Hematological activity (HA) (>50% reduction in blasts and/or improvement in neutrophil and/or platelet counts) was observed in 7 patients (41%). Anti-moxetumomab pasudotox neutralizing antibodies (titer ≥50% neutralization) developed in 3 of 21 (14%) patients. Conclusions: Moxetumomab pasudotox is active in pediatric patients with relapsed and chemotherapy-refractory ALL. The current observed anti-leukemic activity and safety profile at doses up to 40 mcg/kg warrant further development of moxetumomab pasudotox in pediatric ALL. ClinicalTrials.gov Identifier: NCT00659425 This study was sponsored by MedImmune, LLC, and supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Disclosures: Wayne: NCI: Co-inventor on patents assigned to the NIH for the investigational product. Off Label Use: This is a Phase I trial of an investigational agent. Bhojwani:MedImmune, LLC: Research Funding. Silverman:Enzon: Consultancy, Honoraria; EUSA: Consultancy, Honoraria. Jeha:Genzyme: Honoraria, Research Funding. Pui:EUSA: Honoraria; Enzon: Honoraria; Sanofi: Honoraria. Buzoianu:MedImmune, LLC: Employment. FitzGerald:NCI: Co-inventor on patents assigned to the NIH for the investigational product. Kreitman:NCI: Co-inventor on patents assigned to the NIH for the investigational product. Ibrahim:MedImmune, LLC: Employment.

Abstract 2516 Currently no adequate therapies exist for patients with refractory hairy cell leukemia (HCL). The CD22 antigen is commonly expressed on the cell surface of B cells and B-cell malignancies. CD22 is highly expressed in HCL, even in chemoresistant disease, and is therefore a potential target for antibody-directed therapy in refractory/resistant HCL. We developed CD22-specific targeted immunotoxins composed of the variable domains of a monoclonal anti-CD22 antibody fused to a truncated form of a Pseudomonas exotoxin. The first anti-CD22 immunotoxin we studied was CAT-3888 (BL22), which demonstrated remarkable activity in patients with relapsed/refractory HCL. Moxetumomab pasudotox (also known as CAT-8015 or HA22) is a derivative of CAT-3888 with a higher affinity to CD22 and an improved cytotoxicity profile ex vivo against HCL and other B-cell malignancies. A phase 1 dose-escalation study is being conducted to determine the maximum tolerated dose (MTD) of moxetumomab pasudotox and to assess its safety and immunogenicity profiles and clinical activity in HCL. Preliminary safety data and antitumor activity from the study were previously reported (Kreitman et al, ASH 2009, ASCO 2010). Eligibility criteria include age ≥18 years, ≥2 previous systemic therapies (including purine analogs), ECOG performance status of 0–2, and the presence of cytopenia or symptomatic splenomegaly. A standard 3+3 dose-escalation design with an expanded cohort at the MTD or highest dose level is being used. Patients receive moxetumomab pasudotox as a 30-min IV infusion administered on days 1, 3, and 5 (QODx3) of a 28-day treatment cycle, up to a total of 10 cycles. Patients receive prophylaxis with low-dose aspirin (if platelets >100,000/μL) and IV hydration to prevent hemolytic uremic syndrome (HUS), the dose-limiting toxicity (DLT) in clinical studies with CAT-3888. Additionally, patients receive hydroxyzine, ranitidine, and acetaminophen to avoid infusion reactions. Response is evaluated before cycle 2 and thereafter before even-numbered cycles. Enrollment and treatment are ongoing. At the time of reporting, a total of 32 patients had received moxetumomab pasudotox at one of five dose levels: 5, 10, 20, and 30 μg/kg (n=3 in each cohort), and 40 μg/kg (n=4), and in an expanded cohort at the 50-μg/kg dose level (n=16). Median age was 59 years (range, 40–77 years). Patients were heavily pretreated; 19 (59%) were previously treated with rituximab. No DLTs have been observed, and an MTD has not been reached. Adverse events have been mostly mild and consistent with those seen in clinical studies with CAT-3888. The most frequently reported drug-related adverse events have been hypoalbuminemia (53%), peripheral edema (44%), pyrexia (34%), elevated ALT (34%) and AST (31%), fatigue (28%), headache (28%), localized edema (22%), and nausea (22%). Capillary leak syndrome (grade 2) has developed in 7 patients (22%); 5 patients were in the 50-μg/kg dose cohort. Reversible grade 2 HUS has been reported in 2 patients (6%; 30 μg/kg, n=1; 50 μg/kg, n=1); both began on day 8 of the treatment cycle. Treatment-related toxicities ≥grade 3 have been observed in 3 patients (30 μg/kg, n=1; 50 μg/kg, n=2) and include decreased haptoglobin levels, elevated gamma-glutamyltransferase, and lymphopenia. Moxetumomab pasudotox has shown clinical activity in 47% of recipients, and responses have been observed in both the dose-escalation and expansion cohorts. Overall response (OR), complete response (CR), and partial response (PR) rates observed to date are listed in the table. The median time to response is currently 2.79 months. Neutralizing antibodies have developed in 14 patients (44%). Moxetumomab pasudotox appears to have a safety profile similar to that of CAT-3888 (BL22), and with a lower incidence of HUS at the highest dose tested (50 μg/kg QODx3). Moxetumomab pasudotox has demonstrated antitumor activity in patients with relapsed/refractory HCL, including 44% of patients in the expansion cohort. Further studies in HCL and other B-cell malignancies expressing CD22 are warranted. Clinical Activity 5 μg/kg (n=3) 10 μg/kg (n=3) 20 μg/kg (n=3) 30 μg/kg (n=3) 40 μg/kg (n=4) 50 μg/kg (n=16) Total (N=32) OR 33% 67% 67% 33% 50% 44% 47% CR 0% 33% 67% 33% 50% 25% 31% PR 33% 33% 0% 0% 0% 19% 16% ClinicalTrials.gov Identifier: NCT00462189 This study was sponsored by MedImmune, LLC. Disclosures: Kreitman: NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties. Robak: MedImmune, LLC: Research Funding. Noel: NIH: Patents & Royalties. FitzGerald: NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties. Buzoianu: MedImmune, LLC: Employment. Lechleider: MedImmune, LLC: Employment. Pastan: NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties.

Abstract 3246 Although most children and adolescents with ALL are cured, treatment-associated side effects are significant and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce toxicities of chemotherapy. CD22 is a B-lymphoid differentiation antigen expressed on most B-lineage ALL blasts. We recently conducted a Phase I trial of the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) in children with ALL or non-Hodgkin lymphoma (NHL). The safety profile was acceptable and transient clinical activity was seen; however, no objective responses were observed (Clin Cancer Res 2010;16:1894). We are conducting a Phase I trial of a modified agent with higher CD22 binding affinity (moxetumomab pasudotox, also known as CAT-8015 or HA22). Methods: Patients 6 months to 24 years of age with relapsed or refractory CD22+ B-lineage ALL or NHL are eligible for enrollment. Moxetumomab pasudotox is being administered at doses of 5, 10, 20, or 30 μg/kg every other day for 6 doses, every 21 days for up to 6 cycles. All patients receive pre-medication with acetaminophen, ranitidine, and diphenhydramine and prophylaxis for central-nervous-system leukemia with intrathecal hydrocortisone, cytarabine, and methotrexate. An initial completed cohort (A) consisted of one patient each treated at the first 3 dose levels (5, 10, 20 μg/kg) followed by standard 3+3 dose escalation commencing at 30 μg/kg. In attempt to reduce the incidence of vascular leak syndrome (VLS), a second ongoing cohort (B) is also receiving dexamethasone 2.5 mg/m2 every 12 hours around doses of moxetumomab pasudotox during cycle 1 with standard 3+3 dose escalation commencing at 20 μg/kg. Results: At the time of reporting, 14 patients 5 to 22 years of age (median, 11) with ALL (13 precursor-B, 1 mature B-cell) have been treated in the clinical trial. Patients had received a median of 4 prior regimens (range, 2–8), 12 were chemotherapy refractory, and 7 had received prior stem cell transplantation. Thirteen had leukemia-associated baseline cytopenias and thus were not evaluable for hematologic toxicities. The most common adverse events observed to date have been hyperbilirubinemia, transaminase elevations, hypoalbuminemia, elevated creatinine, febrile neutropenia, hypertension, microscopic proteinuria, hemoglobinuria, hypoxia, and