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

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 sustained for

3 to 4 days The maximum tolerated dose was 40 pglkgld

for 7 days (total 280 pglkg) The dose-limiting toxicities

LTHOUGH HIGH-DOSE myeloablative therapy fol-

A lowed by autologous bone marrow (BM) reinfusion can

induce a clinical complete remission in the vast majority of

patients with relapsed B-cell non-Hodgkin’s lymphoma

(NHL), between 50% and 85% of these patients ultimately

relapse.13’ Tumor recurrence primarily is attributable to the

presence of clones of lymphoma cells resistant to high-dose

therapy, but reinfusion of lymphoma cells harbored within

the autologous marrow probably also contributes to r e l a p ~ e ~ , ~

In an attempt to overcome tumor cell resistance, a number

of investigators have intensified the myeloablative regimen

Although complete remission rates may be increased with

this approach, the morbidity and mortality of therapy also

significantly We and others have also attempted

to decrease the number of relapses by purging lymphoma

cells from the harvested autologous Unfonmately,

purging has been effective in removing residual lymphoma

cells from the marrow in only 50% of patients; therefore,

small numbers of residual lymphoma cells may continue to

contribute to r e l a p ~ e ~ In an attempt to overcome these ob-

stacles to autologous BM transplantation (ABMT), a number

of investigators have begun to treat patients after ABMT with

therapies designed to overcome lymphoma cell resistance and

eradicate residual neoplastic cells transferred in the reinfused

BM Because of the myelosuppressive side effects attendant

to high-dose therapy, traditional chemotherapeutic agents

cannot be used early after ABMT In contrast, new agents

with nonoverlapping toxicity, such as immunotoxins or cy-

tokines, both may be delivered safely in this setting and may

be capable of killing resistant residual lymphoma

Over the past 3 years, we have used a novel immunotoxin

to treat patients with relapsed B-cell NHL The immunotoxin,

AntLB4-blocked ricin (Anti-B4-bR) combines the B-cell

specificity of the anti-B4 (CD19) monoclonal antibody

(MoAb) with a toxin, termed “blocked ricin.”12 In blocked

ricin, which is derived from the potent protein toxin ricin,

the binding of ricin is attenuated by attaching affinity ligands

to the galactose binding sites that mediate nonspecific bind-

ing.I3 The resultant immunotoxin is highly cytotoxic to cells

that express the CD 19 antigen and effects its cytotoxicity by

inhibiting protein synthesis j 4 Therefore, Anti-B4-bR poten-

tially may kill lymphoma cells resistant to chemotherapy

I

Blood, Vol81, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBANo 9 (May 1 ), 1993: pp 2263-227 1

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

treating patients with relapsed B-cell NHL In the first trial, Anti-B4-bR was administered by daily bolus infusion for 5

dose (MTD) of 50 pg/kg/d (total 250 pg/kg) and the dose-

limiting toxicity (DLT) was defined by transient grade IV

increases in hepatic transaminases and thromb~cytopenia.’~

Because additional preclinical studies suggested that higher doses of Anti-B4-bR could be administered safely by 7-day continuous infusion, we also conducted a phase I trial in a similar patient population using a 7-day continuous infusion.I6 This treatment schedule allowed potentially therapeutic serum levels to be sustained in serum for up to 4 days, and a higher MTD of 50 pg/kg/day X 7 days (total 350 p g / kg) was achieved In this trial, the DLT was identical to that

of the bolus infusion trial although continuous infusions also led to mild, reversible capillary leak syndrome

Although clinically significant responses, including complete remissions, were seen in both phase I trials, the majority

of responses were observed in patients with lower tumor burdens This suggested that one major obstacle to effective immunotoxin therapy might be the delivery of these agents to all of the lymphoma cells Therefore, we hypothesized that administration of Anti-B4-bR after ABMT might lead to improved tumor cell delivery to a small number of residual tumor cells and potentially eradicate remaining resistant lymphoma cells In this study, we report a phase I trial of

