Clinical Cancer Research The Science of Cancer Health Disparities Stand Up to Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Robbins, D. H.
Right arrow Articles by Kreitman, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Robbins, D. H.
Right arrow Articles by Kreitman, R. J.
Clinical Cancer Research Vol. 6, 693-700, February 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Hairy Cell Leukemia, a B-Cell Neoplasm That Is Particularly Sensitive to the Cytotoxic Effect of Anti-Tac(Fv)-PE38 (LMB-2)

David H. Robbins, Inger Margulies, Maryalice Stetler-Stevenson and Robert J. Kreitman1

Laboratory of Clinical Pathology, Division of Cancer Therapy, National Cancer Institute, NIH, Bethesda, Maryland 20892


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-Tac(Fv)-PE38 (LMB-2) is a recombinant, single-chain immunotoxin composed of the variable domains of the anti-Tac (anti-CD25) monoclonal antibody fused to a truncated form of Pseudomonas exotoxin (PE). Until now, this agent has been reported to be very cytotoxic toward T-cell but not B-cell leukemic cells freshly obtained from patients and is being tested clinically in patients with CD25+ malignancies of both B- and T-cell origin. Hairy cell leukemia (HCL) is a B-cell malignancy in which the cells are usually CD25+ and their ex vivo sensitivity to LMB-2 was unknown. Malignant cells from the first HCL patient to be tested were very sensitive to the cytotoxic effect of LMB-2 in vitro (IC50, 1.1 ng/ml), and this patient responded clinically to LMB-2 administered systemically. Therefore, we decided to assess the potential clinical utility of LMB-2 in other patients with HCL. We tested fresh leukemic cells from nine additional CD25+ HCL patients. LMB-2 was very cytotoxic ex vivo in all patients with IC50s as low as 0.5 ng/ml. Malignant cells freshly obtained from patients with chronic lymphocytic leukemia were also sensitive to LMB-2 but not as sensitive as cells from HCL patients. These results indicate that CD25+ HCL is a B-cell neoplasm that is particularly sensitive to LMB-2, and this agent may be useful in patients who have failed standard therapies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCL2 is a B-cell malignancy that comprises 2% of all leukemias (1 , 2) . Although not curative, the purine analogues 2'-deoxycoformycin and 2-chlorodeoxyadenosine can each induce long-term complete remissions in most patients, but 10–20% of patients are initially or eventually refractory to chemotherapy (3, 4, 5, 6, 7) . These patients can live for many years with considerable morbidity attributable to pancytopenia, requiring frequent transfusions of blood and platelets, and antibiotics for opportunistic infections. The malignant cells in 80% of cases express high surface levels of the {alpha} subunit of the IL2R (IL2R{alpha}), also referred to as CD25, Tac, or p55 (8) . We decided to target these malignant cells using a recombinant immunotoxin, anti-Tac(Fv)-PE38 (LMB-2), which binds to CD25, internalizes into the target cell, and results in cell death.

LMB-2 is a Mr 63,000 single-chain protein containing the variable heavy domain of the monoclonal antibody anti-Tac (9) fused via a 15-amino acid linker to the variable light domain, which in turn is fused to a Mr 38,000 truncated form of PE (10) . The truncated toxin, PE38, is devoid of the domain that binds to normal cells and contains the translocating and ADP-ribosylating domains. On the basis of structural (11 , 12) and functional (13) studies, intoxication by LMB-2 has been shown to require binding to CD25, internalization and processing of the toxin within its translocation domain (14, 15, 16) , binding of the Mr 35,000 COOH-terminal fragment of the toxin to the intracellular KDEL receptor, which carries it to the endoplasmic reticulum (17 , 18) , translocation of the toxin into the cytoplasm (19 , 20) , and finally ADP-ribosylation of elongation factor 2, leading to apoptosis and cell death (21 , 22) . The native COOH terminus of the toxin consists of the residues REDLK, and after removal of the terminal lysine residue, the sequence REDL has been shown to bind to the KDEL receptor (18) . When the REDLK sequence is changed to KDEL, the resulting recombinant toxin anti-Tac(Fv)-PE38KDEL binds with higher affinity to the KDEL receptor and consequently has higher cytotoxic activity but also higher animal toxicity (17 , 23) . Preclinical studies with LMB-2 showed that it produced complete regressions of CD25+ tumors in mice (10) , and toxicology studies showed that blood levels causing tumor regression of mouse xenografts are well tolerated by monkeys (24) . LMB-2 binds to both human and primate CD25 but not murine CD25. LMB-2 is very cytotoxic toward malignant cells freshly isolated from patients with adult T-cell leukemia (25, 26, 27, 28) . However, LMB-2 is not very cytotoxic toward cells from most patients with B-CLL, unless the COOH terminus REDLK is changed to KDEL (17 , 29) . To determine whether LMB-2 would result in clinical responses in patients, we began Phase I testing with LMB-2 in patients with CD25+ hematological malignancies of both B- and T-cell origin.