pleural effusion. Two of 7 patients in Cohort A treated at 30 μg/kg experienced Grade 3 and Grade 4 VLS. None of 7 patients treated in Cohort B developed VLS. All toxicities attributed to moxetumomab pasudotox were reversible. Twelve patients were evaluable for response: 3 (25%) achieved complete remission by morphology and flow cytometry after 1, 1, and 2 cycles, respectively; 5 (42%) had hematologic activity (blood count improvement, blast reduction); and 3 (25%) had stable disease. One patient treated at the lowest dose level had progressive disease. Two patients developed high-titer neutralizing antibodies. Conclusions: Moxetumomab pasudotox has shown clinical activity against chemotherapy-refractory pediatric ALL, with complete remissions achieved in 3 of 12 patients. The demonstration of clinical activity and the safety profile exhibited by this agent to date support further study in ALL. ClinicalTrials.gov Identifier: NCT00659425 This study was sponsored by MedImmune, LLC. Disclosures: Off Label Use: Moxetumomab pasudotox is an investigational agent. Bhojwani:MedImmune: Research Funding. Silverman:Enzon: Consultancy, Honoraria; EUSA: Consultancy, Honoraria. Jeha:Genzyme: Honoraria, Research Funding. Pui:EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria. McDevitt:MedImmune, LLC: Employment. FitzGerald:NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties. Kreitman:NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties. Lechleider:MedImmune, LLC: Employment. Pastan:NCI: Co-inventor on patents assigned to the NIH for the investigational product., Patents & Royalties.

Abstract 839 Although most children with ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. The anti-CD22 immunotoxin RFB4(dsFv)-PE38 CAT-3888 (BL22) was recently shown to have clinical activity with an acceptable safety profile in children with ALL (Blood 2007;110:262a). We undertook a Phase I trial of a modified agent with higher CD22 binding affinity (CAT-8015 or HA22). Methods: Patients 6 months to 24 years of age with relapsed or refractory CD22 + B-lineage ALL or non-Hodgkin lymphoma were eligible for enrollment into this Phase I trial. CAT-8015 was administered at doses of 5, 10, 20, or 30 mcg/kg every-other-day for 6 doses every 21 days for up to 6 cycles. One patient was enrolled at each of the first 3 dose levels (5, 10, 20 mcg/kg) with standard 3+3 dose escalation commencing at 30 mcg/kg. All patients received acetaminophen, ranitidine and diphenhydramine to mitigate infusion-related symptoms, and prophylaxis for central-nervous-system leukemia with intrathecal hydrocortisone, cytarabine and methotrexate. Patients at high risk for tumor lysis syndrome received standard prophylaxis. Results: Seven patients with ALL (6 precursor-B, 1 mature B-cell) 5 to 17 years of age (median, 10) were treated on the clinical trial. All patients had been heavily pre-treated and had baseline cytopenias due to active malignancy and thus were not evaluable for hematologic toxicities. The most common adverse events observed to date have been hyperbilirubinemia, transaminase elevations, hypoalbuminemia, elevated creatinine, febrile neutropenia, abdominal pain, pyrexia, hypertension, microscopic proteinuria, hemoglobinuria, hypoxia and pleural effusion. Two of 4 patients treated at 30 mcg/kg experienced Grade 3 or greater toxicity consistent with capillary leak: 1 with Grade 3 pleural effusion and hypoxia and 1 with Grade 4 vascular leak syndrome. All toxicities attributed to CAT-8015 were reversible. Clinical activity was demonstrated in 4 of 7 subjects. One patient treated at 10 mcg/kg had a complete remission by morphology and flow cytometry. Three patients met the protocol definition for hematologic activity (blood count improvement). One of these patients developed high-titer neutralizing antibodies. Two patients met the protocol definition for stable disease. The patient treated at the lowest dose level had progressive disease. Conclusions: CAT-8015 appears to be active against chemotherapy-refractory ALL. Strategies to predict and/or prevent vascular leak syndrome are currently being developed. Disclosures: No relevant conflicts of interest to declare.

