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 expression 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

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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 corticosteroids were allowed for patients who had been previously 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 scheme was were used for xenograft studies. This cell line was estab- modified to shorten the treatment interval from 28 to 21 d lished and authenticated as previously described (18), and to increase the number of doses per cycle from three and phenotype was reconfirmed by serial flow cytometric to six (Table 1). If dose-limiting toxicity (DLT) was analyses including at the time of the xenograft studies. encountered in one subject in a cohort of three, an EU-1 cells were injected by tail vein into 5-wk-old female additional three patients were entered at that dose level. C.B-17 severe combined immunodeficient mice−/− If DLT occurred in two or more patients at any given dose (107 cells per mouse). Seventy-two hours after injection, level, the maximum tolerated dose was considered to have cohorts of 10 (treatment) or 5 (control) xenografts were been exceeded. Re-treatment required the recovery of treated with BL22 at dose levels of 1.5, 3, or 4.5 μg/dose, BL22-related toxicity to lower than grade 2 and the or control agents through i.p. injection every other day for absence of DLT, high-titer neutralizing antibodies, and nine doses. Xenograft recipients were euthanized at hind- disease progression.

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Table 1. Subject characteristics

ID Dx Age (y) Disease status Steroids CD22 CD22 site density PB blast count expression (%) (per cell) (per microliter) Pre/peak Post/nadir

1 ALL 9 Relapse no. 5, refractory, nine prior regimens 100 6,478 43 17 2 ALL/ 14 Relapse no. 6, refractory, eight prior regimens, 100 1,661 0 0 LBL allo-SCT, extramedullary chloromas 3 ALL 13 Relapse no. 1, refractory, seven prior regimens + 100 4,341 2,401 3,160 4 ALL 22 Relapse no. 1, refractory, four prior regimens 100 2,303 96,660 69,540 5 ALL 19 Relapse no. 1, refractory, six prior regimens 100 1,835 61,661 26,358 6 ALL 9 Relapse no. 2, two prior regimens 100 7,978 33 29 7 ALL 16 Relapse no. 1, refractory, four prior regimens + 100 5,978 27 4 8 ALL 3 Relapse no. 1, refractory, four prior regimens 100 4,546 67,815 187,000 9 ALL 17 Relapse no. 4, six prior regimens, allo-SCT × 2 100 1,448 876 401 10 LBL 14 Relapse no. 1, refractory, three prior regimens 100 3,856 4 3

11 ALL 5 Relapse no. 2, refractory, + 100 3,053 5,568 37,474 six prior regimens, allo-SCT 12 ALL 16 Relapse no. 1, refractory, four prior regimens 100 4,371 46,540 26,640 13 BL 17 Refractory, four prior regimens, auto-SCT 100 ND 0 0 14 ALL 4 Relapse no. 2, refractory, five prior regimens 100 3,402 1 588 15 ALL 11 Relapse no. 2, refractory, seven prior regimens + 75-82 5,640 34 2,385 16 ALL 18 Relapse no. 2, refractory, five prior regimens + 100 9,380 12,880 33

17 ALL 19 Relapse no. 2, refractory, + 100 7,070 276 2 four prior regimens, allo-SCT

18 ALL 4 Relapse no. 1, refractory, five prior regimens + 100 6,577 2 0

19 ALL 11 Relapse no. 2, refractory, six prior regimens + 100 4,721 164 136

20 ALL 9 Relapse no. 2, refractory, four prior regimens 100 7,549 6 2

21 ALL 8 Relapse no. 2, refractory, four prior regimens 100 3,849 988 268 22 ALL 10 Relapse no. 2, refractory, three prior regimens 100 731 22,735 200

23 ALL 18 Relapse no. 2, four prior regimens, allo-SCT 100 7,585 1,398 10

Abbreviations: ANC, absolute neutrophil count; ID, identification number; Dx, diagnosis; PB: peripheral blood; BM, bone marrow; Nab, neutralizing antibody development; pre, pretreatment; post, posttreatment; allo-SCT, allogeneic stem cell transplant; auto-SCT, autologous stem cell transplant; QOD, every other day; Q, every; PD, progressive disease; BL, Burkitt lymphoma; ND, not done; CNS, central nervous system; LBL, lymphoblastic lymphoma; PET, positron emission tomography; LDH, lactate dehydrogenase; hypo, hypocellular. *Progressive extramedullary disease (renal, intraorbital masses) determined retrospectively after cycle no. 2.

