1179.Full.Pdf

Vol. 1. 1 179-i 187, October 1995 Clinical Cancer Research 1179

Elimination of Human Leukemia by Monoclonal Antibodies in an Athymic Nude Mouse Leukemia Model’

Yang Xu and David A. Scheinberg2 pies can be evaluated. Current models are extremely limited: (a) Memorial Sloan-Kettering Cancer Center, New York, A true mouse nonlymphoid leukemia, such as Friend on Raus- cher leukemia (3, 9), is not useful, because the antigen targets of New York 10021 human leukemias are not expressed on mouse cells or in other small animals. (b) Xenogmafted nude mouse tumors have been ABSTRACT proposed (10-13), but these models are not relevant as a model A human acute myeboid leukemia model has been de- of leukemia, because the cells grow as a solid tumor or as veboped by i.v. transplantation of HL-60 myeloid leukemia ascites, and many of the biological and pharmacological issues cells into Swiss nude mice pretreated with cycbophospha- are not addressed. (c) The growth of human cells from AML mide. HL-60 cells disseminated into hematopoietic tissues as patients in the hematopoietic tissues of immunodeficient mice determined by flow cytometric analysis, fluorescence ml- has been described (14, 15). These human leukemia cells were croscopy, fluorescence in situ hybridization analysis, and grown in irradiated SCID mice. This model mimics the human colony formation assay. Passive immunotherapy using mu- disease, but it is less reproducible because it uses fresh human rine anti-CD13 (F23) or anti-CD33 (M195) mAbs was able to cells as opposed to a cell line. Moreover, the mice are heavily eliminate completely the HL-60 cells in the mice, as deter- immunocompromised, which renders their cane and mainte- mined by fluorescence in situ hybridization analysis, colony nance difficult and expensive. Therefore, we developed an al- formation assay, and culture of mouse blood and tissue cells temnative human leukemia model in cycbophosphamide-treated in vitro. Although F23 is abbe to inhibit completely CD13/ athymic nude mice. Although there are also limitations to this aminopeptidase N enzymatic activity, actinonin, another po- model, it adequately mimics disseminated human leukemia from tent inhibitor of CD13/aminopeptidase N, was not active as a pharmacological standpoint; has appropriate antigen targets; is an antibeukemic agent. HL-60 cell surface antigens, includ- reproducible; has low morbidity and mortality; is inexpensive; ing CD13 (aminopeptidase N) and CD33 (p67), down-regu- and requires little technical expertise. Immunotherapy with bated over time, and murine anti-HL-60 antibody was gen- mAbs was evaluated in this model. erated while the cells grew in the mice. This response was suppressed by cyclophosphamide. These data suggest that leukemia cell ebimination was antibody mediated. MATERIALS AND METHODS Animals. Six-week-old female outbmed Swiss nu/nu mice INTRODUCTION were purchased from Sloan-Kettering Institute (New York, NY). All bedding material was sterilized before use; the cages Human AML3 remains a largely incurable disease that is resistant to conventional therapies (1). Immunotherapies, in were covered with an air filter and maintained in isolation particular, mAb therapies, are under investigation as alternatives cabinets. Animal handling and experiments were performed in or adjuncts to chemotherapy (2). Phammacokinetics are favorable sterilized atmosphere using a laminar flow hood. Swiss nude to the efficient delivery of mAbs to leukemia cells (3), and mice, 6-8 weeks of age, required i.p. injections with 3 mg therapeutic effects have been observed in murine and human cycbophosphamide/mouse 3 days before iv. injection of HL-60 leukemia (3-5). mAbs that react with CD33, a Mr 67,000 cells. The cycbophosphamide dose was based on preliminary gbycopmotein expressed on early myeboid progenitor cells and experiments that showed that HL-60 cells did not engraft webb in myeboid leukemia cells, but not normal stem cells, have been mice without previous injection of cycbophosphamide: 16% of used in Phase I trials for the treatment of AML (4, 6-8). An Swiss nude mice (4/25 mice) without pretreatment of cyclo- animal model could allow rapid therapeutic development of phosphamide had no palpable tumor nodules at 8 weeks after these immunotherapies, but, at present, theme are no useful local s.c. injection, whereas all (10/10) nude mice developed models of human myeboid leukemia in which new mAb thera- local tumors at 4 weeks with pretreatment with 3 mg cycbophosphamide. Similar results were observed in mice that received i.v. injections by assaying CD13 and CD33’ cells in the lungs, where leukemia cells were initially trapped (16). This suggested that the nude mice retain limited ability to prevent HL-60 Received 2/2/95; revised 5/22/95; accepted 6/26/95. xenografting. I This work was supported by NIH Grant ROl CA55349 and by the Markey Charitable Trust. D.A.S. is a Lucille P. Markey Scholar. HL-60 Human Leukemia Cell Line. The HL-60 cell 2 To whom requests for reprints should be addressed, at Memorial line is a well-characterized human acute myeboid leukemia Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY derived from peripheral blood blasts (17). The HL-60 cells 10021. expressed both CD13 and CD33, two human myeloid mark- 3 The abbreviations used are: AML, acute myeboid leukemia; FISH, ems (6, 18, 19). The numbers of CD13 and CD33 antigens/cell fluorescence in situ hybridization; MPF, mean peak fluorescence; SCID, severe combined immunodeficient; APN, aminopeptidase N; HuM 195, were 20,000 (20) and 10,000 (21), respectively. The HL-60 cell humanized M195. line was maintained routinely in our laboratory. Cells were