Abstract 888 Novel therapies are needed for patients with relapsed or refractory hairy cell leukemia (HCL), particularly those who have failed purine analogs. CD22 is expressed in the majority of B-cell malignancies and universally in HCL, making it an ideal therapeutic target. CAT-8015 is a targeted immunotoxin composed of an anti-CD22 antibody fused to a modified form of Pseudomonas exotoxin A. It has a novel mechanism of action compared with other CD22 targeted antibodies, as CAT-8015 is internalized upon binding to CD22, inhibiting protein translation and prompting apoptosis. CAT-8015 has shown significant antitumor activity in B-cell tumor cell lines and malignant cells isolated from patients with HCL. CAT-8015 (HA22) is a high-affinity derivative of CAT-3888 (BL22) that displays higher CD22-binding and inhibitory activity due to mutation of 3 amino acids. A multicenter, dose-escalation, phase I study is being conducted to estimate the maximum tolerated dose (MTD) of CAT-8015, and evaluate its safety, efficacy and immunogenicity profiles in HCL. Adult HCL patients who previously received at least 2 systemic therapies (including purine analogs) and had cytopenias or symptomatic splenomegaly requiring treatment are eligible to participate. A standard 3+3 dose-escalation design is being employed at doses of 5, 10, 20, 30, 40, and 50 ug/kg. CAT-8015 is administered as a 30-min IV infusion on days 1, 3, and 5 (QODx3) of each 28-day cycle for up to a total of 10 cycles until disease progression, intervening toxicity [e.g. dose-limiting toxicity (DLT)], completion of two cycles of treatment beyond documentation of complete response (CR), or other reason for which the patient might become ineligible. Patients receive premedication with hydroxyzine, ranitidine, and acetaminophen to prevent infusion reactions; and low-dose aspirin and IV hydration to prevent hemolytic uremic syndrome (HUS), which has been observed in association with CAT-3888. Data are electronically archived by each investigator and were pooled for analysis. The study is ongoing with 2 more patients expected to be treated at 50 ug/kg. A total of 26 patients have received CAT-8015 to date. Three patients enrolled at each of the 5, 10, 20, and 30 ug/kg dose levels; 4 patients at the 40 ug/kg dose level; and 10 patients at 50 ug/kg dose level. The median age is 59 years (range 40-77), and the majority (84.6%) are male. Patients were heavily pretreated (median number of prior therapies: 3, range 2-7). Among the 26 patients in total, 14 received prior rituximab (53.8%); among 10 patients in cohort 50 ug/kg, 7 received prior rituximab (70.0%). Patients have received a median of 3 treatment cycles (range 1-8). No DLTs have been observed and an MTD has not been reached. Expanded enrollment at 50 ug/kg has been undertaken to better characterize the safety profile and antitumor activity. The most common drug-related toxicities have been of grade 2 or lower severity: hypoalbuminemia (57.7%), peripheral edema (42.3%), pyrexia (38.5%), elevated ALT (34.6%) and AST (30.8%), headaches (26.9%), and nausea (26.9%). Four patients (15.4%) developed grade 2 vascular leak syndrome (VLS). One treatment-related serious adverse event occurred, a reversible grade 2 HUS that was reported in the 30 ug/kg dose cohort. Anti-drug antibodies developed in 10 patients (38.5%). CAT-8015 was highly active in HCL. Among the 26 patients treated, the objective response (OR) rate was 73.1% (19/26), with a CR rate of 34.6% (9 patients) and a partial response (PR) rate of 38.5% (10 patients). Responses were observed at all dose levels. Specific OR rates at the 5,10, 20, 30, 40, and 50 ug/kg/dose cohorts were 100%, 100%, 33%, 33%, 75% and 80%, respectively. At the time of this report, none of the patients achieving a CR has relapsed. Four of 9 (44.4%) patients with CR have a duration of response of >12 months. CAT-8015 exhibited an acceptable safety profile when administered up to 50 ug/kg QOD × 3, and demonstrated substantial antitumor activity in patients with relapsed/refractory HCL. These data demonstrate that CAT-8015 is a promising new product candidate for patients with advanced HCL. These data support further investigation in newly diagnosed patients with HCL and suggest that CAT-8015 may have clinical activity in other B-cell malignancies Disclosures: Kreitman: NIH: Patents & Royalties. Off Label Use: Recombinant immunotoxin HA22 for targeting CD22+ cells. Robak:MedImmune, LLC: Research Funding. FitzGerald:NIH: Patents & Royalties. Pastan:NIH: Patents & Royalties.