Toxicity grading and definition. Version 3.0 of the NCI the following exceptions: tumor lysis syndrome, abnormal Common Terminology Criteria for Adverse Events6 was electrolytes responding to supplementation, grade 3 he- used for toxicity and adverse event reporting. DLT was de- patic dysfunction with resolution before the next cycle, fined as a nonhematologic toxicity of grade 3 or more with and grade 3 fever, hypertriglyceridemia, hypercholesterol- emia, and hypoalbuminemia in the absence of vascular leak syndrome. Hematologic DLT was defined as grade 4 6 http://ctep.cancer.gov/reporting/ctc.html hematologic toxicity (with the exception of lymphocytes)

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Table 1. Subject characteristics (Cont'd)

BM blasts Cycles Dose level Clinical activity Response Nab

Pre Post

>90% >90% 1 1 (10 μg/kg QOD × 3 Q 28 d) PD − <5% <5% 1 1 (10 μg/kg QOD × 3 Q 28 d) PD −

>90% ND 1 1 (10 μg/kg QOD × 3 Q 28 d) ⇓ pain PD − >90% ND 1 2 (20 μg/kg QOD × 3 Q 28 d) ⇓ PB blasts PD − >90% ND 1 2 (20 μg/kg QOD × 3 Q 28 d) ⇓ PB blasts PD − 72% 90% 1 2 (20 μg/kg QOD × 3 Q 28 d) PD − >60% 63% 1 3 (25 μg/kg QOD × 3 Q 28 d) PD (CNS) + >90% ND 1 3 (25 μg/kg QOD × 3 Q 28 d) PD − 79% >90% 1 3 (25 μg/kg QOD × 3 Q 28 d) ⇓ PB blasts PD − 30-70% 23-90% 3 4 (30 μg/kg QOD × 3 Q 28 d) Improved PET scan No response → PD + (skeletal sites), platelet (post no. 3) (post no. 3) count normalization 78% ND 1 4 (30 μg/kg QOD × 3 Q 28 d) PD −

>90% ND 1 4 (30 μg/kg QOD × 3 Q 28 d) ⇓ PB blasts PD − <5% ND 1 5 (30 μg/kg QOD × 3 Q 21 d) ⇓ pain PD − >90% 90% 1 5 (30 μg/kg QOD × 3 Q 21 d) ⇓ BM cellularity (50-70%) PD − 25% >90% 1 6 (30 μg/kg QOD × 6 Q 21 d) PD − >90% 60-90% 1 6 (30 μg/kg QOD × 6 Q 21 d) ⇓ PB and BM blasts, No response + BM cellularity (0-85%) LDH, hepatosplenomegaly, renal infiltration >90% 30-50% 3 6 (30 μg/kg QOD × 6 Q 21 d) ⇓ PB and BM blasts, No response − uric acid, ⇑ platelet and reticulocyte counts >90% Hypo 2 7 (40 μg/kg QOD × 6 Q 21 d) ⇓ BM cellularity (5-20%) No response − >90% splenomegaly, ⇑ neutrophil count >90% >90% 1 7 (40 μg/kg QOD × 6 Q 21 d) ⇓ BM cellularity (focal) No response − (three doses) 80-90% 70-80% 2 7 (40 μg/kg QOD × 6 Q 21 d) ⇓ BM blasts (focal), regenerating No response → PD − trilineage hematopoiesis, ANC (post no. 2) and platelet count normalization >90% >90% 1 7 (40 μg/kg QOD × 6 Q 21 d) ⇓ PB blasts PD − >90% >90% 2 7 (40 μg/kg QOD × 6 Q 21 d) ⇓ PB blasts, LDH, splenomegaly, PD* − pain, testicular masses, adenopathy >90% >90% 1 7 (40 μg/kg QOD × 6 Q 21 d) ⇓ PB blasts No response −

