A Novel Raji-Burkitt's Lymphoma Model for Preclinical and Mechanistic Evaluation of CD52-Targeted Immunotherapeutic Agents

Cancer Therapy: Preclinical

A Novel Raji-Burkitt’s Lymphoma Model for Preclinical and Mechanistic Evaluation of CD52-Targeted Immunotherapeutic Agents Rosa Lapalombella,1Xiaobin Zhao,1, 2 Georgia Triantafillou,1Bo Yu,3,4 Yan Jin, 4 Gerard Lozanski,5 Carolyn Cheney,1Nyla Heerema,5 David Jarjoura,6 Amy Lehman,6 L. James Lee,3,4 Guido Marcucci,1Robert J. Lee,2,4 Michael A. Caligiuri,1 Natarajan Muthusamy,1and John C. Byrd1, 2

Abstract Purpose:Todate, efforts to study CD52-targeted therapies, such as alemtuzumab, have beenlim- ited due to the lack of stable CD52 expressing transformed B-cell lines and animal models.We describe generation and utilization of cell lines that stably express CD52 both in vitro and in vivo. Experimental Design: By limiting dilution, we have established several clones of Raji-Burkitt’s lymphoma cell line that express surface CD52. Immunophenotype and cytogenetic charac- terizationof these clones was done. In vivo usefulness of the CD52high cell line to evaluate the ther- apeuticefficacyofCD52-directedantibody wasinvestigatedusingaSCIDmousexenograftmodel. Results: Stable expression of CD52 was confirmed in cells cultured in vitro up to 52 weeks of continuous growth. The functional integrity of the expressed CD52 molecule was shown using alemtuzumab, which induced cytotoxic effects in vitro in the CD52high but not the CD52low clone. Compared with control antibody, alemtuzumab treatment in CD52high inoculated mice resulted in significantly increased median survival. Comparable levels of CD52-targeted direct cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity and anti-CD52 immunoliposome-mediated delivery of synthetic oligodeoxyribo nucleotides in CD52high clone and primary B-chronic lymphocytic leukemia cells implicated potential in vivo application of this model for evaluation of CD52-targeted antibody and immunoliposomes encapsulating therapeutic agents. Conclusions:These results show the in vitro utility of the cloned Raji cell lines that stably express high levels CD52. The disseminated leukemia-lymphoma mouse model described herein using these stable cell lines can serve as an excellent system for in vivo therapeutic and mechanistic evaluation of existing and novel antibodies directed against CD52 molecule.

CD52 is a 21-kDa to 28-kDa glycosyl-phosphatidylinositol poietic stem cells, erythrocytes, plasma cells, and platelets do not anchored membrane glycoprotein (1) that is highly expressed on express this antigen (6). CD52 antigen is also expressed on vary- all normal B and T lymphocytes, monocytes, macrophages, eosi- ing subsets of tumor cells, including T-prolymphocytic leukemia, nophils, natural killer cells, and dendritic cells (2–5). Hemato- hairy cell leukemia, multiple myeloma, non–Hodgkin lymphomas, acute lymphoid leukemia, and B-chronic lymphocytic leukemia (B-CLL). CD52 has been shown to be expressed at Authors’ Affiliations: 1Division of Hematology-Oncology, Department of Internal higher density in leukemic B and T cells compared with normal Medicine, 2Division of Pharmaceutics, College of Pharmacy, 3Department of T and B lymphocytes, and at least for CLL, the expression of the Chemical and Biomolecular Engineering, 4Center for Affordable Nanoengineering CD52 is virtually uniform on all leukemic cells (7). Although 5 6 of Polymeric Biomedical Devices, Department of Pathology, and Center for the biological function of CD52 in these cells is elusive, recently Biostatistics, Ohio State University, Columbus, Ohio Received 4/26/07; revised 9/6/07; accepted 9/28/07. a costimulatory role for CD52 in T cells contributing to the Grant support: Specialized Center of Research from Leukemia and Lymphoma induction of regulatory T cells has been proposed (8). Society, NIH grants P01 CA95426 and P01 CA101956, and National Science Alemtuzumab (Campath 1-H), a humanized IgG1n antibody Foundation grant EEC-0425626. directed against CD52, mediates effective depletion of both The costs of publication of this article were defrayed in part by the payment of page normal and transformed lymphocytes in vitro and in vivo charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (9, 10). Initially used clinically in treatment of autoimmune Note: N. Muthusamy and J.C. Byrd contributed equally to the work described disease, alemtuzumab was subsequently found to be highly herein. effective in treating lymphoproliferative disorders, such as Requests for reprints: Natarajan Muthusamy, 455E, OSU CCC, 410 West 12th B-CLL (11–13). Despite the extensive use in clinical trials, it Avenue, Ohio State University, Columbus, OH 43210. Phone: 614-292-8135; Fax: 614-293-7526; E-mail: [email protected]. is unclear how cross-linking CD52 by alemtuzumab mediates F 2008 American Association for Cancer Research. growth inhibition and apoptosis. Preclinical studies have doi:10.1158/1078-0432.CCR-07-1006 shown that alemtuzumab acts mostly through immunologic