cultured in 10% FCS/10% Serum Plus/RPM! 1640 and 1% determined by our laboratory. After hypotonic treatment in

peniciblin-streptomycin at 37#{176}Cin a humidified atmosphere of 0.075 M KC1 and fixation in 3:1 fixative (three parts absolute 5% CO,/air. Cell viability was always higher than 95%, and methyl abcohol:one part glacial acetic acid), slides were incu- cells were free of Mvcoplasma contamination. bated in 2X standard SSC/0.5% NP4O at 37#{176}Cfor 30 mm; mAbs. mAbs F23 (IgG2a), reactive with CD13 antigen, dehydrated successively in 70, 80, and 95% ethanol at room and M195 (IgG2a) and HuM195 (human IgGi), reactive with temperature for 2 mm each; and then dried in air. The chromo- CD33 antigen, were prepared at Sloan-Kettering Institute (22). somes were denatured by incubation at 70#{176}Cfor 2 mm in 70% MY7 (IgGl), reactive with CD13 antigen, and MY9 (IgG2b), formamide/2X SSC (pH 7.0) and dehydrated as described reactive with CD33 antigen, were purchased from Coubter Elec- above. FITC-conjugated direct-labeled specific human chromo- tronics. some 17 a-satellite DNA probe D17Z1 (Oncon; Ref. 26) was Xenotransplantation. A 0.2-mb aliquot containing 3 X denatured by incubation at 70#{176}Cfor 5 mm and cooled on ice. l0 HL-60 cells from suspension culture was transplanted i.v. For hybridization, 30 jib of the probe (1 ng/jib) were used under into the tail veins of nude mice. Mice were killed after trans- a 22 X 50-mm coverslip. The covenslip was seabed with rubber plantation at the times indicated. For tissue studies, pieces of cement, and the slides were incubated at 37#{176}Cfor 30 mm in a organs were minced, and intact single cells were isolated on a prewarmed humidified chamber. After hybridization and coven- Ficoll-Hypaque density gradient or after passage through a slip removal, excess probe was removed by incubation in 0.5 X 70-p.m nylon filter (Spectrum). Preliminary work showed that SSC at 72#{176}Cfor 5 mm, followed by two changes of 2 mm each HL-60 cells could grow in nude mice after i.p. injection. At 7-8 of PBS plus 0.1% Triton X-100. The chromosomes were coun- weeks after injection of 3 X i0 HL-60 cells without pretreat- terstained by incubation for 2 mm in 0.3 jig/mb propidium ment with cycbophosphamide, CD33 cells could be found iodide and immediately photographed with a Zeiss microscope. outside of the penitoneal cavity. We found 31% of the cells in At least 200 cells were scored for each slide. the liver, 15% of the cells in the lung, 9% in the spleen, and 3% in the bone marrow. However, one third of the mice died at 5 RESULTS weeks. The poor leukemia distribution and the rapid deaths made this i.p. model less useful for studying therapy, and it was Detection of CD13 and CD33 HL-60 Cells in Mice by not pursued. Flow Cytometry and Fluorescence Microscopy. The model Flow Cytometry Analysis and Fluorescence Micro- was initiated by injection of 3 X i0 HL-60 cells via the tail scopic Assay. Flow cytometry were done as described previ- vein. Four weeks after the transplant, the bone marrow, spleen, ousby (22). For direct immunofluorescence, 1 ,000,000 cells peripheral blood, lung, and lymph node contained 8-90% beu- were incubated in a 0.1-mb final volume with the fluonescein- or kemia cells (Fig. 1 and Table 1). The number of cells was rhodamine-conjugated mAb for 60 mm on ice, washed twice, determined by the percentage