Increased Efficiency in Selective Elimination of Leukemia Cells by A

[CANCER RESEARCH 45, 69-75, January 1985]

Increased Efficiency in Selective Elimination of Leukemia Cells by a Combination of a Stable Derivative of Cyclophosphamide and a Human B-Cell-specific Immunotoxin Containing Pokeweed Antiviral Protein1

F. M. Uckun,2 S. Ramakrishnan, and L. L. Houston3

University of Heidelberg, Heidelberg, West Germany [F.M.U.], and Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045 [S.R., L.L.H.]

ABSTRACT (19). ABMT5 avoids acute graft-verst/s-host disease and has become an alternative approach to the use of allogeneic marrow Leukemia cells were mixed with normal human bone marrow in the treatment of acute leukemia. The problem that prevents cells to simulate bone marrow from leukemia patients; the mix the more general use of autologous marrow is the presence of ture was then treated with a combination of stabilized derivative occult tumor cells in the remission marrow (14). In acute leuke- of cyclophosphamide [Mafosfamide (ASTA Z 7557)] and poke- mias where ABMT is performed in the second or subsequent weed antiviral protein-containing immunotoxin. The ability of this remission, ex vivo purging of the autologous marrow graft seems protocol for selective elimination of B-ALL cells was evaluated to be essential for therapeutic efficiency. by clonogenic assay. The monoclonal antibody (843) portion of Contaminating tumor cells can be eliminated from human the immunotoxin was directed against human B-cells and was marrow either by immunological or pharmacological methods. linked to pokeweed antiviral protein by a disulfide bond. The One pharmacological method involves the use of 4-HC, a syn combination of ASTA Z 7557 and immunotoxin was superior to thetic derivative of cyclophosphamide (29). In a clinical phase I either ASTA Z 7557 or the immunotoxin alone and produced study (27), patients receiving autologous marrow treated with 4- nearly 7 logs of elimination of leukemia cells from the cell mix HC (up to 100 itg/ml) after myeloablative pretransplant therapy tures. About 5 logs of contaminating tumor cells were eliminated showed successful hematological recovery, despite the inhibition from a 200-fold excess of normal marrow under conditions where of committed stem cells [colony-forming units in culture, burst- fewer than 50% of pluripotent stem cells were lost. Moreover, forming units (erythroid)]. There is a significant correlation be this manipulation did not inhibit subsequent production of pluri tween the fractional tumor cell elimination and the pretreatment potent stem cells in long-term bone marrow cultures, indicating proliferation index of target cells (32), indicating the 4-HC is most that the more primitive progenitors were not harmed. cytotoxic to rapidly cycling tumor cells. This cell cycle-dependent antitumor activity of 4-HC might protect noncycling pluripotent marrow progenitors, but quiescent noncycling tumor cells might INTRODUCTION also escape the cytotoxic effects of the drug (33). In this case, another reagent interfering at a different step (e.g., translation) Chemotherapy is the primary treatment for childhood and adult in the cellular machinery in combination with 4-HC may improve lymphoblastic leukemia. The disease-free survival for children is the efficacy of treatment. 50% (12), but adults have lower rates of initial achievement of Effective tumor cell purging is also possible using either com complete remission, shorter duration of complete remission, and plement-mediated cell lysis (1) or immunotoxins (18, 35). Immu- shorter survival time (36). The 2-year disease-free survival in notoxins are conjugates of monoclonal antibodies and toxins or adults varies between 10 and 40% (28). However, for patients toxin-related enzymes (hemitoxins) that specifically inhibit the who relapse while on maintenance therapy or shortly after elec growth of leukemia cells In vitro (17, 21, 22, 34) and In vivo (3, tive cessation of chemotherapy, subsequent treatment has been 25,23). In contrast to the relative nonspecificity of 4-HC cytotox- unsatisfactory. Although complete second remissions can usu icity, immunotoxin-mediated cytotoxicity is highly selective and ally be attained, the median duration of such remissions has is restricted to tumor cells bearing the target antigen. Tumor usually been less than 6 months (26). Allogenic bone marrow cells are eliminated regardless of their proliferation index if they transplantation during second or third remission results in greater bear the relevant antigen. than 2-year disease-free survival in 35 to 60% of patients (31).4 The present study was performed to compare the ability of However, only 35 to 40% of patients have HLA-identical sibling mafosfamide (ASTA Z 7557) by itself and in combination with a donors available; acute graft versus host disease develops in 30 target cell selective immunotoxin to eliminate leukemia cells from to 70% of patients transplanted with HLA identical marrow and a synthetic mixture of leukemia and normal bone marrow cells. is an indirect cause of death in 20 to 40% of affected individuals Mafosfamide is a new stabilized oxazophosphorine derivative of 4-HC that shows comparable In vitro effects of 4-HC (27). A 1This work was supported in part by Grant CA 29889 from the National Cancer highly sensitive in vitro clonogenic assay, capable of measuring Institute. 1 Present address: Department of Therapeutic Radiology, University of Minne 8 logs of elimination of tumor cells, was used to follow the tumor sota, Minneapolis, MN 55455. 'Present address: Cetus Corporation, 1400 Fifty-third Street, Emeryville, CA 5 The abbreviations used are: ABMT, autologous bone marrow transplantation; 94608. PAP, pokeweed antiviral protein from spring leaves; B-ALL, Burkitt's acute lym- 4 G. Santos, personal communication. phocytic leukemia; 4-HC, 4-hydroxycyclophosphamide; FMF, flow microfluorome- Revived May 30, 1984; accepted September 21, 1984 try; CFU-GEMM, pluripotent stem cells.