From the Division of Tumor Immunology, Dana-Farber Cancer Institute; the Department of Medicine, Haward Medical School, Bos- ton, MA; and ImmunoGen, Inc, Cambridge, MA

Submitted September 14, 1992; accepted December 14, 1992

Supported by National Institute of Health Grant Nos CA34183

stitute Clinical Oncology Research Career Development Award (1 K12CAOl730)

Address reprint requests to Lee M Nadler, MD, Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney St, Boston,

MA 021 15

The publication costs of this article were defayed in part by page charge payment This article must therefore be hereby marked

indicate this fact

0 1993 by The American Society of Hematology

0006-49 71/93/8109-0024%3.00/0

2263

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2264 GROSSBARD ET AL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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

Anti-B4-bR

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 for-

mulated as a sterile injectable solution containing 100 &mL of Anti-

B4-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 ad-

ministration 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)

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 che-

mosensitive 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 ir-

radiation (TBI) in fractionated doses (200 cGy) twice daily on 3 con-

secutive days (total 1,200 cGy) Within zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA18 hours after the completion

of TBI, all patients received a re-infusion of purged autologous mar-

row.'

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 ob-

taining computer tomography zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(CT) scans of previous sites ofdisease,

gallium scans in patients with prior gallium avid disease, chest ra-

diographs, 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 r 3 0 , 0 0 0 / ~ 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-occlusive disease, 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 pro-

tocol was approved by the Institutional Review Board of the DFCI,

and all patients signed an informed consent form approved by that

committee

Study Design

After the documentation of complete remission, patients were ad- mitted to the DFCI and received a continuous infusion of Anti-B4-

bR 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 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI11 or IV myelosuppression of less than 7 days

duration was not considered a dose-limiting toxicity 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

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 Re- search Laboratories) was used, allowing the use of highly diluted serum samples and reducing the nonspecific background signals

HAhiA/HARA Detection

HAMA and HARA were measured by established ELISA tech- niques 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 per- formed 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 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 lym-

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

Patient Selection and Characteristics

Between 6/29/90 and 81219 1 , 2 1 patients with B-cell NHL

in sensitive relapse attained a minimal disease state and un-

derwent anti-B-cell MoAb-purged ABMT Of these 21 pa-

tients, 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 el-

evated 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 eligibility criteria post-ABMT, three patients were

treated more than 104 days post-ABMT The first two pa-

tients, treated at day 151 and 140, respectively, both were

eligible to receive Anti-B4-bR by day 90 post-ABMT Un-

fortunately, 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-

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 Al- though 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 trans-

location 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

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-B4-

bR and 2 patients were retreated at the same dose level as their initial therapy Patients were not retreated for the fol-

Table 1 Patient Characteristics

Dose AntikB4-bR

Patient No Disease Pre-ABMT at ABMT at ABMT Date of ABMT Translocation AntikB4-bR (rg/kg/d)

1

2

3

4

5

6

7

8

9

10

11

12

FSCCL

SLL

FML

DSCCL

DML

FSCCL

ILL

FML

FSCCL

4

3

2

3

5

3

2

5

2

5

3

PR

BM LN

BM, LN

LN

BM, LN

LN, EN

BM, LN

LN

BM, LN

LN

BM, LN

LN

6/29/90

711 1/90 9/28/90

21719 1

411 1/91 5/3/91 6/14/91 5/31 191 7/3/91

212219 1

8/2/91

1011 1/90

Yes Yes Yes

No Yes Yes Yes

No Yes

No Yes Yes

151

140

8 0

85

62

78

81

61

95

62

208

104

20

4 0

50

4 0

40

4 0

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

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2266 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAGROSSBARD ET AL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

lowing the initial course (n = I), posttherapy macular hem-

orrhage (n = l), HAMA/HARA development before the sec-

ond course of therapy (n = 3), and refused a second course

of therapy (n = 1)

