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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3340-3348
Responses in Refractory Hairy Cell Leukemia to a Recombinant
Immunotoxin
By
Robert J. Kreitman,
Wyndham H. Wilson,
David Robbins,
Inger Margulies,
Maryalice Stetler-Stevenson,
Thomas A. Waldmann, and
Ira Pastan
From the Laboratory of Molecular Biology, the Laboratory of Clinical
Pathology, the Metabolism Branch, and the Medicine Branch, National
Cancer Institute, National Institutes of Health, Bethesda, MD.
 |
ABSTRACT |
We report major responses in 4 of 4 patients with hairy cell
leukemia (HCL) who have recently been treated on a phase I trial with
the recombinant immunotoxin LMB-2. The immunotoxin, designed to target
CD25+ malignancies, is composed of the Fv portion of the
anti-Tac (anti-CD25) antibody, fused to a 38-kD truncated form of
Pseudomonas exotoxin A, and has previously been called
anti-Tac(Fv)-PE38. All 4 HCL patients were resistant to standard and
salvage therapies for HCL, including 2-chlorodeoxyadenosine (CdA) and
interferon  , and all patients responded to LMB-2 after a single
cycle. One patient treated with 2 cycles had a complete remission (CR),
with regression of HCL cells from the blood and marrow and resolution of splenomegaly and pancytopenia. As is typical for patients in CR
after treatment with CdA, minimal residual disease was detectable by
flow cytometry of the bone marrow aspirate. This patient has not
relapsed after 11 months. Three other patients had 98% to 99.8%
reductions in malignant circulating cells. These results represent a
proof of principal that targeted therapy with recombinant Fv-containing
proteins can be clinically useful. LMB-2 may be an effective new
therapy for patients with chemotherapy-resistant CD25+ HCL.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
VARIABLE DOMAINS make up a small
percentage of the molecular weight of an antibody but contain all the
amino acids that bind to antigen. In 1988, genetic engineering was used
to produce recombinant Fv fragments in which the variable domains of
the heavy chain and light chain of the antibody were connected by a
peptide linker.1,2 Since then, more than 1,000 reports have
described single-chain Fv molecules engineered to target a variety of
antigens, many of which are antigens selectively expressed on cancer
cells. It has been hoped that these agents, connected to effector
molecules such as toxins or radionuclides, could cause regression of
malignant disease in patients. Although radiolabeled single-chain Fvs
were shown to successfully image tumors in patients,3 no
major responses have yet been reported in clinical therapy trials.
Antibodies have been used to target protein toxins to cancer cells as
immunotoxins; the toxins are potent enough so that only one or a few
molecules need to reach the cytoplasm of target cells to produce cell
death.4 Single-chain Fvs fused to toxins are termed
recombinant immunotoxins, and are produced by recombinant DNA
techniques in Escherichia coli. The molecules are extremely active and have been shown to kill cells with only a few hundred or
thousand binding sites.5 The first recombinant immunotoxin produced contained the variable domains of anti-Tac, the monoclonal antibody to the interleukin-2 (IL-2) receptor,6 fused to a truncated form of the bacterial toxin Pseudomonas exotoxin that is
devoid of its binding domain.7 For clinical development, a
closely related molecule, anti-Tac(Fv)-PE38 (LMB-2), was produced that
contains the variable heavy domain (VH) of anti-Tac fused via a 15 amino acid linker to the variable light domain
(VL), which in turn is fused to the amino terminus of a
38-kD truncated form (amino acids 253-364 and 381-613) of the
toxin.8 LMB-2 has been shown to be cytotoxic toward
malignant cells expressing CD25 that were either established as cell
lines or directly obtained from patients with hematologic
malignancies.9-12 The mode of cytotoxicity appears to
include binding to CD25, internalization and processing of the toxin
within its translocation domain,13-15 binding of the 35-kD
carboxyl terminus of the toxin intracellularly to the KDEL receptor
that carries it to the endoplasmic reticulum,16,17 translocation of the toxin into the cytoplasm,18,19 and
catalytic ADP-ribosylation of elongation factor 2, leading to apoptosis and cell death.20,21 In vivo, LMB-2 produced complete
