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: 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.

Description: 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.

Description: FCRL1 (Fc receptor–like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC50s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL.

Description: 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.

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: Severe T cell depletion required for allogeneic hematopoietic stem cell transplantation from haplo-identical donors results in poor immune reconstitution and leads to high rates of mortality from infections, and relapse. One approach to overcome this problem is to infuse T cells depleted of alloreactivity. Selective depletion (SD) of alloreactive T cells is achieved by elimination of activated T cells after ex-vivo stimulation with recipient cells. To determine optimum selective depletion conditions, we have investigated the factors that modify alloreactivity of T cells. Methods: Alloreactivity was measured by one-way mixed lymphocyte reaction (MLR) using 3H thymidine uptake. PBMCs were used as responders and either irradiated expanded T cells (expT) or dendritic cells (DCs) as stimulators. T cells were expanded using anti-CD3 coated beads. DCs were generated from monocytes by GM-CSF and IL-4 stimulation. Selective depletion was performed by co-incubation of responder and stimulator cells for 72 hours and depletion of activated cells by an immunotoxin, LMB-2 (Anti-Tac (Fv)-PE-38), which was added to the culture at 24 and 48 hours. Effectiveness of the depletion was tested by a secondary MLR utilizing the original stimulator cells and unrelated third part cells. Results: Expansion of T cells has resulted in increase of HLA-DR, CD80 and CD86 expression compared to resting T cells (52.5% vs. 6%, 20.9% vs. 0.9%, and 32.9% vs. 20.9%, respectively), resulting in better stimulation in MLR (6505 cpm vs 1620 cpm). In one-way MLR using either PBMCs or CD25 depleted PBMCs as responders and expanded T cells and DCs as targets, with or without anti-CD28 in the culture media. DCs were better stimulators than expT cells (6636 vs. 4308). However, most dramatic effect was seen when anti-CD28 was added to the culture, increasing response to both expT cells and to a lesser extent DCs (40,169 and 19,303). Removal of CD25 positive cells also improved alloreactivity in all culture conditions (6636 in expT, 16,644 in DC, 57,363 in expT+CD28, and 30,943 in DC+CD28). To better define the effect of the target, we have performed Vbeta repertoire analysis of responding cells after expT cell, DC and expT cell+anti-CD28 stimulation. Flow cytometry revealed expansion of discrete Vbeta families, in addition to shared ones. We have then performed selective depletion using PBMCs or CD 25 depleted PBMCs as stimulators and expT cells, expT cell+anti-CD28, and DCs as stimulators. Residual alloreactivity after expT cell stimulation against original stimulators, DCs and third party cells were 7%, 147% and 99% respectively. Interestingly, after SD utilizing DCs as stimulators, there was substantial residual activity against expT cells (69%). When SD was performed using expT cells as stimulators with anti-CD28, combined with CD25 depletion, the depletion against both original stimulators and DCs was improved (2% and 54%, respectively). Conclusion: Depletion of regulatory T cells, and co-stimulation with ant-CD28 improves alloreactivity and selective depletion. Whether improvement in in-vitro selective depletion will result in better clinical outcome will be tested in a clinical trial.

Description: 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