Serum levels of immunoconjugate were determined by ELISA

in all patients by separate detection of the blocked ricin and

anti-B4 moieties of the conjugate There was a highly repro-

ducible relationship between the dose of Anti-BCbR admin-

istered 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

toxicity assays, sustained exposure of malignant B cell to con-

centrations of Anti-B4-bR above I nmol/L would be pre-

Patient 3 (20 pg/kg/d)

Patient 5

2 - 0 1 (40 pngikgld) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0 2 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 rep-

resents 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

Table 2 Serum Levels of Anti-E4-bR

Dose Day 7 Serum Level

Patient No lualkaldl lnmol ? SE)

1

2

3

4

5

6

7

8

9

10

1 1

12

20

40

50

40

0.13 +0.01 0.37 + 0.04

Undetectable 0.27 i zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA0

1.10 k 0.02 1.44 f 0.05

Undetectable

1.78 t 0.20 2.51 t 0.23' 1.71 i.0.17 2.63 f 0.1 1"

1.20 f 0.10 1.12 f 0.1 1

1 1 1 f0.15

Patients 2 and 5 received two courses of therapy Levels for the second

course were undetectable

of therapy

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

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 electrocar-

Overall toxicity

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2 2 6 7 Table 3 Anti-B4-bR Post-ABMT: Clinical Toxicities

Systemic Gastrointestinal Capillary Leak Hematologic Patient No (pg/kg/d) Fever Status (ECOG) Headache Myalgias Nausea Vomiting Transaminases Dose Performance Hepatic zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAHypoalbuminemia (>20% decrease) Edema Dyspnea Leukocytosis Anemia zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

1 20 2 0 0 0 2 1 2 Yes 0 0 No Yes

2 20 1 0 0 0 0 0 3 No 0 0 No Yes

2 0 0 0 0 0 0 0 1 No 0 0 No Yes

3 2 0 1 0 0 2 0 0 3 No 2 0 No No

4 40 2 0 0 1 0 0 3 Yes 2 0 No No

5 40 1 1 1 0 0 0 3 No 0 0 No No

40 1 0 0 0 0 0 1 No 0 0 No No

6 40 1 3 1 2 0 0 3 No 3 0 No No

7 50 2 3 0 2 2 0 2 Yes 2 0 No Yes

9 40 0 2 2 1 2 2 4 No 0 0 No No

10 40 2 3 0 0 2 0 3 No 0 0 Yes No

1 1 40 1 1 1 2 0 0 2 Yes 0 0 No No

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) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

or change in creatinine immediately after therapy Five pa-

tients treated at doses of 40 and 50 pg/kg/d developed head-

aches 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

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 asso-

ciated 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 el-

evated prothrombin time (PT) or partial thromboplastin time

(PTT)

Leukopenia was not apparent in any patient, but leuko-

cytosis occurred in two patients with white blood cell (WBC)

counts increasing to above lO,OOO/pL in the absence of in-

fection (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

Hematologic

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 Pa- tient 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 hemolytic- uremic syndrome, in this case 90 days after Anti-B4-bR therapy Again, this was characterized by self-limited thrombocytopenia, anemia, and renal dysfunction

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

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) Hypo- albuminemia 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-

Hepatotoxicity

Hypoalbuminemia and capillary leak syndrome

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2268 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAGROSSBARD ET AL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Table 4 Thrombocytopenia and Hepatoxicity

Patient Pre Anti-64-bR Post Anti-64-bR Days t o Recovery' SGOT/ULN SGPT/ULN Days t o Recovery'

1 65 36 14 4.4 4.3 14

2 257

202

3 71

4 50

5 34

32

6 129

7 32

8 102

9 62

10 141

1 1 96

12 102

97

95

50

20

26

46

38

20

16

52

35

50

7

15

14

13

t

21

14

22

*

22

18

5.4 1.5 5.6 11.4

6 1.2 9.8 3.1 8.4 26.3

19 3.4 13.4

5.8 1.3 7.1 11.6 6.9 0.8 8.2

2 6.3

28 16.6 3.8 17.5

25

21

13

12

7

14

22

*

14

15

Days to recovery to baseline value

t Follow-up information not available until day 48 Platelets fully recovered at that time