The experiments described in the present study were prompted by the results of a cytotoxicity assay in which fresh malignant cells from an HCL patient who had failed standard and salvage therapy were exposed to LMB-2. This assay showed that the malignant B-cells were very sensitive to LMB-2. This patient was subsequently treated with LMB-2 and had a rapid response with virtual clearing of malignant cells from the peripheral blood. The purpose of the present study was to examine HCL cells ex vivo from patients in a variety of stages of disease to determine whether such patients might also be candidates for LMB-2 therapy. The cells were also incubated with control molecules to investigate whether the cytotoxic activity of LMB-2 was specifically mediated by CD25 on the surface of the malignant cells and whether the cell death was toxin mediated.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Cells.
Malignant cells from patients were obtained as part of approved protocols at NIH. Anticoagulated venous blood was obtained from patients and used within 24 h. The blood was diluted 1:1 with PBS, centrifuged over Ficoll, and incubated with recombinant toxins or control molecules in 100-µl aliquots in media consisting of leucine-free RPMI, RPMI, and fetal bovine serum (88:2:10). The cell concentration was 106/well. After 3 days of incubation with immunotoxin, the cells were incubated with either [3H]leucine (1 µCi/well) or WST-1 reagent (Boehringer-Mannheim, Gaithersburg, MD; 10 µl/well) for 4–8 h. Cells labeled with [3H]leucine were frozen and thawed, harvested onto protein-binding glass-fiber filters, and counted on a Betaplate scintillation counter (Pharmacia-LKB, Gaithersburg, MD) to determine inhibition of protein synthesis. Cells labeled with WST-1, which reacts with mitochondrial dehydrogenases, were read at 450 nm (A450) after subtracting the absorbance at 680 nm.

Recombinant Toxins and Negative Control Molecules.
Anti-Tac(Fv)-PE38 (LMB-2), anti-Tac(Fv)-PE38KDEL, and the control molecules anti-Tac(Fv)-PE38KDELAsp553 and B1(dsFv)-PE38 were described previously (10 , 30) . Anti-Tac(Fv)-PE38 and anti-Tac(Fv)-PE38KDEL differ only in that the former molecule ends in REDLK, whereas the latter ends in KDEL. Anti-Tac(Fv)-PE38KDELAsp553 (LMB-2Asp553) is identical to anti-Tac(Fv)-PE38KDEL, except that it contains a Glu553Asp mutation that inactivates the ADP-ribosylation activity of PE without affecting other properties of the immunotoxin (10 , 21 , 31) . B1(dsFv)-PE38 was used as a control molecule because it binds to the Ley antigen, which is not present on hematopoietic cells but has the same toxin residues as LMB-2.

Binding Assay.
Cells from patients were washed with binding buffer (DMEM containing 0.2% sodium azide and 0.1% BSA), resuspended in binding buffer, and added in 0.15-ml aliquots to 96-well U-bottomed plates. For each patient sample, each well contained the same number of cells. Among the patients studied, the number of cells/well ranged from 2 x 106 to 1 x 107. Varying amounts of [125I]-labeled anti-Tac were added to the cells in 0.05-ml aliquots. After 45–90 min of incubation at 4°C with intermittent (20–30 min) suspension of the cells, the 96-well plate was centrifuged (4°C for 5 min at 2000 rpm), and the cells were washed twice with 0.2 ml of cold binding buffer, the radioactivity associated with the resuspended cells was counted, and the numbers of sites/cell were calculated using Scatchard plots.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ex Vivo Malignant Cell Sensitivity to LMB-2 in a Patient with HCL.
To determine whether HCL cells would be sensitive to the cytotoxic effect of LMB-2, we partially purified HCL cells from the peripheral blood of patients by Ficoll centrifugation, incubated the cells with LMB-2 for 3 days, labeled the cells with [3H]leucine, and measured the decrease in protein synthesis. Fig. 1ACitation shows that cells from the first patient with HCL were very sensitive to LMB-2, with a concentration-dependent inhibition of protein synthesis. The concentration of LMB-2 required for half-maximal inhibition of protein synthesis (IC50) was 1.1 ng/ml, and protein synthesis was inhibited 99.5% at 10 ng/ml.



View larger version (42K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Ex vivo response of HCL cells to LMB-2. Malignant cells from HCL patients 1–10 (A–J, respectively) were incubated with the indicated concentrations of either LMB-2 (•) or the control molecules B1(dsFv)-PE38 ({circ}) and LMB-2Asp553 ({blacktriangledown}). The cells were labeled with [3H]leucine to determine inhibition of protein synthesis. Dashed line, 50% of the protein synthesis (in cpm) in the absence of toxin. Bars, SDs from the means of triplicate experiments.

 
Ex Vivo Sensitivity of HCL Cells from Other Patients to LMB-2.
The result in the first patient prompted us to obtain data on more patients with HCL. The clinical characteristics of the patients studied are summarized in Table 1Citation . Patients varied greatly with respect to years since diagnosis; patients 3 and 9 were newly diagnosed, patient 5 had a 1-year history but was not treated, and patient 7 had a 37-year history with a variety of previous therapies. The average age at the time of diagnosis was 47 ± 10, and the male:female ratio was 4, which is similar to those values (52–54 years and 3.6–3.9, respectively) reported previously in reviews of 211 and 725 patients (32 , 33) . All of the patients with high WBC counts (>15,000/mm3) were treated previously with splenectomy. Most of the patients were pancytopenic, and nearly half were transfusion dependent. All 10 patients had HCL cells in the peripheral blood. The results of FACS analyses are also summarized in Table 1Citation . FACS analyses in all patients were diagnostic of HCL with strongly positive staining for CD11c and at least positive or strongly positive staining for CD103 and B-cell antigens. Thus, the patients used for this study had characteristics typical for HCL, although patients with very low WBC counts were underrepresented because of insufficient numbers of malignant cells to test.