regressions of CD25+ tumors in mice.8
Toxicology studies showed that blood levels causing tumor regression in
mouse xenografts are well tolerated by monkeys.22 We began
phase I testing with LMB-2 in patients with hematologic malignancies in
1996. In this ongoing trial, 4 patients with hairy cell leukemia (HCL)
have been treated. The present report focuses on the responses in these
4 HCL patients.
HCL is a malignancy of well-differentiated B lymphocytes that is
diagnosed in 500 to 600 patients per year in the United
States.23,24 Recent advances in the development of
nucleoside analogs to treat HCL have led to long-term clinical complete
remissions (CRs) in a high percentage of patients, particularly those
treated with deoxycoformycin (DCF) or 2-chlorodeoxyadenosine
(CdA).25-28 CRs do not result in a cure, because minimal
residual disease is detectable by flow cytometry or by polymerase chain
reaction.29,30 Moreover, based on large trials of these
agents, 10% to 20% of patients are or will become refractory to
chemotherapy and many will die from complications of pancytopenia due
to progressive bone marrow involvement. The 4 HCL patients treated with
LMB-2 had disease resistant to CdA as well as other established forms
of treatment for HCL and had pancytopenia to a degree that necessitated
at least palliative therapy. The response in 1 patient (no. 30) was most valuable clinically due to resolution of pancytopenia, lack of
infection after treatment, lack of immunogenicity that allowed repeated
dosing, and lack of dose-limiting toxicity.
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MATERIALS AND METHODS |
Patients with HCL were treated as part of a phase I trial of LMB-2 in
patients with hematologic malignancies. To be eligible, patients had to
have disease refractory to conventional chemotherapy, evidence of CD25
on the surface of the malignant cells, and adequate organ function and
had to be resistant to standard chemotherapy. LMB-2 was produced by the
Monoclonal Antibody and Recombinant Protein (MARP) facility of the
National Cancer Institute (NCI; Frederick, MD) and the
investigational new drug (IND) application is held by the Cancer
Therapy and Evaluation Program (CTEP) of the NCI. LMB-2 was diluted
into 50 mL of 0.2% albumin in 0.9% NaCl and administered as a
30-minute intravenous (IV) infusion administered every other day for 3 doses (QOD × 3). Patients without neutralizing antibodies or
progressive disease could be retreated after restaging at monthly
intervals. Assessment of disease was performed using
fluorescent-activated cell sorting (FACS) analysis of blood and
radiological studies. CR was defined as disappearance of evaluable
disease lasting at least 4 weeks. A negative FACS analysis was not
required. A partial response (PR) required reduction in tumor burden by
at least 50% lasting at least 4 weeks, including a  50% decrease in
malignant cell count and a 50% decrease in the sum of the products
of perpendicular measurements of malignant solid masses. Responding
patients could be retreated providing that they did not develop
neutralizing antibodies to LMB-2 as assessed by cytotoxicity
assay.31 Patients with greater than 75% neutralization of
1 µg/mL of LMB-2 in the serum were ineligible for further therapy.
The maximum number of cycles allowed was arbitrarily set at 10 for
patients in PR, and patients attaining CR could receive 2 additional
cycles after demonstration of a CR. The 4 HCL patients received LMB-2
at 3 different dose levels (30, 40, and 63 µg/kg IV QOD × 3. The beginning dose in the overall phase I clinical trial was 2 µg/kg
QOD × 3, which was chosen because it was 10% of the dose in
monkeys, which resulted in no significant toxicity (transaminase
elevations). Dose escalation in the phase I trial involved doses of 2, 6, 10, 20, 30, 40, 50, and 63 µg/kg IV QOD × 3 to determine the
maximum tolerated dose (MTD). The MTD (40 µg/kg IV QOD × 3) was
defined as the dose that is greater than 20% below that at which 2 of
2 to 6 patients incur dose-limiting toxicity (DLT). Patients were not
premedicated to prevent fever. The Common Toxicity Criteria of the NCI
were used to grade toxicity. DLT was defined as grade III-IV with some
exceptions. As in other trials of chimeric toxins,32-36
transaminase elevations of 5.1 to 20 times normal were not considered
as DLT in the absence of evidence of impaired hepatic function.
Hematologic abnormalities were non-dose-limiting in leukemic patients
and only grade IV hematologic abnormalities constituted DLT in
nonleukemic patients. Fever, which was well tolerated and did not
result in suspending therapy, was also not considered dose-limiting.