of CD13’ and CD33 cells; and fixed with I % panaformaldehyde before analysis. Cells were neither of these antigens is expressed on mouse cells. Mice that analyzed on an EPICS flow cytometen (Coulter) or evaluated by received no injections showed no positive cells and were used as fluorescence microscopy. HL-60 cells were preincubated with negative controls. After 6 weeks, the proportion of HL-60 cells 2% heat-inactivated rabbit serum/10% FCS/RPMI 1640 at room rose to 17% in bone marrow, 26% in the spleen, 28% in the temperature for 15 mm to reduce nonspecific binding. blood, 92% in the lung, and 91% in the lymph node (Table 1). Mouse Anti-HL-60 IgG Analysis. The sera from nude The high percentage of HL-60 cells seen in the lungs is similar mice bearing leukemia were collected at various times after to previous observations in the SCID mouse and may be caused HL-60 transplant. HL-60 cells (fresh cells derived from tissue by leukemia cells trapped in the lungs initially (16). The per- culture stocks) were preincubated with 2% heat-inactivated rab- centage of HL-60 cells in mouse bone marrow, spleen, and bit serum/10% FCS/RPMI 1640 at room temperature for 15 mm peripheral blood also was evaluated by fluorescence micro- to reduce nonspecific binding. Thirty p.1 of test nude mouse scopy. Both results were similar (Table 1), suggesting that either serum were incubated with 5 X io HL-60 cells on ice for 60 of these two assays may be used. mm, then washed, and 50 jib goat antibodies to mouse IgG-FITC The percentage of human leukemia cells in peripheral (50 jig/mb) were added for 30 mm. Washed cells were analyzed blood of the mice also was confirmed by a human a-satellite by flow cytometry as described above. Serum from untreated, probe specific for human chromosome 17 that does not hybrid- untnansplanted Swiss nude mice was used as negative control. ize with mouse DNA (Refs. 16, 24, 26; Table 1). At 10 weeks, The MPF of cells was determined relative to the background HL-60 cells were present in the blood and bone marrow (Fig. 2), MPF of cells stained with isotype-matched control mAb on as well as in the liver, lung, kidney, and spleen (data not shown), normal nude mouse serum, in which a value of 1.0 reflects MPF as determined by FISH using a human chromosome 17 a-sat- equivalent to background. Under these conditions, the MPF eblite probe, D17Z1. These HL-60 cells were also two to three represents a quantitative value for the maximum number of times larger than were the mouse cells (Fig. 2) and could be bound munine Ig on the surface of the leukemia cells. readily detected morphobogicably. In addition, the D17Z1 probe FISH Analysis. FISH analysis was performed using did not hybridize with mouse cells from blood, liven, kidney, standard techniques (23-25) with some modifications and is and spleen (data not shown), confirming that the probe is spe- described in brief below. Single tissue cells (10) were cultured cific for human cells. Therefore, the widespread distribution of in 10% FCS/l0% serum/RPMI 1640 for 24 h and treated with HL-60 leukemia cells in Swiss nude mice into multiple organs, 10 ng/ml Colcemid for the final 4 h. At this condition, all HL-60 as well as hematopoietic tissue, makes it a useful model for cells were arrested in the metaphase on intemphase stage, as evaluating immunotherapy and other therapeutic approaches.