Abstract 4119 While the majority of pediatric patients with newly diagnosed B-lineage acute lymphoblastic leukemia (ALL) are cured with standard chemotherapy regimens, treatment is associated with multiple toxicities, and ALL remains the most frequent cause of cancer mortality in childhood. CD22, a B-lineage surface glycoprotein involved in B cell signaling and adhesion, is expressed in most cases of B-lineage ALL. We are conducting clinical trials of anti-CD22 immunotoxins [RFB4(dsFv)-PE38] for pediatric ALL. To assess eligibility for such targeted therapy, CD22 expression by ALL cells was studied in peripheral blood and/or bone marrow aspirate samples from 50 patients with relapsed ALL. The level of CD22 expression by ALL cells was quantitated by measuring mean anti-CD22 antibody binding per ALL cell (ABC) under saturating conditions using flow cytometry and the BD Biosciences QuantiBRITE system for fluorescence quantitation. Patients ranged in age from 3 to 22 years (median 10 years) and included 27 males and 23 females. CD22 expression was detected in all samples, and the vast majority of cases demonstrated expression of CD22 in 100% of leukemic blasts. CD22 antigen density in ALL cells varied widely among patients at baseline (range 451 - 14,519; mean 4276; median 3824; standard deviation 2976; see graph). CD22-directed immunotoxin therapy was initiated in 29 of the 50 patients, 19 of whom had samples quantitated for CD22 expression levels both before and after immunotoxin therapy. Most patients exhibited limited variation in the mean number of anti-CD22 molecules bound per ALL cell when comparing multiple specimens. In conclusion, CD22 expression varies widely in pediatric B-lineage ALL and persists despite repeated exposure to CD22-directed therapy. (MedImmune, LLC, sponsored the clinical studies of anti-CD22 immunotoxin CAT-8015.) Disclosures: No relevant conflicts of interest to declare.

For the treatment of B cell malignancies, we have developed a high affinity recombinant immunotoxin (HA22), which is composed of the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A. HA22 is an improved form of immunotoxin BL22, which produced a 50–60% complete remission (CR) rate in cladribine-resistant hairy-cell leukemia (HCL). A partial response (PR) rate of 17% was observed with BL22 in chemoresistant chronic lymphocytic leukemia (CLL). The higher response rate of BL22 in HCL compared to CLL is related to its higher cytotoxicity for HCL than CLL cells ex vivo (median IC50 4.3 ng/ml in 33 HCL patients vs 430 ng/ml in 70 CLL patients, p < 0.0001), and the higher CD22 expression on HCL than on CLL cells (median 44,000 vs 1250 sites/cell).To increase cytotoxic activity, the affinity of the Fv of BL22 was increased about 15-fold by phage display by mutating 3 amino acids in HCDR3 from SSY to THW. Patients with HCL and CLL were treated on 2 separate multicenter phase I protocols. To be eligible, patient required at least 2 prior standard therapies and a medical indication for treatment. HA22 was administered by 30 min infusion every other day for 3 doses (QOD ×3) and patients could be retreated at the same dose level every 4 weeks unless progressive disease or immunogenicity occurred. A standard 3–6 dose escalation scheme was used, allowing new patients to be enrolled at a higher dose level if 0 of 3 or 1 of 6 patients at the current level had dose limiting toxicity (DLT). A total of 16 HCL patients were treated with HA22 at 5, 10, 20, 30, and 40 ug/Kg QOD ×3. Three patients were treated at each of the first 4 dose levels, and a 4th patient received 40 ug/Kg QOD ×3 because one patient at that dose level received only 1 cycle. Of 14 evaluable patients who received a total of 45 cycles, 4 (29%) developed high levels of neutralizing antibodies. No DLT was observed on any cycle, although 1 patient developed a grade II hemolytic uremic syndrome (HUS) during cycle 3 of 30 ug/Kg QOD ×3. Major responses included 6 (38%) CRs and 6 (38%) PRs out of 16 evaluable HCL patients (unmonitored). Responses were observed at all dose levels including 3 of 3 PRs at the lowest dose level, and 1–2 CRs at each of the next dose levels. Like BL22, peak plasma levels of HA22 were inversely related to tumor burden as assessed by circulating malignant cells or spleen size, and increased with repeated doses as patients responded. Phase I testing of HA22 in CLL is at an earlier stage with 11 patients enrolled at dose levels of 5, 10, and 20 ug/Kg QOD ×3, and decreases in circulating CLL cells have been observed. In conclusion, HA22 has not yet shown DLT in phase I testing and is highly active in HCL. Compared to BL22, HA22 shows no evidence of increased toxicity due to enhanced binding affinity to CD22.