Plasma levels of LMB-2 were determined by incubating dilutions of
plasma with CD25+ SP2/Tac cells37 and comparing
cytotoxicity as assessed by {3H}-leucine incorporation
with that obtained by an LMB-2 standard.10 Assessment of
neutralizing antibodies to LMB-2 was performed by incubating LMB-2 with
patient sera and then testing its cytotoxicity against SP2/Tac
cells.31 For cytotoxicity analysis of the HCL cells from
patients, peripheral blood mononuclear cells were obtained from the
blood by Ficoll centrifugation and incubated with different concentrations of LMB-2 at 37°C, and {3H}-leucine
incorporation was determined. For binding assays to determine numbers
of CD25 sites/cell from Scatchard plots, the Ficoll-purified
mononuclear cells were incubated with 0.125, 0.25, 0.5, 1, 2, or 4 nmol/L {125I}-humanized anti-Tac for 1 to 2 hours in
the presence or absence of a 100-fold excess of unlabeled LMB-2, as
reported previously.5
| |
RESULTS |
To test the clinical activity of LMB-2, patients with refractory
CD25+ hematologic malignancies were treated in a phase I
trial, 4 of whom had HCL. Table 1
summarizes the age, years since diagnosis, previous therapy, disease
status before LMB-2, CD25 expression, dose of LMB-2, drug-related
toxicity encountered, immunogenicity, and response in these patients.
All 4 patients had typical HCL based on morphologic and
immunocytochemical criteria. Patients had HCL for 12 to 17 years and
had each been treated with CdA and interferon  (IFN). Patients no.
15, 32, and 35 had 5, 5, and 2 cycles each of CdA, with decreasing
extent and durability of response to later cycles. Patient no. 30 had
no response to 1 cycle of CdA. All patients received IFN either before
or after CdA. For patient no. 15, IFN was effective before but not
after he became resistant to CdA. Of the 3 patients previously treated with splenectomy (patients no. 15, 32, and 35), patients no. 15 and 35 had high circulating HCL counts.
Clinical response of patient no. 30.
Patient no. 30 is a 47-year-old man diagnosed with HCL after presenting
with severe anemia (hemoglobin level [Hgb], <5 g/dL). IFN
maintained the Hgb at 8 to 10 g/dL for 4 years, but treatment was
stopped due to progressive granulocytopenia. He was then treated with
CdA without response. He was subsequently treated with frequent blood
transfusions for progressive disease-related anemia. Before study
entry, his lowest Hgb before transfusion was 3.8 g/dL. He also was
treated with imipenem and clarithromycin for pulmonary and cutaneous
Mycobacterium chelonae. Physical examination was significant
for old hyperpigmented skin lesions and an enlarged spleen that was
palpable 5 cm below the left costal margin. The white blood cell count
(WBC) was 2,070/µL, the granulocyte count was 360/µL, the platelet
count was 47,000/µL, and the Hgb was 10.0 g/dL with transfusion.
Hairy cells, visible in the peripheral blood film, were quantitated at
480/µL by FACS analysis. Computed tomography (CT) showed an enlarged
spleen and precarinal lymphadenopathy. Biapical scarring was observed
consistent with chronic mycobacterial infection. He received LMB-2 at
63 µg/kg on days 1, 3, and 5. By day 3, HCL cells were no longer
visible in the peripheral blood. By day 8, the spleen was no longer
palpable and the marrow biopsy was no longer positive for HCL. By day
31, the spleen size by CT had normalized and thrombocytopenia resolved.
Cycle 2 of LMB-2 was begun on day 32 of the first cycle.
Granulocytopenia and anemia resolved after cycle 2. The patient
achieved a CR with minimal residual disease detectable by FACS and no
evidence of progression 13 months after beginning LMB-2.