lasting >5 d or any platelet transfusion. Subjects with ab- nential or biexponential model based on Aikake's rule normal blood counts due to bone marrow infiltration (20). For biexponential kinetics, the β half-life was used were not evaluable for hematologic toxicity. in analyses. Pharmacokinetics. Plasma levels of BL22 were deter- Neutralizing antibody assay. To assay for the presence of mined by incubating dilutions of plasma with Raji cells neutralizing antibodies, mixtures containing 90% serum and comparing cytotoxicity as assessed by [3H]leucine and 10% BL22 (final BL22 concentration, 1,000 ng/mL) incorporation to that obtained by a BL22 standard as pre- were incubated at 37°C for 15 min, diluted, and cultured viously described (15). Samples were obtained before, im- with Raji cells, and the percentage of inhibition in the mediately after, and at 1/2, 1, 1 1/2, 2, 4, 8, 12, and 18 h presence of subject serum was calculated as previously de- after each day 1 dose, and before and immediately after scribed (15). High-titer neutralizing antibodies were de- subsequent doses. Area under the curve was calculated fined as levels that resulted in >75% neutralization of on the first dose of the cycle from either a monoexpo- 1,000 ng/mL of BL22 in this assay.

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Response criteria. Standard disease-specific clinical and laboratory response criteria were used (21, 22). Statistical analyses. For mice evaluated in preclinical studies, the probability of survival as a function of time, according to treatment administered, was determined by the Kaplan-Meier method with a log-rank test used to determine the difference of the probability of survival between the three pooled control groups and the combined BL22-treated groups. For the dose effect, pooled data from the two higher dose level groups were analyzed compared with the lowest dose. Results were not adjusted for multiple comparisons. The relationship between clearance and peripheral blast count per microliter in subjects treated on the clinical trial was determined using Spearman rank correlation, with ∣r∣ > 0.70 indicating a strong correlation and the P value indicating a result from a test of whether r =0. Changes in peak BL22 levels between the first and last dose were evaluated using a paired t test after verifying that the differences followed a normal probability distribution. Although patients received a varying number of doses, the difference is meant to illustrate the effect associated with maximal dosing on the trial. All P values were two-tailed and are reported without adjustment for multiple comparisons. Human subjects protections. All studies were approved by the Investigational Review Boards of the NCI (preclinical studies, clinical trial NCT00077493) or Emory University School of Medicine (preclinical studies).

Results

CD22 expression. To assess the frequency of CD22 expression in B-lineage ALL, blasts from 93 children with pre-B or Burkitt-type ALL were evaluated by flow cytometry. All cases were CD22+. CD22 expression distribution and density were further quantified in 54 of these cases. One hundred percent of the blasts within individual cases expressed CD22 in 52 of 54 cases. Minor populations of blasts without demonstrable CD22 expression were de- tected in two individuals (∼80% and 90% CD22+). Deter- mination of antibody binding capacity per cell revealed that the average CD22 site density within cases ranged from 451 to 16,523 sites per blast (median, 4,062), and only four cases showed <1,000 CD22 sites per cell (Fig. 1). Cytotoxicity assays. In vitro cytotoxicity assays were conducted on blast samples obtained from 42 children with B-precursor ALL (Fig. 1). BL22-induced killing was

Fig. 1. Activity of BL22 against CD22-expressing human ALL blasts. A, CD22 was expressed by primary patient blasts. Anti-CD22 antibody–binding capacity per cell quantified in 54 primary patient samples revealed an average CD22 density of 451 to 16,523 sites per cell (median, 4,062). B, BL22 was cytotoxic against primary patient samples in vitro in a dose-responsive manner. A representative cytotoxicity curve is presented. C, murine xenografts treated with BL22 had significant prolongation of leukemia-free survival in a dose-responsive fashion (P < 0.05). RFB4, anti-CD22 MoAb; LMB2, anti–CD25-PE immunotoxin.