www.aacrjournals.org 569 Clin Cancer Res 2008;14(2) January 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical mechanisms, such as complement-dependent cytotoxicity and approved by Ohio State University Institutional Review Board. (CDC; refs. 14–16) and/or antibody-dependent cellular cyto- Primary CD19-positive cells were isolated using Rosette-Sep (StemCell toxicity (ADCC; refs. 17–19) by virtue of its IgG1 Fc region. Technologies). Cells were maintained in RPMI 1640 (Invitrogen) Two recent studies have shown that this agent induced supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, and penicillin (100 units/mL)/streptomycin (100 Ag/mL; enhanced apoptosis in primary CLL cells in vitro, alone or in Invitrogen) at 37jC, 5%CO2, and high humidity. combination with a cross-linking anti-Fc antibody, in the Immunophenotyping studies. Cells (1 106) were washed twice in absence of complement or immune effector cells, through PBS (Invitrogen) and stained with fluorochrome-labeled antibodies a nonclassic caspase-independent pathway (20, 21). There specific for human CD52 (Serotec), CD19, CD23, FcgIIb receptor is also evidence to suggest that alemtuzumab may trigger (CD32), HLA-DR, CD50, CD20, CD37 (BD Biosciences), CD55, CD59 caspase-dependent cell death in CLL cells (22, 23), as well as (Invitrogen), and CD40 (Beckman-Coulter) for 30 min at 4jC. The cells B-lymphoid Wien 133 (24) and Ramos cell lines (21). Con- were rinsed twice in PBS and analyzed by fluorescence-activated cell trasting with these, another study has shown that alemtuzumab sorting (FACS) on a Beckman Coulter EPICS XL (Beckman Coulter). alone did not induce apoptosis in serum-free medium (14). Ten thousand events were collected under list mode and analyzed using Despite its extensive use, alemtuzumab has shown limita- Windows Multiple Document Interface for Flow Cytometry analysis program. tions in clinical use in B-cell malignancies because of its cyto- Assessment of antibody-binding capacity by Quantum Simply Cellular toxic activity on T and other cell types, resulting in immuno- Method. Quantitative analysis of CD52 was done using the Quantum compromised state associated with susceptibility to secondary Simply Cellular Microbeads kit (Bangs Laboratories) according to infections. To overcome this, a concerted effort is being made to manufacturer’s instructions. The kit consists of a mixture of four micro- improve the existing therapeutic agents directed against CD52 sphere populations coated with different amounts of goat anti-human and to delineate the mechanisms by which CD52-targeted immunoglobulin with a precalibrated antibody-binding capacity reagents mediate cytotoxicity (25). Detailed analysis of in vivo (ABC). Briefly, the beads were stained with labeled antihuman CD52 and in vitro mechanisms to elucidate CD52-mediated killing antibody, and based on the different ABC, each type of microbeads of malignant B cells and testing of novel combination stra- bound a known amount of antibody. By plotting each population’s tegies have been limited due to the absence of valuable cell fluorescence intensity versus its assigned ABC value, a standard ABC curve was generated. Cells were stained with antihuman CD52 antibody lines and animal models. Although many of in vitro maintained and analyzed in a similar method by flow cytometry. QuickCal software lymphoid-derived tumor cells express CD52, the level of expres- was used to convert the mean fluorescence intensity of each cell sample sion is very low and the stability of the expression is unpre- to the number of molecules of antigen expressed per cell. For each dictable. To overcome this and to develop a model system to sample, the ABC value of the isotypic control was subtracted from the evaluate the CD52-directed antibody and immunoliposomal ABC value of the positive cells (26, 27). formulations containing cytotoxic agents for targeted delivery, p53 mutational analysis. Mutations for the p53 gene were assessed we describe isolation and evaluation of a Raji-Burkitt’s lym- by sequencing genomic DNA. Briefly, genomic DNA was extracted phoma B-cell line that stably expresses high level of CD52 using the QIAmp kit according to the manufacturer’s instructions p53 (CD52high Raji) in vitro and in vivo. The functional integrity of (Qiagen). Each exon was amplified individually from genomic the expressed CD52 molecule was shown using alemtuzumab, DNA and subjected to automated sequencing by standard methods at Ohio State University (Comprehensive Cancer Center) Sequencing which can promote rapid cell death of the CD52high cells Core facility. The sequence was then compared with reported p53 through the same immunologic mechanisms, such as CDC, sequence (Genbank accession no. AB017815).7 ADCC, and direct apoptosis active in primary CLL cells. Fur- Cytogenetic analysis. Exponentially growing cells were treated with high thermore, CD52 Raji cells can be specifically targeted by 0.77 Ag/AL colcemid (Invitrogen) for 2 h at 37jC in the presence of 5% alemtuzumab-coated immunoliposomes comparable with pri- CO2 and then fixed using standard laboratory procedures. Briefly, cell mary B-CLL cells, making this cell line model suitable for suspension was spun down for 10 min at 1,000 rpm, the supernatant studying the targeted delivery of antibody-conjugated liposo- was discarded, and the cell pellet was slowly resuspended in 5 mL of mal drug carriers. In vivo usefulness for the CD52high cell line 0.075 mol/L KCl. After incubation at 37jC, the cell suspension was for evaluating therapeutic efficacy of CD52 -directed antibody is spun down as above and cells were resuspended in freshly prepared j shown using a xenograft SCID mouse model of disseminated Carnoy fixative (3:1, methanol/acetic acid) stored at 4 C for 30 min to overnight and then washed twice with fresh fixative. The cell suspension leukemia/lymphoma. was then dropped onto precleaned, warm, wet slides. The slides were aged at 90jC for 1 h, banded with trypsin, and stained with Wright Materials and Methods stain (Fisher Scientific). Banded metaphases were analyzed using a Zeiss Axioskop 40 microscope. For each cell line, 10 metaphases were Raji cell isolation. Several clones of Raji Burkitt’s lymphoma B cells karyotyped using an Applied Imaging Karyotyping System. Human expressing different levels of CD52 were selected by limiting dilution. cytogenetic nomenclature (ISCN, 1995; ref. 28) was used to describe Briefly, Raji cells (American Type Culture Collection) were plated in karyotypes. 6 96-well plates at three and one cells per well. Resulting clones were Analysis of direct cytotoxicity. Cells (1 10 cells/mL) were treated tested for surface expression of CD52 by flow cytometry, and promis- with alemtuzumab (anti-CD52), rituximab (anti-CD20), or trastuzu- ing candidates were recloned in the same manner. Cells were main- mab (anti-HER2) at a concentration of 10 Ag/mL. The cross-linker, goat tained in RPMI 1640 supplemented with 10% heat-inactivated fetal anti-human IgG (Fc specific; Jackson ImmunoReasearch Laboratories), bovine serum, 2 mmol/L L-glutamine (Invitrogen), and penicillin was added to the cell suspension 5 min after adding the primary anti- (100 units/mL)/streptomycin (100 Ag/mL; Invitrogen) at 37jC, bodies at a concentration of 50 Ag/mL. In addition, a group of samples