CANCER RESEARCH VOL. 45 JANUARY 1985 69

cell elimination. The combination of ASTA Z 7557 and immuno- of Germany) equipped with a sheath flow chamber and a mercury high- toxin was superior to either ASTA Z 7557 or immunotoxin alone pressure lamp. For fluorescence excitation, a BG38/UG 5 filter combi and produced nearly 7 logs of elimination of B-ALL cells. The nation was inserted. Prior to recording of FMF histograms, the cell suspensions were adjusted to approximately 1 x 105 cells/ml with lgG1 monoclonal antibody B43 used for immunotoxin synthesis detects a human B-cell-specific antigen. fluorochrome solution. Pulse height analysis was performed with a multichannel analyzer (MCA 8100, Canberra Industries, Meriden, CT) displaying the distribution in 256 channels. Collection of pulses was MATERIALS AND METHODS ended automatically when any of the channels had a count of 10,000 pulses. Computerized correction and evaluation of FMF distributions was Stem Cell Toxicity Assay. Stem cell toxicity of in vitro "purging" performed as described previously (8). procedures was evaluated by a modification of the in vitro clonal assay B43-PAP Immunotoxin. A pan-B monoclonal antibody (B43) was described by Fauser and Messner (5). 1.5 x 105 density gradient sepa conjugated to PAP using A/-succinimidyl-3-(2-pyridyldithio)propionate rated bone marrow mononuclear cells or 1 x 103 to 1 x 104 purified (23). PAP is an enzyme (M, 29,000) that catalytically inactivates the 60S stem cells from normal marrow were suspended in Iscove's modified Dulbecco's medium containing 0.9% methylcellulose, 30% fresh plasma, subunit of mammalian ribosomes and inhibits cellular protein synthesis. PAP was isolated from spring leaves of pokeweed (10). B43 detects a 5% phytohemagglutinin-lymphocyte-conditioned medium and 65 Â¡Â¿M2- B-cell-specific antigen expressed on the earliest known B-lymphocyte mercaptoethanol. Triplicate 1-ml samples (1.5 x 105 mononuclear cells and is strongly expressed throughout B-cell differentiation but not ex or 103 to 104 purified stem cells/ml) were cultured in 35-mm Retri dishes pressed at the terminal stage of differentiation, the plasma cell. B43 for 14 days in a humified 5% CO2 atmosphere. Erythropoietin [1 unit/ml reacts with the pre-B-cell line NALM-6 (50%), with all B-ALL cell lines (Connaught Step III)] was added at the start of culture. Stem cells were (>95%), with non-Hodgkin's lymphomas (70%), with null cell-ALL, and purified from normal bone marrow using a modification of a previously with normal B-cells. It does not react with T-cell acute lymphocytic described 2-step technique (7, 9). Briefly, immature myeloid cells, B- leukemia, leukemic T-cell lines such as HSB-2, CEM, or MOLT 3, or with cells, pre-B-cells, T-cells, and monocytes/macrophages were depleted myeloid and erythroid cells or their progenitors [colony-forming units in the first step using a mixture of monoclonal antibodies OKT 11, OKT (granulocyte-macrophage), colony-forming units (erythroid), or burst- 3 (T-cells), B43, B-1 (B- and pre-B-cells), anti-MY8, and anti-MOL (mye forming units (erythroid)]. B43 does not react with the CFU-GEMM from loid cells). Cells that bound the antibodies were removed by rosetting normal bone marrow. B43-PAP immunotoxin was purified by adsorption with sheep erythrocytes coated with purified rabbit immunoglobulin and to Protein A-Sepharose. The ratio of antibody to PAP (1:2.0) was subsequent density gradient separation. Antibody-negative interface determined by radioimmunoassay (20, 22). cells were further purified using an anti-la monoclonal antibody in a Protein Synthesis Assay. The incorporation of [3H]leucine into protein second rosetting step, la-positive cells were highly enriched (60-fold) in was measured as described previously (22) using B-ALL cells. Assays pluripotent stem cells. were done in triplicate. Clonogenic