Despite advances in curative treatment, childhood ALL remains a leading cause of cancer-related mortality in pediatrics and survivors are at risk of multiple late effects. Targeted therapies that overcome chemotherapy resistance and decrease nonspecific toxicities are needed. The B-lineage differentiation antigen CD22 represents a relevant target for pre-B ALL. Monotherapy with the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) has been well tolerated in a pediatric Phase I trial. To date however, only transient clinical activity has been observed with this agent in children with ALL. We conducted cytotoxicity studies to evaluate combination regimens of a higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) with standard ALL chemotherapeutic agents. Methods: CD22+ human malignant cell samples studied included cell lines of Burkitt lymphoma (CA46) and pre-B ALL (REH and KOPN-8), as well as primary lymphoblasts from 2 children with multiply relapsed pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. Asparaginase, cytarabine, doxorubicin, methotrexate, and vincristine were added to cells individually and in various combinations with CAT-8015. Total incubation time was 2 days for cell lines and 5 days for primary patient blasts. Cell viability was assessed using WST-8. The Chou-Talalay median-effect method was used for data analysis (Chou TC, Talalay P. Adv Enzyme Regul1984;22:27–55). Results: All samples were sensitive to CAT-8015 and to most chemotherapy agents tested. Simultaneous addition of a fixed concentration of CAT-8015 with varying concentrations of each chemotherapy drug led to a 2- to 4-fold reduction in IC50 in comparison to individual agents. Assays with simultaneous addition of equipotency ratios of CAT-8015 and chemotherapy agents had synergistic or additive interactions. These interactions were sequence dependent, and the addition of CAT-8015 followed by chemotherapy led to greater positive interactions than the reverse order. Further assays with simultaneous addition of CAT-8015 and doublet chemotherapy drugs showed enhanced synergistic and additive interactions compared to assays with only doublet agents. Conclusions: CAT-8015 combined with standard chemotherapy agents results in synergistic or additive cytotoxicity against CD22-positive Burkitt lymphoma and pre-B ALL cell lines and primary blasts from children with pre-B ALL. High affinity anti-CD22 recombinant immunotoxins have the potential to overcome chemotherapy resistance and to decrease nonspecific treatment-associated toxicities. In addition, CAT-8015 might synergistically enhance chemotherapy-induced cytotoxicity, thus improving efficacy and allowing the use of less toxic regimens in the future. Clinical trials of recombinant immunotoxins targeting CD22 in combination with standard ALL chemotherapy regimens are planned.

Although most children with pre-B ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. We evaluated the B-lineage differentiation antigen CD22 and the anti-CD22 immunotoxin RFB4(dsFv)-PE38 (CAT-3888 or BL22) in childhood ALL. Methods: Blasts were obtained from children with pre-B ALL. Flow cytometry was used to determine CD22 expression and anti-CD22 antibody binding using the BD Quantibrite System. 72 hour in vitro protein synthesis cytotoxicity assays were conducted. Murine xenografts (human ALL cell line EU-1 into CB17 SCID mice) were treated with varying doses of CAT-3888. A pediatric Phase I trial of CAT-3888 was initiated. Results: All cases (n=69) were CD22 positive. Median CD22 site density (studied in 28 cases) was 4,099 sites/cell (range, 451 – 9,380). In vitro cytotoxicity to CAT-3888 was assessed in 43 cases and the median IC50 was 10 ng/ml. Murine xenografts treated with CAT-3888 had significant prolongation of leukemia free survival in a dose responsive fashion (p Conclusions: CD22 represents a relevant target for childhood pre-B ALL. All cases tested were CD22+ and most blasts were highly sensitive to CAT-3888 in vitro at concentrations below clinically achievable levels. CAT-3888 showed a dose response effect and prolonged leukemia free survival in murine ALL xenografts. CAT-3888 is well tolerated in children and activity has been seen in most subjects treated on the Phase I trial. A new higher affinity anti-CD22 immunotoxin (CAT-8015 or HA22) is in development.