5%CO2, and high humidity. B-CLL isolation. Peripheral blood was obtained from patients with B-CLL through the CLL Research Consortium from Ohio State 7 National Center for Biotechnology Information. EntrezNucleotide. http:// University. Written, informed consent was obtained to procure blood www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide. Accessed February 16, samples from CLL patients according to the Declaration of Helsinki 2006.

Clin Cancer Res 2008;14(2) January 15, 2008 570 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. CD52+ B-Cell Lymphoma Model with no treatment was collected as media control. The apoptosis of cells was assayed in triplicate, and the experiments were repeated twice. at 24 and 48 h posttreatment was measured using Annexin V–FITC/ The housekeeping gene TATA-binding protein was used for normal- propidium iodide staining followed by FACS analysis according to ization purposes due to its lowest variability among different types the supplier’s instructions (BD Biosciences). Results were expressed of lymphoid cells (30). The following primer sets were used: CD52 as percentage of total positive cells over media control [% positive (121 bp) 5¶-CAGCCTCCTGGTTATGGTACAG-3¶,5¶-GGCCACGAAGA- cells = (% Annexin V and/or propidium iodide–positive cells of AAAGGAAA-3¶ and TATA-binding protein (170 bp) 5¶-AGGAGCCAAG- treatment cell sample) - (% Annexin V and/or propidium iodide– AGTGAAGAACAG-3¶,5¶-GTTGGTGGGTGAGCACAAG-3¶. Reaction con- positive cells of media control)]. FACS analysis was done using EPICS ditions were 95jC 10 min, 40 cycles of 95jC 30 s, 64jC 30 s, XL cytometer (Beckman-Coulter). 72jC 1 min. The amplicon size was confirmed by 1% agarose gel CDC. For CDC assay, Raji cell clones at 106 cells/mL were sus- electrophoresis. Real-time PCR was run in parallel with both CD52 and pended in media alone, media with 30% human serum, or media with TATA-binding protein primers to normalize data to the housekeep- 30% heat-inactivated (56jC, 30 min) human serum. Cells were then ing gene. The expression of CD52 relative to the housekeeping gene A treated with alemtuzumab, rituximab, or trastuzumab at 10 g/mL. TATA-binding protein was calculated by plotting the Ct (cycle number), After incubation at 37jC for 1 h, the extent of cytotoxicity was mea- and the average relative expression for each group was determined using C sured by FACS analysis of Annexin V–FITC/propidium iodide fluor- the comparative method (2-D t; ref. 31). escence as described above. Development of a disseminated leukemia-lymphoma model using Raji Analysis of ADCC. ADCC activity was determined by standard cell clones. Female CB17 SCID mice (Taconic Farm), 4 to 6 weeks of 4-h 51Cr-release assay. 51Cr-labeled target cells (1 105 Raji cells/mL) age, were housed in pathogen-free, isolated cages. Raji cell clones frozen were incubated with media alone or in the presence of various in cryovials (1 107 cells per vial) and stored in liquid nitrogen antibodies (10 Ag/mL) at 37jC for 30 min. Unbound antibody was (-180jC) were thawed 10 days before the in vivo engraftment. To ensure washed off, and the cells were plated at 1 104 cells per well. Effector the consistency of engraftment, cells were examined for the viability by cells (peripheral blood mononuclear cells from healthy donors) Annexin V–FITC/propidium iodide staining followed by FACS analysis, were then added to the plates at indicated effector-to-target ratios. CD52/CD19/CD20 expression, and in vitro sensitivity to antibody After 4-h incubation, supernatants were removed and counted in a treatments on the same day of inoculation. Only cells with >90% Gamma counter. The percentage of specific cell lysis was determined viability (as evidenced by Annexin V-//propidium iodide-) were used for by % lysis = 100 (ER - SR)/(MR - SR). ER, SR, and MR represent injection. Cells were resuspended in 0.9% sodium chloride for injection experimental, spontaneous, and maximum release respectively. Data (USP, Hospira, Inc.) at a density of 107 cells/mL at room temperature, were normalized to the media control. and 200 AL(2 106 cells) were inoculated through tail vein using a Preparation of immunoliposome. 3h-[N-(N¶,N¶-Dimethylamino- mouse tail illuminator (Braintree Scientific). Tissue samples obtained ethane)-carbamoyl]cholesterol and distearoyl phosphatidylethanolamine from tumor-bearing SCID mice that showed early signs of paralysis (DSPE)–polyethylene glycol (PEG; molecular weight, 2,000 Da)– were submitted to Ohio State University Pathology Core Facility for maleimide were purchased from Avanti Polar Lipids, Inc. Methoxy- histologic analysis. The presence of disease was confirmed by the DSPE-PEG and Egg phosphatidylcholine were obtained from Lipoid. presence of human leukemic cells in the tissue sections. Bone marrow 2-Iminothiolane (Traut’s reagent), 5,5¶-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent), and other chemicals were purchased from Sigma Chemical Co. A carboxyfluorescein (FAM)–terminus modified ODN (5¶-(6) FAM-TAC CGC GTG CGA CCC TCT) was custom synthesized by Alpha DNA, Inc. An ethanol dilution method was modified and used to prepare the FAM-ODN loaded immunoliposomes (29). Briefly, protamine sulfate in citrate acid (20 mmol/L, pH 4) was mixed with lipids composed of (3h-[N-(N¶,N¶-Dimethylaminoethane)-carbamoyl]- cholesterol/Egg phosphatidylcholine/PEG-DSPE molar ratio = 33.5: 65:1.5) at lipids/protamine mass ratio of 12.5:0.3, followed by addition of oligonucleotide in citrate acid (20 mmol/L, pH 4) at oligonucleotide/ lipids/protamine weight ratio of 1:12.5:0.3. The complexes were then dialyzed against citrate acid (20 mmol/L, pH 4) for 1 h and then further dialyzed against HBS buffer [145 mmol/L NaCl, 20 mmol/L HEPES (pH 7.4)] overnight, using a DispoDialyzer (Spectrum Laboratories) with a molecular weight cutoff of 10,000 Da. Liposome size distribution was analyzed on a NICOMP Particle Sizer Model 370 (Particle Sizing Systems). Volume-weighed analysis showed particle size of f54 nm. A post- insertion method was adopted to incorporate antibody ligands into preformed liposomes containing FAM-ODN. In this method, alemtuze- mab (anti-CD52) was reacted with 20 Traut’s reagent (2 h, room temperature) to yield thiolated antibody (anti-CD52-SH). The anti-CD52- SH was then reacted to micelles of maleimide-PEG-DSPE at a molar ratio of 1:10 and then incubated with FAM-ODN–loaded liposomes for 1hat37jC. Targeted liposomes with alemtuzumab-PEG-DSPE–to-lipid ratios of 1:500 (0.2 mol%) was prepared. Nontargeted control liposomes were prepared by coupling the isotype control antibody trastuzumab to the liposomes using the same method. Quantitative reverse transcription–PCR assay. RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s directions, DNase treated, and converted to cDNA with SuperScript Fig. 1. Differential CD52 expression in Raji clones. Cells from parental, CD52low First-Strand Synthesis System for reverse transcription–PCR (Invitro- high and CD52 Raji clones were stained with PE-labeled anti-CD52 (A), CD20 (B), gen). Real-time PCR was done using the LC FastStart DNA Master SYBR or isotype-matched antibodies and analyzed on FACS. Shaded histogram profile Green 1 kit (Roche Diagnostics) in a Roche Lightcycler. Each sample indicates the isotype control, and open histogram indicates the specific antibody.