Assay. Lymphoma cells were grown on a monolayer of irradiated human bone marrow cells in 96-well culture dishes and counted by limiting dilution after treatment with ASTA Z 7557 as reported previ RESULTS ously.6 Briefly, the treated cell suspension was diluted and, from each dilution, a 100-^1 volume was placed in several wells. After 14 days, the Effect of ASTA Z 7557 Incubation on Protein Synthesis. Our previous data6 showed that about 1 ng B43-PAP/ml inhibited wells were examined for Clonogenic growth using an inverted phase microscope. Estimates of the most probable number of the remaining protein synthesis of target B-ALL cells by 50% after 16 hr of Clonogenic units were made with a modification of the Spearman-Karber incubation. At 10 Â¿tg/ml,more than 95% of protein synthesis method (11,13) by counting the number of wells showing growth. Under was inhibited selectively. The presence of chloroquine (40 /tw) normal conditions, this assay can measure elimination of almost 6 logs further increased the sensitivity of B-ALL cells to B43-PAP by of Clonogenic tumor cells and has been adjusted so that a 1-log difference about 9-fold. ASTA Z 7557 was not completely inhibitory to in Clonogenic growth is statistically significant (p < 0.001). Using a labor- protein synthesis at any concentration (25, 50, and 100 Â¿ig/ml) intensive modification (100 culture plates per single test), we can measure tested, and all 3 concentrations inhibited protein synthesis by 8 logs of elimination. The mean Clonogenic fraction of B-ALL cells in the about 60% (data not shown). However, parallel measurement of presence of irradiated bone marrow cells was about 95% in 5 experi ments. In the absence of feeder cells, the mean Clonogenic efficiency DNA synthesis indicated that nearly 77% inhibition was elicited was 52% (range, 30 to 65%). by 40 Mg/ml of the drug. At 25 ÃŸgonly 7.6% synthesis was Long-Term Marrow Culture. Long-term cultures were also performed observed, and at 50 ^g/ml almost complete inhibition of DNA at 33Â°(3% CO2) in complete medium with 12.5% fetal calf serum, 12.5% synthesis was noticed. Chloroquine (40 ^M) did not enhance the horse serum, 10 /IM mercaptoethanol, and 0.1 ^M hydrocortisone 21- protein synthesis inhibition achieved with ASTA Z 7557. hemisuccinate as described previously (6). Effect of ASTA Z 7557 on Cell Cycle Kinetics. The evaluation ASTA Z 7557 Treatment. ASTA Z 7557 is cytotoxic in vitro and in of the cell cycle distribution of B-ALL cells treated with ASTA Z vivo (2), and its activity probably requires hydrolysis to release 4-HC and 7557 was complicated by varying portions of background debris. mercaptoethane sulfonate. ASTA Z 7557 (1 mg/ml) was dissolved in By means of computerized subtraction of exponentially decreas tissue culture Medium 199, and target cells were incubated with it (10 to 100 iig/ml) for 30 min at 37Â°.The cells were then washed twice at ing corrections (8), it was possible to clear the original FMF 10Â°and resuspended in freshly prepared Clonogenic medium. distributions. Chart 1 demonstrates the effects of short-term Flow Microfluorometric DNA Analysis. B-ALL cells were washed incubation with ASTA Z 7557 on cell cycle distribution of B-ALL twice with phosphate-buffered saline and fixed with 96% ethanol to cells. Cell cycle distribution of controls (untreated or incubated produce a monodisperse suspension. The DNA-specific fluorochrome with medium 199 at 37Â°for 30 min) did not show any significant 4',6-diamidino-2-phenylindol was used for fluorescence staining. The time dependent variation and was identical with the cell cycle nuclear fluorescence of the specifically stained DNA was measured with distribution of B-ALL cells treated with ASTA Z 7557 at Time 0 an ICP 22 pulse cytophotometer (Phywe AG, Gottingen, Federal Republic or after incubation for 30 min at 37Â°(G0Gi = 45 Â±2% [S.D.]; S