Before study entry, HCL cells from patient no. 30 by FACS were positive
for  light chains and CD103 and stained brightly for CD11c, CD19,
CD20, CD22, and CD25, but were negative for CD5, CD10, and CD23. To
quantitate the patient's circulating malignant cells, peripheral blood
was examined by 2-color flow cytometry. Figure 1 shows
CD19+/CD25+ and
CD11c+/CD103+ populations before LMB-2, on day
8, and on day 177 (cycle 2, day 146). The malignant population,
originally comprising 35% of the lymphocytes, decreased greater than
90% in 2 days, as shown in Fig 2A, from
480/µL pretreatment to 43/µL on day 3. This effect was from 1 dose
of LMB-2. On days 8, 22, and 31, the HCL counts were 3.0, 1.0, and
3.0/µL, respectively, indicating a maximal reduction of 99.8% in the
peripheral blood. Flow cytometry on day 8 (shown in Fig 1C and D)
indicated a selective depletion of the malignant cells of nearly 3 logs. The CD11c+/CD103+ malignant clone
identified in Fig 1D still was brightly positive for CD25, as shown in
Fig 1C. After the first 2 doses of cycle 2, the patient's HCL count
decreased further on day 5 to 1.8/µL. After the final dose of cycle
2, on day 8 the HCL count was less than 0.37/µL, which is too low to
confirm the presence of malignant cells. The count was 1.9 and 1.1/µL
on days 21 and 35 of the second cycle, respectively. Because of the
lack of symptoms and limited drug supply, the patient was not
retreated. The HCL cell count remained low, being 1/µL on day 117 of
cycle 2. By cycle 2, day 146, the HCL cells by flow cytometry were too
few to be diagnostic of HCL (Fig 1E and F). Results from light
chain staining were unable to confirm the presence of circulating
malignant cells. Malignant circulating cells could still not be
confirmed 29 days later, although by flow cytometry a small population
of cells comprising less than 0.01% of lymphocytes was suggestive of
persistent HCL (Fig 1E and F). The quantity of these cells, if
malignant, would be approximately 0.05 HCL cells per microliter, or
nearly 5 logs less than the pretreatment level.
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| Fig 1.
Flow cytometry (FACS) analysis of circulating cells in
HCL patient no. 30. At 3 different time points, pretreatment (A and B),
cycle 1 day 8 (C and D), and cycle 2 day 145 (E and F), data from
2-color analyses are shown to identify
CD25+/CD19+ cells (A, C, and E) or
CD11c+/CD103+ cells (B, D, and F).
Perpendicular lines were drawn to differentiate malignant (upper-right
quadrants) from nonmalignant cells. Horizontal lines in (B), (D), and
(F) are drawn above nonmalignant cells that are dimly positive for
CD11c.
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| Fig 2.
Response of patient no. 30 to LMB-2. Patient no. 30 received LMB-2 at 63 µg/kg IV QOD × 3 for 2 cycles. Cycle 1 began
on day 1 and cycle 2 began on day 32. In (A), the number of HCL cells
per microliter ( ) as determined by FACS analysis is shown. Normal
blood cells are represented in (B) as absolute granulocyte count in
cells per microliter × 10 1 ( ), platelet count per
microliter × 103 ( ), and hemoglobin in grams per
deciliter × 10 ( ).
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With respect to hematologic function before treatment, patient no. 30 was dependent on red blood cell transfusions and had preexisting
thrombocytopenia (47,000/µL) and granulocytopenia (360/µL). With
LMB-2, the patient became independent of transfusions. The platelet
count increased during cycle 1 to 145,000/µL, but the patient
remained granulocytopenic, with a granulocyte count of 400/µL (Fig
2B). During cycle 2, the pancytopenia improved, with a platelet count
of 187,000/µL, a granulocyte count of 1,530/µL, and Hgb of 11.2 g/dL. The patient's pancytopenia fully resolved during the 6 months
off treatment. As shown in Fig 2B, the patient's hematologic recovery
was not associated with even transient hematologic toxicity to LMB-2.
Figure 3A shows the patient's splenomegaly
in axial section before treatment with LMB-2. The cranial-caudal
diameter was 16 cm, and after 1 cycle of LMB-2 decreased to a normal
size of 11 cm by day 31 (Fig 3B). The spleen size was 9 cm after cycle
2 and showed no evidence of progression during 5 months off treatment, as shown in Fig 3C. The patient had abnormally enlarged precarinal adenopathy (up to 1.6 cm) before LMB-2, which also resolved after the
first cycle. The pretreatment bone marrow biopsy was infiltrated with
HCL cells, and liquid marrow could not be aspirated. The marrow biopsy
by day 8 was no longer positive for HCL and was negative on day 206, showing hypocellularity with adequate normal hematopoietic precursors.
However, the marrow aspirate on day 206, which could be obtained for
the first time since the patient's diagnosis, contained 0.15%
malignant cells by FACS. Thus, malignant cells outside blood vessels
responded to LMB-2, but minimal residual HCL could be detected by FACS
in the bone marrow aspirate.
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| Fig 3.