PBL

0 .0 .0 E E z z

0 0 C) Fig. I Flow cytometry analy- 10 100 10 00 sis of the blood and tissues Log Fluorescence from Swiss nude mice that received human HL-60 leukemia cells after 4 weeks. Single-cell suspensions from peripheral blood leukocytes (PBL), bone BM marrow (BFvf), spleen (SPL), lungs (LU), and lymph nodes 0 0 (LN) of representative mice, .0 .0 E E which were pretreated with cy- z clophosphamide and into which z were transplanted 3 x l0 0 0 HL-60 cells, were incubated C) C) with MY9-FITC, and were assayed as described in ‘‘ Materi- abs and Methods. ‘‘ Dotted line, control mice; solid line. mice 44444;, 1000 that received HL-60 cells. Log Fluorescence HL-60 cells from tissue culture are shown as a comparison (dotted line. cells plus IgG2b- FITC; solid line. cells plus MY9-FITC).

0 0 .0 .0 E E z z

0 0 0 C)

100 10 Log Fluorescence Log Fluorescence

Table I Flow cytometnic, fluorescence microscopic, and FISH analysis of Swiss nude mice transplanted with HL-60 leukemia cells as a human AML modela

% of HL-60 cells by fluorescence % of Human 0/ of HL- 60 cells by flow cytometry” microscopic assay cells (FISH)

Weeks (no. of Peripheral blood Peripheral blood Peripheral blood mice) Bone marrow Spleen leukocytes Lungs Lymph node Bone marrow Spleen leukocytes leukocytes

Control (n = 7)” - - - - - 0 0 0 0 4(n7) 8±4 10±3 15±5 90±8 90±11 7±4 11±3 16±7 17±7 6(n=7) 17±9 26±4 28±6 92±6 91±12 19±9 28±7 32±9 30±10

10 (,i = 6) - - - - - 0 0 0 39±14 a The Swiss nude mice bred in our defined-flora colony (Memorial Sloan-Kettering Institute) were given injections i.p. of 3 mg cyclophosphamide and, 3 days later, were given 3 X i07 HL-60 cells via tail vein injection. At the times indicated, the bone marrow, spleen, peripheral blood leukocytes, lungs, and lymph node were analyzed by flow cytometry for CD1Y (MY7-RD1) and CD3Y (MY9-FITC) cells. The control murine organs served as negative control. For fluorescence microscopic assay, cells were stained with MY9-FITC or MY7-RD1 . Percentages of human cells were determined by human chromosome 17 probe. At least 200 cells were scored for each slide. The results are the means ± SE. 6 For CD13 and CD33 HL-60 cells.

C Control mice were not given injections of HL-60 cells.

‘I-, <1%.

A B

S I.. 0

C D w I

#{149}4s1’P’‘‘* 0 .. g- #{149},.. 5 j

Fig. 2 FISH analysis of Swiss nude mice 10 weeks after transplantation of HL-60 leukemia cells. HL-60 leukemia cells (3 X I0) were transplanted

into the mice, and metaphase chromosomes or interphase nuclei from single-cell suspensions of representative mice were probed with DI7ZI , a human

a-satellite probe specific for sequences on chromosome 17, as described in ‘‘ Materials and Methods. ‘‘ Positive cells showed two fluorescent green

spots, but only one spot may be visible in these photographs because of the single focal length in the photograph. A. HL-60 cells showed 100% stained cells: B. mouse peripheral blood cells at 10 weeks after HL-60 transplantation: C, normal mouse control bone marrow cells showed no stained cells; and D. mouse bone marrow cells at 10 weeks after HL-60 transplantation.