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Fig. 2. Adverse events attributed to BL22. Maximum toxicity grade per patient per cycle (n = 23 subjects, 30 cycles). AST, aspartate aminotransferase; ANC, absolute neutrophil count; WBC, white blood count; PT, prothrombin time; PTT, partial thromboplastin time.

observed in all samples, with IC50s that ranged from 0.5 subject at dose level 7 received three of six doses. All to 100 ng/mL (median, 9.8). subjects were evaluable through the DLT evaluation Xenograft studies. Murine xenografts treated with BL22 period for the primary study end points (i.e., toxicity, had a significant prolongation of leukemia-free survival pharmacokinetics, and immunogenicity). During the (Fig. 1). Treatment cohorts were analyzed against the con- course of the trial, the treatment schedule was amended trols (using grouped data for both), and differences (treat- to escalate the dose intensity (Table 1) based on safety ment versus control) were significant (P <0.001).To and activity data from preceding cohorts. Cohort 5 evaluate dose response, 3 and 4.5 μg cohorts were com- was terminated early to increase the number of doses bined after determining that they had similar survival, administered per cycle from three to six. and were analyzed compared with the 1.5 μgcohort. Toxicity. BL22 treatment was associated with an accept- The difference was significant (P < 0.05). able safety profile. No subject experienced infusion Phase I trial. Twenty-three subjects ranging in age from reactions, allergic events, vascular leak syndrome, or 3 to 22 years (median, 13) were treated on the phase I hemolytic uremic syndrome. All adverse events were trial. Twenty had ALL with marrow relapse, and one each self-limited and most were grades 1 and 2 (Fig. 2). Grades had ALL with extramedullary relapse, stage 4 lymphoblas- 3 and 4 events were rapidly reversible. Two subjects treated tic lymphoma, and Burkitt lymphoma. All had been heavi- at dose level 7 experienced grade 4 ALT elevation of 1 to ly pretreated, having received a median of four prior 2 days in duration, which met the original protocol regimens (range, 2-9) and 20 were refractory to chemo- definition of DLT. However, both of these resolved to therapy at the time of enrollment (Table 1). Twenty-two levels required for ongoing treatment as scheduled subjects completed at least one cycle of BL22 and one supporting a revision in the DLT definition.

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Table 2. Cycle 1 pharmacokinetic parameters

Dose (μg/kg) Subjects Peak day 1 (ng/mL) T1/2 (min) AUC (μg * min/mL) Vd (L/kg) Clearance (mL/min/kg)

10 3 65 (61-107) 68 (40-146) 7 (4-22) 0.12 (0.10-0.14) 1.45 (0.45-2.11) 20 3 139 (64-341) 85 (35-120) 11 (7-36) 0.09 (0.02-0.14) 2.13 (0.55-2.98) 25 3 186 (112-413) 110 (101-136) 17 (12-66) 0.06 (0.02-0.08) 1.52 (0.38-2.14) 30 8 356 (85-500) 118 (43-248) 52 (5-116) 0.11 (0.03-0.28) 0.60 (0.27-343) 40 6 469 (287-589) 108 (78-167) 53 (26-84) 0.10 (0.06-0.58) 0.81 (0.39-1.31)

NOTE. All values represent median (range).