Reduction of spleen size in HCL. Axial sections are shown
from computed tomography performed on patient no. 1 before (A) and
after (B) the first cycle of LMB-2 and then on day 175 of the second
cycle (C).
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Pharmacokinetics of LMB-2.
Plasma levels of LMB-2 were quantitated by cytotoxicity assay to
determine the amount of cytotoxic activity that was delivered to
patient no. 30 by treatment with LMB-2.
Figure 4 displays the plasma levels of
LMB-2 during the first and second cycles of treatment. The peak plasma
levels were 600 to 1,100 ng/mL during cycle 1 and 750 to 1,000 ng/mL
during cycle 2. By simple monoexponential decay, the t1/2
for day 1 was 380 minutes for cycle 1. The decrease in plasma level for
other doses followed a biexponential pattern, and after the first dose
of cycle 2, the t1/2 was 77 minutes and the
t1/2 was 420 minutes. Levels of LMB-2 remained
detectable throughout each cycle, with minimum drug concentrations,
obtained 2 days after the first dose, of 1.6 and 2.2 ng/mL for cycles 1 and 2, respectively. These minimum plasma levels are several-fold greater than the concentration of LMB-2 required for 50% inhibition of
HCL cells from this patient ex vivo (IC50 = 0.5 ng/mL, see below). Thus, significantly cytotoxic plasma levels of LMB-2 were maintained during the week of treatment by the administration of the
immunotoxin as a 30-minute IV infusion QOD × 3.
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| Fig 4.
LMB-2 concentrations in the plasma of patient no. 30 before and after each IV infusion of LMB-2 (63 µg/kg). Plasma levels
for cycle 1 (A) and cycle 2 (B) were quantitated by cytotoxicity assay
of diluted plasma on SP2/Tac cells. Error bars indicate standard
deviations of means of triplicate experiments.
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Response to LMB-2 in 3 additional HCL patients.
Figure 5 depicts partial responses in 3 additional patients with HCL treated with LMB-2. Each of these 3 patients (no. 15, 32, and 35) did not respond or relapsed after
splenectomy, IFN, and CdA, and patient no. 32 also was unresponsive to
DCF. Peripheral blood malignant cells, as quantitated by examination of
the peripheral blood film and by flow cytometry, constituted evaluable
and measurable disease in these patients. As shown in Fig 5A, patient
no. 15 had a WBC of 70,200/µL, a granulocyte count of 280/µL, and
an HCL count of 63,900/µL before treatment. The platelet count and Hgb were 42,000/µL and 7.2 g/dL, respectively, both
transfusion-dependent. He received 30 µg/kg IV QOD × 3 beginning on day 1. The effect of the first dose was observed on day 3, when the WBC decreased to 23,000/µL and the HCL count decreased to
20,100/µL. By day 8, the WBC was 640/µL, with an HCL count of only
115/µL, a reduction of 99.8% from the pretreatment level. The
patient was restaged on day 30, with a 95% reduction of HCL from
pretreatment. This patient was not retreated because of infections,
including an infected intravenous catheter. Patient no. 35 had a WBC
count of 65,300/µL, a neutrophil count as low as 838/µL, a platelet count of 47,000/µL, a Hgb of 8.7 g/dL, and an HCL count of
60,700/µL before treatment (Fig 5B). After receiving 40 µg/kg IV
QOD × 3, the effect of the first dose, assessed on day 3, was a
reduction in the HCL count to 34,900/µL. The maximal response was
observed on day 13 with a 98% reduction in HCL cells to 1,380/µL.
After 1 cycle of LMB-2 the Hgb increased from 8.7 to 11.4 g/dL and the platelet count increased from 47,000 to 129,000/µL. This patient's serum before treatment had evidence of a low level of pre-existing antibodies that neutralized 50% of the activity of 200 ng/mL of LMB-2.
After the first cycle of LMB-2, the antibody titer increased, and by
day 21, it completely neutralized 1,000 ng/mL of LMB-2. A second cycle
was begun on day 22, but no biologically active LMB-2 was detected in
the blood and no further response was observed with the neutralizing
antibodies. Patient no. 32 had a pretreatment WBC of 1,430/µL, a
neutrophil count of 470/µL, and an HCL count of 350/µL. The
pretransfusion Hgb and platelet count were 7.9 g/dL and 15,000/µL.