Immunotherapy in the AML Model. Immunotherapy The elimination of cells by use of an antibody infused at of the HL-60 leukemia using myeboid-specific mAb was tested this early time point may reflect prevention of engraftment as in the model to determine its usefulness as a model for mAb- well as killing. Therefore, additional mice were allowed to fully based therapies. The test animals were treated with 200 jig mAb engraft before treatment began. F23 was able to protect mice i.p., three times a week for 5 weeks, beginning on the same day from death 4 weeks after HL-60 leukemia cells had been trans- as the initiation of the model. On a weight basis, the doses used planted (Fig. 3B). This suggested that passive immunotherapy here are not unlike doses of mAb in human trials. could eliminate AML engrafted previously, because leukemia Approximately 60% of the untreated control animals were dissemination was clearly seen by 4 weeks (Table 1). dead at 10 weeks after the HL-60 cell injection (Fig. 3A). The Effects of Actinonin on Metastasis of Leukemia Cells mice treated with F23, M195, and F23 plus M195 were all alive in Vivo. F23 is a potent inhibitor of CD13/APN (EC 3.4.11.2) at 10 weeks, whereas the mortality of the groups treated by in vitro (20, 27, 28). CD13/APN inhibitors have been examined control antibody IgG2a (TA99) was similar to the untreated in clinical studies and could inhibit various tumor metastasis in control groups. This suggests that F23 and M195 can specifi- vito (29-31). Anti-CD13/APN mAb could also inhibit the in- cabby protect mice from HL-60 leukemia. In the groups treated vasion of human metastatic tumor cells into reconstituted base- with F23 or M195 for 5 weeks and tested at 10 weeks, there ment membrane (32). Therefore, the antileukemic action of F23 were no detectable human cells, as determined by FISH analysis might be attributable to its potent effect on CD13/APN rather (Table 2). In the untreated and TA99-tneated groups, 28-41% than to its immunological actions. Therefore, we tested whether human cells were found in bone marrow, spleen, and peripheral the most potent inhibitor of CD13/APN, actinonin, could inhibit blood (Table 2). In addition, a lymph node from the untreated HL-60 leukemia cell dissemination in vivo in mice. Swiss nude group at 10 weeks showed nearby 100% human chromosome mice were treated with 250 jig (an estimated initial serum 17-positive cells, as determined by FISH (data not shown). concentration of 0.3 mM) actinonin i.p., three times a week for

A nude mouse might contribute to the rejection of the HL-60 cells, 100 the bevels of mouse anti-HL-60 lgG in these two sets of mice . M195 -.-.-.-.- p3+M195 were compared. IgG anti-HL-60 levels in the leukemia-negative V mice were approximately three times higher than those in mice .1 that did not reject the leukemia cells (Fig. 4A). This result E suggested that a fraction of nude mice that had not been pre- V : treated with cycbophosphamide has an ability to reject HL-60 4... - 3 0 40 _____ cells and that specific Ig may contribute to this effect. After a- No mamentl V pretreatment with cycbophosphamide, the lungs from all seven .0 ----- Actinonin L..__ E mice were positive for CD33-staining cells. In addition, the z 20 anti-HL-60 IgG levels in these mice were approximately 8-fold ______lower than those in the mice that had not been pretreated with

0 2 4 6 8 10 cycbophosphamide, which had rejected the HL-60 cells (Fig. Weeks 4A). These data suggested that cycbophosphamide reduced the level of immune response to HL-60 cells, in this instance, as measured by IgG levels; therefore, this response appeared to be important in the rejection of HL-60 cells. Similar results were obtained from mice that received s.c. V injections of HL-60 (Fig. 4B). Of the Swiss nude mice, 16% (.4 E (4/25) that were not pretneated with cycbophosphamide had no V palpable tumor nodules at 8 weeks after local s.c. injection, whereas all (10/10) nude mice developed local tumors at 4 ‘4- 0 weeks when pretreated with 3 mg cycbophosphamide. The rel- a- V .0 ative anti-HL-60 levels at 6 weeks in these groups of mice E showed the same distribution as had been described with mice z that received iv. injections. Mice that rejected the HL-60 cells had the highest bevels (27.6), whereas mice with tumor growth 0 2 4 6 8 10 12 and mice treated with cycbophosphamide had bevels of 1 1 .7 and Weeks 4.2, respectively (Fig. 4B).

Fig. 3 Survival curves of Swiss nude mice after transplantation of Modulation of Cell Surface Antigen Targets on HL-60 HL-60 leukemia cells. HL-60 cells (3 X 10) were transplanted into Cells in Vivo. Analysis of MPF of human leukemia cells in the Swiss nude mice as described in ‘‘ Materials and Methods. ‘‘ After the mice showed a time-dependent decrease in the expression of transplantation, the mice were treated with 200 .a.g mAb or 250 p.g the cell surface markers CD13 and CD33. At 10 weeks after the actinonin i.p., three times a week for 5 weeks. A, treatment beginning at the first day of the transplantation: no treatment ( 15 mice); actinonin (7 injection, CD33 antigen on HL-60 cells was nearby undetectable mice); TA99 (7 mice); F23 (7 mice); M195 (7 mice); and F23 plus (Fig. 5) and no CD3Y cells were found (Table 1). CD13 M195 (7 mice). The latter three treatments all resulted in 100% survival antigen down-regulation was similar to CD33 down-regulation, and are, therefore, shown as overlapping lines. B. treatment beginning at as determined by flow cytometmy using MY7-RDI (data not the 28th day after the transplantation: no treatment (7 mice); F23 shown). In this group of mice pretreated with cycbophospha- treatment (7 mice). mide, low levels of anti-HL-60 IgG still were produced (Fig. 5). The bevels peaked at 6 weeks, then fell, coincident with CD33 and CD13 surface antigen down-regulation. The boss of CDI3 and CD33 expression may be attributed to development of a