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Table 1. Surface immunostaining of Raji cell clones

Raji cell clone %CD52 %CD20 %CD55 %CD59 %CD50 %CD23 %HLA-DR %CD32 %CD40 %CD19 %CD37 (MFI) (MFI) (MFI) (MFI) (MFI) (MFI) (MFI) (MFI) (MFI) (MFI) (MFI) Parental Raji cell line 43 (4) 100 (161) 20 (92) 29 (64) 2 (30) 85 (952) 100 (945) 100 (39) 100 (590) 100 (89) 97 (16) CD52low Raji clone 10 (2) 100 (300) 24 (163) 23 (117) 2 (34) 41 (590) 100 (520) 100 (48) 100 (347) 100 (105) 97 (11) CD52high Raji clone 99 (36) 100 (237) 99 (308) 99 (205) 93 (89) 36 (666) 100 (480) 100 (52) 100 (330) 100 (143) 97 (13)

Abbreviation: MFI, mean fluorescence intensity.

cells (1 106/mL) flushed from femurs with cold PBS within 0 to expressed varied levels of surface CD52 not exceeding 50% at 3 days after hind-limb paralysis were stained with PE-labeled anti- any given time. By limiting dilution cloning, we established hCD52 and FITC-labeled anti-hCD19 to confirm the existence of clones that stably expressed high or low level of surface CD52 human leukemia cells. Antibody treatment was started 3 days (Fig. 1A). Cells of different clones were collected and analyzed postinoculation of target Raji cells. Alemtuzumab dissolved in PBS for different surface protein expression by immunostaining. (1 mg/mL) was injected i.v. via tail vein and maintained every other day schedule for 2 weeks (5 mg/kg/injection, seven injections each mouse). The immunophenotypic characteristics of the original Raji- Placebo (saline) and isotype control (trastuzumab) were given at the Burkitt’s lymphoma cell line (parental cell line) compared with same schedule and dose. Animals were monitored daily for signs of two selected clones expressing high or low levels of CD52 high low illness and sacrificed immediately if hind-limb paralysis, respiratory (respectively CD52 and CD52 Raji clones) are reported distress, or 30% body weight loss was noted. Body weight was mea- in Table 1. Phenotypically, these lines seem to be similar to sured once every week. Survival time (paralysis time) was used as the parental Raji B-cell line expressing comparable high level primary end point for evaluation. of human leukocyte antigens HLA-DR, CD19, CD20, CD37, All animal experiments were carried out under protocols approved by CD40, and CD32. Compared with the parental and CD52low Ohio State University Institutional Laboratory Animal Care and Use clones, the CD52high clone expresses higher levels of CD52 Committee. (227,538 versus 21,280 ABC) in higher percentage of cells Statistical analysis of data. All analyses were done by statisticians in (99% versus 10%). The CD52 mRNA level, as assessed by real- Center for Biostatistics, Ohio State University. Mixed effects models were fitted to the cytotoxicity data. In all cases, the primary hypothesis time reverse transcription–PCR analysis of RNA, indicated a high tests were for interactions between cell type and conditions. Random 1.8-fold increases in CD52 mRNA in CD52 clone compared low effects associated with the interactions were always included in the with CD52 clone (data not shown). In addition, the levels models to ensure that error was not underestimated. of expression of other glycosyl-phosphatidylinositol–linked Kaplan-Meier estimates of the survival function for both treatments proteins, such as CD55 and CD59, showed variation between (alemtuzumab versus trastuzumab) and engraftments (CD52high Raji clones in a manner directly associated to the expression of low versus CD52 Raji) were plotted, and the median survival times for CD52 (99 F 1%, 99 F 1%). Unlike other B-cell lines, this each treatment engraftment combination were calculated with 95% pattern of surface antigen expression remained stable with confidence intervals. A Cox proportional hazards model was applied to continuous cell culture over several months (data not shown). the data with the factors of treatment, engraftment, and their inter- Molecular cytogenetic characterization of the isolated clones action in the model. A significance level of a = 0.05 was used for all tests. SAS software version 9.1 (SAS Institute, Inc.) was used for all the were done to confirm the lineage connection to Raji Burkitt’s statistical analyses. lymphoma as well as to exclude possible contamination by other cell lines. The chromosomal rearrangements of the cell Results lines are shown in Table 2. The Raji genome is relatively stable in these sublines, which Physical characteristics. The parental Raji-Burkitt’s lympho- all share three of the structural aberrations previously ma cell line obtained from American Tissue Culture Collection described, including the t(8;14) translocation, typical in