She received only 1 dose of LMB-2 at 63 µg/kg IV due to DLT,
including diarrhea on day 2 and reversible cardiomyopathy from day 5 to
7. As shown in Fig 5C, the malignant count transiently increased from
350 to 4,600/µL on day 3, but by day 16, FACS identified a large
number of dead malignant cells in addition to 9.4/µL HCL cells. The
maximum response was 3.4/µL HCL cells on day 22, which was less than
1% of the pretreatment count. During the transient increase in WBC and
HCL after the first dose, the neutrophil and platelet counts
transiently increased from the pretreatment level of 470 and
15,000/µL to a maximum of 1,780 and 155,000/µL, respectively, on
day 3.
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| Fig 5.
Response of other HCL patients to LMB-2. For patients no.
15 (A), 35 (B), and 32 (C), the concentrations of circulating WBCs
() and HCL cells determined by FACS and morphology () are shown.
Treatment days are shown () for the indicated cycles. Patients no.
15 and 35 in (A) and (B) received LMB-2 at 30 and 40 µg/kg IV QOD × 3, respectively. Patient no. 32 received 1 dose of LMB-2 at 63 µg/kg
on day 1. In (C), the Y-axis is depicted showing the log of the WBC and
HCL counts to show the wide fluctuation in the counts after the first
dose.
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Toxicity of LMB-2 in the HCL patients.
Toxicity in all patients was transient, lasting up to 2 to 3 days after
onset. Patient no. 15, who received 30 µg/kg × 3, had grade I
transaminase elevations and grade II fever, with peak aspartate
transaminase (AST) and alanine transaminase (ALT) values of 44 and 62, respectively, and a maximum temperature (Tmax) of 38.6°C. In patient no. 30, the transaminases were elevated before LMB-2 administration up to an AST of 45 U/L and an ALT of 54 U/L. With
1 cycle of LMB-2 at 63 µg/kg QOD × 3, mild increases in these abnormalities were noted, with a peak AST of 69 U/L and ALT of 66 U/L.
The Tmax was only 37.7°C. With the higher plasma levels of LMB-2
for cycle 2 (Fig 4B), toxicity to cycle 2 included non-dose-limiting grade III AST and ALT elevations, up to 241 and 200, respectively, a
Tmax of 37.8°C, and transient grade II nausea and vomiting and rash. In patient no. 35, treated at 40 µg/kg QOD × 3, toxicity was limited to transient grade I nausea, rash, and weight gain. The
dose in patient no. 32 was 63 µg/kg, but treatment was stopped after
1 dose due to dose-limiting diarrhea (grade III) and grade II nausea
and vomiting. Grade I fever was observed with a Tmax of 38.1°C.
This patient also had a transient cardiomyopathy without evidence of
cardiac necrosis, which became evident on day 5 and resolved in 1 to 2 days. It was not determined whether toxicity in this patient was
mediated directly by LMB-2 or by cytokines38,39 produced by
normal or malignant cells. However, cardiomyopathy was not observed in
preclinical toxicology studies. Hematologic toxicity could not be
easily evaluated in patients no. 15 and 32, because these pancytopenic
patients were transfusion-dependent before treatment and had no
significant change in transfusion requirements during their response.
Patients no. 30 and 35 had no significant hematologic toxicity.
CD25 expression by HCL cells and sensitivity to LMB-2 ex vivo.
All 4 HCL patients had malignant cells that were positive for CD25 by
flow cytometry. To quantitate the level of CD25 expression on the
malignant cells, binding assays were performed using
{125I}-anti-Tac(IgG) in the presence or absence of an
excess of unlabeled LMB-2. Patients no. 15, 30, and 35 had sufficient
numbers of malignant cells in the peripheral blood to perform such
studies on fresh malignant cells isolated by Ficoll centrifugation. The
number of CD25 sites per cell, as determined from Scatchard plots, is depicted in Table 1. Patient no. 30, who responded best to LMB-2, had
6,200 ± 32 sites/cell. HCL cells from patient no. 15 had 7,200 ± 180 sites/cell. This patient had the same maximal percentage response to 1 cycle as patient no. 30 (99.8% reduction) but far greater numbers of cells were killed. Patient no. 35, whose 98% response was the lowest of the HCL patients treated, had significantly lower CD25 expression at 1,900 ± 180 and 1,400 ± 35 sites/cell measured on day  5 and day 0, respectively. To
determine whether the malignant cells in the responding patients were
sensitive to LMB-2, they were incubated with LMB-2, as reported
previously for chronic lymphocytic leukemia (CLL)
cells.5 In patients no. 15, 30, and 35, the concentrations
of LMB-2 required for 50% inhibition of protein synthesis
(IC50s) were 1.1 ± 0.09, 0.05 ± 0.15, and 3 ± 0.08 ng/mL, respectively. Cytotoxicity required both binding to CD25
and ADP-ribosylation of elongation factor 2 after internalization,
because LMB-2 mutants deficient in either binding or ADP-ribosylation
activity were inactive on these cells (data not shown). Thus, CD25
expression in these few patients correlated with sensitivity of the
malignant cells to LMB-2, which also correlated with clinical response.