5 weeks, beginning on the same day as the initiation of the pobycbonal munine anti-HL-60 immune response (see above), model. This schedule was identical to that of F23 described which bound to and modulated the antigens from the surface. above, and the experiment was done concurrently with the F23 Other antigens on the HL-60 cell (CD29 and CD71) also down- experiments as a control. Actinonin was unable to inhibit HL-60 regulated (data not shown). Rapid modulation of human hema- cells grown in the mice. The mice treated with actinonin were topoietic antigens has been found in vitro and in t’:s’o (3-5, 33, not protected, and the mortality of the group was approximately 34). 70%, which is similar to that seen in the untreated control groups (Fig. 3A). This finding suggests that actinonin could not protect mice from HL6O leukemia. DISCUSSION Mouse Anti-HL-60 IgG Levels. In an attempt to explain We describe a leukemia model in nude mice using HL-60 the possible mechanisms of immune rejection in these animals, cells injected i.v. after cycbophosphamide pretreatment. Cells

10 mice that received injections of 3 X i0 HL-60 cells iv. engrafted and spread rapidly throughout the body. Although this without pretreatment with cycbophosphamide were assayed at 6 model is neither appropriate non proposed as a biological model weeks for the presence of leukemia. Eight of these mice had of leukemia cell growth in t’ivo, it is appropriate for the study of CD33 cells in the lungs, whereas two of the mice had no CD33 antibody-based immunotherapy with mAbs reactive with anti- cells in the lungs. To assess whether an immune response by the gens on HL-60 cells, such as CD33, CD15, and CDI3, all of

Table 2 FISH analysis, colony formation, and culture assays of Swiss nude mice 10 weeks after transplantation with the HL-60 leukemia cell line with or without mAb treatment”

Culture assay/ % of Cells with human chromosome 17 HL-60 colonies/10’ cells io cells

Treatment Bone Peripheral blood Bone Peripheral blood Bone (no. of mice) marrow Spleen lymphocytes marrow Spleen lymphocytes marrow Spleen

Control”/none (‘a = 7) 0 0 0 0 0 0 0 0

None (a = 6) 30 ± 12 35 ± 12 39 ± 14 TNTC’ TNTC TNTC >io >i07

TA99 (a = 3) 28 ± 1 1 3 1 ± 10 41 ± 20 TNTC TNTC TNTC > i0 > l0

F23 (a = 7) 0 0 0 0 0 0 0 0

Ml95 (ii = 7) 0 0 0 0 0 0 0 0 F23 +M195(n 7) 0 0 0 0 0 0 0 0

“ HL-60 leukemia cells (3 X l0) were transplanted into Swiss nude mice as described in Table I. Immunotherapy was described in Fig. 2. At 10 weeks, the bone marrow, spleen, and peripheral blood leukocytes were analyzed by FISH for human chromosome iT’ cells; colony formation assay; and cx vito culture in liquid phase. For colony formation assay, 106 mononuclear cells were plated in 0.6% agar containing 10% FCS/10% serum plus/RPMI 1640; colonies were scored after 2 weeks. For culture assay, l0 bone marrow or spleen mononuclear cells were cultured in 10 ml 10% FCS/l0% Serum Plus/RPMI 1640; live cells were counted after 3 weeks. Data are the means ± SE.

I, Control mice were not given injections of HL-60 cells.

‘ TNTC, too numerous to count (>30(X) colonies).

which are under investigation in human clinical trials. We used cabby to various mouse cells. As a result, indirect assay cannot be this model to study immunotherapy by use of passively admin- used easily for detection of human cells. istered mAbs. The model is not appropriate for study of T-cebl- Immunotherapy of the HL-60 leukemia using myeboid- mediated events nor for long-term therapy because of the down- specific mAb F23 and M195 was abbe to completely eliminate 3 regulation of target antigens. x i07 HL-60 cells and prevent death in this model (Fig. 3A and Although leukemia cells were detected readily by flow Table 2). We further confirmed the ability of antibody-mediated cytometry at 4 and 6 weeks in these mice, surprisingly, the nude antibeukemic effects in additional mice in which complete en- mice contained no detectable CD13 on CD33 cells in bone graftment of HL-60 cells (4 weeks) had occurred. At this later marrow, spleen, and peripheral blood at 10 weeks (Table 1). time point, the antibeukemic effects could not be attributed to However, FISH analysis of these organs at 10 weeks showed prevention of engraftment by the mAb. To further determine that they still contained human chromosome 17 cells (Table 2). whether there were viable residual HL-60 cells in these mice, we The steady loss of cell surface markers over a 8-10-week period cultured i05 bone marrow and spleen cells taken at 10 weeks in rendered the use of immunophenotyping inadequate to study the 10 ml liquid medium containing 10% FCS. model at later time points. The FISH results reflected more After 3 weeks of culture, there were approximately i0 adequately the HL-60 cell distribution in the mice. FISH data cells in the cultures from bone marrow and spleens of the were also consistent with colony formation assays at 10 weeks untreated and TA99-treated mice, but no cells in the mice that (Table 2). did not receive injections of HL-60 or in the F23-, M195-, or The loss of markers over time appeared to be the result of F23 plus M195-treated groups (Table 2). The cells that grew out immune modulation. Some human antigen down-regulation has of the bone marrow and spleens in the untreated or TA99-treated been shown after transplantation of human peripheral blood groups were 100% CD13, CD33 and human chromosome

lymphocytes into SCID mice (35). However, little attention to 17f (data not shown). These various ex vivo assays were, this phenomenon or its linkage to the mouse immune response therefore, consistent with FISH analysis, suggesting that HL-60 has been paid previously. As a result, immunophenotypic anal- cells were completely eliminated from the mice after treatment ysis without DNA analysis on colony formation assays may lead with F23 on M195. to inaccurate conclusions in these AML models. Therefore, the The mechanism by which leukemia cells were eliminated fluorescence assays alone resulted in false-negative results at the by F23 on M195 is unclean. F23 is capable of completely

later time points. Indirect flow cytometmic assay, as described inhibiting CD13/APN enzymatic function in vitro (20, 27). (16), also was attempted to increase the sensitivity of detection CD13/APN has been implicated in tumor metastasis (32). of the HL-60 cells. Washed single-cell suspensions from the Therefore, we tested whether another potent APN inhibitor mice were incubated with munine mAb against CDI3 (F23) and (actinonin), which binds to the same zinc motif of CD13/APN to CD33 (M195) and then FITC-conjugated F(ab’)2 goat antibod- which mAb F23 binds (20, 27, 28), could inhibit HL-60 beuke- ics to mouse IgG, washed, and analyzed by fluorescence micro- mia cell growth in vivo in mice. Actinonin, at a concentration of scope. Control mice that had not received injections of HL-60 approximately 0.3 msi, administered three times a week, was cells showed 30% or more positive cells in the spleen and bone unable to inhibit HL-60 cell growth in the mice. We observed marrow (data not shown). This suggested that there were many that the death rate in the actinonin-treated group was similar to cells with surface-bound mouse IgG in the spleen and bone that in the untreated control group. This suggested that the marrow. Therefore, the secondary antibodies bound nonspecifi- elimination of HL-60 cells in mice by F23 was more likely a

A 10 35, 5. - -6-- D33 ...... Anti-HL6O-IgG 30’ 8

V V 25’ 6 ci 5. 20’ 0 4

.. 15’ . .... 2 ...... #{149} 10’ ...... V 5, V 0 2 4 6 8 10

0’ Weeks Control No growth Growth CPA treated (N = 7) (N=2) (N=8) (N=7) Fig. 5 HL-60 cell surface antigen down-regulation and relative murine anti-HL-60 IgG levels in the mice after transplantation of HL-60 cells CPA untreated groups with pretreatment with cycbophosphamide and without mAb treatment. B HL-60 leukemia cells (3 x 10) were transplanted into Swiss nude mice ‘‘ ‘‘ 35 as described in Materials and Methods. At the times indicated, the a-. enlarged lymph nodes and mouse sena were collected. Single-cell sus- 5- pensions (5 X 10) were stained with MY9-FITC on ice for 60 mm. 30 washed, and analyzed by flow cytometry using the EPICS Profile II