6 F. M. Uckun, S. Ramakrishnan, and L. L. Houston. Journal of Immunology, in = 39 Â±1%; G2M = 16 Â±1%; coefficient of variation, <3%). A press. 1985. short exposure to 50 ng ASTA Z 7557/ml prevented nuclei from

CANCER RESEARCH VOL. 45 JANUARY 1985 70

10" B-ALL cells with 50 ng ASTA Z 7557/ml of B43-PAP pro 100 duced 6.95 logs of tumor cell killing; only 11 B-ALL clones could be detected compared to (a) 9.6 x 107 clones in the untreated control, (b) 9.5 x 107 clones in the control treated with 20 ng B43 antibody/ml; (c) 4.8 x 103 clones when 50 p.gASTA Z 7557/ ml was used without immunotoxin, and (d) 9.3 x 107 clones in the control treated with 10 ng irrelevant immunotoxin/ml (T3- 3A1-PAP). T3-3A1-PAP is an immunotoxin directed against the antigen P41 expressed on T-cells and not found on B-cells and is effective at about 50 ng/ml against target T-cells (21). When 1 x 10" target B-ALL cells were treated with 100 ^9 ASTA Z 7557/ml in combination with 10 Â¿Â¿gB43-PAP,no clonogenic units could be detected, indicating that almost 8 logs of B-ALL cells were eliminated. 20 40 60 The combination of treatment with 100 ng ASTA Z 7557/ml HOURS followed by treatment with unconjugated PAP (5 /Â¿g/ml)for 9 hr Chart 1. Target B-ALL cells (1 x 10Â«;2 x 107 cells/ml) were treated with 10 was not more toxic to B-ALL cells than was ASTA Z 7557 alone; (O), 25 (A), or 50 (â€¢)iig ASTA Z 7557/ml. After 30 min, the cells were washed twice with clonogenic medium, resupsended at 5 x 10s cells/ml, and distributed both treatments produced 4.7 logs of elimination. The additive into 5 flasks (7.5 x 106 B-ALL cells/flask) in clonogenic medium. At 0,12, 48, and effects of combined ASTA Z 7557 and B43-PAP treatment 72 hr. all cells from one flask were washed twice with phosphate-buffered saline and fixed in 96% ethanol. DNA-FCM measurements were preformed as in "Mate appears not to be restricted to B43-PAP because 2 other PAP- rials and Methods." The coefficient of variation was <3% in all measurements. A, containing immunotoxins (T3-3A1-PAP and 5E9-11-PAP) di percentage of B-ALL cells in GO/1 ; B, percentage of cells in S; C, percentage of rected against T-cells were also potentiated by 4-HC treatment.7 cells in G2M. Points, means of 20 replicate experiments; S.D. was less than 5% of the mean values. To determine if immunotoxin escaping from dying cells could inhibit other cells, 1 x 106 B-ALL cells were treated with 100 ng dividing and reentering G0/i; furthermore, it also partially syn ASTA Z 7557/ml followed by treatment with 20 M9 B43-PAP/ml. chronized the cell population and increased the proportion of After washing of the treated cells, cells were combined with 1 x cells moving from G0/i into S phase. By 12 hr, a partially syn 106 untreated B-ALL cells and assayed for clonogenic growth. chronous population of cells moving into S phase was evident 9.5 x 105 clones could be detected after 14 days of incubation. with a concomitant decrease in the number of cells in G0/i. By When a parallel treatment of 2 x 106 B-ALL cells (all cells received 24 hr, 68 Â±2% of the cells were in S phase, and only 18 Â±1% the combination treatment) was compared for clonogenic were in G0/i. Cells continued to move into and through S phase growth, no clones could be detected. This indicates that cells and accumulated in G2M. By 72 hr, 96 Â±3% of the cells were in dying from the combination treatment do not affect the clono G2M, 4% were in S phase, and virtually none were in G0/i phase. genic growth of other cells present in the culture. The effects of short-pulse exposure to 100 ng ASTA Z 7557/ml Elimination of B-ALL Cells from Human Bone Marrow by were identical to those of 50 Â¿Â¿gASTAZ 7557/ml. In contrast, ASTA Z 7557. To simulate a remission marrow with occult cells treated with a lower concentration of ASTA Z 7557 (25 ng/ leukemic cells, a 200-fold excess of irradiated (5000 R) normal, ml) partially recovered after 48 hr. This could be due to recovery density gradient separated bone marrow cells were added to 1 of the treated cells from the perturbing effects of ASTA Z 7557 x 106 B-ALL cells. The cell mixture was incubated with different or to the proliferation of cells that escaped or were partially concentrations of ASTA Z 7557 for 30 min at 37Â°, and the resistant to its effect. An almost complete recovery occurred clonogenic growth was measured. As can be seen in Chart 2, a when cells were incubated with 10 fig ASTA Z 7557/ml; by 72 dose-dependent elimination of B-ALL cells resulted and a maxi hr, the DMA histogram was nearly identical with the control. mum kill of 4.5 logs was obtained with 100 ng ASTA Z 7557/ml. Effect of 4-HC and B43-PAP on Clonogenic Growth of B- The extent of kill is not significantly different from the 4.7-log kill ALL Cells. B-ALL target cells (1 x 106) were treated either with obtained when 1 x 106 B-ALL cells were treated with 100 ^9 ASTA Z 7557 alone (30 min, 37Â°)or with ASTA Z 7557 (30 min, 37Â°)and then with B43-PAP (9 hr, 37Â°).After treatment, cells ASTA Z 7557/ml in the absence of added bone marrow cells, indicating that the antitumor activity of ASTA Z 7557 was not were washed twice with clonogenic medium and assayed for affected by the presence of normal bone marrow. clonogenic growth by limiting dilution. A dose-dependent killing Chart 2 also shows that the combination of ASTA Z 7557 and of B-ALL cells was observed with a maximum kill of 4.7 logs at immunotoxin is more effective than is ASTA Z 7557 alone in 100 tig ASTA Z 7557/ml (Table 1). tumor cell elimination. No clonogenic growth was observed after If the ASTA Z 7557 treatment was followed by incubation with treatment with ASTA Z 7557 at concentrations higher than 50 immunotoxin, more effective kill was observed at all ASTA Z Â¿tg/mlin combination with immunotoxin, indicating that the im 7557 concentrations. Treatment with 25 /Â¿gASTA Z 7557/ml in combination with 10 /Â¿gB43-PAP/ml eliminated more than 4 logs munotoxin can efficiently find its target in the presence of a large of clonogenic B-ALL cells. This treatment was not toxic to CFU- number of nontarget cells. The increased cytotoxicity in the GEMM and did not inhibit the production of CFU-GEMM in long- presence of immunotoxin is specific, since no added cytotoxicity was observed when ASTA Z 7557 was combined with an term bone marrow cultures as shown below. To better quantify irrelevant immunotoxin (T3-3A1-PAP) at a concentration of 10 the antitumor activity of higher concentrations of ASTA Z 7557 combined with B43-PAP, a very labor intensive modification of Mg/ml. Furthermore, the continuous presence of free B43 anti- the conventional clonogenic assay was used. Treatment of 1 x 7F. M. Uckun, S. Ramakrishnan,and L. L. Houston, unpublisheddata.

