The Novel Calicheamicin-Conjugated CD22 Antibody Inotuzumab Ozogamicin (CMC-544) Effectively Kills Primary Pediatric Acute Lymphoblastic Leukemia Cells

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The novel calicheamicin-conjugated CD22 antibody inotuzumab ozogamicin (CMC-544) effectively kills primary pediatric acute lymphoblastic leukemia cells

JF de Vries1, CM Zwaan2, M De Bie1, JSA Voerman1, ML den Boer2, JJM van Dongen1 and VHJ van der Velden1

1Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands and 2Department of Pediatric Oncology/Hematology, Erasmus MC, Sophia Children’s Hospital, Rotterdam, The Netherlands

We investigated whether the newly developed antibody (Ab) - One of these new approaches is antibody (Ab)-targeted targeted therapy inotuzumab ozogamicin (CMC-544), consisting chemotherapy, where the Ab-part of the compound is used to of a humanized CD22 Ab linked to calicheamicin, is effective in specifically deliver the cytostatic drug to the target cell, resulting pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters in less or no bystander cell death. One of the first examples of Ab- determine its efficacy. CMC-544 induced dose-dependent cell based delivery is gemtuzumab ozogamicin (GO, Mylotarg kill in the majority of BCP-ALL cells, although IC50 values varied or CMA-676), which consists of a humanized CD33 Ab substantially (median 4.8 ng/ml, range 0.1–1000 ng/ml at 48 h). covalently linked to a derivative of the potent DNA-damaging The efficacy of CMC-544 was highly dependent on calicheami- cytotoxic agent calicheamicin.7,8 Calicheamicin induces cell cin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 death in its target cells by interaction with double-helical DNA in expression. Although CD22 expression was essential for the minor groove causing site-specific double-stranded DNA uptake of CMC-544, a repetitive loop of CD22 saturation, cleavage at very low concentrations.9 GO was initially developed CD22/CMC-544 internalization and renewed CD22 expression for the treatment of patients with CD33-positive acute myeloid was not required to achieve intracellular threshold levels of leukemia (AML) and the single-agent maximum tolerated dose calicheamicin sufficient for efficient CMC-544-induced apopto- was two infusions of 9 mg/m2 with a 14-day interval. Later sis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (My- regimens in combination with chemotherapy usually applied a 2 lotarg) in acute myeloid leukemia (AML) patients, in which single dose of GO at a dose of 3–4.5 mg/m . Despite the initial complete and prolonged CD33 saturation was required for accelerated approval, confirmatory studies failed to demonstrate apoptosis induction. These data suggest that CMC-544 treat- sufficient benefit and revealed liver toxicity, which led to ment may result in higher response rates in ALL compared with voluntary withdraw of the market authorization in the United response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low States (http://www.fda.gov/Safety/MedWatch/SafetyInformation/ CMC-544 dosages, are warranted. SafetyAlertsforHumanMedicalProducts/ucm216458.htm). In chil- Leukemia (2012) 26, 255–264; doi:10.1038/leu.2011.206; dren, however, the risk–benefit ratio may be more promising.10 published online 26 August 2011 Recently, a novel Ab-based therapy has been developed, Keywords: acute lymphoblastic leukemia; CMC-544 (inotuzumab called inotuzumab ozogamicin or CMC-544, which consists of a ozogamicin); CD22; calicheamicin; immunoconjugate; pediatric humanized CD22 Ab (G5/44, selected because of the highest internalization capacity upon stimulation11) conjugated to a derivative of calicheamicin (CalichDMH). CMC-544 is intended to be studied for the treatment of CD22-positive B-cell Introduction 12 malignancies. The human B-lymphocyte-restricted antigen (Ag) CD22 is expressed early in B-cell development from the Acute lymphoblastic leukemia (ALL) is the most common cancer pro-B-cell stage onwards and persists on B-cells until they diagnosed in children, representing around 30% of cancer 13 1 differentiate into plasma cells. Mature and immature B-cell diagnoses among children younger than 15 years of age. In the 14 malignancies generally express CD22. majority of ALL cases (85%), the B-cell precursors are affected CMC-544 has already been shown to be effective in B-cell (BCP-ALL), while in 15% of cases the malignant cells are of 12,15 lymphoma cell lines and BCP-ALL cell lines. Moreover, also T-cell origin (T-ALL).2 ALL treatment generally comprises in murine models, CMC-544 showed strong anti-tumor activity chemotherapy consisting of different combinations of cytostatic 11,12 against multiple subcutaneous or systemically disseminated drugs depending on the collaborative group protocols. Currently 16 human CD22 þ B-cell lymphoma xenografts. Recently, the at least 80–85% of childhood ALL patients are cured with first results of a phase I trial with CMC-544 in patients with non- conventional chemotherapy,1,3,4 but these drugs may result in Hodgkin’s B-cell lymphoma have been published demonstrating several adverse acute and long-term side effects, such as that CMC-544 has activity in lymphoma patients, with reversible neurocognitive late effects, infertility, secondary malignancies 17 thrombocytopenia as the main toxicity. In these studies or cardiotoxicity.5,6 Therefore, nowadays, new approaches are CMC-544 was administered as single-agent at a dose of not only aiming at achieving higher survival rates, but also at 2 1.8 mg/m with a 28-day interval. diminishing long-term side effects and improving quality of life. We investigated whether CMC-544 is also effective in primary BCP-ALL cells in vitro. Based on the structural homology Correspondence: Dr VHJ van der Velden, Department of Immunology, between CD22 and CD33 receptors (Siglec family) and the Erasmus MC, University Medical Center Rotterdam, Dr Molewater- similarity of the two immuno-conjugates, we hypothesized that plein 50, 3015 GE Rotterdam, The Netherlands. E-mail: [email protected] CMC-544 has a similar mechanism of action as GO. In previous Received 3 May 2011; revised 1 July 2011; accepted 6 July 2011; GO studies, we showed that prolonged maximal saturation of published online 26 August 2011 CD33 Ags on AML cells by GO is required for efficient Efficacy of CMC-544 in BCP-ALL JF de Vries et al 256 GO-induced cell death, both in-vitro and in-vivo. Furthermore, epitope located in the same CD22 protein domain.11 CD22 and a repeated loop of binding of GO, saturation of CD33 Ags, CD33 expression levels on ALL cells were expressed as CD22 internalization of the complex and renewed expression of CD33 and CD33 ratio, defined as MFI of ALL cells divided by MFI of Ags was essential for intracellular accumulation of calicheami- CD22- or CD33-negative lymphocytes (T and NK cells) from the cin and for the efficacy of GO.18,19 In the clinical trials with GO same patient, respectively. in AML patients only low dosages of GO were added to the existing AML treatment protocols to assure a reasonable tolerability of the combination of drugs. With these low In vitro incubation of cells with CMC-544, free concentrations the criteria of prolonged and maximal saturation calicheamicin, GO or G5/44 Ab will not be fulfilled, which might in part explain the low For continuous-labeling experiments, ALL cells were incubated response rates observed in these clinical trials. Moreover, it was at a cell concentration of 1 Â 106 cells/ml (100 000 cells/well) in shown that AML cells may be relatively resistant to the cytotoxic a 96-wells flat-bottom microtiter plate with different concentra- compound calicheamicin.20 In this study, we evaluated which tions of CMC-544 (0–1000 ng/ml), free calicheamicin (0–100 ng/ parameters determine the efficacy of CMC-544 in vitro in ml), GO (0–10 000 ng/ml) or G5/44 Ab (0–10 000 ng/ml) at 37 1C primary BCP-ALL. These data can be taken into account for the for different periods of time (0, 24, 48, 72 or 96 h). design of a clinical phase I/II trial with CMC-544 in children For pulse-labeling experiments, cells were incubated with with relapsed BCP-ALL. various concentrations of CMC-544 for 1 h, 6 h or 24 h at 37 1C, after which non-bound CMC-544 was removed by washing the cells twice with RPMI. The cell pellet was suspended in RPMI/ Materials and methods 10% fetal calf serum (1 Â 106 cells/ml) and cells were incubated at 37 1C for 48 h. Cells and culture conditions For determination of internalization and renewed expression, All human ALL cell lines (MIK-ALL, ALL-P0, REH, RCH-ACV cells were incubated on ice with CMC-544 (5000 ng/ml) for and 697) used were obtained from the German Collection of 15 min, after which non-bound CMC-544 was removed by Microorganisms and Cell cultures (Prof Dr HG Drexler, DSMZ washing the cells twice with ice-cold RPMI. The cell pellet GmbH, Braunschweig, Germany). Cell lines were cultured in was suspended in RPMI/10% fetal calf serum (1 Â 106 cells/ml) RPMI (Lonza, Basel, Switzerland) supplemented with 10% fetal and cells were either stored on ice or incubated at 37 1C for 0.5, calf serum, penicillin G sodium (100 U/ml) and streptomycin 1or3h. sulfate (0.1 mg/ml), at 37 1C in a humidified atmosphere containing 5% CO2. Peripheral blood and bone marrow (BM) samples from pediatric patients with BCP-ALL (n ¼ 20) or AML (n ¼ 3) were Detection of cell survival obtained in accordance to the declaration of Helsinki, with the Quantitative flow cytometric analysis of absolute numbers of approval of the local Medical Ethics Committee (Erasmus MC, viable cells was performed using a modified carboxyfluorescein Rotterdam), after patients had given written informed consent. diacetate succinimidyl ester-based cytotoxicity assay, as de- Mononuclear cells were obtained by Ficoll-Paque (Pharmacia, scribed previously.22,23 Target cells were not labeled with Uppsala, Sweden) density centrifugation of peripheral blood or carboxyfluorescein diacetate succinimidyl ester, but to distin- BM, and cultured as described for cell lines. guish different cell populations in BM-mononuclear cell of ALL We selected BCP-ALL patients based on variable expression patients (malignant B, mature B and non-B-cells) specific levels of CD22 (median fluorescence intensity (MFI) CD22- antibodies were used, such as CD10, CD19 and CD20 Ab (all label: 13–233). Fully negative BCP-ALL are extremely rare,21 purchased from BD BioSciences). AML blasts were gated on the (Supplementary Figure 1) and therefore were not included in basis of forward/side scatter, and CD45 and CD34 expression. To this study. each tube 10 000 Flow-Count Fluorospheres (Coulter Corpora- tion, Miami, FL, USA) were added, and 2000 beads were acquired, enabling quantitative analysis of surviving target cells. Antibody conjugates 7-amino-actinomycin D (Alexis Corp, Lausen, Switzerland) or CMC-544, N-acetyl g-calicheamicin dimethyl hydrazide propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) was used (CalichDMH, inactive form of calicheamicin), GO and G5/44 to exclude dead cells. Ab were kindly provided by Pfizer Inc. (New York, NY, USA). The percentage of survival was defined as ((mean absolute CMC-544 is comprised of a humanized IgG4 CD22 mAb number of viable target cells in control mediumÀ (G5/44), linked to N-acetyl g-calicheamicin dimethyl hydrazide absolute number of viable target cells (experimental))/(mean (CalichDMH) via an acid-labile AcBut linker with an average absolute number of viable target cells in control med- drug loading of 73 mg of CalichDMH per mg of G5/44 protein ium)) Â 100. Normalized dose-response curves were determined (B6 mol/mol).12 Concentrations of CMC-544 and GO are by non-linear regression analysis (Graphpad software, La Jolla, expressed as equivalents of CalichDMH and that of unconju- CA, USA), and from these curves IC50 values (defined as the gated Ab are expressed as the quantity of Ab protein, unless concentration resulting in 50% survival) were calculated. If no indicated else. curve could be calculated, data were expressed as 4the highest concentration of the test. Moreover, if less than 300 viable cells were left after culturing of cells in medium, samples were CD22 and CD33 expression levels excluded from the analysis. CD22 and CD33 expression levels were determined by flow As a control for the flow cytometric assay, for some patients, cytometry using PE-conjugated G5/44 Ab or PE- or APC- also an MTT-based assay was performed, as described conjugated CD22 Ab (clone HCL-1) from BD Biosciences previously.24 Both methods yielded highly comparable results (San Jose´, CA, USA) and PE-conjugated CD33 Ab (clone P67.6) (for CMC-544: Spearmann’s correlation coefficient ¼ 0.85; from BD, respectively. Both CD22 antibodies recognize an P ¼ 0.02; data not shown).

