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Anti-CD22 Immunotoxin RFB4(Dsfv)-PE38 (BL22) for CD22-Positive Hematologic Malignancies of Childhood: Preclinical Studies and Phase I Clinical Trial

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Anti-CD22 Immunotoxin RFB4(Dsfv)-PE38 (BL22) for CD22-Positive Hematologic Malignancies of Childhood: Preclinical Studies and Phase I Clinical Trial Published OnlineFirst March 14, 2010; DOI: 10.1158/1078-0432.CCR-09-2980 Cancer Therapy: Clinical Clinical Cancer Research Anti-CD22 Immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-Positive Hematologic Malignancies of Childhood: Preclinical Studies and Phase I Clinical Trial Alan S. Wayne1, Robert J. Kreitman2, Harry W. Findley5, Glen Lew5, Cynthia Delbrook1, Seth M. Steinberg3, Maryalice Stetler-Stevenson4, David J. FitzGerald2, and Ira Pastan2 Abstract Purpose: Although most children with B-lineage acute lymphoblastic leukemia (ALL) and non– Hodgkin lymphoma are cured, new agents are needed to overcome drug resistance and reduce toxicities of chemotherapy. We hypothesized that the novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), would be active and have limited nonspecific side effects in children with CD22-expressing hematologic malignancies. We conducted the first preclinical and phase I clinical studies of BL22 in that setting. Experimental Design: Lymphoblasts from children with B-lineage ALL were assessed for CD22 expres- sion by flow cytometry and for BL22 sensitivity by in vitro cytotoxicity assay. BL22 was evaluated in a human ALL murine xenograft model. A phase I clinical trial was conducted for pediatric subjects with CD22+ ALL and non–Hodgkin lymphoma. Results: All samples screened were CD22+. BL22 was cytotoxic to blasts in vitro (median IC50, 9.8 ng/mL) and prolonged the leukemia-free survival of murine xenografts. Phase I trial cohorts were treated at escalating doses and schedules ranging from 10 to 40 μg/kg every other day for three or six doses repeated every 21 or 28 days. Treatment was associated with an acceptable safety profile, adverse events were rapidly reversible, and no maximum tolerated dose was defined. Pharmacokinetics were influenced by disease burden consistent with rapid drug binding by CD22+ blasts. Although no responses were observed, transient clinical activity was seen in most subjects. Conclusions: CD22 represents an excellent target and anti-CD22 immunotoxins offer therapeutic promise in B-lineage hematologic malignancies of childhood. Clin Cancer Res; 16(6); 1894–903. ©2010 AACR. There has been great progress in the curative treatment approaches that can overcome chemotherapy resistance of hematologic malignancies in childhood (1). Acute lym- and decrease nonspecific toxicities are needed to improve phoblastic leukemia (ALL), the most common pediatric the outcome for children with hematologic malignancies. cancer, is highly curable and 80% of children with B- CD22 is a B-lineage–restricted surface molecule that precursor ALL achieve long-term relapse-free survival (2). modulates B-cell receptor signaling and mediates cellular However, the outlook remains guarded for individuals adhesion (7). Immunotoxins are proteins that consist of with certain high-risk features at diagnosis and for those two primary components: a targeting moiety responsible who relapse. Hematologic malignancies remain a leading for cell binding and a bacterial or plant toxin that induces cause of cancer-related mortality in pediatrics (3, 4). Addi- cell death upon internalization (8). The recombinant im- tionally, current therapies carry the risk of treatment- munotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), contains associated morbidity and mortality (5, 6). Thus, novel the variable domains of the anti-CD22 monoclonal antibody (MoAb) RFB4 fused to a 38-kDa fragment of Pseudomonas aeruginosa exotoxin A (PE; refs. 9, 10). BL22 Authors' Affiliations: 1Pediatric Oncology Branch and 2Laboratory of is cytotoxic toward CD22+ cell lines and malignant cells Molecular Biology, 3Biostatistics and Data Management Section, Office of the Clinical Director, and 4Laboratory of Pathology, Center for Cancer from patients, and it is active in murine xenograft models Research, National Cancer Institute, NIH, Bethesda, Maryland; and 5Aflac (11–13). In phase I and II human clinical trials, BL22 in- Cancer Center and Blood Disorders Service, Emory University School of duced complete remissions in adults with hairy cell leuke- Medicine/Children's Healthcare of Atlanta, Atlanta, Georgia mia resistant to purine analogue therapy and exhibited Corresponding Author: Alan S. Wayne, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Building 10, Room a safety profile conducive to continued development 1-3750, 9000 Rockville Pike, MSC 1104, Bethesda, MD 20892-1104. (14–16). We hypothesized that this novel anti-CD22 im- Phone: 301-496-4256; Fax: 301-451-7010; E-mail: [email