CANCER RESEARCH VOL. 45 JANUARY 1985 71

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. DRUG/IMMUNOTOXIN ELIMINATION OF LEUKEMIA CELLS days of culture, 11.3 Â± 1.53 CFU-GEMM/105 cells could be Tabte3 detected in control cultures compared to 11.6 Â± 1.15 CFU- flecovery of CFU-GEMM after treatment with ASTA Z 7557 and B43-PAP Highly purified stem cells (1x10*) were treated in the presence and absence GEMM/106 cells, 1.6 Â±0.57 CFU-GEMM/105 cells, and 0 Â±0 of 1 x 10* irradiated B-ALL cells with ASTA Z 7557 (30 min, 37Â°).After treatment, CFU-GEMM/105 cells in samples treated with 25, 50, and 100 the cells were washed once and incubated with 10 ^9 B43-PAP/ml (9 hr, 37Â°)and washed twice; 104 cells were assayed for stem cell colony formation in culture as nQ ASTA Z 7557/ml, respectively. (In both experiments, triplicate described in "Materials and Methods.' Results are the means of 10 replicated samples were plated for each mafosfamide dose.) To preclude experiments. Controls averaged 65 Â±5 CFU-GEMM/104 cells in the absence and that the stem cell toxicity is secondary to inhibition of some 30 Â±2 CFU-GEMM/104 cells in the presence of 1 x 10* irradiated B-ALL cells. The standard deviations are due to donor variations between experiments. regulatory cells or to bystander effects from dying cells other % recovery of CFU-GEMM than marrow progenitors, we tried to evaluate the sensitivty of CFU-GEMM to ASTA Z 7557 by in vitro clonal assays using ASTA Z75570 1 x 10* irradiated irradiated B-ALLcells60 highly purified stem cell suspensions. The results, shown in Table B-ALLcells57 Â±5" 2, were expressed as the percentage of progenitor cells re Â±10 covered per 103 to 104 treated cells compared to untreated cells 10 50 Â±10 55 + 5 25 60 Â±10 65 Â±10 cultured under the same conditions (see legend of Table 2). The 50 0Â±0 0Â±0 maximum ASTA Z 7557 concentration that was not toxic to 100With 1 Â±1Without 0Â±0 " Mean Â±S.D. CFU-GEMM has been determined to be 25 fig/ml. Higher con centrations of ASTA Z 7557 were highly toxic to CFU-GEMM, and nearly all measurable GFU-GEMM were lost after treatment treated marrow cultures. About 40% of CFU-GEMM were lost with 50 to 100 Mg ASTA Z 7557/ml (30 min, 37Â°). under conditions in which 4.4 logs of B-ALL cells were eliminated. Effect of ex Vivo Treatment of B43-PAP/ASTA Z 7557 on Addition of an equal number of irradiated B-ALL cells (1x10* Pluripotent Stem Cells. In 2 experiments 1 x 107 mononuclear cells) did not affect significantly the stem cell toxicity of the bone marrow cells were treated with 25, 50, or 100 ^g mafos- combined treatment. This suggests that target leukemic cells famide/ml (30 min, 37Â°)followed by 10 /ig B43-PAP/ml (9 hr, dying from the combination treatment would not affect the in 37Â°).A fixed 10-/ig/ml concentration of B43-PAP was chosen vivo repopulation capacity of the manipulated autologous mar as our previous studies showed that after treatment with higher row grafts. concentrations of B43-PAP more than 50% of pluripotent stem Long-term cultures were also used to evaluate the marrow cells were lost.6 toxicity of the combination treatment of 25 ^g ASTA Z 7557/ml After 14 days culture of triplicate samples 11.3 Â±1.53 GFU- and 10 tig B43-PAP/ml. In 2 experiments, 2 x 107 normal bone GEMM/105 cells were observed in control cultures compared to marrow cells were treated with 25 /tg ASTA Z 7557/ml, washed 7.7 Â±1.17 CFU-GEMM/105 cells, O Â±O CFU-GEMM/105 cells, once, incubated for 9 hr at 37Â°with 10 nQ B43-PAP/ml, washed, and O Â±O CFU-GEMM/105 cells in cultures treated with 25, 50, and then kept in culture. Subsequent production of CFU-GEMM and 100 ^g mafosfamide/ml in combination with 10 ^g B43-PAP/ was evaluated by plating 1.5 x 10s nonadherent mononuclear ml, respectively. As it is impossible to determine whether there cells per Retri dish every 2 weeks. After 4 weeks of culture, are bystander effects from dying B- and pre-B-cells, which react untreated controls showed 15 CFU-GEMM per 1 x 105 nonad with B43-PAP or other cells sensitive to mafosfamide, above and herent mononuclear cells, and the treated samples showed 10. beyond the effect of the combination protocol on pluripotent After 8 weeks of culture, controls and treated cells showed 11 and 9 CFU-GEMM per 1 x 105 mononuclear cells, respectively. stem cells in the conventional assay system, we also used highly purified stem cell suspensions to evaluate the effects of the combination treatment on the pluripotent marrow progenitors. Highly purified stem cell suspensions (1 x 107 cells/ml) were DISCUSSION treated with different concentrations of ASTA Z 7557 (30 min, A critical requirement for therapeutic efficiency of ABMT in 37Â°)followed by 10 ^g B43-PAP/ml (9 hr, 37Â°).Table 3 compares acute leukemia is some effective method of purging the autolo the CFU-GEMM recovered from 104 cells of treated and un- gous marrow graft of occult leukemic cells by either pharmaco logical or immunological methods. This paper describes a com Tabte2 bination of chemoseparation and immunoseparation to eliminate Recovery of CFU-GEMM after treatment with ASTA Z 7557 Highly purified stem cells (1 x 10") in 1 ml were treated with ASTA Z 7557 (30 leukemic cells from human marrow. The clonogenic assay used min, 37Â°), washed twice in medium 199 at 19Â°, and resuspended in Iscove's in this study is highly sensitive and can measure 8 logs of modified Dulbecco's medium. 103 to 10* cells were cultured for 14 days as elimination of tumor cells. We demonstrate that the combination described in "Materials and Methods.* Results are expressed as the percentage of of mafosfamide and B-cell-directed immunotoxin (B43-PAP) can progenitor cells recovered per 103 to 104 treated cells compared to untreated cells cultured under the same conditions. Results are the mean of 10 replicated experi eliminate nearly all tumor cells added to a large excess of normal ments. Control cultures for all CFU-GEMM experiments shown averaged 7 Â±1 human bone marrow cells. Addition of chloroquine also enhances CFU-GEMM/103 cells and 65 Â±5 CFU-GEMM/104 cells, indicating an approximately 60-fold enrichment of pluripotent stem cells. The standard deviations are due to the action of immunotoxin cytotoxicity. Immunotoxins have now donor variations between experiments. been shown to be selective in their action by several groups; ASTA Z 7557 CFU-GEMM there is little doubt that their use will be of clinical significance in the future. 0102550100100 Â±0a91 We have shown previously that ex vivo treatment with B43- Â±15100 PAP can selectively and effectively eliminate contaminating B- Â±202Â±10Â±0 ALL lymphoblasts from human marrow and have demonstrated that less than 50% of pluripotent stem cells were lost under Mean Â±S.D. conditions in which 6 logs of B-ALL cells were eliminated by