Leukemia Efficacy of CMC-544 in BCP-ALL JF de Vries et al 257 Detection of double-strand DNA breaks expression at t ¼ 0 h)] Â 100. In pulse labeling experiments, After exposure of cells for 24 h to various concentrations of the percentage of renewed CD22 expression was calculated as CMC-544, cytospin slides were made (50 000 cells per slide). [(specific MFI of total CD22 expression at t ¼ x)À(specific MFI of Calicheamicin is known to induce DNA double strand breaks.9 CMC-544-bound CD22 at t ¼ x)]/[(specific MFI of total CD22 Histone H2AX is rapidly phosphorylated at serine 139 in the expression at t ¼ 0)À(specific MFI of CMC-544-bound CD22 at chromatin micro-environment surrounding a double strand t ¼ x)] Â 100. break.25 Analysis of g-H2AX foci was performed as described No detectable CMC-544 was present in cell supernatants after previously,26 using monoclonal mouse anti-phospho-histone incubation of CMC-labeled cells at 37 1C (data not shown), H2A.X Ab (2 ng/ml; Upstate Biotechnology, Waltham, MA, assuring that all the CMC-544 that disappears from the cell USA) and FITC-conjugated rabbit anti-mouse (1:40) secondary membrane has been internalized, and has not been dissociated antibodies (DAKO, Glostrup, Denmark). Cell nuclei were from the cell membrane into the culture medium. stained with DAPI (Sigma). g-H2AX foci were analyzed under a Zeiss Axioplan fluorescence microscope, and analyzed by Axiovision software (Zeiss, Go¨ttingen, Germany). Confocal laser microscopy MIK-ALL cells were stained with either G5/44-PE or CD22-APC (HCL-1, BD) for 20 min on ice. Cells were washed (cold) Cell cycle analysis and either directly fixed (15 min at RT in PFA 0.5%) or incubated For cell cycle analysis, cells were washed with phosphate- for various times at 37 1C and subsequently fixed. After buffered saline, fixed and permeabilized for 10 min at room washing with phosphate-buffered saline/BSA cytospin slides temperature using FACS lysing solution (BD BioSciences). Cells (50 000 cells/slide) were prepared. Cells were embedded in were stained for 30 min at 4 1C with FITC-conjugated anti-Ki-67 Prolong Gold (Invitrogen, Breda, The Netherlands) supplemen- monoclonal antibodies (1:10; Zebra Bioscience B.V/DAKO). ted with DAPI (Sigma). The suspension preparations were After washing, cells were incubated with RNase (20 mg/ml; analyzed on a Leica SP5 laser-scanning confocal microscope Sigma-Aldrich) for 5 min at 37 1C, and finally stained with (Leica Microsystems BV, Rijswijk, The Netherlands). Analysis 50 mg/ml propidium iodide. Cells were subjected to was performed using Leica Application Suite (LAS) AF Lite flow cytometric analysis of DNA content using a FACSCalibur software (Leica Microsystems). flow cytometer (BD BioSciences). The percentages of cell Lysotracker Red DND-99 (Invitrogen-Molecular Probes, cycle distribution were calculated by CellQuest software Paisley, UK) was used as a lysosomal marker27 according to (BD BioSciences). the manufacturer’s instructions.

Detection of apoptosis Statistics Apoptosis was analyzed by labeling the cells with annexin The nonparametric Mann–Whitney U-test was used to deter- V-FITC and propidium iodide, using the IQP apoptosis kit mine differences in sensitivity between AML and ALL cells. (ImmunoQuality Products, Groningen, The Netherlands) Wilcoxon paired t-tests were applied to compare ALL and according to the manufacturer’s instructions. normal cell fractions within one ALL patient. Pearson’s correlation test was used to calculate the strength of linear dependence between different parameters and the efficacy of Analysis of CD22 Ag saturation, internalization of CMC-544. In all tests, a P-value of less than 0.05 was considered CMC-544 and renewed expression of CD22 Ags significant. Analysis of CD22 saturation, internalization and renewed expression was performed using a flow cytometric assay as described previously for GO.19 Briefly, to determine the amount Results of CD22 Ags bound by CMC-544 (CMC-544-bound CD22) cells were incubated with biotin-conjugated mouse anti-human IgG4 Mechanism of CMC-544-induced cell death in ALL cell antibodies (BD BioSciences), followed by streptavidin-PE (BD lines BioSciences) as a second step reagent. To detect total CD22 CMC-544-induced dose- and time-dependent cell death in expression (that is, maximal CMC-544 binding), cells were first various ALL cell lines, with IC50 values ranging from 0.15 to incubated with excess CMC-544 (final concentration: 5 mg/ml), 4.9 ng/ml (in calicheamicin equivalents; Figure 1a and Supple- followed by successive incubation with biotin-conjugated mentary Figure 2). CMC-544-induced cell death was CD22- mouse anti-human IgG4 antibodies and streptavidin-PE. As a dependent, as excess G5/44 (the Ab part of CMC-544) blocked negative control, cells were incubated with streptavidin-PE CMC-544-induced cell death (Figure 1b). Incubations with G5/ alone. To detect CD22 antigenic sites not occupied by CMC- 44 Ab alone did not affect cell survival (Figure 1c) indicating 544, cells were incubated with CD22-PE (clone G5/44) or, as a that CMC-544-induced cell death is not mediated via CD22 negative control, IgG1-PE (BD Biosciences). Analysis was signaling. performed on ALL blast cells, as defined by scatter character- After binding of CMC-544 to CD22 Ags at the cell surface istics and CD10 and/or CD19 expression levels. All incubations (Figure 2a), internalization of the CD22 receptor/CMC-544 were performed at 4 1C to prevent internalization during complex takes place. Within 30 min after binding, virtually all immunostaining. Data were acquired on a FACSCalibur flow CD22 antibodies were relocated to the cytoplasm of the cell cytometer and analyzed using CellQuest software. (Figure 2b), illustrating rapid internalization of the CD22 Ag/Ab The percentage of saturation was calculated as (specific MFI complex. This is followed by fusion of the CMC-544-containing of CMC-544-bound CD22)/(specific MFI of total CD22 endosome with a lysosome (Figure 2c, yellow), resulting in a expression) Â 100. In pulse labeling experiments, the percentage lower internal pH of the vesicle, subsequent degradation of the of internalization was calculated as [1À(specific MFI of acid-labile linker between G5/44 Ab and calicheamicin, and CMC-544-bound CD22 at t ¼ x)/(specific MFI of total CD22 release of calicheamicin.

Leukemia Efﬁcacy of CMC-544 in BCP-ALL JF de Vries et al 258

Figure 2 Mechanism of CMC-544-induced cell death. (a–c) Binding and internalization of CMC-544, followed by fusion of endosomes with lysosomes, as visualized by confocal laser microscopy. In these figures, CMC-544 is mimicked by CD22-APC (green) to enable counterstaining with Lysotracker Red (red). Using G5/44-PE antibodies, similar binding and internalization kinetics were observed as with CD22-APC. DAPI (blue) was used to stain the nuclei; phase- contrast was used to visualize cell morphology (not shown). (a) Binding of CD22-APC antibodies after 20 min incubation on ice. CD22-APC signals (green) are mainly observed at the cell surface. (b) Internalization of CD22-APC after 30 min incubation at 37 1C. CD22-APC is primarily located in the cytoplasm as illustrated by counterstaining with DAPI. (c) Fusion of a CD22-APC containing endosome with a lysosome results in colocalization of CD22-APC and Lysotracker Red, resulting in yellow signals. (d) Induction of ds DNA breaks (immunofluorescence) in MIK-ALL after incubation with 0, 10 or 100 ng/ml CMC-544 for 24 h. (e) Induction of apoptosis in MIK-ALL after incubation with 0 or 100 ng/ml CMC-544 for 24 h or 48 h. Apoptosis was measured by flow cytometry using annexin V- propidium iodide staining. Viable cells are shown in the lower left quadrant, early apoptotic cells in the lower right quadrant and late apoptotic/necrotic cells in the upper right quadrant.

After DNA damage is detected, the cell will try to repair its DNA Figure 1 CMC-544 induces effective and CD22-specific cell kill in damage during the G2 phase of the cell cycle. In rapidly dividing ALL cell lines. (a) Dose-survival curves of various ALL cell lines after ALL cell lines, a concentration dependent G2/M cell cycle arrest exposure for 48 h to 0–1000 ng/ml CMC-544. (b) Inhibitory effect of was observed after 24 h exposure to 10 or 100 ng/ml CMC-544 various concentrations of G5/44 Ab on cell death of MIK-ALL cells (data not shown). Finally, ALL cells became apoptotic induced by 48 h exposure to 10 ng/ml of CMC-544. (c) MIK-ALL cells were exposed to various concentrations of CMC-544 (n ¼ 4) or G5/44 (Figure 2e). antibody alone (n ¼ 2). Percentages of cell survival were determined after 48 h of incubation, and mean values±s.d. were plotted. CMC-544 concentrations were expressed in ng/ml; 1000 ng of Impact of intracellular accumulation of calicheamicin CMC-544 consists of 73 ng calicheamicin and 927 ng G5/44 Ab. on efficacy of CMC-544 in ALL cell lines We previously showed that accumulation of intracellular levels of calicheamicin by a continuous loop of binding/internalization We next examined the actual execution pathway induced by of GO and renewed expression of CD33 Ags highly contributed released and activated calicheamicin. After 24 h of incubation, to the efficacy of GO in AML cells.18,19 Prolonged maximal CMC-544 already induced a few ds DNA breaks at a saturation of CD33 Ags by GO was crucial to obtain sufficiently concentration of 10 ng/ml, but severe DNA damage was high intracellular GO levels. To investigate whether this was observed at concentrations of 100 ng/ml or higher (Figure 2d). also the case for CMC-544, we first examined the relationship

Leukemia Efficacy of CMC-544 in BCP-ALL JF de Vries et al 259 between CMC-544 concentration and saturation of CD22 Ags in various ALL cell lines. Saturation was dependent on CMC-544 concentration, but not on CD22 expression, as cell lines with differential CD22 expression showed a similar relationship between concentration and saturation. By combining the concentration–saturation data with the concentration–survival data (Figure 1a), the relation between saturation and cell survival could be calculated. As shown in Figure 3a, efficient cell death could already be observed at low CD22-saturation levels, indicating that, in contrast to previous results with GO, no prolonged and maximal saturation is required. To further investigate whether intracellular accumulation of calicheamicin affects the efficiency of CMC-544-induced cell death, we exposed MIK-ALL cells (CD22high) and ALL-P0 cells (CD22low), both showing equal sensitivity to free calicheamicin (data not shown), either continuously (48 h) or for 1, 6 or 24 h to various concentrations of CMC-544, and measured cell survival after 48 h. In both cell lines, but especially in the CD22low ALL- P0 cells, continuous exposure to CMC-544 was more effective than pulse exposure (Figures 3b and c), suggesting that a continuous loop of internalization and renewed expression of CD22 contributes to the efficacy of CMC-544, especially in cells with low CD22 expression. Preliminary data suggest that rapid renewed CD22 expression is due to the presence of an intracellular pool of CD22 receptors (data not shown). These data collectively show that only a limited amount of calicheamicin is required to induce cell death, but that intracellular accumulation of calicheamicin by continuous internalization and renewed expression potentiates CMC-544 efficacy and leads to more rapid and efficient cell death.