Pharmacokinetics. There was a dose-related increase in Clinical activity. Most subjects had high disease burden the peak plasma levels of BL22 with wide interpatient var- and rapidly progressive disease at the time of protocol en- iation in pharmacokinetic parameters (Table 2). BL22 was rollment. No responses were observed, however, transient rapidly cleared from the circulation with a plasma half-life clinical activity was seen in 16 of 23 subjects (70%), on cycle 1 that ranged from 35 to 248 minutes (median, which in some cases was dramatic and clinically signi- 106). Cycle 1 clearance correlated with increased peripher- ficant (Table 1; Fig. 4). For example, four subjects had al blast count (Spearman correlation, r = 0.73; 95% confi- >2 log10 reduction in circulating blasts and four had recov- dence interval, 0.45-0.88; P < 0.0001; Fig. 3). Peak plasma ery of normal blood counts. Decreased blast infiltration of levels increased with progressive dosing in individuals bone marrow (6) and extramedullary sites (3) were also who experienced blast reduction treated at the highest observed. Notably, the most significant clinical activity doses (30 and 40 μg/kg; P = 0.01; data not shown). This was seen at the highest dose levels, including the four in- was in contrast to subjects without peripheral blast count dividuals with the largest reduction in peripheral blast reduction. Thus, pharmacokinetics seemed to be influ- counts (two each treated at dose levels 6 and 7) and all enced by disease burden, consistent with rapid BL22 bind- those with normalization of blood counts (treated at ing by CD22+ blasts. doses of 30 μg/kg or higher). Of the seven subjects without Immunogenicity. Only 3 of 23 subjects (13%) developed disease progression, two were ineligible to remain on the neutralizing antibodies. One had preexisting low-titer study (neutralizing antibodies, grade 4 ALT elevation), two antibodies that increased to 93% after cycle 1. Two devel- chose to discontinue treatment after two to three cycles, oped de novo antibodies with 78% neutralization after cy- and three developed progressive disease after two to three cle 1 and 65% after cycle 3. cycles, one of which was associated with the development of neutralizing antibodies.

Discussion

Despite significant progress in the curative treatment of childhood hematologic malignancies, relapse remains one of the greatest challenges in pediatric oncology (3). Furthermore, survivors have life-long risks of treatment- associated morbidity and mortality (6). New therapeutic approaches are needed to overcome chemotherapy resistance and to reduce side effects (23). CD22 is rapidly internalized upon antibody or immunotoxin binding (24). As shown, this antigen is expressed in high frequency in childhood ALL. Unconjugated MoAbs may induce cytotoxicity by direct and indirect (e.g., im- mune mediated) mechanisms, the latter of which are expected to be defective in individuals with ALL (25). Unconjugated MoAb against CD22 (epratuzumab) has re- cently been studied in childhood ALL and its activity as a single agent in the setting of relapsed ALL seems to be limited (26). Notably, anti-RFB4 control showed no activity against ALL xenografts or in cytotoxicity assays with prima- Fig. 3. The effect of disease burden on BL22 pharmacokinetics. Cycle 1 BL22 clearance correlated with the absolute peripheral blood ry samples from children with ALL (Fig. 1). blast count (Spearman correlation r = 0.73; 95% confidence interval, The activity of MoAbs can be dramatically increased by 0.45-0.88; P < 0.0001). linkage to toxic moieties. Plant and bacterial toxins cause

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Fig. 4. Hematologic improvement after BL22. A, improvement in the absolute neutrophil count (ANC) and platelet count during therapy. Arrows, BL22 treatment days. B, magnification, ×100 (Olympus BX51); C, oil magnification, ×1,000 (Olympus BX51). Bone marrow biopsies reveal a decrease in blast infiltration and an increase in normal hematopoietic precursors. Terminal deoxynucleotidyl transferase immunohistochemistry (blasts stain brown; subject no. 20).