V (Coulter). Normal Swiss nude mouse cells were stained with MY9-FITC 25 V as negative control. For anti-HL-60 IgG assays, 30 p.1 mouse serum

ci were incubated with 5 x 10’ HL-60 cells as described in ‘‘ Materials and 20’ Methods. ‘‘ MPF was shown. C

S V 10 V

V 5. V these untreated mice retained a partial immune response that was adequate to occasionally reject HL-60 cells. Because these mice back functional T lymphocytes, the elimination may be Control No growth Growth CPA treated caused by a humonal response, NK cells, on macnophage-based (N = 7) (N = 4) (N = 21) (N = 10) opsonization. Even in SCID mice, NK and bymphokine-acti- CPA untreated groups vated killer cell activity exist (36, 37). Cycbophosphamide fun- them depressed the immune response so that mice were unable to Fig. 4 Relative murine anti-HL-60 lgG levels were influenced by previous cyclophosphamide treatment A, HL-60 leukemia cells (3 X reject the HL-60 cells. A comparison of the mice that did not

10) were transplanted iv. into Swiss nude mice as described in ‘‘ Ma- reject HL-60 cells with and without pretreatment with cycbo- tenials and Methods, ‘‘ except with or without previous cyclophospha- phosphamide, for bevels of specific polycbonal anti-HL-60 anti- mide (CPA) treatment. At 6 weeks, 10 mice that did not require body, showed that the specific immunogbobulin response de- cyclophosphamide were killed, and CD1Y and CD33 HL-60 cells in the lungs were assayed. Two mice rejected the HL-60 cells and were creased at beast 50% after the treatment (Fig. 4). This finding negative for cells in the lungs, whereas eight mice engrafted and were suggested that cycbophosphamide can reduce directly the ability positive for cells in the lungs. The sera from all 10 mice were incubated of mice to generate a specific response to HL-60. Reductions of with 5 X i0 HL-60 cells followed by goat antibodies to mouse other arms ofthe immune response are also likely. F23 or M195 IgG-FITC as described in “Materials and Methods.” A group of seven may ebiminate the HL-60 cells in the mice as a result of specific mice that were pretneated with cycbophosphamide and a group of mice that did not receive HL-60 are shown for comparison. B, twenty-five IgG-mediated tumor suppression via neutraphib-, macnophage-, mice received 3 X i07 HL-60 cells s.c. without previous cyclophosph- or NK-mediated antibody-dependent cell-mediated cytotoxicity amide treatment. Four mice had no palpable tumor nodules at 6 or 8 or via organization. A robe for the participation of a bow-level weeks. Ten mice pretreated with 3 mg cyclophosphamide developed polycbonab immune response in the elimination of HL-60 cells in local tumors at 4 weeks. All sera were collected at 6 weeks and assayed as described in A. Bars, SD of the mean. A comparison of the data from conjunction with the passive infusion cannot be excluded, how- each group by Student’s t test showed significant differences between ever. On the basis of these model data, a trial using high doses groups (P < 0.05). of HuM195 has been initiated at Memorial Hospital (New York, NY). In addition, this model will allow the efficient evaluation of a series of constructs of HuM195 that have been prepared. These include ‘31I-HuM195, 213Bi-HuM195, #{176}Y-HuM195, HuM195-gebonin, and HuM195 in combination with cytokines, result of its immunological effects rather than of inhibition of such as interleukin 2. Moreover, combinations of mAb that are APN. now in human use alone (such as anti-CD33, anti-CD15, and A fraction of the nude mice that was pretneated with anti-CD13), but that cannot be infused together feasibly in cycbophosphamide rejected HL-60 cells (Fig. 4), suggesting that humans because of restrictions in the development of investi-

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Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. Elimination of human leukemia by monoclonal antibodies in an athymic nude mouse leukemia model.

Y Xu and D A Scheinberg

Clin Cancer Res 1995;1:1179-1187.

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