Effect of CMC-544 on cell survival of primary ALL and various CD22-negative cells To investigate whether CMC-544 is also effective in primary BCP-ALL cells, BM-mononuclear cells from 20 patients with BCP-ALL with variable CD22 expression (CD22 ratio ranging from 2.5. to 26.4) were tested for their sensitivity to CMC-544, as shown for one ALL patient sample in Figure 4a. Patient data are summarized in Supplementary Table 1. CMC-544 induced both dose-dependent and time-dependent cell death in primary þ þ ALL cells (Figure 4b). IC50 values of ALL cells (CD10 CD19 ) varied enormously (Figure 4c), but were generally B1000-fold À À À lower than IC50 values of normal cells (CD10 CD19 CD22 , mainly T-cells) derived from the same patients (median IC50 values of 4.8 and 2928 ng/ml calicheamicin equivalents, respectively). To further define the specificity of CMC-544, five BCP-ALL and three AML patient samples were exposed to free calicheamicin, CMC-544 and GO (Supplementary Figure 3). AML patient cells were equally sensitive to calicheamicin and Figure 3 Accumulation of calicheamicin has only a minor role in the GO, and the least sensitive to CMC-544. Primary BCP-ALL cells efficacy of CMC-544. (a) Relationship between saturation of CD22 were over 1000 times as sensitive to calicheamicin as primary antigens by CMC-544 and cell survival after 48 h exposure of MIK-ALL AML cells, and B100 times as sensitive to CMC-544. ALL cells to CMC-544. Mean values of three independent experiments are high low patient cells were more sensitive to CMC-544 than to GO, shown. (b) MIK-ALL (CD22 ) and (c) ALL-P0 (CD22 ) cells were demonstrating CD22-specific uptake of CMC-544. incubated for 1, 6, 24 or 48 h to various concentrations of CMC-544. Percentages of cell survival were measured after 48 h, and mean % survival ±s.d. of three independent experiments are shown. Important parameters for efficacy of CMC-544 To unravel the heterogeneity in sensitivity of ALL blasts to CMC-544, we examined which parameters are important for for CMC-544, IC50 values for calicheamicin varied substantially efficient CMC-induced cell death, based on the mechanism of between different ALL patients (Figure 5a). A strong correlation action presented before. (r ¼ 0.8, P ¼ 0.0004) was found between sensitivity to calicheamicin and efficacy of CMC-544, indicating that efficacy of Sensitivity to calicheamicin. We first analyzed the sensi- CMC-544 is highly dependent on cellular sensitivity towards 20 tivity of ALL cells to free calicheamicin. Similar to IC50 values calicheamicin. In line with previous studies, primary ALL cells

Leukemia Efﬁcacy of CMC-544 in BCP-ALL JF de Vries et al 260 (n ¼ 20) were signiﬁcantly more sensitive to calicheamicin (median: 0.17 ng/ml) than primary AML cells (n ¼ 3, median: 1023 ng/ml; Figure 5b), suggesting that lower intracellular concentrations may already be effective in ALL compared with AML cells.

Figure 5 Sensitivity to calicheamicin is crucial for efficacy of CMC-544. (a) Relationship between sensitivity of BCP-ALL cells to calicheamicin and efficacy of CMC-544 after 48 h of incubation. The correlation between calicheamicin sensitivity and efficacy of CMC-544 was highly significant (P ¼ 0.0004; Pearson’s r ¼ 0.80). (b) Differential sensitivity of BCP-ALL (n ¼ 17) and AML cells (n ¼ 3) for calicheamicin. Median IC50 values (gray bars) for BCP-ALL and primary AML cells were 0.2 and 1021 ng/ml calichDMH equivalents, respectively.

Figure 4 CMC-544 is effective and specific in primary BCP-ALL cells. (a) Example of flow cytometric analysis of CMC-544-induced cell death in BM-mononuclear cells (MNC) of a BCP-ALL patient. Untreated (CMC 0 ng/ml) cells are shown in the upper panel, CMC- 544-treated (10 ng/ml) cells are shown in the lower panel. For each test 2000 flowcount beads were acquired and the corresponding absolute numbers of surviving cells were calculated. Propidium iodide was used to specifically gate on viable BM-MNC (first plot). Different populations within the BM-MNC could be identified by immunostaining with APC-conjugated CD10 antibodies and FITC-conjugated CD19 antibodies. CD10 þ CD19 þ cells (in blue) are ALL cells, CD10ÀCD19 þ cells are mature B cells, CD10ÀCD19À cells (in red) are remaining normal cells, mostly T cells. (b) Example of dose– survival curves of BCP-ALL cells from one patient after 24, 48, and 72 h of treatment with CMC-544. From these curves IC50 values were þ calculated. (c)IC50 values of ALL cells (CD10 ; n ¼ 19) and normal cells (CD10À; n ¼ 14) isolated from BM-MNC of the same ALL patients after 48 h of treatment with CMC-544 are shown. Median IC50 values (gray bars) for BCP-ALL cells and normal cells were 4.8 and 2928 ng/ ml calichDMH equivalents, respectively. If no IC50 value could be calculated from the dose–survival curves, IC50 was indicated as 4maximum concentration (in calichDMH equivalents) tested in the experiment.

Leukemia Efﬁcacy of CMC-544 in BCP-ALL JF de Vries et al 261

Figure 6 The level of CD22 expression, internalization of CD22/CMC-544 and renewed CD22 expression only partly affect CMC-544 efficacy. (a) Relationship between CD22 ratio and efficacy of CMC-544 after 48 h of incubation in BCP-ALL cells of 20 patients. CD22 ratio is defined as MFI of CD22 expression on ALL cells divided by MFI of CD22 expression on T-cells from the same patient. The correlation between CD22 ratio and efficacy of CMC-544 was not significant (P ¼ 0.48; Pearson’s r ¼À0.19). (b) Relationship between CD22 ratio and efficacy of CMC-544 after correction for calicheamicin sensitivity of BCP-ALL cells (n ¼ 15). This correction was made by taking the ratio of the IC50 values for calicheamicin and CMC-544 after 48 h of incubation, and plotting logarithmic values of this ratio against log CD22 ratio. The correlation between these two parameters was almost significant (P ¼ 0.09; Pearson’s r ¼ 0.45). (c, d) BCP-ALL cells were pulse labeled with CMC-544 (5 ug/ml) for 20 min on ice, and then incubated in medium at 37 1C. After 0 h and 3 h of incubation, levels of CMC-544 bound to the cell surface (CMC-544-bound CD22) and total CD22 expression at the cell surface (maximal CMC-544 binding) were determined by flow cytometry. Percentages of internalization and renewed CD22 expression were calculated as described in the Material and methods. (c) Relationship between internalization and efficacy of CMC-544. The correlation between these two parameters was significant (P ¼ 0.05; Pearson’s r ¼À0.63). For two BCP-ALL samples no IC50 value could be determined, but in line with the linear relationship, these CMC-544-insensitive BCP-ALL cells showed a relatively low internalization capacity. (d) Relationship between renewed CD22 expression and efficacy of CMC-544. The correlation between these two parameters was not significant (P ¼ 0.85; Pearson’s r ¼À0.07).

CD22 expression. In primary ALL cells, no direct relation process of saturation, internalization and consequently in an was found between CD22 expression and efficacy of CMC-544 ongoing intracellular accumulation of calicheamicin, we (Figure 6a), probably owing to the predominant contribution of evaluated whether renewed CD22 expression was related to calicheamicin sensitivity to CMC-544-induced cell kill. The sensitivity to CMC-544. As shown in Figure 6d, no relation was difference in sensitivity for CMC-544 and calicheamicin (that is, observed between both parameters. the ratio between the IC50 for calicheamicin and the IC50 value Altogether, these data show that the efficacy of CMC-544 in for CMC-544) was only weakly related to CD22 expression primary ALL cells is mainly dependent on the cellular sensitivity levels (Figure 6b), (r2 ¼ 0.21; P ¼ 0.09). Therefore, although to calicheamicin, and to a lesser extent on CD22/CMC-544 CD22 expression is required for CMC-544 binding and internalization, whereas CD22 expression levels and renewed internalization, CD22 levels apparently have only a minor role expression do not seem to contribute significantly. in determining CMC-544 efficacy.

Discussion Internalization of CMC-544. We next studied whether the rate of internalization of CMC-544 contributes to CMC-544 This study demonstrates that CMC-544 effectively kills primary efficacy. A significant correlation was found between inter- þ CD22 BCP-ALL cells in vitro. The proposed mechanism of nalization and efficacy of CMC-544 (Figure 6c). Moreover, two action of CMC-544 is highly comparable to the CD33 patients who were relatively insensitive to CMC-544 (IC 50 immunotoxin GO, involving binding of CMC-544 to CD22 values 41000 ng/ml after 48 h), showed the lowest internaliza- Ags, internalization of the CD22/CMC-544 complex, fusion of tion capacity. Interestingly, for one of these patients (with an the CMC-544 containing endosome with a lysosome, release of internalization of 60%), CMC-induced cell death was only calicheamicin, induction of ds DNA breaks (frequently leading delayed, since after 96 h of exposure less than 50% leukemic to G /M cell cycle arrest) and finally induction of apoptosis cell survival was observed. 2 (Figure 7). However, in this study we also demonstrate that major differences exist between GO in AML and CMC-544 in Renewed expression of CD22 Ags. As renewed expres- ALL, which may have impact on the clinical development of sion of CD22 Ags at the cell surface may result in an ongoing these compounds. Particularly, the high sensitivity of ALL cells

Leukemia Efficacy of CMC-544 in BCP-ALL JF de Vries et al 262 before intracellular threshold levels of calicheamicin are obtained after exposure to CMC-544. This was further illustrated by the finding that in ALL-P0 (CD22low), but not in MIK-ALL (CD22high), cells after 24 h of incubation, a difference in efficacy between free calicheamicin and CMC-544 was observed, whereas no difference was found anymore after 72 h of incubation (data not shown), showing that CMC-544-induced cell death was only delayed in CD22low ALL cells. These data are in clear contrast to studies with GO in AML patients, in which complete and prolonged CD33 saturation was required for apoptosis induction, which may be related to a high drug efflux of AML cells and reduced calicheamicin sensitivity.30 Although CD22 expression is required for CMC-544’s activity, in primary BCP-ALL cells the efficacy of CMC-544 was not dependent on CD22 expression levels, as no significant correlation was found between these two parameters. Appar- ently, even low CD22 expression levels can already result in sufficiently high intracellular calicheamicin levels. In addition, Figure 7 Proposed mechanism of action of CMC-544. (1) Binding of CD22-independent uptake of CMC-544 by ALL cells may CMC-544 to CD22 receptors at the cell surface of target cells. (2) contribute to its cytotoxic effect, in a similar way as we Internalization of the CMC-544-CD22 receptor complex. (3) Renewed published previously for CD33-independent endocytosis of GO expression of CD22 receptors at the cell surface, which enables by BCP-ALL cells.35 Our data indicate that CMC-544-induced binding and internalization of new CMC-544 leading to intracellular cell death was completely mediated via calicheamicin-induced accumulation of calicheamicin. (4) Fusion of the CMC-544 containing endosome with a lysosome, which will lead to degradation of the acid- apoptosis and not by CD22 signaling. This is in contrast to GO, labile linker, and release of inactive calicheamicin. Via a thiol- where expression of the protein kinase Syk was essential for modification step active calicheamicin is formed. (5) Active calichea- efficacy of GO in AML cells, suggesting that CD33 signaling micin may be removed from the cell by drug efflux pumps. (6) DNA contributed to GO-induced cell death.36 For future clinical trials intercalation and ds DNA break formation by free calicheamicin with CMC-544, preferably CD22-positive patients should be entering the nucleus. (7) Apoptosis induction due to irreversible DNA included, but our data suggest that the level of CD22 expression damage. does not need to be taken into account (although high CD22 expression on ALL cells will enhance/facilitate CMC-544- induced cell death). However, given the CD22-independent for CMC-544 and calicheamicin will allow specific and efficient uptake of CMC-544, it may be interesting to evaluate the in vivo killing of ALL cells at relatively low dosages. efficacy of CMC-544 in CD22-negative ALL patients as well. A major finding of our studies is that the intracellular Although CD22 expression levels only limitedly affect CMC- threshold of calicheamicin required for cell death induction is 544 efficacy, the capacity of internalization of the CD22/CMC- much easier reached in BCP-ALL cells (with CMC-544) than in 544 complex was important for CMC-544 efficacy. Two patients AML cells (using GO). The intracellular threshold level is showed ineffective internalization after 3 h (Figure 6c), which dependent on at least three processes. First, calicheamicin needs was accompanied by ineffective CMC-544 response. One of to be targeted to the cells by binding of CMC-544 to the CD22 these patients only showed a delayed CMC-544 response, Ag and subsequent internalization of the CMC-544/CD22 Ag whereas the other was resistant. As the latter patient was also complex. Second, intracellular calicheamicin can be pumped resistant to calicheamicin, it is difficult to determine the out of the cell by various drug efflux pumps. Finally, the contribution of inefficient internalization to the CMC-544 intracellular threshold level is dependent on the intrinsic resistance. Comparable to the differential internalization of sensitivity of the target cell for calicheamicin. Although Rituximab in certain subgroups of mature B-cell lymphomas,37 expression of drug efflux pumps like P-glycoprotein (Pgp)28 BCP-ALL subgroups with differential internalization of CMC-544 and MRP29 may have a major role in the unresponsiveness of may exist. However, the strong relation between CMC-544 adult patients to GO30 and CMC-544,31 drug efflux does not sensitivity and calicheamicin sensitivity indicates that the uptake seem to be a general problem in pediatric ALL.32–34 Conse- of CMC-544 (including binding, internalization, and renewed quently, the intracellular accumulation of calicheamicin in ALL expression) is generally not limiting the efficacy of CMC-544. cells is mainly dependent on the binding and internalization of This is in contrast to GO in AML patients, in which the efficacy CMC-544. In line with this, the sensitivity of BCP-ALL cells to of GO is highly dependent on the intracellular accumulation of CMC-544 was mainly determined by the cell’s sensitivity to GO via the ongoing process of saturation, internalization and calicheamicin. Although CD22 expression was essential for renewed expression of CD33.18,19 Consequently, a positive CMC-544 binding and internalization, a repetitive loop of CD22 correlation was found between CD33 expression and the saturation, CD22/CMC-544 internalization and renewed CD22 efficacy of GO.38 expression was not required to achieve intracellular threshold Goemans et al.,39 previously postulated that the large levels of calicheamicin sufficient for efficient CMC-544-induced interpatient differences in calicheamicin efficacy in pediatric apoptosis in BCP-ALL cells. Nevertheless, high CD22 expression AML are caused by differences in intrinsic sensitivity of these levels may accelerate CMC-544-induced cell death. Especially target cells to calicheamicin. Similarly, the heterogeneity in in CD22low ALL cells ongoing CD22/CMC-544 binding and response to calicheamicin in BCP-ALL cells may also be internalization and renewed CD22 expression contributed to the explained by differences in intrinsic calicheamicin sensitivity, efficacy of CMC-544, as demonstrated by the higher efficacy of which is dependent on the level of DNA accessibility, DNA CMC-544 after continuous (48 h) than after pulse (1, 6 or 24 h) repair capacity or functionality of the apoptotic machinery exposure. Apparently, in CD22low ALL cells it takes longer (expression of pro- and anti-apoptotic proteins) of the target