View this table:
[in this window]
[in a new window]

  		Table 1 Clinical characteristics of HCL patients at the time of cytotoxicity assay

 
As shown in Fig. 1Citation , malignant cells from patients 1–10 were all sensitive to the cytotoxic effect of LMB-2. The level of protein synthesis used to determine IC50s was halfway between the maximal incorporation in the absence of toxin and the minimum (background) amount of incorporation in the presence of cycloheximide (10 µg/ml). IC50s are listed in Table 2Citation . For comparison, control cells were obtained from two patients with variant HCL where CD25 was not expressed (patients 11 and 12). Maximum inhibition for the CD25+ HCL patients varied, with the percentage of HCL cells making up the sample of cells tested. When nearly all of the cells in the sample were HCL cells as in patients 1 and 6, inhibition of the sample was >99%. Thus, there was no evidence of a LMB-2-resistant population of malignant cells. The IC50s in most patients varied from 0.5 to 6 ng/ml (8–95 pM), except in patient 9 (Fig. 1I),Citation where the IC50 was higher, at least in part, because of the benign cells that made up most of the sample; normal peripheral blood mononuclear cells contain mostly resting T-cells, which are CD25 negative and have been shown previously to be resistant to LMB-2 (26 , 29) . Although preparative sorting techniques could not yield sufficient numbers of HCL cells to test from patients with low malignant cell counts, trypan blue analysis confirmed that it was the malignant cells in these samples that were being killed (data not shown). Thus, all of the CD25+ HCL patients tested had malignant cells that were significantly sensitive to LMB-2 ex vivo.


View this table:
[in this window]
[in a new window]

  		Table 2 Cytotoxicity of LMB-2 toward fresh HCL cells

 
Specificity of the Cytotoxicity of LMB-2 on HCL Cells.
Several control experiments were performed to determine whether the cytotoxic activity of LMB-2 on the HCL cells was attributable either to the cells nonspecifically internalizing LMB-2 or because of a toxin-independent pathway, where the binding of ligand to CD25 on the cells triggers cell death. To determine the cellular susceptibility to nonspecific internalization of toxin, the HCL cells were incubated with the control molecule B1(dsFv)-PE38, which binds to the Ley antigen present on solid tumors and not on hematopoietic cells (30) . This protein, also Mr 63,000, has the same toxin domain as LMB-2. B1(dsFv)-PE38 was not significantly cytotoxic toward any of the 10 HCL cell samples (Fig. 1)Citation , indicating that the HCL cells do not nonspecifically internalize PE38. The toxin PE38KDEL without a binding domain (34) was also tested and was not cytotoxic to the HCL samples (data not shown). To determine whether a toxin domain is necessary for LMB-2-induced cytotoxicity, HCL cells from patients 2 to 10 were incubated with the LMB-2 mutant LMB-2Asp553, which lacks ADP ribosylation activity. As shown in Fig. 1Citation , HCL cells from patients 2 to 10 showed no sensitivity to LMB-2Asp553. Thus, the cytotoxicity of LMB-2 on the HCL cells requires both binding to CD25 on the cell surface, internalization, and ADP-ribosylation activity of the toxin within the cytoplasm of the HCL cell. A further control regarding the specificity of the LMB-2 is the fact that HCL cells, which were CD25 negative, were resistant to the cytotoxic effect of LMB-2 (Table 2)Citation .

Correlation of CD25 Expression with LMB-2 Cytotoxicity.
To determine the relationship between cytotoxicity and number of CD25 sites/cell, radiolabeled binding assays were performed to quantitate CD25 expression. Cells at 4°C in the presence of azide were incubated with increasing concentrations of [125I]-labeled-anti-Tac (IgG) in the presence or absence of an excess of unlabeled LMB-2, the washed cells were counted, and numbers of sites/cell were computed from Scatchard plots. As shown in Table 2Citation , the CD25+ HCL cells expressed between 1250 and 7200 sites/cell, whereas the CD25-negative HCL cells had <200 sites/cell. Patients 1 and 4 had the highest CD25 expression and also the lowest IC50s. IC50s and CD25 expression in the other patients were very similar (3.1–6.1 ng/ml and 1250–4000 sites/cell). The binding studies indicate that HCL cells with high CD25 expression should be very sensitive to LMB-2.

Effect of the KDEL Mutation on the Cytotoxic Activity of LMB-2 toward HCL Cells.
It was determined previously that freshly obtained ATL cells were sensitive to LMB-2, but B-CLL cells were generally not unless the COOH terminal residues of the toxin, REDLK, were mutated to KDEL (17 , 26, 27, 28, 29) . To determine whether the KDEL COOH terminus would increase the cytotoxicity of LMB-2 toward HCL cells, HCL cells were tested simultaneously with either LMB-2 or anti-Tac(Fv)-PE38KDEL. As shown in Table 3Citation , it was unexpectedly found that the KDEL COOH terminus had very little effect on cytotoxicity. In none of the seven samples tested with both immunotoxins was there more than a 2-fold difference in cytotoxicity between the two immunotoxins. The HCL cells were incubated under identical conditions as reported previously for CLL cells (29) , except that to enhance cell viability during short-term culture, the cells were incubated at a concentration of 107/ml instead of 106/ml (both 100 µl/well).


View this table:
[in this window]
[in a new window]

  		Table 3 Effect of KDEL terminus on CD25-mediated cytotoxicity toward HCL and other leukemias.