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cellular cytotoxicity through the inhibition of protein syn- This trial shows that BL22 can be administered at doses thesis after internalization. These are highly potent and ac- of up to 40 μg/kg every other day for 6 doses in children tive in minute quantities, such that even a single molecule with ALL. No maximum tolerated dose was defined. in the cytoplasm is sufficient to kill a cell (27). There have Although two subjects treated at the highest dose level been limited studies of immunotoxins in childhood he- developed brief grade 4 ALT elevations, this was not matologic malignancies, and previously evaluated agents dose-limiting given the short duration (1-2 days). have been associated with severe adverse events and a high We subsequently chose to close the trial and apply the incidence of immunogenicity (28, 29). The clinical devel- schedule developed in this study (every other day for opment of immunotoxins in general has been hampered 6 doses every 21 days ) to phase I testing of a modified by nonspecific toxicities, immunogenicity, and production BL22 construct with higher affinity for the CD22 antigen. complexities. Serial modifications in the Pseudomonas- This second-generation agent, HA22 or CAT-8015, was en- based immunotoxin constructs used at the NCI have gineered to replace three amino acid residues in the heavy reduced nonspecific toxicities, increased stability, chain complementary determining region 3 of the BL22 enhanced tissue penetration, and improved targeted cellu- binding domain. This modification increased the binding lar killing (30). affinity for CD22 by 14-fold, which resulted in an approx- BL22 is a potent immunotoxin that targets CD22, which imately 1 log10 improvement in cytotoxicity against a as shown, is expressed in relatively high density on the sur- variety of CD22+ malignancies (31, 32). face of 100% of the blasts in the vast majority (96%) of In summary, CD22 represents an excellent target for pe- cases of childhood B-lineage ALL. BL22 was shown to have diatric B-lineage hematologic malignancies. These studies clinical activity with acceptable toxicity in adults with re- offer proof-of-principle that anti-CD22 Pseudomonas-based lapsed and refractory hairy cell leukemia, in which a max- immunotoxins can be administered to children and have imum tolerated dose of 40 μg/kg every other day for the potential to overcome chemotherapy resistance and in- 3 doses every 28 days was defined (15). This pediatric duce cytotoxicity of CD22+ blasts refractory to standard phase I trial extends those observations and establishes therapy. Anti-CD22 immunotoxins hold therapeutic that activity can be achieved in highly resistant childhood promise in this common subtype of pediatric cancer. ALL with acceptable toxicity. Notably, BL22 was tolerated at a greater dose intensity (i.e., six doses every other day Disclosure of Potential Conflicts of Interest every 21 days) compared with adults, and hemolytic ure- R.J. Kreitman, D.J. FitzGerald, and I. Pastan are coinventors on patents mic syndrome, which was the DLT in adults, was not ob- assigned to the NIH for the investigational product used in this research. served. Importantly, antileukemia activity was seen at all dose levels; however, clinical benefits in this highly refrac- Acknowledgments tory population were modest and transient at the doses tested. There are several possible explanations for the lim- We thank the clinical trial participants and their families, referring ited observed activity. Higher doses are likely required to physicians, Dr. David Waters, Dr. Lubing Gu, CURE Childhood Cancer, Inc., and the staff of the NCI, NIH Clinical Center, Aflac Cancer Center achieve maximal benefit. Furthermore, although peak le- and Blood Disorders Service, Emory University/Children's Healthcare of vels at the upper doses exceeded the concentrations re- Atlanta, Genencor, Inc., and MedImmune, LLC. quired for in vitro cytotoxicity, drug exposure was limited in most subjects due to the rapid clearance associated with Grant Support large disease burden. CD22 expression has been shown to Intramural Research Program of the NIH, National Cancer Institute, be a determinant of response to BL22 in vitro (12), al- the Center for Cancer Research, and a Cooperative Research and Devel- though there was no obvious influence of antigen density opment Agreement between the NIH, National Cancer Institute, and on clinical activity in this trial (Table 1). However, small MedImmune, LLC. The costs of publication of this article were defrayed in part by the numbers preclude definitive conclusions in this regard, payment of page charges. This article must therefore be hereby marked and it is notable that all subjects without progressive dis- advertisement in accordance with 18 U.S.C. Section 1734 solely to ease had site densities that exceeded 3,000 sites per cell, indicate this fact. whereas 6 of 16 with progressive disease had lower levels Received 11/10/2009; revised 01/08/2010; accepted 01/09/2010; of expression. published OnlineFirst 03/09/2010.

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www.aacrjournals.org Clin Cancer Res; 16(6) March 15, 2010 1903

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

Alan S. Wayne, Robert J. Kreitman, Harry W. Findley, et al.

Clin Cancer Res 2010;16:1894-1903. Published OnlineFirst March 14, 2010.

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