Leukemia Efficacy of CMC-544 in BCP-ALL JF de Vries et al 263 cells. Decreased DNA accessibility had a role in GO efficacy, as 3 Pieters R, Carroll WL. Biology and treatment of acute lympho- addition of valproic acid, an agent causing histone hyperace- blastic leukemia. Hematol Oncol Clin North Am 2010; 24: 1–18. tylation that opens the chromatin structure, had a beneficial 4 Pui CH, Campana D, Pei D, Bowman WP, Sandlund JT, Kaste SC effect on GO-induced cell death in AML patients.40 We are et al. Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med 2009; 360: 2730–2741. currently investigating whether these mechanisms have a role in 5 Elliott P. Pathogenesis of cardiotoxicity induced by anthracyclines. CMC-544 efficacy as well. Semin Oncol 2006; 33 (Suppl 8): S2–S7. To confirm our in-vitro results, clinical studies with CMC-544 6 Lipshultz SE. Exposure to anthracyclines during childhood causes in BCP-ALL patients are warranted. As shown in previous cardiac injury. Semin Oncol 2006; 33 (Suppl 8): S8–S14. clinical trials with GO in AML patients, consumption of GO by 7 Hamann PR, Hinman LM, Hollander I, Beyer CF, Lindh D, circulating peripheral CD33 þ cells affected GO concentrations Holcomb R et al. Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody-calicheamicin conjugate for treatment of within the BM, thereby hampering the efficacy of GO. These acute myeloid leukemia. Bioconjug Chem 2002; 13: 47–58. problems could be partially solved by administration of multiple 8 Sievers EL, Larson RA, Stadtmauer EA, Estey E, Lowenberg B, dosages of GO and by erasing the leukemic burden by Dombret H et al. Efficacy and safety of gemtuzumab ozogamicin in chemotherapy before infusion of GO.41 We expect that patients with CD33-positive acute myeloid leukemia in first circulating CD22 þ cells will only limitedly hamper the efficacy relapse. J Clin Oncol 2001; 19: 3244–3254. of CMC-544 in ALL patients, as no complete saturation of 9 Zein N, Sinha AM, McGahren WJ, Ellestad GA. Calicheamicin gamma 1I: an antitumor antibiotic that cleaves double-stranded CD22 Ags on ALL cells in the BM is required to obtain efficient DNA site specifically. Science 1988; 240: 1198–1201. CMC-544-induced cell death. 10 Zwaan CM, Reinhardt D, Zimmerman M, Hasle H, Stary J, Stark B In conclusion, most of the drawbacks with GO in AML likely et al. Salvage treatment for children with refractory first or second can be avoided for CMC-544 treatment in BCP-ALL patients. relapse of acute myeloid leukaemia with gemtuzumab ozogami- Our data suggest that clinical efficacy may be obtained cin: results of a phase II study. Br J Haematol 2008; 148: 768–776. even when smaller dosages are given in combination with 11 DiJoseph JF, Popplewell A, Tickle S, Ladyman H, Lawson A, chemotherapy, and that, for instance, a fractionated schedule Kunz A et al. Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody during a typical 4-week ALL-induction regimen may against CD22. Cancer Immunol Immunother 2005; 54: 11–24. be effective, rather than a single-agent high-dose regimen. 12 DiJoseph JF, Armellino DC, Boghaert ER, Khandke K, Dougher MM, Therefore, although in the just started clinical trial of CMC-544 Sridharan L et al. Antibody-targeted chemotherapy with CMC-544: in adult ALL CMC-544 is given as one single high dosage a CD22-targeted immunoconjugate of calicheamicin for (1.8 mg/m2) per 4-week cycle (NCT01134575), other clinical the treatment of B-lymphoid malignancies. Blood 2004; 103: studies will be performed applying repetitive low dosages of 1807–1814. 13 Tedder TF, Tuscano J, Sato S, Kehrl JH. CD22, a B lymphocyte- CMC-544, which can later be easily combined with existing specific adhesion molecule that regulates antigen receptor multi-agent chemotherapy regimens. signaling. Annu Rev Immunol 1997; 15: 481–504. 14 Cesano A, Gayko U. CD22 as a target of passive immunotherapy. Semin Oncol 2003; 30: 253–257. Conflict of interest 15 Dijoseph JF, Dougher MM, Armellino DC, Evans DY, Damle NK. Therapeutic potential of CD22-specific antibody-targeted The authors declare no conflict of interest. chemotherapy using inotuzumab ozogamicin (CMC-544) for the treatment of acute lymphoblastic leukemia. Leukemia 2007; 21: 2240–2245. Acknowledgements 16 DiJoseph JF, Goad ME, Dougher MM, Boghaert ER, Kunz A, Hamann PR et al. Potent and specific antitumor efficacy of CMC- 544, a CD22-targeted immunoconjugate of calicheamicin, against The authors want to acknowledge Dr Patrick Kelly and Dr Erik systemically disseminated B-cell lymphoma. Clin Cancer Res Vandendries (both employees of Pfizer) for their comments 2004; 10: 8620–8629. and discussions, and Pfizer (legacy Wyeth) for providing us with 17 Advani A, Coiffier B, Czuczman MS, Dreyling M, Foran J, Gine E CMC-544, calicheamicin and CD22 antibody (G5/44). The et al. Safety, pharmacokinetics, and preliminary clinical activity of authors would like to thank Dr Adriaan Houtsmuller and Nicole inotuzumab ozogamicin, a novel immunoconjugate for the Larmonie for their assistance with the confocal microscopy, and treatment of B-cell non-Hodgkin’s lymphoma: results of a phase Mathilde Broekhuis for her support with the MTT assays. I study. J Clin Oncol 2010; 28: 2085–2093. 18 van der Velden VH, Boeckx N, Jedema I, te Marvelde JG, Hoogeveen PG, Boogaerts M et al. High CD33-antigen loads in peripheral blood limit the efficacy of gemtuzumab ozogamicin Author contributions (Mylotarg) treatment in acute myeloid leukemia patients. Leukemia 2004; 18: 983–988. JFdV designed and performed research, analyzed and inter- 19 van Der Velden VH, te Marvelde JG, Hoogeveen PG, Bernstein ID, preted data, and wrote the manuscript; CMZ designed research Houtsmuller AB, Berger MS et al. Targeting of the CD33- and critically reviewed the manuscript; MdB and JSAV calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internaliza- performed research and analyzed data; MLdB provided patient tion by leukemic and normal myeloid cells. Blood 2001; 97: samples and analytical tools, and critically reviewed the 3197–3204. manuscript; JJMvD and VHJvdV designed research, interpreted 20 Zwaan CM, Reinhardt D, Jurgens H, Huismans DR, Hahlen K, data and wrote the manuscript. Smith OP et al. Gemtuzumab ozogamicin in pediatric CD33- positive acute lymphoblastic leukemia: first clinical experiences and relation with cellular sensitivity to single agent calicheamicin. 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