Table 2. Genetic characterization of Raji cell clones

Raji cell clone Chromosomal rearrangement p53 mutation Codon Variation AA substitution Parental 47, XY, der(1)t(1;6)(p36.3;q13)ins(6;?)(q13;?), 72 CCC!CGC Pro!Arg der(4)t(4;1)(q35;p36.11), +7, 213 AGC!AAC Arg!Gln der(8)(12pter>12p11.2::8q22>8cen>8q24.1::14q32.3>14qter), 234 CAC!CAT His!Tyr del(9)(p23), der(14)t(8;14)(q24.1;q32.3), del(18)(q11.1)[10] CD52low Raji clone 48, XY, der(4)t(1;4)(p36.1;q35), ins(6;?)(q13;?), +7, 72 CCC!CGC Pro!Arg der(8)(12pter>12p11.2::8q22>8cen>8q24.1::14q32.3>14qter), 213 AGC!AAC Arg!Gln der(14)t(8;14)(q24;q32), add(15)(p11.2), +20 234 CAC!CAT His!Tyr CD52high Raji clone 49,XY,+3, der(4)t(1;4)(p36.1;q35), ins(6;?)(q13;?), +7, 72 CCC!CGC Pro!Arg der(8)(12pter>12p11.2::8q22>8cen>8q24.1::14q32>14qter), 213 AGC!AAC Arg!Gln der(14)add(14)(p11.2)t(8;14)(q24;q32), +15 234 CAC!CAT His!Tyr

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Burkitt’s leukemia/lymphoma, and one numerical chromo- clones in the presence or absence of human serum as source somal aberration (+7; ref. 32). In addition, consistent with of complement. Treatments with alemtuzumab but not tras- the previous report, we were able to identify in all the clones tuzumab at 10 Ag/mL in the presence of 30% human serum the arginine/proline polymorphism in the p53 gene at position resulted in cytotoxicity in CD52high Raji cells. Results indi- 72 (33) and two missense mutations causing amino acid cated that alemtuzumab mediates CDC on the CD52high clone substitutions at residues 213 and 234 in the p53 gene (34) as (19.8 F 7.6%) but not on the CD52low Raji clone (3 F 2.3%, summarized in Table 2. P < 0.0001) or the parental Raji clone (10.9 F 4.9%, CD52high Raji cells are susceptible to CDC. To determine if P < 0.0001; Fig. 2). No significant cytotoxicity was observed the CD52high Raji cells are sensitive to CDC, we tested the effect in any of the cell types tested with heat-inactivated human of alemtuzumab on the parental, CD52high, and CD52low Raji serum which lacks active complement.

Fig. 2. Alemtuzumab-mediated cytotoxicity of Raji clones.Top, differential CDC in Raji clones (A) and primary B-CLL cells (B). Parental, CD52low,andCD52high Raji cell clones and B-CLL cells were incubated with 10 Ag/mL of alemtuzumab or trastuzumab in the presence of 30% of either active or heat-inactivated human serum as a source of complement. Relative cytotoxicity was determined after 1h of incubation at 37jC by Annexin V/propidium iodide staining. Alemtuzumab induced higher cytotoxicity on CD52high compared with CD52low Raji cell clone; *, P < 0.0001. Middle, differential ADCC in Raji clones (C) and primary B-CLL cells (D). ADCC was measured using freshly isolated peripheral blood mononuclear cells from normal volunteers and Raji cell clones at 12.5:1, 25:1, 50:1effector-to-target ratio (E/T) in presence or absence of 10 Ag/mL alemtuzumab. Columns, average of triplicate wells and were representative of three independent experiments; bars, SD.The overall CD52high versus CD52low cytotoxicity was significantly higher for alemtuzumab (P = 0.0236). Bottom, differential cytotoxicity in Raji clones (E) and primary B-CLL cells (F). Direct cytotoxicity. Raji cell clones were incubated with 10 Ag/mL of alemtuzumab or trastuzumab with or without 50 Ag/mL cross-linking goat anti-human Fc antibody (aFc).The percentage of apoptosis was determined byAnnexinV/propidium iodide staining after 24 h. For alemtuzumab, there was significant synergy with cross-linking for CD52high versus CD52low (P < 0.0001).