More patients will need to be treated to determine the significance of
this correlation.
| |
DISCUSSION |
Four patients with HCL have been treated in a phase I trial of the
anti-CD25 recombinant single-chain immunotoxin LMB-2 in patients with
CD25+ hematologic malignancies. We report here that major
responses occurred in all 4 patients; all patients were resistant to
standard and salvage chemotherapy, including CdA. The reductions of
malignant cells in the peripheral blood varied from 98% to at least 5 logs. The most durable response met the criteria for CR used in
clinical trials of purine analogs in HCL,27,28 although
minimal residual disease is known to be present. These are, to our
knowledge, the first major responses in cancer patients to a cytotoxic
agent that is targeted by an Fv fragment of an antibody.
Although a full report of the phase I trial of LMB-2 in patients with
other hematologic malignancies will be made separately, we found that
the toxicity, pharmacokinetics, and immunogenicity of LMB-2 in the 4 HCL patients are representative of the other 31 patients so far
treated. LMB-2 has been well tolerated without DLT in all 9 patients
treated at the 40 µg/kg × 3 dose level with toxicities,
including transaminase elevations (8), fever (7), nausea (5), alkaline
phosphatase elevations (3), vomiting (2), proteinurea and increased
creatinine (2), thrombocytopenia (2), and weight gain (1). Only 6 of 35 patients developed levels of neutralizing antibodies after the first
cycle that were high enough to disqualify them from additional cycles
of treatment. It is interesting that, whereas immunogenicity is low in
HCL, it may be lower in CLL, because no evidence of immunogenicity was
observed in 8 patients after a total of 16 cycles. Eliminating
immunogenicity in all HCL patients might require using
immunosuppressive agents. A clinical trial is currently under way to
determine if the B-cell-depleting agent Rituximab40 can
prevent patients with solid tumors from becoming immunized to the
anti-Ley immunotoxin LMB-1.31 In addition to
HCL, partial responses have been observed in adult T-cell leukemia,
cutaneous T-cell lymphoma (CTCL), Hodgkin's disease, and CLL.
HCL is a very treatable albeit incurable malignancy, with long-term
complete or partial responses observed with DCF or CdA in most
patients. However, up to 10% to 20% of patients become refractory and
are in need of more effective salvage therapy.27,28,30 The
management of such patients is often problematic, with both disease and
chemotherapy-related bone marrow damage limiting therapeutic options.
LMB-2 may be particularly useful for chemotherapy-refractory patients
with impaired bone marrow function, due to lack of hematopoietic toxicity observed (Fig 2B). LMB-2 could also be combined with conventional chemotherapeutic agents to improve responses in refractory patients. It has recently been reported that a recombinant toxin can be
combined with either doxorubicin or ARA-C to synergistically target
multiply drug-resistant malignant cells in culture.41 The
activity of LMB-2 combined with chemotherapeutic drugs is currently
being evaluated ex vivo to explore whether LMB-2 might improve the
clinical efficacy of chemotherapeutics against HCL.
Compared with other B-cell leukemias such as CLL, HCL is a particularly
sensitive disease to LMB-2 at least in part due to the high CD25
expression on the malignant cells in most patients. However, even cells
from patient no. 35, which had relatively low CD25 expression (Table
1), were still quite sensitive to LMB-2. HCL cells could be
particularly efficient at trafficking the active fragment of the toxin
intracellularly to the cytosol or other steps that are part of the
intoxication process. Other mechanisms of cell killing cannot be
excluded, particularly in the case of patient no. 30, who had a
prolonged and improving response. It is possible that a several-log
response produced by the cytotoxic effect of LMB-2 corrected the
patient's immune deficiencies, which in turn led to a further multilog
reduction of HCL cells several months later. It is notable that the
clinical response in HCL patients treated with chemotherapy can occur
after many months and improve with time. Because cytoreduction from 1 to 2 cycles of LMB-2 usually did not select for CD25
tumor cells, it should be possible to use additional cycles of LMB-2 to
determine whether minimal residual disease can be further reduced or eliminated.