 
To determine whether the improved culturing conditions were related to the lack of difference between LMB-2 and anti-Tac(Fv)-PE38KDEL on HCL cells, B-CLL samples were also tested at 107/ml. As shown in Table 3Citation , the sensitivity of B-CLL samples to LMB-2 (which does not have the KDEL COOH terminus) was much greater than reported previously, with 6 of 13 samples being sensitive to LMB-2 with IC50s <50 ng/ml. However, in most (9 of 13) samples, anti-Tac(Fv)-PE38KDEL was over 10-fold more cytotoxic than LMB-2, and in all but B-CLL sample 1, the difference was >2-fold. In contrast, Table 3Citation shows that in patients with T-cell leukemias, including two with T-CLL (nos. 1 and 2) and three with ATL (nos. 3–5), the difference in sensitivity with or without the KDEL COOH terminus was always <10-fold and usually <2-fold. All of the IC50s shown in Table 3Citation represent specific cytotoxicity that could not be produced with control molecules used in Fig. 1Citation (data not shown). Thus, the sensitivity of HCL cells to LMB-2 was similar to that observed in T-cell leukemias and was similar to the high sensitivity that B-CLL cells displayed only to the more active molecule anti-Tac(Fv)-PE38KDEL.

Correlation of Inhibition of Protein Synthesis to Cell Death.
The clinical response of patient 1 to LMB-2 was direct evidence that protein synthesis inhibition as measured by [3H]leucine incorporation leads to cell death. Nevertheless, it was necessary to determine quantitatively whether the protein synthesis inhibition assay was a relevant measure of the capacity of LMB-2 to kill HCL cells. Thus, when possible, cells from patients with HCL were incubated with LMB-2 and reacted with WST-1, which is a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in viable cells and is thus a measure of cell viability. As shown in Fig. 2Citation , all of the four HCL samples tested in this manner demonstrated the same concentration-dependent inhibition by the viability assay, as was observed by the protein synthesis inhibition assay. The background in the assay was the absorbance after treatment of the HCL cells with cycloheximide 10 µg/ml and varied from 0.23 to 0.69. The IC50s for HCL cells from patients 2, 3, 6, and 7 were very similar to those achieved by testing inhibition of protein synthesis (Fig. 1Citation and Tables 2Citation and 3Citation ). The control molecules B1(dsFv)-PE38 and LMB-2Asp553 were not cytotoxic toward HCL cells in the WST-1 assay, indicating that the inhibition in viability caused by LMB-2 on HCL cells required both binding to CD25 and ADP-ribosylation activity within the cytosol. Thus, protein synthesis inhibition caused by LMB-2 in HCL leads to cell death.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Correlation of inhibition of protein synthesis with HCL cell death to LMB-2. HCL cells from patients 2, 3, 6, and 7 (A–D, respectively) were incubated with LMB-2 (•), B1(dsFv)-PE38 ({circ}), and LMB-2Asp553 ({blacktriangledown}) as in Fig. 2Citation and pulsed with WST-1 reagent to determine cell viability. The dashed lines indicate the level of cell viability (in absorbance units), which is halfway between the maximal amount in the absence of toxin, and the minimum amount (background as indicated by the dotted line) in the presence of 10 µg/ml cycloheximide.

 
Time Course of LMB-2-induced Cytotoxicity on HCL Cells.
The 3-day incubation of HCL with recombinant immunotoxin was used in the above assays to allow sufficient time for the toxin to internalize, undergo intracellular transport to the cytosol, and inhibit protein synthesis. This long time of incubation could theoretically overestimate the likelihood that LMB-2 would kill HCL cells after bolus injection into patients, because after bolus administration, plasma LMB-2 concentration declines with a half-life of 4–8 h. To examine this possibility, HCL cells from patient 7 were incubated with LMB-2 or anti-Tac(Fv)-PE38KDEL for only 4 h, washed to remove recombinant toxin, and incubated with fresh media for a total of 7 days before measuring inhibition of protein synthesis. As shown in Fig. 3Citation A, LMB-2 was still cytotoxic toward HCL cells after just 4 h of exposure, with an IC50 of 60 ± 3 ng/ml. The IC50 was 18 ± 1.3 ng/ml if the cells were incubated for the same period of time (7 days) without washing out the toxin. Thus, a 40-fold increase in incubation time (7 days versus 4 h) led to a <5-fold increase in cytotoxicity, indicating that LMB-2 binds to HCL cells rather quickly and its cytotoxicity does not require prolonged time for receptor binding and recycling. Fig. 3BCitation shows the same for anti-Tac(Fv)-PE38KDEL, with the IC50 increasing only 4-fold from 5 ± 1.1 ng/ml after 7 days of incubation to 20 ± 3 ng/ml after 4 h of incubation. We compared the IC50s of LMB-2 and anti-Tac(Fv)-PE38KDEL after 3-day and longer incubations with HCL cells from patients 1, 2, and 5–7. Unlike B-CLL cells, which had 3–10-fold increased sensitivity with an additional day of incubation (29) , HCL cells usually had <2-fold increased sensitivity with increased incubation time (data not shown). Thus, the exposure time required for anti-CD25 immunotoxins to kill cells is shorter for HCL than B-CLL.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Relationship of time of incubation to cytotoxicity on HCL cells. HCL cells from patient 7 were incubated with LMB-2 (A) or anti-Tac(Fv)-PE38KDEL (B) for either 3 (•) or 7 ({blacktriangledown}) days, pulsed 6 h with [3H]leucine, harvested, and counted. Alternatively, the cells were incubated with toxins for 4 h, washed free of toxin, and then incubated with media for 7 days prior to pulsing ({circ}).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our goal in the present study was to determine whether a major clinical response in a patient with chemotherapy-refractory HCL was a unique case of B-cell sensitivity to LMB-2 or rather was indicative that in this B-cell malignancy, the cells are often very sensitive to LMB-2. To accomplish this, HCL cells freshly obtained from the responding patient were compared with HCL cells from other patients in terms of ex vivo sensitivity. We found that in all 10 patients with CD25+ HCL, the freshly obtained malignant cells were sensitive to LMB-2.

The cytotoxicity of LMB-2 on the HCL cells was found to be mediated by binding to CD25, which is strongly expressed on malignant cells in ~80% of patients with HCL (8) . The existence on HCL cells of high-affinity IL2Rs, which require {alpha}, ß, and {gamma} chain IL2R subunits, is not clear. It was reported originally that HCL cells express only the {alpha} chain (CD25) and that IL2 binding to HCL cells is predominantly low affinity (35 , 36) . More recently, the ß (p75) chain has been detected on HCL cells (37) , and in fact patients with variant HCL are reported to express ß (CD122) without {alpha} (CD25; Ref. 38 ). Nevertheless, freshly obtained HCL cells were reported by Bulger et al. (39) to be resistant to the recombinant IL2-toxin DAB486IL-2, which requires high-affinity IL2Rs for optimal cytotoxicity. In the current study, we tested malignant cells from CD25+ HCL patients 2, 6, and 7 and from the CD25-negative variant-HCL patient 11 with the improved recombinant IL2-toxin DAB389IL2 (40) and found all samples to be resistant (data not shown). Thus, immunotoxins that can bind to CD25 alone with high affinity, such as LMB-2 or other anti-CD25 immunotoxins, such as RFT5-dgA (41) and RFT5(scFv)-ETA' (42) , may have unique potential for treating this disease.

HCL cells appear to be particularly sensitive to the cytotoxic effect of LMB-2, similar in sensitivity to T-cell leukemias (Table 3)Citation . Several patients with CD25+ B-CLL were identified who also had malignant cells that were very sensitive to LMB-2, but HCL was more consistently sensitive. High surface expression of CD25 on HCL cells can only partly explain this phenomenon. For example, HCL patients 6–8 had malignant cells with only 1250–1900 sites/cell, and these HCL cells were much more sensitive to LMB-2 than were leukemic cells from B-CLL patients 3 and 7 with higher numbers of sites/cell. Thus, HCL cells may be particularly efficient, more so than some B-CLL cells, in internalizing and intracellularly transporting the immunotoxin after it binds to the cell surface. It was interesting that HCL cells differed from B-CLL cells in the lack of difference of their sensitivity to LMB-2 and anti-Tac(Fv)-PE38KDEL. The KDEL COOH terminus, which improves binding of the toxin to the intracellular KDEL receptor by 100-fold (17) , is not necessary for efficient HCL killing. This may be because the concentrations of internalized toxin and KDEL receptor may be extremely high within the compartment where toxin and KDEL receptor molecules bind, so that concentrations of these components are not limiting. In view of the similar cytotoxic activities of LMB-2 and anti-Tac(Fv)-PE38KDEL on HCL, LMB-2 would be the more appropriate agent for this disease because of its lower toxicity in nonhuman primates.

The results of 3-day assays should be relevant in predicting the sensitivity of HCL cells in patients to LMB-2, not only if given by continuous infusion but also if given by bolus injection. After 30-min i.v. infusion, the half-life of LMB-2 in patients with a variety of hematological malignancies usually ranges from 3 to 7 h. The sensitivity of HCL cells was only ~4-fold less if exposed to toxin for 4 h than if exposed for 7 days. Continuous infusion of other immunotoxins has not resulted in a dramatic increase in response rate and in some cases may be associated with higher rates of vascular leak syndrome because of prolonged exposure to endothelial cells (43 , 44) . The results presented in this study would argue that HCL patients, at least those whose major disease component is in the peripheral blood, might be effectively treated by bolus infusion.

Patient 1 discussed here was the first major response to LMB-2 and also to our knowledge the first major response of any cancer to an Fv-containing protein. Single-chain Fvs have previously shown utility for imaging and other diagnostic applications (45) rather than for the therapy of cancer. At this time, a total of 35 patients have been treated with LMB-2, with seven additional responses. Like patient 1, HCL patients 4 and 6 in this study also met the entry criteria of the clinical trial, which required failure of both standard and salvage therapy, and both went on to respond. Patient 4, in fact, met National Cancer Institute criteria for a complete remission, which is ongoing 18 months after beginning LMB-2. Patients 1, 4, and 6 correspond to patients 15, 30, and 35, respectively, whose clinical responses have been detailed recently (46) . A fourth patient (patient 32 in the Phase I trial), whose HCL cells constituted too small a percentage of her peripheral blood malignant cells to include in this ex vivo study, also was treated with LMB-2 and responded. In our Phase I trial, patients 15, 30, 32, and 35 began with circulating HCL counts of 63,900/µl, 478/µl, 350/µl, and 60,700, and the maximum percentage decrease with LMB-2 was 99.8%, >5 logs, 99%, and 98%, respectively (46) . In addition to major responses in HCL, LMB-2 has induced responses in patients with ATL, CLL, CTCL, and Hodgkin’s disease. All drug-related toxic effects were reversible and most commonly consisted of transaminase elevations and fever.