protected]. munotoxin would be active and have limited nonspecific doi: 10.1158/1078-0432.CCR-09-2980 side effects in children with CD22-expressing hematologic ©2010 American Association for Cancer Research. malignancies. We conducted the first preclinical studies 1894 Clin Cancer Res; 16(6) March 15, 2010 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 14, 2010; DOI: 10.1158/1078-0432.CCR-09-2980 BL22 for Pediatric Hematologic Malignancies limb paralysis and evaluated for the presence of human Translational Relevance leukemia by histopathology. BL22 and control agents. Recombinant immunotoxins B-lineage hematologic malignancies remain a lead- were produced as previously described (11, 12, 19). Clin- ing cause of cancer-related mortality in pediatrics and ical grade BL22 for human use was produced by the current therapies are associated with a wide array of Monoclonal Antibody and Recombinant Protein Facility toxicities. New agents are needed to overcome drug of the National Cancer Institute (NCI) and provided by resistance and reduce nonspecific adverse effects. We MedImmune Cambridge (formerly Cambridge Antibody report the results of the first preclinical studies and Technology, Inc., a subsidiary of MedImmune, LLC). Con- phase I clinical trial of a novel anti-CD22 immuno- trol reagents for preclinical studies included PBS, anti- toxin, RFB4(dsFv)-PE38, in the setting of childhood CD22 MoAb (RFB4-IgG provided by the Developmental hematologic malignancies. An acceptable toxicity pro- Therapeutics Program of the NCI), and the anti–CD25- file and transient clinical activity was observed. This PE immunotoxin anti–Tac(Fv)-PE38 (LMB2). phase I clinical trial serves as proof-of-principle that this Phase I clinical trial. A phase I trial was conducted at immunotoxin construct is cytotoxic to chemotherapy- the NIH Clinical Center (ClinicalTrials.gov no. resistant CD22+ blasts and that it can be administered NCT00077493). to children in doses that achieve serum levels which ex- Eligibility. Individuals between 6 mo and 24 y of ceed the expected in vitro IC50. The trial established a age with relapsed or refractory ALL or non–Hodgkin lym- dose and schedule for subsequent testing of RFB4 phoma who had exhausted available curative therapies (dsFv)-PE38 with a modified Fv sequence that confers were eligible. Measurable or evaluable disease that was higher binding affinity for CD22. Future trials are CD22+ by flow cytometry (>30%) or immunohistochem- planned in combination with standard chemotherapy istry (>15%) was required. Subjects must have been off agents. other investigational agents for at least 30 d and systemic chemotherapy for at least 14 d. Individuals with isolated testicular relapse and active central nervous system malig- and phase I clinical trial of BL22 for pediatric ALL and nancy were excluded, but concurrent prophylactic non–Hodgkin lymphoma. intrathecal chemotherapy was permitted. Concurrent cor- ticosteroids were allowed for patients who had been pre- viously treated with such to reduce the likelihood of rapid Materials and Methods disease progression during the time required to travel to the NIH and undergo eligibility screening. The doses of Patient samples. Fresh bone marrow or peripheral blood corticosteroids could not have been increased for at least blasts were collected from children with B-lineage ALL. 7 d before trial enrollment and patients were required to In vitro cytotoxicity. Seventy-two–hour in vitro cytotoxi- have persistent or progressive (i.e., not decreasing) disease city assays were conducted using protein synthesis inhibi- burden. Eligibility required aspartate aminotransferase tion ([3H]leucine incorporation) and colorimetric viability and alanine aminotransferase (ALT) of five or more times (WST-1). Results were expressed as the IC50 value (concen- the upper limit of the normal, total bilirubin of ≤2 mg/dL, tration of BL22 required to reduce viability/protein synthe- and age-adjusted normal creatinine. sis by 50% compared with nontreated controls) as BL22 administration. BL22 was administered i.v. over previously described (12). 30 min every other day for three or six doses. Intravenous Flow cytometry and antigen binding site determination. hydration was initiated 6 h before BL22 using 5% dextrose CD22 antigen expression and absolute peripheral blast and 0.45% sodium chloride at a rate of 90 mL/m2/h. Pre- counts were determined by flow cytometry. Antigen site medication consisted of acetaminophen, diphenhydra- density was quantified by determining the anti-CD22 an- mine, and ranitidine. tibody binding capacity per cell (17) using the BD Bio- Study design. Cohorts of three to six subjects were sciences QuantiBRITE system for fluorescence quantitation. treated at doses of 10, 20, 25, 30, and 40 μg/kg/dose. Murine xenografts. Cells from the human ALL line EU-1 During the course of the trial, the escalation
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