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CD52high Raji cells are sensitive to ADCC. We further close to 1) indicating that alemtuzumab had no noticeable investigated whether CD52high Raji clone also showed a higher effect on death, which further indicated the lack of CD52 sensitivity to alemtuzumab-mediated ADCC compared with expression in these cells. However, for CD52high clone, the CD52low Raji clone, using peripheral blood mononuclear cells hazard ratio is 0.12, indicating that alemtuzumab decreases the as effector cells (Fig. 2). Standard 51Cr release assays were done rate of death by roughly eight times (1/0.12) compared with at different effector-to-target ratios. First, we compared the trastuzumab (P = 0.0005). Mice inoculated with CD52high Raji overall CD52high versus CD52low cytotoxicity (averaging over clones and treated with alemtuzumab survived >80 days longer all of the effector/target ratios) between the alemtuzumab and (note that only 29% of the CD52high Raji mice given alem- trastuzumab conditions. Whereas the relative cytotoxicity was tuzumab died by the end of the study, so we cannot provide an significantly higher for CD52high compared with CD52low with estimate of the median survival time for that group) than mice alemtuzumab (P = 0.0236) and not significantly different with inoculated with CD52low Raji clones receiving the same trastuzumab (P = 0.9763). However, the comparison of the treatment. Tissue obtained from sacrificed trastuzumab-treated CD52high versus CD52low difference between alemtuzumab tumor-bearing SCID mice showed massive distribution of neo- and trastuzumab was only borderline significant (P = 0.0521) plastic cells in multiple sites, including liver, kidney, mesenteric based on the interaction test. In addition, we did not find any strong differences in effector concentration trends between the CD52low and CD52high cell lines with alemtuzumab (P = 0.0844). Direct cytotoxicity. To determine if CD52high Raji clone exhibited increased sensitivity to alemtuzumab-mediated direct cytotoxicity, parental, CD52high, and CD52low Raji clones were subjected to alemtuzumab or trastuzumab treatment in media without plasma or effector cells. Alemtuzumab treatment of CD52high Raji clone resulted in 80 F 1% cell death compared with 19.5 F 1.1% and 17.5 F 1%, respectively, in parental and CD52low Raji clones after 24 h of treatment in the presence of a secondary cross-linking antibody (P < 0.0001; Fig. 2). A significant synergy with cross-linking for CD52high versus CD52low was found. Specifically, without cross-linking, the average percentage of cytoxicity for CD52high was 16.4% higher than for CD52low, and with the addition of cross-linking, the difference between CD52high and CD52low increased by 63%. Extended incubation up to 48 h resulted in a similar trend (data not shown). In vivo applicability of CD52high Raji cells for evaluation of CD52-targeted therapy. To evaluate the expression stability of CD52 molecules and the suitability of the Raji variants for in vivo evaluation of CD52-targeted reagents, we established xenograft leukemia/lymphoma SCID mouse models using CD52high and CD52low Raji clone cells. Inoculation of each of the cell lines showed tumorigenic activity in mice character- ized by symptomatic metastasis at multiple sites, including central nervous system, liver, and lung, resulting in a progressive body weight loss, as well as hind-limb paralysis, followed by death of nearly 100% mice from massive tumor burden 17 to 30 days postinoculation. Mice inoculated with CD52high and CD52low Raji cells were randomly divided into three groups and treated with alemtuzumab, trastuzumab, or saline solution at an early stage of disease, i.e., on day 3 after low cell inoculation. All the CD52 Raji cells–inoculated mice Fig. 3. Demonstration of the use of CD52high clone for in vivo evaluation of treated with saline (data not shown) or trastuzumab died CD52-targeted therapeutic agents. A, SCID mice were inoculated with CD52high (n = 29) or CD52low (n = 19) Raji cells and treated with alemtuzumab (n =14 within 18 to 22 days (median survival times 20 days), whereas and n = 10, respectively) in parallel with trastuzumab (n =15andn =9, high >93% of the CD52 Raji cells–inoculated mice receiving the respectively). A Cox model was applied to the data with the factors treatment, same treatment died within 20 to 40 days (median survival engraftment, and their interaction in the model (P < 0.0024 for engraftment treatment interaction between CD52high and CD52low alemtuzumab-treated mice; times 34 days; Fig. 3A). P = 0.0005 between CD52high alemtuzumab-treated or trastuzumab-treated mice). As expected, a significant engraftment treatment interaction Neoplastic cells in spinal cord of a trastuzumab-treated CD52high-inoculated B high C was found (P = 0.0024) demonstrating a difference in the effect mouse ( ) and alemtuzumab-treated CD52 -inoculated mouse ( )withonlya few neoplastic cells in the meninges (arrow). D and E, expression of human CD52 of alemtuzumab compared with trastuzumab, depending on positive cell in mouse bone marrow from engrafted mice within 0 to 3 days after the cell type given (CD52high versus CD52low). For CD52low hind-limb paralysis. D, CD52high-inoculated trastuzumab-treated mouse at day 24 after inoculation and f10 % CD19 +/CD52+ cell population was observed. clone, no difference was found between trastuzumab-treated In contrast, no CD19+/CD52+ (<0.5%) was observed in a representative and alemtuzumab-treated groups (P = 0.6410; hazard ratio, CD52high-inoculated alemtuzumab-treated mice at day 24 (E).

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Fig. 4. CD52high and primary B-CLL cells are targeted with alemtuzumab-coated immunoliposomal FAM-ODN. Raji CD52high (A) or B-CLL (B) cells were incubated for 1h at 37jC with either alemtuzumab-coated or trastuzumab-coated immunoliposomal FAM-ODN, washed twice in PBS, and analyzed by FACS. Shaded histogram profile indicates the unstained cells, and open histogram indicates the specific immunoliposomal formulation. Cells from CD52high Raji (C)and B-CLL (D) were stained with PE-labeled anti-CD52 or isotype-matched antibodies and analyzed on FACS. Shaded histogram profile indicates the isotype control, and open histogram indicates the specific antibody. Uptake of alemtuzumab-coated immunoliposomal FAM-ODN. Raji CD52high (E)orB-CLL(F) cells were treated with alemtuzumab-coated or trastuzumab-coated immunoliposomal FAM-ODN for 1h at 37jC, washed thrice with PBS and visualized on a Nikon fluorescence microscope (magnification, 400). Analysis of unstained cells for both the cell type was included as a control.

and tracheobronchial lymph node, and spinal cord (Fig. 3B). susceptibility to alemtuzumab-mediated direct cytotoxicity, Corresponding tissue obtained from alemtuzumab-treated CDC, and ADCC functions and in vivo sensitivity as described CD52high cells–inoculated mice showed absence of neoplastic previously by us and others (20, 21). To determine the possible cells except for spinal cord where a few residual neoplastic cells use of this model for CD52-targeted delivery of therapeutic (Fig. 3C) were observed in the meninges compared with con- agents, the delivery of FAM-labeled oligodeoxyribo nucleotide trol mice. (FAM-ODN) containing liposomes was compared in Raji Bone marrow obtained within 0 to 3 days of hind-limb paral- CD52high and primary B-CLL cells in vitro. CD52high Raji ysis from trastuzumab-treated mice that had received CD52high clones or primary B-CLL cells were incubated for 1 h at 37jC Raji cell inoculation had a 10% human CD19+/CD52+ cell with either alemtuzumab-coated or trastuzumab-coated immu- population (Fig. 3D). In contrast, no human CD19+/CD52+ noliposome loaded with FAM-ODN and washed twice in cell population (<0.5%) was observed in alemtuzumab-treated PBS. Flow cytometric and fluorescence microsopic analyses CD52high Raji–inoculated mice (Fig. 3E). Analysis of bone mar- of both CD52high and B-CLL showed higher uptake of alem- row cells from similarly treated CD52low Raji clone–inoculated tuzumab-coated immunoliposome-FAM-ODN compared with mice exhibited presence of human CD19+/CD52- cells (data not trastuzumab-coated control liposomes (Fig. 4). These studies shown). suggest potential utility for CD52high Raji clones in in vivo Targeted delivery of alemtuzumab-conjugated immunoliposo- evaluation of CD52-targeted liposomes for therapy in CLL and mal oligodeoxyribo nucleotide to CD52high Raji cells and primary other B-cell malignancies. B-CLL cells. The above-described properties of CD52high clone Effect of rituximab on CD52high Raji cells. The surface resembled several features of primary B-CLL cells, including staining analyses of the isolated clones revealed the presence

www.aacrjournals.org 575 Clin Cancer Res 2008;14(2) January 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical of other molecules, such as CD20, CD40, and CD37, that can be targeted by other B-cell specific antibodies and provide a model for such therapeutics. To further investigate the possibility of extending the use of the Raji-Burkitt’s lymphoma model to the evaluation of immunotherapeutic agents target- ing molecules different from CD52, the in vitro and in vivo preliminary effects of the anti-CD20 monoclonal antibody rituximab were evaluated. Despite comparable percentage of CD20-expressing cells in CD52high and CD52low Raji clones (i.e., all the clones expressed CD20 in >99% cells), rituximab mediated 2-fold higher levels of CDC on the CD52low Raji clones (80 F 15% versus 35 F 16%), perhaps due to the presence on this clone of lower levels of complement resistance surface proteins, such as CD55 and CD59, that inhibit CDC on tumor cells (Fig. 5A; refs. 35, 36). The direct cytotoxic effect of rituximab upon parental (66 F 3%) and CD52high (60 F 4%) Raji clones were 2- fold higher compared with the CD52low Raji clones (33 F 3%; Fig. 5C). CD52high Raji clone showed the highest susceptibility (57 F 11%) to rituximab-mediated ADCC when compared with the parental and CD52low Raji clone, respectively 22% F 15% and 36% F 15% at the highest E/T ratio (50:1; Fig. 5B). Interestingly, consistent with in vitro studies, preliminary study using CD52high Raji clone–inoculated mice showed a higher sensitivity to rituximab treatment (50% survival at 100 days after injection) compared with the CD52low Raji clone–injected mice that received the same treatment, in which all mice died within 40 to 60 days after inoculation (data not shown). The increased capability of rituximab to mediate ADCC of the CD52high clone compared with CD52low and parental clones could be explained by the fact that CD52high clone, but not CD52low and parental clones, expresses high-level CD50 (ICAM3) mRNA as detected by microarray analysis (data not shown) and surface expression confirmed by flow cytometry. Surface expressed ICAM3 on target cells serves as an important ligand for LFA1 expressed on natural killer cells in the initiation of the immune response (37). Although the differential effect of ICAM3 may explain the differential effects of CD20-mediated ADCC, the molecular changes that attribute to the differences in direct cytotoxic effect observed for CD52high and parental cell line compared with CD52low remain to be determined.

Fig. 5. Rituximab-mediated cytotoxicity in Raji clones. A, complement-mediated Discussion cytototoxicity. Parental, CD52low,andCD52high Raji cell clones were incubated with 10 Ag/mL of rituximab or trastuzumab in the presence of 30% of either active Herein, we have established Raji cell line models expressing or heat-inactivated human serum as a source of complement. Relative cytotoxicity was determined after 1h of incubation at 37jC by Annexin V/propidium iodide high or low levels of CD52 useful for evaluating both in vitro staining (n =10).B, ADCC was measured using freshly isolated peripheral blood and in vivo effects of CD52-targeted antibody reagents, such mononuclear cells from normal volunteers and Raji cell clones at 12.5:1, 25:1, 50:1 effector-to-target ratio (E/T) in presence or absence of 10 Ag/mL rituximab or as alemtuzumab. By limiting dilution of the Raji Burkitt’s trastuzuamb. Columns, average of triplicate wells and were representative of three lymphoma cell line, which expresses high levels of CD52 only independent experiments; bars, SD. C, rituximab-mediated directed apoptosis. low high in a small percentage of cells (43%), we isolated clones Parental, CD52 ,orCD52 Raji cell clones were incubated with 10 Ag/mL of rituximab or trastuzumab with or without 50 Ag/mL cross-linking antibody expressing either high or low levels of CD52 in >90% of the cell (goat anti-human Fc).The percentage of apoptosis was determined byAnnexinV/ population and comparable high level of HLA-DR, CD19, propidium iodide staining after 24 h. CD40, CD32, and CD20. The pattern of surface expression of CD52, as well as other glycosyl-phosphatidylinositol–linked protein was found stable with continuous cell culture over as well as rituximab, can induce in vitro cell lysis in CD52high several months. The surface level of CD52 was found directly Raji cells. Furthermore, these cells are sensitive to complement related to the CD52 mRNA level as real-time PCR analysis of or antibody-mediated cytotoxicity. In addition, both CD52- RNA-exhibited 1.8-fold increases in CD52 mRNA in the targeted and CD20-targeted antibodies induced direct cyto- CD52high clone. The data clearly show that alemtuzumab, toxicity in the presence of a cross-linking antibody. Clearly,

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the CD52 density was important in alemtuzumab-mediated 24 days in vivo. Consistent with the elimination of alemtuzu- killing, as it was able to induce cytotoxic effects in vitro on mab-targeted cells, no sign of hCD19+/CD52+ cells were found the CD52high clone but not on the parental and CD52low Raji in bone marrow of similarly treated CD52low inoculated mice. clone. Despite the ability of alemtuzumab to mediate compa- In addition to the bare antibody, the CD52high Raji clone can rable levels of CDC, natural killer cell–mediated ADCC, and serve as a model for other CD52-targeted therapeutics, such direct cytotoxicity in CD52high Raji clones and CD19+ B cells as alemtuzumab immunoliposomes loaded with a therapeutic from CLL patients (Fig. 2B), the biochemical basis of alem- agent. tuzumab-mediated direct cytotoxicity remains to be estab- Alemtuzumab has been shown to be very effective in eli- lished. The CD52high Raji model described here will serve as an minating CD52+ cells and is currently used to target leukemic B amenable tool for biochemical characterization of alemtuzu- and T cells, as well as in immunosuppressive treatment mab-mediated cytotoxicity. It is interesting to note that despite (41–44). Despite extensive use of alemtuzumab in clinical comparable high percentage of CD20-positive cells, the trials, the mechanism of its action remains to be clarified, due CD52high clone showed a particularly limited sensitivity to mostly to the lack of reproducible model cell lines and animal rituximab-mediated CDC compared with CD52low clone. This models to study the in vitro and in vivo effect of this mono- could be attributed to the lower density of CD20 antigen on the clonal antibody. In fact, many of in vitro maintained lymphoid- high expression clone. Studies of haptens have, in fact, derived tumor cells only express CD52 at very low levels (1) indicated that increasing the density of the target molecule and the stability of the molecule in vivo is less predictable. determine increased sensitivity to complement mediated lysis Recently a xenotransplant model of multiple myeloma in (38–40). It is also possible that the increased levels of NOD/SCID mice using KMS-11 cells has been developed to expression of complement inhibitory proteins, such as CD55 investigate the in vivo activity of alemtuzumab. In this expe- and CD59, in CD52high Raji clone cells may contribute to the rimental condition, probably due to the low expression of reduced CDC effects. This is inconsistent with the inhibition CD52 on KMS-11 cells, alemtuzumab treatment initiated at an of monoclonal antibody–mediated CDC on tumor cells by early stage of disease substantially delayed the in vivo growth CD55 and CD59 (35). Further support comes from the limited of myeloma cells rather than eradicating the disease (45). sensitivity of CD52high clone to alemtuzumab-mediated CDC The Raji Burkitt’s lymphoma cell clones expressing high compared with increased direct cytotoxicity and ADCC. levels of CD52 stably both in vitro and in vivo will overcome the Therefore, ADCC and direct cytotoxicity represent, at least in limitations of previously described Raji-xenograft models that this model, the major mechanisms of alemtuzumab-mediated showed drastic modulation of the expression of the CD52 killing in vitro, and these may act synergistically in vivo. molecules in vivo. The stable CD52high B-cell lines and the Alemtuzumab treatment in CD52high-inoculated mice in- disseminated leukemia-lymphoma mouse model described creased significantly the median survival (71% of the mice here can serve as an excellent system for in vitro and in vivo were still alive by the end of the study). Human CD19+/CD52+ therapeutic and mechanistic evaluation of existing and novel cells were found in bone marrow from trastuzumab-treated, antibodies directed against CD52 molecule and antibody-based but not alemtuzumab-treated, CD52high-inoculated mice after targeted therapeutics, such as immunoliposomes.

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Rosa Lapalombella, Xiaobin Zhao, Georgia Triantafillou, et al.

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