CANCER RESEARCH VOL. 45 JANUARY 1985 73

B43-PAP in the presence of 40 nu chloroquine. In this study, we by long-term marrow cultures was not inhibited significantly after asked whether the extent of tumor cell elimination by immuno- this combination treatment. The better recovery of stem cells toxin could be enhanced by combination with mafosfamide. One during the later treatment schedule is due to the decreased potential advantage of the use of immunotoxins is that the concentration of ASTA Z 7557 used. efficiency may not depend on the proliferation rate of target cells High concentrations (50 to 100 M9/ml) of ASTA Z 7557 are and quisescent noncycling tumor stem cells can be effectively highly toxic to CFU-GEMM, but CFU-GEMM is probably not the eliminated if they bear the antigen. As we used rapidly cycling pluripotent stem cell absolutely necessary for successful engraft- tumor cells in our experimental system, we may have overesti ment (30).8 This follows from the observation of full hematological mated the ability of ASTA Z 7557 to inhibit the growth of leukemic recovery in all patients receiving autologous marrow grafts cells. On the other hand, some antigens may appear and disap treated ex vivo with 100 ^g 4-HC/ml (14), which has comparable pear within the cell cycle, and it is conceivable that cell cycle effects to ASTA Z 7557 (4). Therefore, CFU-GEMM inhibition at dependency will be seen with immunotoxins directed against this level does not necessarily preclude the use of ASTA Z 7557 such antigens. Other antigens, such as common acute lympho- at 50 to 100 Mg/ml concentration. The same is true for combi blastic leukemia antigen, may undergo antigenic modulation (24, nation of B43-PAP with high concentrations of ASTA Z 7557 25). Lymphoblasts may be encountered in practice that are more that resulted in elimination of 7 to 8 logs of contaminating target difficult to be inhibited by B43-PAP than B-ALL cells. Differences B-ALL cells. A clinical study is clearly needed to verify the in target cell sensitivity independent of antigen density have been repopulation capacity of autologous marrow grafts treated with reported for T-cell-directed immunotoxins (21). These potential immunotoxin and high concentrations of ASTA Z 7557. obstacles can be removed, however, using a cocktail of immu This study indicates that the combined treatment with B43- notoxins (34). Our preliminary data indicate that combination of PAP and ASTA Z 7557 has clinical potential for ABMT. Combined 2 PAP-containing immunotoxins are not more toxic to pluripotent use of immunotoxins and drugs, such as ASTA Z 7557, is likely stem cells than are the single immunotoxins. to improve therapy over immunotoxins alone or protocols using In this study, most stem cell toxicity assays are done on a antibody and complement. All these systems will have particular highly purified stem cell suspension. One advantage of this model advantages and disadvantages based on the characteristics of system is the higher probability that mixed colonies observed the tumor cells involved. As variations would undoubtedly be are derived from single pluripotent stem cells, as the cultures encountered when these studies are transferred into clinical were initiated with more dilute cell suspensions (103 to 104 cells/ settings, it is of importance to increase the number of reagents ml) compared to our conventional assay conditions, in which 1 available. to 2 x 10s cells/ml are seeded. Studies of the distributions of isoenzymes of glucose-6-phosphate dehydrogenase among he- ACKNOWLEDGMENTS mopoietic colonies have shown that these are true clones only We thank Dr. Dietrich Haag, Institute for Experimental Pathology, Heidelberg, when cultures are started with very dilute cell suspensions (16). for DNA-FCM analysis. Dr. Hans Burkert (ASTA Werke, Federal Republic of A second advantage is the measurement of the direct effects of Germany)for a generous gift of ASTA Z 7557, and Dr. J. D. Griffin, Dana FÃ¤rber the purging methods on the pluripotent stem cells. Despite these Cancer Institute, Boston, MA, for monoclonalantibodies for stem cell purification. advantages and the higher sensitivity of the assays due to the We also wish to thank Dr. R. C. Bast (Harvard Medical School. Boston, MA) for higher frequency of mixed colonies (65 CFU-GEMM/10" cells), his advice in establishingthe limiting dilution assay. there is a question of whether this system is an appropriate model for ex vivo "purging" of autologous marrow grafts, as the REFERENCES presence of the many elements in remission marrow, in addition 1. Bast, R. C., Jr., Ritz, J., Upton, J. M., Feeney, M., Sallan, S. E., Nathan, D. to the pluripotent stem cells, may significantly impair or potentiate G., and Schlossman, S. F. Elimination of leukemic cells from human bone marrow using monoclonalantibody and complement Cancer Res., 43: 1389- the effects of the purging methods against normal and/or leu 1394,1983. kemic cells. This will largely depend on the different agents used 2. Brock, N., Niemeyer,U., Pohl, J., and Scheffler, G. Stabilized primary metab and/or the target leukemic cells involved. With B43-PAP immu- olites of oxazophosphorine cytostatics with high cytotoxic specificity and cancerotoxic selectivity needingno enzymatic activation. Proceedingsof Third notoxin and mafosfamide, however, there was no significant NCI-EDRTC Symposium on New Drugs in Cancer Therapy (Institut Joules difference in stem cell toxicity measured by conventional CFU- Bordet, Brussels),Abstract 47,1981. 3. Blythman, H. E., Casellas, P., Gros, 0., Gros, P., Jansen, F. K., Paolucci, F., GEMM assay when compared to stem cell toxicity measured in Pau, B., and Vidal, H. Immunotoxins:hybrid moleculesof monoclonalantibod our model system which used highly purified bone marrow stem ies and toxin subunit specifically kill tumor cells. Nature (Lond.), 290: 145- cells. 146,1981. 4. Douay. L., Gorin, N. C., Gerota, I., Likforman, J., Najman, A., Baillou, C., This work indicates that the combination of ASTA Z 7557 and Leguen, M. C., and Duhamel, G. In vitro treatment of leukemic bone marow B43-PAP immunotoxin is significantly superior to immunosepa- for autologous transplantation: the interest of cyclophosphamidederivatives and lysosomotropic agents. Exp. Hematol., 11:152-154,1983. ration or chemoseparation alone. Treatment with 50 to 100 //g 5. Fauser, A. A., and Messner, H. A. Identification of megakaryocytes, macro ASTA Z 7557/ml followed by treatment with 10 ^g B43-PAP/ml phages,and eosmophils in colonies of human bone marrow containingneutro- eliminated 7 to 8 logs of target B-ALL cells. Complete CFU- philic granulocytes and erythroblasts. Blood, 53: 1023-1027, 1979. 6. Gartner, S., and Kaplan,H. S. Long-termculture of human bone marrow cells. GEMM inhibition was observed after this treatment, and further Proc. Nati. Acad. Sci. USA, 77: 4756-4759, 1980. study is needed to evaluate the repopulation capacity of autolo 7. Griffin, J. D., Beveridge. R. P., and Schlossman, S. F. Isolation of myetoid progenitor cells from peripheral Wood of chronic myetogenous leukemia pa gous marrow grafts after such a protocol. The addition of chlor tients. Blood, 60: 30-37, 1982. oquine to this protocol during the immunotoxin treatment in 8. Haag, D. Flow microfluorometricdeoxyribonucleicacid (DNA)analysissupple creased the extent of kill from 4 to 7 logs when 25 ng ASTA Z menting routine histopathologic diagnosis of biopsy specimens. Lab. Invest., 42: 85-90, 1980. 7557/ml and 10 //g B43-PAP/ml were used. CFU-GEMM recov ery was reduced by 40%, but CFU-GEMM recovery as measured " G. Santos and H. Kaizer, personal communication.

CANCER RESEARCH VOL. 45 JANUARY 1985 74

9. Hokland, P., Rosenthal, P., Griffin, J. D., Nadler, L. M., Datey,J., Hokland, M., 24. Raso, V., Ritz, J., Basala, M., and Schlossman, S. F. Monoclonal antibody- Schlossman, S. F., and Ritz, J. Purification and characterization of fetal ricin-A chain conjugate selectively cytotoxic for cells bearing the common hematopoietic cells that express the common acute lymphoblastic leukemia acute lymphoblasticleukemiaantigen. Cancer Res., 42: 457-464,1982. antigen (CALLA). J. Exp. Med., 157: 114-129, 1983. 25. Ritz, J., Pesando,J. M., Notis-McConarty,J. and Schlossman,S. F. Modulation 10. Houston, L. L., Ramakrishnan,S., and Hermodson, M. A. Seasonalvariations of humana acute lymphoblastic leukemia antigen induced by monoclonal in different forms of pokeweed antiviral protein, a potent inactivator of ribo- antibody in vitro. J. Immunol., 725:1506-1514,1980. somes. J. Biol. Chem., 25: 9601-9604, 1983. 26. Sallan,S. E., and Hitchcock-Bryan,S. Relapsein childhoodacute lymphoblastic 11. Hubert, J. J. Bioassay, pp. 93-95. Dubuque:Kendall/Hunt Publishing Co., leukemia after elective cessation of initiall treatment: failure of subsequent 1980. treatment. Med. Pediatr.Oncol., 9: 455-462,1981. 12. Inali, A., Sallan,S. E., Cassady, T. R., Hitchcock-Bryan,S., Clavell,L. A., Belli, 27. Santos, G. W., and Kaizer, H. In: B. Lowenberg and A. Hagenbeck (eds.), J. A., and Sollee, N. Efficacy and morbidity of central nervous system "prophy MinimalResidualDiseasein Acute Leukemia, pp. 165-181. Boston: Martinus laxis" in childhood acute lymphoblastic leukemia:eight years' experience with Nijhoff Publishers,1984. cranial irradiation and intrathecal methotrexate. Blood, 61: 297-303,1983. 28. Schauer, P., Artin, Z. A., Mertelsmann, R., Cirrincione, C., Friedman,A., Gee, 13. Johnson, E. A., and Brown, B. W. The Spearman estimator for serial dilution T. S., Dowling, M., Kempin,S., Straus, D. J., Koziner, B., McKenzie,S., Thaler, assays. Biometrics, 17: 79-88,1961. H. T., Dufour, P., Little, C., Dellaquila,C., Ellis, S. and Clarkson, B. Treatment of acute lymphoblastic leukemia in adults: results of the L-10 and L-10M 14. Kaizer, H., and Santos, G. W. Bone marrow transplantation in acute leukemia. Semin. Hematol. 19: 227-240,1982. protocols. J. Clin. Oncol., 7: 462-470, 1983. 15. Krolick, K. A., Uhr, J. W., Slavin, S., and Vitetta, E. S. In vivo therapy of a 29. Sharkis,S. J., Santos, G. W., and Colvin, M. Eliminationof acute myelogenous murine B cell tumor (BCL,) using antibody-neinA chain immunotoxins.J. Exp. leukemic cells from marrow and tumor suspensions in the rat with 4-hydro- peroxycyclophosphamide.Blood, 55. 521-523,1980. Med., 755:1797-1809, 1982. 30. Siena, S., Castro-Malaspina,H., Gulati, S., Lu, L., Cartagena, T., O'Reilly, R. 16. McCulloch, E. A. Stem cells in normal and leukemia hemopoiesis. Blood, 62: J., Clarkson, B. D., and Moore, M. A. S. In vitro purging with hydroperoxycy- 1-13, 1983. clophosphamide(4-HC)and its effects on hematopoieticand stromal elements 17. Muirhead, M., Martin, P. J., Torok-Storb, B., Uhr, J. W., and Vitetta, E. S. Use of human bone marrow. Second International Conference on Malignant Lym- of an antibody-nan A chain conjugate to delete neoplasticB cells from human bone marrow. Blood. 62: 3237-3332,1983. phoma, Lugano, Abstract 92, 1984. 18. Olsnes,S., and Pihl,A. Chimerictoxins. Pharmacol.Ther, 75: 355-381,1982. 31. Thomas, E. D. Bone marrow transplatation: a lifesaving applied art. J. Am. 19. O'Reilly, R. J. Altogenicbone marrow transplantation:current status and future Med. Assoc., 249: 2528-2536, 1983. 32. Uckun, F., Korbling, M., and Hunstein, W. Assessment of clonogenic tumor directions. Blood, 62: 941-964, 1983. stem cell eliminationfrom the autologous marrow graft. InternationalSympos 20. Ramakrishnan, S., Eagle, M. R., and Houston, L. L. Radioimmunoassayof ium on Detection and Treatment of Minimal Residual Disease in Acute Leu ricin A- and B-cnains applied to samples of ricin A-chain prepared by chroma- kemia(TheDr. DanielDenhoedCancerCenter),Rotterdam,Abstract 38,1983. tofocusing and by DEAE Bio-GelA chromatography. Biochim. Biophys. Acta, 33. Uckun, F., Korbling, M., and Hunstein,W. In vitro chemotherapy as a prelude 779:341-348,1982. to autologous marrow transplantation (ABMT) in high-grade malignant non 21. Ramakrishnan,S., and Houston, L. L. Inhibitionof humanacetatelymphoblastic Hodgkin's lymphomas.J. Cancer Res. Clin. Oncol., 707:47, 1984. leukemia cells by immunotoxins: potentiation by chloroquine. Science (Wash. 34. Vallera,D. A., Ash, R. C., Zanjani, E. D., Kersey,J. H., LeBien,T. W., Beveriey, DC), 223: 58-61, 1984. P. C. L., Neville, D. M., Jr., and Youle, R. J. Anti-T-cell reagents for human 22. Ramakrishnan, S., and Houston, L. L. Comparison of the selective cytotoxic bone marrow transplantation: ricin linked to three monoclonal antibodies. effects of immunotoxins containing ricin A chain or pokeweed antiviral protein Science(Wash. DC),512-515, 1983. and anti-Thy 1.1 monoclonalantibodies. Cancer Res., 44: 201-208,1984. 35. Vitetta, E. S., Krolick, K. A., Miyama-lnaba, M., Cushly, W., and Uhr, J. W. 23. Ramakrishnan, S., and Houston, L. L. Prevention of growth of leukemia cells Immunotoxins:a new approach to cancer therapy. Science (Wash. DC), 209: in mice by monoclonal antibodies directed against thy 1.1 antigen disulfide 644-650,1983. linked to two ribosomal inhibitors: pokeweed antiviral protein or ricin A chain. 36. Woodruff, R. The managementof adult ALL. Cancer Treat. Rev., 5: 95-113, Cancer Res., 44: 1398-1404,1984. 1978.

CANCER RESEARCH VOL. 45 JANUARY 1985 75 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1985 American Association for Cancer Research. Increased Efficiency in Selective Elimination of Leukemia Cells by a Combination of a Stable Derivative of Cyclophosphamide and a Human B-Cell-specific Immunotoxin Containing Pokeweed Antiviral Protein

F. M. Uckun, S. Ramakrishnan and L. L. Houston

Cancer Res 1985;45:69-75.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/1/69

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/45/1/69. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.