Objectives: CD30 is overexpressed on the cell surface by a variety of malignant diseases including Hodgkin’s lymphoma (HL). It is cleaved from the cell surface releasing the soluble ectodomain (sCD30), which may interfere with a monoclonal antibody’s (MAb) ability to reach its target. Recently we identified two epitopes on membrane-associated CD30 that are missing on sCD30. To investigate the relevance of soluble antigen for CD30 mediated tumor targeting, in vitro, biodistribution and γ-imaging studies of three radiolabeled anti-CD30 MAbs (HeFi-1, T105, T408) targeting the CD30 antigen at different epitopes were performed. Materials and Methods: In vitro: HeFi-1 was labeled with 125I and 111In and incubated with CD30+ L540 cells. The pellet and supernatant was analyzed for cell associated radioactivity at various time points. For binding studies, HeFi-1, T105 and T408 were radiolabeled with 131I, incubated with recombinant human CD30/Fc, serum of L540 tumor bearing mice (MS) and L540 cell culture supernatant (CCS) and analyzed by HPLC-SEC. In vivo: L540 tumor bearing nude mice were injected i.v. with dual-labeled (125I and 111In) HeFi-1, T105, or T408. At various timepoints tumors, blood and major organs were collected and analyzed for radioactivity. Serum samples were assayed for complex formation by HPLC-SEC. For γ-imaging studies L540 tumor bearing mice received radiolabeled HeFi–1, T105, or T408 and serial whole body gamma camera images were acquired. Results and Conclusions: Iodine vs. Indium: 111In-HeFi–1 showed higher cell associated retention in vitro than 125I-HeFi-1 as a function of time. In vivo, all anti-CD30 MAbs showed superior tumor accretion when radiolabeled with 111In (25–35% ID/g at 72h; 125I: 6–11%), probably due to internalization of the radioiodinated MAbs, intracellular catabolism and subsequent release of degraded products. HPLC analyses: HeFi-1 showed in vitro complex formation when incubated with CD30/Fc, MS and CCS, whereas T105 and T408 only bound CD30/Fc, indicating their inability to bind sCD30. In vivo, complex formation was observed for T105, although in less amounts compared to HeFi-1, suggesting that T105 binds to the membrane antigen, and the immune complex is subsequently cleaved and released from the cell surface. It also suggests that the T105 epitope may be shared between the membrane and the soluble antigen part, or undergoes conformational changes upon shedding. T408 was found to bind to the cell membrane without evidence of immune complex formation. Biodistribution: There were no significant differences in tumor and normal organ uptake between the three 111In-labeled MAbs. T408 showed an accelerated blood clearance with 18% of the activity left at 72h compared to T105 (34%) and HeFi-1 (55%), probably due to the lack of complex formation, which may prolong MAb circulation. The rapid blood clearance of T408 results in a favorable tumor:blood (therapeutic) ratio at 72h with 4.3, compared to T105 (2.3) and HeFi-1 (1.9). In summary, we have shown that radiometals are more suitable for targeting CD30; we have also demonstrated the impact of soluble antigen on the pharmakokinetics of MAbs, which target CD30 at distinct epitopes. T408 with its lack of binding sCD30 and therefore rapid blood clearance and enhanced therapeutic ratio may be a promising and useful new candidate for RIT of CD30+ lymphoma.