At the present time, 2'-deoxycoformycin and 2-chlorodeoxyadenosine are considered the most active agents for HCL, each capable of inducing complete clinical responses with minimal residual disease in most patients and long-term (4–8-year) relapse-free survival rates of ~75–85% (3 , 5 , 47) . Many of the HCL patients who fail these agents are refractory to chemotherapy, suffer great morbidity from infections and multiple transfusions, and are in need of new agents for treatment. There is limited clinical or preclinical data regarding biological or targeted therapy in HCL other than the IFNs. Clinical trials are also beginning to test the anti-CD20 monoclonal antibody Rituximab in this disease because of high surface expression of CD20. It is expected in the future that the unique surface antigen properties of HCL will lead to recognition of biological agents similar to LMB-2 that can effectively target this disease.


   ACKNOWLEDGMENTS
 
We thank Dr. Ira Pastan for helpful discussions and review of the manuscript, Dr. Thomas Waldmann for providing anti-Tac, and Dr. Q. C. Wang for producing some of the immunotoxins used in the study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Laboratory of Molecular Biology, Division of Cancer Biology, National Cancer Institute, NIH, 37/4B27, 37 Convent Drive, MSC 4255, Bethesda, MD 20892. Phone: (301) 496-6947; Fax: (301) 480-0843; E-mail: kreitmar{at}mail.nih.gov Back

2 The abbreviations used are: HCL, hairy cell leukemia; IL2R, interleukin 2 receptor; PE, Pseudomonas exotoxin; FACS, fluorescence-activated cell sorting; CLL, chronic lymphocytic leukemia; ATL, adult T-cell leukemia. Back

Received 7/29/99; revised 11/ 5/99; accepted 11/ 9/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bouroncle B. A., Wiseman B. K., Doan C. A. Leukemic reticuloendotheliosis. Blood, 13: 609-630, 1958.[Abstract/Free Full Text]
  2. Cheson, B. D., and Martin, A. Clinical trials in hairy cell leukemia. Current status and future directions [published erratum appears in Ann. Intern. Med., 107: 604, 1987]. Ann. Intern. Med., 106: 871–878, 1987.
  3. Saven A., Burian C., Koziol J. A., Piro L. D. Long-term follow-up of patients with hairy cell leukemia after cladribine treatment. Blood, 92: 1918-1926, 1998.[Abstract/Free Full Text]
  4. Grever M., Kopecky K., Foucar M. K., Head D., Bennett J. M., Hutchison R. E., Corbett W. E., Cassileth P. A., Habermann T., Golomb H., Rai K., Eisenhauer E., Appelbaum F., Cheson B. D. Randomized comparison of pentostatin versus interferon {alpha}-2a in previously untreated patients with hairy cell leukemia: an intergroup study [see comments]. J. Clin. Oncol., 13: 974-982, 1995.[Abstract]
  5. Cheson B. D., Sorensen J. M., Vena D. A., Montello M. J., Barrett J. A., Damasio E., Tallman M., Annino L., Connors J., Coiffier B., Lauria F. Treatment of hairy cell leukemia with 2-chlorodeoxyadenosine via the Group C protocol mechanism of the National Cancer Institute: a report of 979 patients. J. Clin. Oncol., 16: 3007-3015, 1998.[Abstract/Free Full Text]
  6. Carbone A., Reato G., Di Celle P. F., Lauria F., Foa R. Disease eradication in hairy cell leukemia patients treated with 2- chlorodeoxyadenosine. Leukemia (Baltimore), 8: 2019-2020, 1994.[Medline]
  7. Filleul B., Delannoy A., Ferrant A., Zenebergh A., Van Daele S., Bosly A., Doyen C., Mineur P., Glorieux P., Driesschaert P., Sokal C., Martiat P., Michaux J. L. A single course of 2-chloro-deoxyadenosine does not eradicate leukemic cells in hairy cell leukemia patients in complete remission. Leukemia (Baltimore), 8: 1153-1156, 1994.[Medline]
  8. Robbins B. A., Ellison D. J., Spinosa J. C., Carey C. A., Lukes R. J., Poppema S., Saven A., Piro L. D. Diagnostic application of two-color flow cytometry in 161 cases of hairy cell leukemia. Blood, 82: 1277-1287, 1993.[Abstract/Free Full Text]
  9. Uchiyama T. A., Broder S., Waldmann T. A. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J. Immunol., 126: 1393-1397, 1981.[Medline]
  10. Kreitman R. J., Bailon P., Chaudhary V. K., FitzGerald D. J. P., Pastan I. Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin-2 receptor-expressing human carcinoma. Blood, 83: 426-434, 1994.[Abstract/Free Full Text]
  11. Allured V. S., Collier R. J., Carroll S. F., McKay D. B. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0 Angstrom resolution. Proc. Natl. Acad. Sci. USA, 83: 1320-1324, 1986.[Abstract/Free Full Text]
  12. Li M., Dyda F., Benhar I., Pastan I., Davies D. R. Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation. Proc. Natl. Acad. Sci. USA, 93: 6902-6906, 1996.[Abstract/Free Full Text]
  13. Hwang J., FitzGerald D. J., Adhya S., Pastan I. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli.. Cell, 48: 129-136, 1987.[CrossRef][Medline]
  14. Chiron M. F., Fryling C. M., FitzGerald D. J. Cleavage of Pseudomonas exotoxin and diphtheria toxin by a furin-like enzyme prepared from beef liver. J. Biol. Chem., 269: 18167-18176, 1994.[Abstract/Free Full Text]
  15. Fryling C., Ogata M., FitzGerald D. Characterization of a cellular protease that cleaves Pseudomonas exotoxin. Infect. Immun., 60: 497-502, 1992.[Abstract/Free Full Text]
  16. Ogata M., Fryling C. M., Pastan I., FitzGerald D. J. Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol. J. Biol. Chem., 267: 25396-25401, 1992.[Abstract/Free Full Text]
  17. Kreitman R. J., Pastan I. Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem. J., 307: 29-37, 1995.
  18. Hessler J. L., Kreitman R. J. An early step in Pseudomonas exotoxin action is removal of the terminal lysine residue, which allows binding to the KDEL receptor. Biochemistry, 36: 14577-14582, 1997.[CrossRef][Medline]
  19. Theuer C., Kasturi S., Pastan I. Domain II of Pseudomonas exotoxin A arrests the transfer of translocating nascent chains into mammalian microsomes. Biochemistry, 33: 5894-5900, 1994.[CrossRef][Medline]
  20. Theuer C. P., Buchner J., FitzGerald D., Pastan I. The N-terminal region of the 37-kDa translocated fragment of Pseudomonas exotoxin A aborts translocation by promoting its own export after microsomal membrane insertion. Proc. Natl. Acad. Sci. USA, 90: 7774-7778, 1993.[Abstract/Free Full Text]
  21. Carroll S. F., Collier R. J. Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J. Biol. Chem., 262: 8707-8711, 1987.[Abstract/Free Full Text]
  22. Brinkmann U., Brinkmann E., Gallo M., Pastan I. Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1. Proc. Natl. Acad. Sci. USA, 92: 10427-10431, 1995.[Abstract/Free Full Text]
  23. Seetharam S., Chaudhary V. K., FitzGerald D., Pastan I. Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL. J. Biol. Chem., 266: 17376-17381, 1991.[Abstract/Free Full Text]
  24. Kreitman R. J., Pastan I. Targeting Pseudomonas exotoxin to hematologic malignancies. Semin. Cancer Biol., 6: 297-306, 1995.[CrossRef][Medline]
  25. Kreitman R. J., Chaudhary V. K., Waldmann T., Willingham M. C., FitzGerald D. J., Pastan I. The recombinant immunotoxin anti-Tac(Fv)-Pseuodomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia. Proc. Natl. Acad. Sci. USA, 87: 8291-8295, 1990.[Abstract/Free Full Text]
  26. Kreitman R. J., Chaudhary V. K., Waldmann T. A., Hanchard B., Cranston B., FitzGerald D. J. P., Pastan I. Cytotoxic activities of recombinant immunotoxins composed of Pseudomonas toxin or diphtheria toxin toward lymphocytes from patients with adult T-cell leukemia. Leukemia (Baltimore), 7: 553-562, 1993.[Medline]
  27. Saito T., Kreitman R. J., Hanada S-i., Makino T., Utsunomiya A., Sumizawa T., Arima T., Chang C. N., Hudson D., Pastan I., Akiyama S-i. Cytotoxicity of recombinant Fab and Fv immunotoxins on adult T-cell leukemia lymph node and blood cells in the presence of soluble interleukin-2 receptor. Cancer Res., 54: 1059-1064, 1994.[Abstract/Free Full Text]
  28. Kreitman R. J., Batra J. K., Seetharam S., Chaudhary V. K., FitzGerald D. J., Pastan I. Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: relative importance of the two toxin disulfide bonds. Bioconj. Chem., 4: 112-120, 1993.[CrossRef][Medline]
  29. Kreitman R. J., Chaudhary V. K., Kozak R. W., FitzGerald D. J. P., Waldmann T. A., Pastan I. Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the interleukin-2 receptor kill malignant cells from patients with chronic lymphocytic leukemia. Blood, 80: 2344-2352, 1992.[Abstract/Free Full Text]
  30. Benhar I., Pastan I. Characterization of B1(Fv)PE38 and B1(dsFv)PE38: single-chain and disulfide-stabilized Fv immunotoxins with increased activity that cause complete remissions of established human carcinoma xenografts in nude mice. Clin. Cancer Res., 1: 1023-1029, 1995.[Abstract]
  31. Ogata M., Chaudhary V. K., Pastan I., FitzGerald D. J. Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol. J. Biol. Chem., 265: 20678-20685, 1990.[Abstract/Free Full Text]
  32. Flandrin G., Sigaux F., Sebahoun G., Bouffette P. Hairy cell leukemia: clinical presentation and follow-up of 211 patients. Semin. Oncol., 11: 458-471, 1984.[Medline]
  33. Frassoldati A., Lamparelli T., Federico M., Annino L., Capnist G., Pagnucco G., Dini E., Resegotti L., Damasio E. E., Silingardi V. Hairy cell leukemia: a clinical review based on 725 cases of the Italian Cooperative Group (ICGHCL). Italian Cooperative Group for Hairy Cell Leukemia. Leuk. Lymphoma, 13: 307-316, 1994.[Medline]
  34. Kreitman R. J., Hansen H. J., Jones A. L., FitzGerald D. J. P., Goldenberg D. M., Pastan I. Pseudomonas exotoxin-based immunotoxins containing the antibody LL2 or LL2-Fab' induce regression of subcutaneous human B-cell lymphoma in mice. Cancer Res., 53: 819-825, 1993.[Abstract/Free Full Text]
  35. Begley C. G., Burton J. D., Tsudo M., Brownstein B. H., Ambrus J. L., Jr., Waldmann T. A. Human B lymphocytes express the p75 component of the interleukin 2 receptor. Leuk. Res., 14: 263-271, 1990.[CrossRef][Medline]
  36. Barnett D., Granger V., Reilly J. T. Expression of the p75 interleukin-2 receptor (beta subunit) in acute and chronic leukaemia. Br. J. Haematol., 76: 314-315, 1990.[Medline]
  37. Trentin L., Zambello R., Benati C., Cassatella M., Agostini C., Bulian P., Adami F., Carra G., Pizzolo G., Semenzato G. Expression and functional role of the p75 interleukin 2 receptor chain on leukemic hairy cells. Cancer Res., 52: 5223-5228, 1992.[Abstract/Free Full Text]
  38. de Totero D., Tazzari P. L., Lauria F., Raspadori D., di Celle P. F., Carbone A., Gobbi M., Foa R. Phenotypic analysis of hairy cell leukemia: "variant" cases express the interleukin-2 receptor ß chain, but not the {alpha} chain (CD25). Blood, 82: 528-535, 1993.[Abstract/Free Full Text]
  39. Bulger K., Murphy J., Janckila A., Nichols J., McCaffrey R. Peripheral blood hairy cell leukemia cells express only low affinity IL-2 receptors. Leuk. Res., 18: 101-104, 1994.[CrossRef][Medline]
  40. Murphy J. R., vanderSpek J. C. Targeting diphtheria toxin to growth factor receptors. Semin. Cancer Biol., 6: 259-267, 1995.[CrossRef][Medline]
  41. Schnell R., Vitetta E., Schindler J., Barth S., Winkler U., Borchmann P., Hansmann M. L., Diehl V., Ghetie V., Engert A. Clinical trials with an anti-CD25 ricin A-chain experimental and immunotoxin (RFT5-SMPT-dgA) in Hodgkin’s lymphoma. Leuk. Lymphoma, 30: 525 1998.[Medline]
  42. Barth S., Huhn M., Wels W., Diehl V., Engert A. Construction and in vitro evaluation of RFT5(scFv)-ETA', a new recombinant single-chain immunotoxin with specific cytotoxicity toward CD25+ Hodgkin-derived cell lines. Int. J. Mol. Med., 1: 249-256, 1998.[Medline]
  43. Grossbard M. L., Lambert J. M., Goldmacher V. S., Spector N. L., Kinsella J., Eliseo L., Coral F., Taylor J. A., Blattler W. A., Epstein C. L., Nadler L. M. Anti-B4-blocked ricin: a Phase I trial of 7-day continuous infusion in patients with B-cell neoplasms. J. Clin. Oncol., 11: 726-737, 1993.[Abstract]
  44. Sausville E. A., Headlee D., Stetler-Stevenson M., Jaffe E. S., Solomon D., Figg W. D., Herdt J., Kopp W. C., Rager H., Steinberg S. M., Ghetie V., Schindler J., Uhr J., Wittes R. E., Vitetta E. S. Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a Phase I study. Blood, 85: 3457-3465, 1995.[Abstract/Free Full Text]
  45. Divgi C. R., Scott A. M., Dantis L., Capitelli P., Siler K., Hilton S., Finn R. D., Kemeny N., Kelsen D., Kostakoglu L., Schlom J., Larson S. M. Phase I radioimmunotherapy trial with iodine-131-CC49 in metastatic colon carcinoma. J. Nucl. Med., 36: 586-592, 1995.[Abstract/Free Full Text]
  46. Kreitman, R. J., Wilson, W. H., Robbins, D., Margulies, I., Stetler-Stevenson, M., Waldmann, T. A., and Pastan, I. Responses in refractory hairy cell leukemia to a recombinant immunotoxin. Blood, in press, 2000.
  47. Flinn I. W., Kopecky K. J., Foucar M. K., Head D., Bennett J. M., Hutchison R. E., Corbett W. E. N., Cassileth P. A., Habermann T., Golomb H., Rai K., Eisenhauer E., Appelbaum F. R., Cheson B. D., Grever M. Long-term results in hairy cell leukemia treated with pentostatin. Proc. Am. Soc. Clin. Oncol., 90(Suppl.1): 578a 1997.



This article has been cited by other articles:


Home page
 BloodHome page
T. Decker, M. Oelsner, R. J. Kreitman, G. Salvatore, Q.-c. Wang, I. Pastan, C. Peschel, and T. Licht
Induction of caspase-dependent programmed cell death in B-cell chronic lymphocytic leukemia by anti-CD22 immunotoxins
Blood, April 1, 2004; 103(7): 2718 - 2726.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. Decker, S. Hipp, R. J. Kreitman, I. Pastan, C. Peschel, and T. Licht
Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides
Blood, February 15, 2002; 99(4): 1320 - 1326.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
R. J. Kreitman, W. H. Wilson, K. Bergeron, M. Raggio, M. Stetler-Stevenson, D. J. FitzGerald, and I. Pastan
Efficacy of the Anti-CD22 Recombinant Immunotoxin BL22 in Chemotherapy-Resistant Hairy-Cell Leukemia
N. Engl. J. Med., July 26, 2001; 345(4): 241 - 247.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Robbins, D. H.
Right arrow Articles by Kreitman, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Robbins, D. H.
Right arrow Articles by Kreitman, R. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online