Conventional immunotoxins consisting of monoclonal antibodies
chemically connected to toxins have been used to target a variety of
tumor types, with some producing at least several clinical responses in
patients with hematologic malignancies35,42-49 or with
solid tumors.31,50 Recombinant growth factor fusion toxins have also induced responses,51-53 and the IL2-toxin
DAB389IL2 is a recently approved salvage therapy for
CTCL. Recombinant immunotoxins containing approximately
25-kD Fv fused to a 38- to 40-kD truncated bacterial toxin are close in
size to growth factor fusion toxins but can bind to target molecules
other than growth factor receptors.10,54 Several of these
new agents are now in clinical trials to determine their toxicity and
clinical activity. One of these, RFB4(dsFv)-PE38 (BL22),55
has just begun to be tested in patients with CD22+
malignancies and might be particularly appropriate for the
poor-prognosis variant of HCL,56,57 which is usually
CD25 but strongly CD22+. In conclusion,
the responses seen with LMB-2 serve as proof of principal of the
potential clinical use of recombinant immunotoxins and validate efforts
to target new antigens with these potent agents.
| |
ACKNOWLEDGMENT |
The authors thank Steven Giardina, Toby Hecht, and Daniel Coffman at
the MARP (Frederick, MD); Catherine Laurencot, Jay Greenblatt, and
Thomas Davis at CTEP; and David Waters and Vickie Marshall at the SAIC
(Frederick, MD). We also thank clinical personnel at the NIH clinical
center, including research nurses Deborah Pearson, Valerie Dyer, and
Miranda Raggio and pharmacist Barry Goldspiel.
| |
FOOTNOTES |
Submitted March 11, 1999; accepted July 14, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ira Pastan, MD, National Cancer Institute,
National Institutes of Health, 37/4E16, 37 Convent Dr, MSC 4255, Bethesda, MD 20892.
| |
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M. Onda, M. Willingham, Q.-c. Wang, R. J. Kreitman, Y. Tsutsumi, S. Nagata, and I. Pastan
Inhibition of TNF-{alpha} Produced by Kupffer Cells Protects Against the Nonspecific Liver Toxicity of Immunotoxin Anti-Tac(Fv)-PE38, LMB-2
J. Immunol.,
December 15, 2000;
165(12):
7150 - 7156.
[Abstract]
[Full Text]
[PDF]
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Y. Tsutsumi, M. Onda, S. Nagata, B. Lee, R. J. Kreitman, and I. Pastan
Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity
PNAS,
July 5, 2000;
(2000)
140210597.
[Abstract]
[Full Text]
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R. W. Rand, R. J. Kreitman, N. Patronas, F. Varricchio, I. Pastan, and R. K. Puri
Intratumoral Administration of Recombinant Circularly Permuted Interleukin-4-Pseudomonas Exotoxin in Patients with High-Grade Glioma
Clin. Cancer Res.,
June 1, 2000;
6(6):
2157 - 2165.
[Abstract]
[Full Text]
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R. J. Kreitman, I. Margulies, M. Stetler-Stevenson, Q.-C. Wang, D. J. P. FitzGerald, and I. Pastan
Cytotoxic Activity of Disulfide-stabilized Recombinant Immunotoxin RFB4(dsFv)-PE38 (BL22) toward Fresh Malignant Cells from Patients with B-Cell Leukemias
Clin. Cancer Res.,
April 1, 2000;
6(4):
1476 - 1487.
[Abstract]
[Full Text]
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T. A. Waldmann, R. Levy, and B. S. Coller
Emerging Therapies: Spectrum of Applications of Monoclonal Antibody Therapy
Hematology,
January 1, 2000;
2000(1):
394 - 408.
[Abstract]
[Full Text]
[PDF]
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Y. Tsutsumi, M. Onda, S. Nagata, B. Lee, R. J. Kreitman, and I. Pastan
Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity
PNAS,
July 18, 2000;
97(15):
8548 - 8553.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION