Adjuvant Immunotoxin Therapy With Anti-B4-Blocked Ricin After Autologous Bone Marrow Transplantation for Patients With B-Cell Non- Hodgkin’s Lymphoma

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Adjuvant Immunotoxin Therapy With Anti-B4-Blocked Ricin After
Autologous Bone Marrow Transplantation for Patients With B-Cell
Non-Hodgkin’s Lymphoma

By Michael L. Grossbard, John G. Gribben, Arnold S. Freedman, J o h n M. Lambert, Jeanne Kinsella, Susan N. Rabinowe,
Laura Eliseo, James A. Taylor, Walter A. Blattler, Carol L. Epstein, and Lee M. Nadler

A

were reversible grade IV thrombocytopenia and elevation
of hepatic transaminases. Mild capillary leak syndrome was
manifested by hypoalbuminemia, peripheral edema (4 patients), and dyspnea (1 patient). Anti-immunotoxin antibodies developed in 7 patients. Eleven patients remain in
complete remission between 1 3 and 26 months post-ABMT
(median 17 months). These results show that Anti-64-bR
can be administered with tolerable, reversible toxicities to
patients with 6-cell NHL in complete remission following
ABMT.
0 1993 by The American Society of Hematology.

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LTHOUGH HIGH-DOSE myeloablative therapy foltreating patients with relapsed B-cell NHL. In the first trial,
lowed by autologous bone marrow (BM) reinfusion can
Anti-B4-bR was administered by daily bolus infusion for 5

induce a clinical complete remission in the vast majority of
consecutive days to 25 patients with a maximal tolerated
patients with relapsed B-cell non-Hodgkin’s lymphoma
dose (MTD) of 50 pg/kg/d (total 250 pg/kg) and the dose(NHL), between 50% and 85% of these patients ultimately
limiting toxicity (DLT) was defined by transient grade IV
relapse.13’ Tumor recurrence primarily is attributable to the
increases in hepatic transaminases and thromb~cytopenia.’~
presence of clones of lymphoma cells resistant to high-dose
Because additional preclinical studies suggested that higher
therapy, but reinfusion of lymphoma cells harbored within
doses of Anti-B4-bR could be administered safely by 7-day
the autologous marrow probably also contributes to r e l a p ~ e . ~ , ~continuous infusion, we also conducted a phase I trial in a
In an attempt to overcome tumor cell resistance, a number
similar patient population using a 7-day continuous infuof investigators have intensified the myeloablative regimen.
sion.I6 This treatment schedule allowed potentially theraAlthough complete remission rates may be increased with
peutic serum levels to be sustained in serum for up to 4 days,
this approach, the morbidity and mortality of therapy also
and a higher MTD of 50 pg/kg/day X 7 days (total 350 pg/
significantly
We and others have also attempted
kg) was achieved. In this trial, the DLT was identical to that
to decrease the number of relapses by purging lymphoma
of the bolus infusion trial although continuous infusions also
cells from the harvested autologous
Unfonmately,
led to mild, reversible capillary leak syndrome.
purging has been effective in removing residual lymphoma
Although clinically significant responses, including comcells from the marrow in only 50% of patients; therefore,
plete remissions, were seen in both phase I trials, the majority
small numbers of residual lymphoma cells may continue to

of responses were observed in patients with lower tumor burcontribute to r e l a p ~ eIn
. ~ an attempt to overcome these obdens. This suggested that one major obstacle to effective immunotoxin therapy might be the delivery of these agents to
stacles to autologous BM transplantation (ABMT), a number
all of the lymphoma cells. Therefore, we hypothesized that
of investigators have begun to treat patients after ABMT with
administration of Anti-B4-bR after ABMT might lead to
therapies designed to overcome lymphoma cell resistance and
improved tumor cell delivery to a small number of residual
eradicate residual neoplastic cells transferred in the reinfused
tumor cells and potentially eradicate remaining resistant
BM. Because of the myelosuppressive side effects attendant
lymphoma cells. In this study, we report a phase I trial of
to high-dose therapy, traditional chemotherapeutic agents
cannot be used early after ABMT. In contrast, new agents
with nonoverlapping toxicity, such as immunotoxins or cyFrom the Division of Tumor Immunology, Dana-Farber Cancer
tokines, both may be delivered safely in this setting and may
Institute; the Department of Medicine, Haward Medical School, BosI
be capable of killing resistant residual lymphoma
ton, MA; and ImmunoGen, Inc, Cambridge, MA.
Over the past 3 years, we have used a novel immunotoxin
Submitted September 14, 1992; accepted December 14, 1992.
to treat patients with relapsed B-cell NHL. The immunotoxin,
Supported by National Institute of Health Grant Nos. CA34183
AntLB4-blocked ricin (Anti-B4-bR) combines the B-cell
and CA55207. M.L.G. is a recipient of a National Cancer Institute Clinical Oncology Research Career Development Award
specificity of the anti-B4 (CD19) monoclonal antibody
(1K12CAOl730).
(MoAb) with a toxin, termed “blocked ricin.”12 In blocked
Address reprint requests to Lee M. Nadler, MD, Division of Tumor
ricin, which is derived from the potent protein toxin ricin,

Immunology,
Dana-Farber Cancer Institute, 44 Binney St, Boston,
the binding of ricin is attenuated by attaching affinity ligands
MA 021 15.
to the galactose binding sites that mediate nonspecific bindThe publication costs of this article were defayed in part by page
ing.I3 The resultant immunotoxin is highly cytotoxic to cells
charge payment. This article must therefore be hereby marked
that express the CD 19 antigen and effects its cytotoxicity by
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
inhibiting protein synthesis.j 4 Therefore, Anti-B4-bR potenindicate this fact.
tially may kill lymphoma cells resistant to chemotherapy.
0 1993 by The American Society of Hematology.
We have evaluated this immunotoxin in two phase I trials
0006-4971/93/8109-0024%3.00/0

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Anti-6-blocked ricin (anti-B4-bR) combines the specificity
of the anti-64 (CDI 9) monoclonal antibody with the protein
toxin “blocked ricin.” In blocked ricin, affinity ligands are
attached to the ricin 6-chain to attenuate its lectin binding
capacity. In a phase I trial, Anti-B4-bR was administered
by 7-day continuous infusion to 1 2 patients in complete
remission after autologous bone marrow transplantation
(ABMT) for relapsed 6-cell nomHodgkin’s lymphoma (NHL).
Patients were treated at 20,40, and 50 pglkgld for 7 days.
Potentially therapeutic serum levels could be sustainedfor
3 to 4 days. The maximum tolerated dose was 40 pglkgld
for 7 days (total 280 pglkg). The dose-limiting toxicities

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Blood, Vol81, No 9 (May 1), 1993: pp 2263-227 1

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GROSSBARD ET AL

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Anti-B4-bR administered by 7-day continuous infusion to
12 patients with B-cell NHL in complete remission after
ABMT. In the results to be reported below, we show that
Anti-B4-bR can be administered in this setting with tolerable
and reversible toxicity and that potentially therapeutic serum
levels can be obtained consistently.
MATERIALS AND METHODS

Anti-B4-bR

Study Design

After the documentation of complete remission, patients were admitted to the DFCI and received a continuous infusion of Anti-B4bR via a central venous line for 7 consecutive days. Anti-B4-bR was

administered at doses of 20,40, and 50 pg/kg/d for 7 days. The study
was designed to gradually escalate the dose of Anti-B4-bR until grade
111 National Cancer Institute Common Toxicity Criteria toxicity was
reached. For the purpose of this protocol, dose escalation was continued until grade IV elevation of hepatic transaminases were seen.
In addition, grade I11 or IV myelosuppression of less than 7 days
duration was not considered a dose-limitingtoxicity. The dose-limiting
toxicity was defined as that toxicity that resulted in a cessation of
dose escalation. At least three patients underwent therapy at each
dose until the dose-limiting toxicity was reached. If the dose-limiting
toxicity was not reached at a given dose level for all three patients,
the next three patients were treated at the next dose level.
Patients were eligible for retreatment at the same dose every 28
days if they continued to meet protocol eligibility requirements, had
recovered from all toxicities ofgrade 2 or greater incurred by the first
course of therapy, did not develop grade 4 toxicity with the first
c o m e of therapy, failed to develop human anti-mouse antibody
(HAMA) or human antincin antibody (HARA) after their initial
course, and agreed to continue on the protocol.
Blood was drawn daily from each patient and samples were obtained for pharmacologic analysis. Weekly blood samples were obtained for HAMA/HARA determination. Follow-up laboratory
studies were obtained weekly for 4 weeks after therapy. Formal restaging of all patients including CT scans, gallium scans in patients
with prior evidence of gallium-avid disease, and BM biopsies has
been completed at 6-month intervals post-ABMT. Follow-up data
on all patients obtained through 8/3 1/92 are included in this report.

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Patient Selection
Patients were eligible for this study if they had undergone ABMT
for B-cell NHL at the Dana-Farber Cancer Institute (DFCI) between

6/29/90 and 8/2/9 I , the time during which the protocol was open
for accrual. Tumor cells from all patients were required to show
reactivity with the anti-B1 (CD20) or anti-B4 (CD19) MoAbs. Before
ABMT, all patients had disease that had relapsed after one or more
primary or salvage chemotherapy regimens, and all patients had chemosensitive disease as defined by the ability to achieve a minimal
disease state after salvage chemotherapy. Minimal disease was defined
as either a complete or partial remission, as indicated by the reduction
oftumor masses to 2 cm or less, and the degree of marrow infiltration
by lymphoma cells to less than 20% of the intertrabecular space. At
the time of BM harvest, marrow was purged with a cocktail of three
MoAbs (anti-BI, anti-B5, and J5) as previously de~cribed.'.'~
All patients received myeloablative therapy with cyclophosphamide
(60 mg/kg of body weight/d), infused on each of 2 consecutive days.
After completing chemotherapy, all patients received total body irradiation (TBI) in fractionated doses (200 cGy) twice daily on 3 consecutive days (total 1,200 cGy). Within 18 hours after the completion
of TBI, all patients received a re-infusion of purged autologous marrow.'
Patients were eligible for treatment with Anti-B4-bR if they were
in complete remission at least 60 days after re-infusion of autologous
marrow. Complete remission was documented in all patients by obtaining computer tomography (CT)
scans of previous sites ofdisease,
gallium scans in patients with prior gallium avid disease, chest radiographs, and BM biopsies. Patients were required to have an Eastern
Cooperative Oncology Group (ECOG) performance status of 0 to 2
at the time of therapy. At protocol entry, all patients were required
to have hematopoietic engraftment as defined by absolute neutrophil
count r500/pL, hematocrit 2259'0, and platelet count r30,000/~L
independent of transfusion. At entry, all patients were required to
have a total bilirubin <2.0 mg/dL, SGOT <90 IU, SGPT < 140 IU,
and creatinine <2.0 mg/dL. Patients had no prior history of hepatic
veno-occlusivedisease, or hepatitis B or C. In addition, patients were
excluded from therapy if their serum aspartate aminotransferase
(SGOT) or serum alanine aminotransferase (SGPT) increased to

greater than five times the upper limit of normal at any time during
ABMT. No patients had a history of lymphomatous meningitis or
evidence of active infection at the time of therapy. The clinical protocol was approved by the Institutional Review Board of the DFCI,
and all patients signed an informed consent form approved by that
committee.

Pharmacology
Blood samples were obtained for the determination of serum levels
of Anti-B4-bR just before immunotoxin infusion and daily during
immunotoxin infusion. Anti-B4-bR concentration in serum was determined by using two independent enzyme-linked immunosorbent
assay (ELISA) methods. The two ELISAs were sandwich assays in
which the Anti-B4-bR conjugate was captured on plates coated with
sheep anti-mouse IgG (Fc specific) and then assayed with either goat
anti-mouse IgG immunoglobulin conjugated to alkaline phosphatase
or rabbit antiricin Ig followed by goat anti-rabbit Ig conjugated to
alkaline phosphatase. A signal amplification method (Bethesda Research Laboratories) was used, allowing the use of highly diluted
serum samples and reducing the nonspecific background signals.

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Anti-B4-bR was manufactured and supplied by ImmunoGen, Inc
(Cambridge, MA) as previously d e ~ c r i b e d . ' Anti-B4-bR
~~'~
was formulated as a sterile injectable solution containing 100 &mL of AntiB4-bR dissolved in phosphate-buffered saline (PBS), pH 7.3, with I
mg/mL of human serum albumin (Immuno-US, Rochester, MI)
added as a carrier. Anti-B4-bR was stored at 2" to 8°C before administration. Three different lots of Anti-B4-bR were used to treat
patients on this trial: P1901.003 (patients I through 4), P190K.005
(patients 5 through I I), PO191.GO4 (patient 12).

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HAhiA/HARA Detection

HAMA and HARA were measured by established ELISA techniques that take advantage of the multivalency of the Ig molecules.
The antigens anti-B4 or blocked ricin were coated on plates to capture
the specific human antiserum. Biotinylated antigen was then captured
by the bound human antiserum and assayed with streptavidin conjugated to horseradish peroxidase. In both assays, the antigens were
used in excess and the final signals recorded were directly proportional
to the amount of absorbed specific human antibody. HAMA and
HARA were considered positive if the patient's value was greater
than two standard deviations above the value for a negative control.

Polymerase Chain Reaction (PCR) Amplification
Nested oligonucleotide amplification of genomic DNA was performed as previously described at both the major breakpoint and the
minor cluster region of the bc/-2/IgH hybrid gene in BM samples
obtained from patients before BM harvest, at BM harvest, postmarrow
purging with MoAbs and complement, before therapy with Anti-B4-


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ADJUVANT IMMUNOTOXIN THERAPY WITH ANTLB4-bR

2265

bR, and posttherapy with Anti-B4-bR.4~’*~‘g
Standard precautions
were taken against cross-contamination of amplified material. For
each amplification, DNA from a
dilution of the human lymphoma B-cell line DHL-6 (which contains a bcl-2 translocation) in
BM cells served as a weak positive control. DHL-6 was a gift from
Dr A. Epstein (University of Southern California, Los Angeles). PCR
buffer containing no DNA served as a negative control. Each sample
was analyzed at least three times at each breakpoint site. In addition,
in samples without detectable PCR product, PCR amplification was
repeated with oligonucleotide primers specific to the gene encoding
the human B-cell activation antigen B7, to ensure that DNA could
be amplified in all samples.
RESULTS

Between 6/29/90 and 812191 , 21 patients with B-cell NHL
in sensitive relapse attained a minimal disease state and underwent anti-B-cell MoAb-purged ABMT. Of these 21 patients, 16 were eligible to receive post-ABMT therapy with
Anti-B4-bR, and I2 were treated. Reasons for ineligibility
included early relapse (n = l), inadequate platelet engraftment
(n = 2), cyclophosphamide bladder toxicity (n = l), and elevated liver function tests (n = l). Eligible patients were not
treated because of patient refusal (n = 3) and patient not
offered therapy by physician (n = 1).
As indicated in Table I , 12 patients received Anti-B4-bR
between 61 and 208 days after ABMT (median 83 days).
Although we intended to treat patients at the earliest time
they met all eligibilitycriteria post-ABMT, three patients were
treated more than 104 days post-ABMT. The first two patients, treated at day 151 and 140, respectively, both were
eligible to receive Anti-B4-bR by day 90 post-ABMT. Unfortunately, production of Anti-B4-bR was delayed during
this time period, and there was no immunotoxin available
for clinical use until the time of actual treatment. Patient 1 1

was not treated until 208 days post-ABMT. This patient had
delayed platelet engraftment, and therefore was not eligible
for therapy until day I 10. At that time, the patient’s physician
was concerned about possible disease recurrence and elected
to wait 3 months before repeat restaging, confirming a com-

Dose Escalation and Pharmacology
Patients were treated in cohorts of three and the dose of
Anti-B4-bR was escalated with each successive cohort until
the MTD was achieved and the DLT was defined. As seen
in Table I , 3 patients successfully received Anti-B4-bR at
20 pg/kg/d X 7 days, followed by 3 patients treated with 40
pg/kg/d X 7 days. Because grade III/IV toxicity was reached
in the 2 patients at the 50 pg/kg/d dose level (toxicity described
below), the dose was again decreased to 40 pg/kg/d and an
additional four patients were treated to refine estimates of
clinical toxicity. All patients completed the full 7 days of
treatment except one patient at 40 pg/kg/d (patient 9), who
developed grade IV thrombocytopenia and hepatotoxicity
after 5 days of therapy, and one at 50 pg/kg/d (patient 7),
who developed grade IV thrombocytopenia after 6 days of
therapy. Ten patients received only one course of Anti-B4bR and 2 patients were retreated at the same dose level as
their initial therapy. Patients were not retreated for the fol-

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Table 1. Patient Characteristics
Patient No.

Disease

No. Regimens
Pre-ABMT

1
2
3
4
5
6
7
8
9
10
11
12

FSCCL
SLL
FML
DSCCL
DML
FSCCL
ILL
FML
FSCCL
FSCCL
FSCCL
FSCCL

4
3
2
3
3
5
3
2
5
2
5
3

Disease Status
at ABMT

Sites of Disease
at ABMT

PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR

PR

BM. LN
BM, LN
LN
BM, LN
BM, LN
LN, EN
BM, LN
LN
BM, LN
LN
BM, LN
LN

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Patient Selection and Characteristics

plete remission, and refemng the patient for protocol therapy
at day 208.
Table 1 summarizes the pretransplant and posttransplant
characteristics ofthe patients. Eight males and 4 females with
a median age of 47 (range 3 1 to 54) were treated. Ten of 12
patients had low-grade NHL at the time of ABMT, and 2
patients had intermediate grade NHL. All 12 patients received
extensive prior treatment with 75% receiving 3 or more chemotherapeutic regimens before high-dose myeloablative
therapy. All patients had a history of nodal infiltration, 7 had
BM involvement, and only 2 had extranodal disease. Although all patients had achieved a minimal disease state at
the time of harvest, none had achieved a complete clinical

remission. At the time of BM harvest, all 12 patients still had
minimal nodal infiltration and 7 had minimal histologic BM
infiltration representing less than 5% of the intertrabecular
space. In addition, 9 of these patients showed a bcl-2 translocation in their original tumor biopsies and BM samples.
At the time of BM harvest, all 9 patients had residual lymphoma cells in their marrow detected by PCR for the bcl-2
translocation.

Date of ABMT

bc/-2
Translocation

6/29/90
711 1/90
9/28/90
1011 1/90
21719 1
411 1/91
5/3/91
6/14/91
5/31 191
7/3/91
212219 1
8/2/91

Yes
Yes
Yes
No
Yes

Yes
Yes
No
Yes
No
Yes
Yes

Day of
AntikB4-bR

151
140
80
85
62
78
81
61
95
62
208
104

Dose AntikB4-bR
(rg/kg/d)

20
20
20

40
40
40
50
50
40
40
40
40

Day of Anti-B4-bR = days post-ABMT when AntikB4-bR therapy initiated.
Abbreviations: FSCCL, follicular small-cleaved cell lymphoma; SLL, small lymphocytic lymphoma; FML, follicular mixed small-cleaved and large-cell
lymphoma; DSCCL, diffuse small-cleaved cell lymphoma; ILL, intermediate lymphocytic lymphoma: BM, bone marrow; LN, lymph node; EN, extranodal.


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GROSSBARD ET AL

lowing reasons: Grade IV thrombocytopenia or hepatotoxicity
during the initial course (n = 4), pneumonitis 3 weeks following the initial course (n = I), posttherapy macular hemorrhage (n = l), HAMA/HARA development before the second course of therapy (n = 3), and refused a second course
of therapy (n = 1).
Serum levels of immunoconjugatewere determined by ELISA
in all patients by separate detection of the blocked ricin and
anti-B4 moieties of the conjugate. There was a highly reproducible relationship between the dose of Anti-BCbR administered by continuous infusion and the serum level. As seen in
Fig 1 and Table 2, within 48 hours after beginning treatment,

patients who received 20 p w d ofAnti-W-bR attained plateau
serum levels of less than 0.37 nmol/L. Based on in Vitro cytotoxicity assays, sustained exposure of malignant B cell to concentrations of Anti-B4-bR above I nmol/L would be pre-

Table 2. Serum Levels of Anti-E4-bR

Patient No.

1
2

3
4
5

Dose
lualkaldl

Day 7 Serum Level
lnmol ? SE)

20
20
20
20
40
40
40
40
50
50

40
40
40
40

0.13+0.01
0.37+ 0.04

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Undetectable
0.27 i. 0

1.10k 0.02
1.44f 0.05

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Undetectable
1.78t 0.20
2.51 t 0.23'

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dicted to kill more than four logs of cells. When the dose was
escalated to 40 pg/kgld, therapeutic levels of above 1 nmol/L
could be achieved in 72 hours, and plateau serum levels in
the range of 1.1 to 2.6 nmol/L could be attained. The serum
levels of the two patients who received 50 pglkgld increased
more steeply, with potentially therapeutic levels attained
within 36 hours. The patient depicted in Fig 1 who received

50 pg/kg/d achieved a maximal level on day 5 of greater than
2.5 nmol/L. Table 2 displays the day 7 serum level that was
attained by continuous infusion in each patient. The plateau
serum concentrations of Anti-B4-bR observed at each dose
level were similar, consistent with the fact that there are few
circulating B cells as well as minimal numbers of residual
CD19 positive tumor cells after ABMT.

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Toxicity

0

2

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1.71 i.0.17
2.63 f 0.11"
1.20f 0.10
1.12f 0.11
1 . 1 1 f0.15

Patients 2 and 5 received two courses of therapy. Levels for the second
course were undetectable.
Patient 7 received 6 days of therapy and patient 9 received 5 days
of therapy.

Patient 3
(20 pg/kg/d)

Patient 5
2 - 0 1(40 pngikgld)

6
7
8
9
10
11
12

4

6

Time (days)
Fig 1. Serum levelsof anti-B4-bR (in nmol/L) in a representative
patient treated at each dose by a 7-day continuous infusion. Upper
panel represents a patient treated at 20 pg/kg/d, middle panel represents a patient treated at 40 pg/kg/d. and lower panel represents
a patient treated at 50 pg/kg/d. Error bars indicate standard error.

Overall toxicity. Anti-B4-bR administration resulted in
systemic toxicities of grade I and I1 occumng in nearly all
patients (Table 3). Nine patients developed fevers in association with therapy, with five patients developing fevers above
100.5"F. These fevers usually began within 24 to 48 hours
of initiation of therapy, and resolved within 24 hours of the
conclusion of therapy. Tolerable and self-limited nausea and
vomiting were observed in five patients. At 20 pg/kg/d fatigue
was absent, and myalgias occurred in only a single patient.

Fatigue and myalgias were seen in most patients treated at
40 and 50 pg/kg/d. Although they did not define the MTD
or DLT, myalgias reached grade I11 toxicity and persisted for
4 to 8 weeks after the completion of treatment. Nevertheless,
creatine phosphokinase (CPK) elevations were not observed
and no patient manifested rhabdomyolysis. Likewise, many
patients had profound fatigue, and four patients had a decline
in their ECOG performance status to 3. There were no allergic
manifestations of immunotoxin administration, including
anaphylaxis, rash, or immune complex formation. Patients
showed no evidence of cardiac toxicity on serial electrocardiograms. Further, there was no nephrotoxicity, proteinuria,

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ADJUVANT IMMUNOTOXIN THERAPY WITH ANTILB4-bR

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Table 3. Anti-B4-bR Post-ABMT: Clinical Toxicities
Systemic
Patient

No
1
2

3
4
5

11

12

20
20
20
20
40
40
40
40
50
50
40
40
40
40

Fever

2
1

0
1

2
1

Gastrointestinal

Performance
Status (ECOG) Headache

Myalgias Nausea

0
0
0
0
0

0
0
0

1

1

0

0

0

0

0
0
0
0
0

2
1

Vomiting

2

1

0
0
0
0
0
0
0

0
0
0
0
0

0
0
0

Capillary Leak

Hepatic
Hypoalbuminemia
Transaminases (>20%decrease) Edema

2
3
1
3
3
3
1
3
2
3

Yes
No
No
No
Yes
No
No
No
Yes

Yes
No
No
Yes
Yes

0
0
0

Hematologic

Dyspnea

3
2

0
0
0
0
0
0
0
0
0

0
0
0

0
0

0
0
0
0

2
2

Leukocytosis Anemia

No
No
No
No
No
No
No
No
No
Yes
No
Yes
No
No

Yes
Yes

Yes
No
No
No
No
No
Yes
No
No
No
No
Yes

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1
1

2
2

0
2

1

0

3

3
3
2
3
1
1

1

2

2
2
3
1

0

0

1

2

1

0

0

0

2
3
2
2

0
0

3
2

0
0
0

4

3
2
3

0
0

1

Toxicities graded by NCI CTC grade unless otherwise noted below. Leukocytosis = WBC > 10,000. Anemia = decrease in hemoglobin by one
toxicity grade from baseline value. Myalgias: grade 1 = mild, grade 2 = decrease in ability to move, grade 3 = disabled. Edema: grade 1 = 1 +, grade

2 = 2+, grade 3 = 3+, grade 4 = 4+ (anasarca)

or change in creatinine immediately after therapy. Five patients treated at doses of 40 and 50 pg/kg/d developed headaches without evidence of other neurologic toxicity. Toxicity
was not dependent on the duration of time that had elapsed
between ABMT and Anti-B4-bR therapy.
Hematologic. The major toxicity seen in this trial, which
proved dose-limiting and defined the MTD, was grade 1V
thrombocytopenia. In 1 1 of the 12 patients, the platelet count
declined during the course of therapy, with a decrease in
platelets noted within 24 to 48 hours after instituting therapy.
As seen in Table 4, the platelet count at the inception of
treatment widely ranged from patient to patient. Four patients
developed grade IV thrombocytopenia during the course of
therapy with platelet counts decreasing to less than 25,000/
pL. However, two of these patients began therapy with grade
I11 thrombocytopenia, with platelet counts of 50,00O/pL and
32,00O/pL, respectively. Platelet counts returned to baseline
within 22 days of completing therapy in all patients for whom
follow-up data are available. Two patients had minor bleeding
episodes during the course of therapy, which were not associated with grade IV thrombocytopenia. Patient 1 developed
mild hemorrhoidal bleeding on day 7 of therapy, and received
a platelet transfusion despite a nadir platelet count of 36,000/
pL. Patient 12 developed a macular hemorrhage 1 week after
completing therapy, and received a platelet transfusion at
that time. This lesion resolved without a residual visual deficit.
In no patient was thrombocytopenia associated with an elevated prothrombin time (PT) or partial thromboplastin time
(PTT).
Leukopenia was not apparent in any patient, but leukocytosis occurred in two patients with white blood cell (WBC)
counts increasing to above lO,OOO/pL in the absence of infection (Table 3). Anemia is more difficult to assess in this
patient population, because it is a common finding in the

early post-ABMT period even in the absence of therapy with
Anti-B4-bR. Moreover, patients underwent phlebotomy of
up to 250 mL during the week of therapy for the required
laboratory studies. However, five patients did show a decline

in their hemoglobin of one grade from baseline during the
course of therapy.
Two patients developed hemolytic-uremic syndrome 60
to 90 days after completing therapy with Anti-B4-bR.20 Patient 5 was noted to have anemia (hemoglobin [Hgb] 8.3 g/
dL), thrombocytopenia (platelets 63,OOO/pL), and a creatinine
of 2.1 mg/dL. These abnormalities resolved spontaneously
over the next 2 months. Patient 10 also developed hemolyticuremic syndrome, in this case 90 days after Anti-B4-bR
therapy. Again, this was characterized by self-limited thrombocytopenia, anemia, and renal dysfunction.
Hepatotoxicity. Transient elevations of SGOT and SGPT
also occurred and contributed to the definition of the MTD.
As seen in Table 3, grade 111 transient elevations of SGOT
and SGPT occurred in eight patients and grade IV elevations
of SGOT and SGPT occurred in one patient. The increase
in transaminase elevations began within 24 to 48 hours of
initiating therapy, achieved a peak at the conclusion of therapy, and resolved within 22 days (Table 4). Table 4 displays
the elevation of hepatic transaminases seen at each dose. This
is reflected as the ratio of the peak SGOT and SGPT divided
by the upper limit of normal value for those parameters.
Although hepatic transaminases were elevated in most patients on this trial, other parameters of hepatic function, including prothrombin time, partial thromboplastin time, alkaline phosphatase, and bilirubin, remained unchanged. In
prolonged follow-up, no patients have developed evidence of
sustained or recurrent hepatic abnormalities.
Hypoalbuminemia and capillary leak syndrome. All patients on this trial developed reductions in serum albumin
during the course of the infusion, with six patients developing
a decrease of 20% or more from baseline (Table 3). Hypoalbuminemia was accompanied by peripheral edema in four
patients and dyspnea in one patient. The peripheral edema

lasted over 1 year posttherapy in a single patient, and resolved
within 2 to 3 months after therapy in the remainder of the
patients. Dyspnea was not accompanied by abnormalities on
physical examination or radiographic studies, but was as-

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6
7
8
9
10

Dose
(pg/kg/d)

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GROSSBARD ET AL

2268

Table 4. Thrombocytopeniaand Hepatoxicity

Platelets

Patient

1
2
3
4
5

Pre Anti-64-bR

11

12

Days to Recovery'

SGOT/ULN

SGPT/ULN

Days to Recovery'

14
7
7
15
14
13

4.4

5.4
1.5
5.6
11.4
6
1.2
9.8
3.1
8.4
26.3
19
3.4
13.4

4.3
5.8
1.3
7.1
11.6
6.9
0.8
8.2
2
6.3
28
16.6
3.8
17.5

14

14
6
25
21
13

36
97
95
50
20
26
46
38
20
16
16
52
35
50

t
21
14
22

*

22
18

Days to recovery to baseline value.
t Follow-up information not available until day 48. Platelets fully recovered at that time.
Follow-up information not available until day 1 13. Patient developed HUS post Anti-B4-bR.

*

12
7
14
22

*

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14
15

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65
257
202
71
50
34
32
129
32
102

62
141
96
102

6
7
8
9
10

Hepatic Transaminases

Post Anti-64-bR

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sumed to be caused by fluid overload and peribronchiolar
edema. These findings were all consistent with mild capillary
leak syndrome. No patients developed pleural effusions, ascites, or hypotension in association with capillary leak.
HAMA/HARA. Data on HAMA and HARA formation
are shown in Table 5. Even though 8 of these patients were
treated within 95 days after ABMT, 7 of the 12 patients developed anti-immunotoxin antibodies. All patients at 20 pg/
kg/d developed HAMA and/or HARA. At the MTD of 40
pg/kg/d, only 4 of 7 patients developed antibodies. Here the
time to HAMA/HARA formation ranged from 27 to 38 days
posttherapy. The 2 patients treated at 50 pg/kg/d did not

develop HAMA or HARA, suggesting a blunting of the immune response at higher delivered doses of this B-cell immunotoxin. No patient developed HAMA in the absence of
HARA. Because follow-up HAMA/HARA data were not obtained beyond day 28 in most patients, the reported rates of
antibody formation may underestimate the true frequency.
In 10 of the 12 patients, levels of serum Ig were determined
before and after therapy with Anti-B4-bR. All 10 patients
began therapy with low or low-normal levels of serum IgG,
and 9 experienced a decrease in IgG levels during therapy
ranging from 8% to 41% (median decrease 24%). Further
follow-up data were not obtained until approximately 6
months posttherapy, at which time levels had returned to
baseline. It is unlikely that such a rapid decline in IgG levels
represents a decrease in Ig synthesis, but rather more likely

reflects a manifestation of capillary leak syndrome with an
associated decrease in intravascular protein concentration.
Although Ig levels were decreased transiently, there was
no apparent increase in infectious complications in patients
receiving AntLB4-bR. Patient 10 developed pneumonitis
within I month after Anti-B4-bR therapy, but work-up including bronchoscopy failed to show a definite infectious
etiology. Nevertheless, the patient received antibiotic therapy
and his pulmonary infiltrate resolved. No other patients developed infectious complications in the first 90 days after
Anti-B4-bR therapy.
Two patients received a second course of therapy with AntiB4-bR. Both patients developed HAMA and HARA early
during the second course of therapy, and neither patient had
detectable serum levels of Anti-B4-bR, consistent with rapid
clearance of the immunotoxin. In no patient on this study,
including the two who developed antibodies during the second
course of therapy, was there any evidence of allergic manifestations of HAMA/HARA. No patients showed evidence
of immune complex formation.
Clinical Responses

Because all patients were in complete remission at the outset of therapy, clinical responses are impossible to assess on
this phase I study. Of note, 1 I of the I2 patients remain in
clinical complete remission between 13 and 26 months after
ABMT (median 17 months).

Table 5. HAMA and HARA Responses
No Patients
Antr-84-bR Dose
bg/kg/d)

No Patients

20
40
50
Total

3
7
2
12

HAMA
Negative

HAMA
Positive

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HARA
Negative

HARA
Positive

Day to Antibody
POSltlVlty

0
3
2
5

3
4

14, 21, 38
27, 28, 32, 38

0
7


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ADJUVANT IMMUNOTOXIN THERAPY WITH ANTI-64-bR

It may be possible to assess responses on a molecular level
by performing PCR analysis to examine for the presence of
residual lymphoma cells that possess the bcl-2 translocation.
Before BM transplant, tumor samples obtained from 9 of
the 12 patients were identified as containing a PCR amplifiable translocation (Table 6). Unfortunately, data are not
available to assess the efficacy of marrow purging with MoAbs
and complement in all of these patients, but at least two
patients had detectable residual lymphoma cells in the reinfused marrow. After ABMT, all patients had BM biopsies
that were histologically negative for residual lymphoma.
However, four of these patients had residual cells with the
bcl-2 translocation detected by PCR analysis. Post Anti-B4bR therapy BM biopsies in three of these patients show no
evidence of cells with the bcl-2 translocation, and all patients
remain in clinical complete remission. By contrast, patient
7, who had no PCR detectable lymphoma cells at the time
of Anti-B4-bR therapy, did have evidence of lymphoma cells
by PCR posttherapy, and ultimately had a relapse of disease
13 months post-ABMT. Patient 6 had evidence of lymphoma
cells in the bone marrow both before and after Anti-B4-bR
therapy, but continues to remain in a clinical complete remission.

2269

Table 6. PCR Analysis

Patient No.

1

2
3
5
6
7
9
11
12

Pre ABMT

Postlysis

Pre Anti-64-bR

Post Anti-Br-bR

Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos

Postlysis samples were obtained after treatment with MoAbs and
complement.

Abbreviations: NA, not available; Pos, presence of cells with detectable
bcl-2 translocation; Neg, absence of cells with detectable bcl-2 translocation.

DISCUSSION

Despite the high rates of complete remission attained early
after ABMT for B-NHL, the majority of patients undergoing
high-dose therapy ultimately relapse. In an effort to enhance
the durability of these complete remissions, investigators have
attempted to provide additional therapy to patients postABMT. We have reported previously that Anti-B4-bR shows
in vitro and in vivo cytotoxicity against B-cell neoplasms,
and therefore may be considered as an agent to use in posttransplant therapy. Although we have shown that Anti-B4bR can be administered safely to patients with relapsed BNHL, we undertook the present trial to determine whether
Anti-B4-bR can be administered safely to patients early after
ABMT, to determine the toxicity profile in this setting, and
to determine the MTD.
In the present report, we show that Anti-B4-bR can be
administered by continuous infusion for 7 days to patients
in complete remission after ABMT for relapsed B-cell NHL.
The MTD was 40 pg/kg/d (total dose 280 pg/kg) with grade
IV reversible thrombocytopenia and transient elevations of
hepatic transaminases defining the DLT. In addition, systemic
side effects including fever, fatigue, and myalgias occurred
frequently in these patients. Mild reversible capillary leak
syndrome manifested by hypoalbuminemia and edema was
observed at all dose levels, but did not limit dose escalation.
A plateau serum level of Anti-B4-bR was achieved within 3
to 4 days after beginning the infusion in all patients, and that
level could be sustained for the duration of the infusion. In
patients receiving doses of 40 pg/kg/d or above, serum levels
above 1.0 nmol/L could be achieved within 72 hours. In

vitro cytotoxicity studies show that 3 logs of a malignant Bcell line can be depleted after 24 hour exposure to Anti-B4bR at equivalent concentration^.'^ The levels achieved in the
serum of these patients therefore potentially were therapeutic.
Eleven of the 12 patients treated remain in continuous complete remission from 13 to 26 months post-ABMT (median

17 months). Thus, this study shows that continuous infusion
of anti-B4-bR at 40 pg/kg/d for 7 days results in the attainment of potentially therapeutic serum levels of immunotoxin
post-ABMT with transient, tolerable toxicities.
Previous clinical trials using immunotoxins for the therapy
of both hematologic malignanciesand solid tumors have been
conducted in patients with relapsed, bulky t ~ m o r s . ~ lAl-~’
though clinical responses have been observed, most were
partial and transient. One explanation for the limited efficacy
observed for these highly cytotoxic agents is inadequate delivery to the neoplastic cell surface. Immunotoxins are large
molecules and their diffusion into sizable tumor masses is
likely to be hampered.28Moreover, immunotoxins can bind
to normal and neoplastic cells bearing the target antigen that
circulate in the blood stream, leading to rapid clearance of
the i m m ~ n o t o x i n This
. ~ ~ trial was designed to circumvent
these obstacles by administering Anti-B4-bR to patients with
minimal tumor burdens. Early post-ABMT, these patients
have low levels of circulating normal and neoplastic B-lymphocytes that can bind Anti-B4-bR.30 Because all patients
were in clinical complete remission at the time of therapy,
the delivery of Anti-B4-bR to the surface of residual malignant cells should be optimal. This study could not address
directly whether immunotoxin was bound to the lymphoma
cell surface because the number of residual lymphoma cells
post-ABMT are below our present levels of detection. However, the consistent time interval required to achieve steadystate levels of immunotoxin at each dose level as well as the
relatively consistent level of immunotoxin observed at each
dose escalation provide evidence that therapeutic levels of
immunotoxin were available to bind to residual lymphoma

cells. These data contrast with those of our previous studies
where circulating tumor cells led to the achievement of variable serum levels, reflecting the accessibility of the tumor to
Anti-B4-bR.’’,I6 The lower MTD of 280 pg/kg observed in
this trial (as contrasted with the MTD of 350 pg/kg when
Anti-B4-bR was administered by 7-day continuous infusion
to patients with bulky relapsed lymphomas) also supports
the notion of increased availability of Anti-B4-bR for uptake
by both normal and malignant cells. Together, these results
suggest that delivering immunotoxin to patients in complete
remission after ABMT increases the likelihood that thera-

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GROSSBARD ET AL

are significantly suppressed in vitro for the first year after
ABMT.30 Despite this degree of immunosuppression, 7 of
the 12 patients treated developed HAMA and/or HARA.
However, there was a trend toward reduced HAMA and
HARA development with higher doses of Anti-B4-bR. These
data suggest that higher doses of Anti-B4-bR may deplete
normal B cells capable of responding to the immunotoxin,

but the extent of decrease in B cell numbers and function
will need to be examined in future studies. Only two patients
received a second course of therapy, and both patients developed HAMA and HARA during the second course.
Therefore, if multiple cycles prove to be necessary to achieve
prolonged disease-free remissions, a different treatment
schedule or additional immunosuppression may be necessary
to deliver multiple courses. Because most patients at the MTD
did not produce anti-immunotoxin antibody until 4 weeks
posttreatment, one approach might be to treat patients every
14 days rather than every 28 days. In an ongoing pilot study,
we have been able to administer Anti-B4-bR at 14-day intervals at a dose of 30 pg/kg/d without significant toxicity.
The ultimate objective of this study was to administer adjuvant therapy in an effort to prevent relapses after ABMT.
Because most patients achieve clinical complete remission
early after ABMT, occult residual lymphoma cells either in
the reinfused marrow or in the patient must contribute to
relapse. Anti-B4-bR, which exerts its cytotoxicity through
the inhibition of protein synthesis, may provide a non-crossresistant therapy with which to treat these patients. The small
number of patients treated on this trial and the short followup permit no definitive conclusions to be made regarding
the efficacy of this therapy. To date, only one patient on this
trial has relapsed, with follow-up ranging from 13 to 26
months post-ABMT. Of note, all patients on this trial were
in partial remission at the time of ABMT. However, it is
conceivable that many of the patients on this trial were at
low risk of early relapse because they had low-grade NHL,
were in complete remission 5 to 8 months post-ABMT, and
lacked detectable bcl-2 positive cells at the time of therapy.
Considering the small number and heterogeneity of the patients treated on this trial, it is not possible to determine
whether Anti-B4-bR adjuvant therapy has contributed to
the disease-free survival. The possibility that Anti-B4-bR
contributed to the eradication of residual bcl-2 positive cells

in 3 patients on this study is intriguing, but will require validation in future studies designed to address this issue directly.

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peutic levels of immunotoxin will be delivered to the surface
of remaining tumor cells.
A major concern of this study was whether patients could
tolerate additional cytotoxic treatment within the first 6
months after ABMT. Although these patients recently had
received high-dose chemotherapy and total body irradiation,
and were still recovering from transplant related toxicities,
the side effect profile of Anti-B4-bR in the posttransplant
setting was similar to that seen in patients with relapsed bulky
NHL who also received continuous infusion of this drug.I6
In the present trial, the MTD was defined by grade IV thrombocytopenia occurring in 4 of 12 patients. The fact that
thrombocytopenia was the dose-limiting toxicity reflects the
lower baseline platelet counts in patients treated post-ABMT
compared with those of patients on our earlier studies. With
the possible exception of a patient who developed a macular
hemorrhage, no patient had bleeding secondary to the
thrombocytopenia, and reductions in platelets resolved rapidly in nearly all patients. Other toxicities were comparable
with those observed in the previous trial using continuous
infusion Anti-B4-bR. Most importantly, these included
transient hepatic transaminase elevations, nausea and vomiting, fever, fatigue, and myalgias. Although the MTD of AntiB4-bR was lower on this trial, the serum level required to
induce each toxicity was comparable. This again reflects the
higher serum level that can be achieved at any given administered state in the absence of significant numbers of normal
and neoplastic B cells. Importantly, no new toxicities were
observed on this trial, and the anticipated toxicities were not
magnified in patients treated early post-ABMT. This was especially true for capillary leak syndrome, which was clinically
significant in only 5 patients, with persistent peripheral edema

in 4 patients and shortness of breath in I patient. Hemolyticuremic syndrome (HUS) was observed in two patients on
this trial, but this side effect has been reported in 10% of
patients undergoing ABMT at our center.20The small number
of patients treated on this trial render it impossible to determine whether the incidence of HUS is increased after treatment with Anti-B4-bR. Considering the small number of
patients treated in this trial, we cannot resolve the question
of whether toxicity was more pronounced and prolonged in
patients treated closer to the time of high-dose myeloablative
therapy. In future studies, we will attempt to treat all patients
within the first 120 days post-ABMT. Theoretically, it might
be advantageous to treat patients within 30 to 60 days postABMT, but post-ABMT thrombocytopenia and abnormalities of liver function tests will likely make this approach less
feasible.
Another purpose of conducting this study in patients postABMT was to treat patients at a time when they were immunosuppressed in order to reduce the frequency of HAMA
and HARA formation and possibly administer more courses
of Anti-B4-bR. Both the murine MoAb and the protein toxin
are immunogenic when administered to patients. Nearly all
solid-tumor patients who receive immunotoxins develop
HAMA and HARA, and up to 71% of leukemia and lymphoma patients likewise may develop an immune res p o n ~ e . ~Our
' ~ prior
~ ~ . studies
~~
showed that patients do not
recover normal numbers of circulating B cells for 3 to 6
months after ABMT, and that their B cell and T cell functions

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ACKNOWLEDGMENT

We appreciate the assistance of Danny Ducello and Linda McGeary
in data management. We also thank Ginny Braman for technical

assistance. Michael L. Grossbard is on the ImmunoGen speakers
panel and Lee M. Nadler is a consultant to ImmunoGen, Inc.
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