A recombinant immunotoxin, BL22 has shown clinical efficacy against hairy cell leukemia and other B-cell non-Hodgkin’s lymphomas. BL22 contains an antibody-derived domain that recognizes CD22, and PE38, a truncated Pseudomonas exotoxin A domain, for inhibition of protein synthesis. Previous studies have shown that Pseudomonas exotoxin A-based immunotoxins exert their cytotoxicity by apoptosis and additional, yet uncharacterized mechanisms. We investigated the cytotoxicity of BL22 in two mantle cell lymphoma lines, NCEB-1 and Granta-519, and in the diffuse, large-cell lymphoma line SU-DHL-4. Presence of the CD22 target was confirmed by flow cytometry. Treatment of NCEB-1 cells to 1 μg/ml BL22 for 24-32h reduced the numbers of viable cells in comparison to cells exposed to a non-binding immunotoxin. Induction of apoptosis, determined by activation of caspase-3 and exposure of phosphatidylserine, was not detected before 48h of exposure. Incorporation of bromo-deoxyuridine into DNA was, however, almost completely inhibited after 24h. Correspondingly, the numbers of G1 phase cells were increased upon treatment with BL22 as determined by flow cytometry. G1-arrest resulted from decreased enzyme activities of cyclin-dependent kinases (cdk)-2 and 4 as assessed in vitro with respective substrates, histone H1 and the retinoblastoma susceptibility (RB) protein. Immunoblotting revealed markedly reduced amounts of cyclins A, E, D1 and D3 whereas cdk2 and cdk4 protein expression levels remained unchanged. Expression of the RB protein was decreased, and the protein was hypophosphorylated in immunotoxin-treated cells. Pulse-labeling with [35S]-labeled amino acids, followed by immunoprecitation of cyclins E and D1 confirmed that BL22 inhibited synthesis of these cyclins. Thus, immunotoxin-induced cell cycle arrest results from insufficient synthesis of regulatory cyclins required for progression through G1 into S-phase. Similar results were obtained in Granta-519 cells. SU-DHL-4 diffuse, large-cell lymphoma cells were highly sensitive to BL22, and G1 arrest was observed as early as after 8–24h in these cells. Expression of cyclins A, D3 and E was decreased while cyclin D1 was not expressed in SU-DHL-4 cells. Interestingly, cell cycle arrest did not prevent from subsequent induction of apoptosis in lymphoma cells continuously exposed to the immunotoxin. In conclusion, we have characterized cell cycle arrest as a previously unknown mechanism that contributes to the cytotoxicity of Pseudomonas exotoxin-based immunotoxins.

The immunoglobulin superfamily receptor translocation associated 2 (IRTA2) gene encodes a cell surface receptor homologous to the family of Fc receptors. Because of the restricted expression of the mRNA in B cell-linage cells, IRTA2 is a new potential target for the immunotherapy of B cell malignancies. To study the function and expression of the IRTA2 gene product, we produced monoclonal antibodies (MAbs) specific to IRTA2. We successfully made 36 MAbs from a mouse immunized with IRTA2-encoding plasmid DNA. All the MAbs reacted with IRTA2-extracellular domain-Fc fusion protein in ELISA and 293T cells expressing IRTA2 in a fluorescence-activated cell sorter (FACS) analysis. Binding of each MAb to a series of deletion mutants of IRTA2-Fc fusion protein and mutual competition of the MAbs to the binding to the whole extracellular domain of IRTA2-Fc fusion protein revealed that the panel of MAbs recognizes more than 10 epitopes on IRTA2. There are at least 1, 1, 2, 4, and 2 epitopes on extracellular domain 1 (ED1), ED3, ED1-3, ED4-9, and whole extracellular domain, respectively. Twenty five out of the 36 MAbs are specific to IRTA2 but 11 others showed cross reactivity to other members of the IRTA family. Among 36 MAbs produced, 3 MAbs (F25, F56 and F119) were used for further analysis based on their specific reactivity with recombinant IRTA2 expressed on 293T cells and lack of cross reactivity with other IRTA family members. In a FACS analysis, MAbs F56 and F119 detected IRTA2 expression on 6/7 B-cell non-Hodgkin’s lymphoma and 1/6 Burkitt’s lymphoma cell lines. Reverse transcriptase-polymerase chain reaction experiments and Western blotting using MAb F25 confirmed the expression profile. We also analyzed blood samples of 11 patients with hairy cell leukemia (HCL), 11 patients with chronic lymphocytic leukemia (CLL), and 4 patients with follicular lymphomas (FL) by FACS. We detected IRTA2 expression on 100% HCL (11/11), 55% CLL (6/11) and 0% of FL cells (0/4). Our results provide the first evidence that the IRTA2 is expressed on the surface of HCL and CLL cells as well as some human lymphoma cell lines. We expect IRTA2 will be established as a new target for immunotherapy.