ALBERT

Description: 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.

Description: Abstract 3442 Poster Board III-330 BL22 is a 63 kDa anti-CD22 recombinant immunotoxin containing truncated Pseudomonas exotoxin and variable domains from an anti-CD22 antibody. Patients with multiply relapsed/refractory hairy cell leukemia received BL22 and achieved 19 (61%) complete remissions (CRs) and 6 (19%) partial responses (PRs) in phase I testing, and 17 (47%) CRs plus 9 (25%) PRs in phase II testing (n=36), for overall response rates (ORR) of 72-81%. The average dose/cycle was the same for phase I and II (29 vs 33 ug/Kg x3). The dose for phase II was 40 ug/Kg x3 initially and 30 ug/Kg x3 for retreatment, but retreatment was held if patients had hematologic remission (HR, neutrophils ≥ 1500/mm3, Hgb ≥ 11 g/dL, and platelets ≥ 100,000/mm3) after cycle 1. Disease-free survival (DFS, CR duration) for phase II has not yet been reached at a median of 32 (range 4-62) months, with 12 (71%) of 17 CRs still ongoing. Considering all 36 CRs from phase I and II testing, median DFS was 33 (3-112) months with 15 (42%) of 36 CRs ongoing. Patients in CR usually underwent bone marrow biopsy every 6 months for 2 years and yearly thereafter, and after relapsing usually remained in HR. In fact, the median HR duration of these patients has not yet been reached at 42 (range 4-112) months, with 24 (67%) of the 36 patients remaining in HR or CR. Outcomes were better for those with pre-BL22 spleens measuring ≤ 200 mm in height than those with either prior splenectomy or spleens 〉 200 mm, in terms of CR (68% vs 34%, p=0.007), ORR 95% vs 48%, p=0.000003), and DFS (median 69+ vs 27 mo, p=0.002). In contrast, CR rates or DFS was not related to whether patients had 1 (n=8) years of response to their last course of purine analog (p=0.5-0.75). Of 69 patients who received BL22, 8 (12%) had a completely reversible hemolytic uremic syndrome (HUS) and all maintained normal renal function after a median 80 (9-112) months of follow-up. We conclude that BL22 is highly active producing durable remissions in chemoresistant HCL, particularly in patients with limited disease burden. Testing is underway with a high-affinity version of BL22, called HA22 (CAT-8015). Disclosures Kreitman: NIH: Patents & Royalties. Off Label Use: BL22 is a recombinant immunotoxin which targets CD22+ cells. FitzGerald:NIH: Patents & Royalties. Pastan:NIH: Patents & Royalties.

Description: 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.

Description: 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.

Description: Background Mesothelin is a 40 kDa protein present on the surface of normal mesothelial cells and overexpressed in many human tumours, including mesothelioma and ovarian and pancreatic adenocarcinoma. It forms a strong and specific complex with MUC16, which is also highly expressed on the surface of mesothelioma and ovarian cancer cells. This binding has been suggested to be the basis of ovarian cancer metastasis. Knowledge of the structure of this protein will be useful, for example, in building a structural model of the MUC16-mesothelin complex. Mesothelin is produced as a precursor, which is cleaved by furin to produce the N-terminal half, which is called the megakaryocyte potentiating factor (MPF), and the C-terminal half, which is mesothelin. Little is known about the function of mesothelin and there is no information on its possible three-dimensional structure. Mesothelin has been reported to be homologous to the deafness-related inner ear proteins otoancorin and stereocilin, for neither of which the three-dimensional structure is known. Results The BLAST and PSI-BLAST searches confirmed that mesothelin and mesothelin precursor proteins are remotely homologous to stereocilin and otoancorin and more closely homologous to the hypothetical protein MPFL (MPF-like). Secondary structure prediction servers predicted a predominantly helical structure for both mesothelin and mesothelin precursor proteins and also for stereocilin and otoancorin. Three-dimensional structure prediction servers INHUB and I-TASSER produced structural models for mesothelin, which consisted of superhelical structures with ARM-type repeats in conformity with the secondary structure predictions. Similar ARM-type superhelical repeat structures were predicted by 3D-PSSM server for mesothelin precursor and for stereocilin and otoancorin proteins. Conclusion The mesothelin superfamily of proteins, which includes mesothelin, mesothelin precursor, megakaryocyte potentiating factor, MPFL, stereocilin and otoancorin, are predicted to have superhelical structures with ARM-type repeats. We suggest that all of these function as superhelical lectins to bind the carbohydrate moieties of extracellular glycoproteins.

Description: 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.