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, as-

cites, or hypotension in association with capillary leak

Data on HAMA and HARA formation

treated within 95 days after ABMT, 7 of the 12 patients de-

veloped 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

develop HAMA or HARA, suggesting a blunting of the im-

mune response at higher delivered doses of this B-cell im-

munotoxin No patient developed HAMA in the absence of

HARA Because follow-up HAMA/HARA data were not ob-

tained beyond day 28 in most patients, the reported rates of

antibody formation may underestimate the true frequency

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

HAMA/HARA

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 Anti- B4-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

Because all patients were in complete remission at the out- set 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

bg/kg/d) No Patients Negative Positive Negative Positive POSltlVlty

20

40

50

Total

3

7

2

12

14, 21, 38

3 4 27, 28, 32, 38

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ADJUVANT IMMUNOTOXIN THERAPY WITH ANTI-64-bR 2269 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

It may be possible to assess responses on a molecular level

by performing PCR analysis to examine for the presence of

Before BM transplant, tumor samples obtained from 9 of

the 12 patients were identified as containing a PCR ampli-

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 rein-

fused 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-B4-

bR 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

cells in the bone marrow both before and after Anti-B4-bR

therapy, but continues to remain in a clinical complete re-

mission

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 post-

ABMT 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 post-

transplant therapy Although we have shown that Anti-B4-

bR can be administered safely to patients with relapsed B-

NHL, 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 B-

cell line can be depleted after 24 hour exposure to Anti-B4-

bR 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 com-

plete remission from 13 to 26 months post-ABMT (median

Table 6 PCR Analysis

Patient zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBANo Pre ABMT Postlysis Pre Anti-64-bR Post Anti-Br-bR

1

2

5

6

7

9

1 1

12

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 trans-

location

17 months) Thus, this study shows that continuous infusion

of anti-B4-bR at 40 pg/kg/d for 7 days results in the attain- ment of potentially therapeutic serum levels of immunotoxin post-ABMT with transient, tolerable toxicities

Previous clinical trials using immunotoxins for the therapy

of both hematologic malignancies and solid tumors have been conducted in patients with relapsed, bulky t ~ m o r s ~ l - ~ ’ Al- 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.28 Moreover, 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-lym- phocytes 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 How- ever, the consistent time interval required to achieve steady- state 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 vari- able 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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2270 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAGROSSBARD ET zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAAL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 throm-

bocytopenia 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 rap-

idly 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 vom-

iting, fever, fatigue, and myalgias Although the MTD of Anti-

B4-bR was lower on this trial, the serum level required t o

induce each toxicity was comparable This again reflects the

higher serum level that can be achieved at any given admin-

istered state in the absence of significant numbers of normal

and neoplastic B cells Importantly, no new toxicities were

observed o n this trial, and the anticipated toxicities were not

magnified in patients treated early post-ABMT This was es-

pecially true for capillary leak syndrome, which was clinically

significant in only 5 patients, with persistent peripheral edema

uremic 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.20 The small number

of patients treated on this trial render it impossible to deter-

mine whether the incidence of HUS is increased after treat-

ment 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 post-

ABMT, but post-ABMT thrombocytopenia and abnormal-

ities of liver function tests will likely make this approach less

feasible

Another purpose of conducting this study in patients post-

ABMT was to treat patients at a time when they were im-

munosuppressed 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 lym-

phoma patients likewise may develop a n immune re-

s p o n ~ e ~ ' ~ ~ ~ ~ ~ Our prior studies showed that patients d o not

months after ABMT, and that their B cell and T cell functions

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 t o 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 t o 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 M T D did not produce anti-immunotoxin antibody until 4 weeks posttreatment, one approach might be t o treat patients every

14 days rather than every 28 days In a n ongoing pilot study,

we have been able to administer Anti-B4-bR a t 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-cross- resistant therapy with which to treat these patients The small number of patients treated o n this trial and the short follow-

up 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 t o 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

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 val- idation in future studies designed to address this issue directly

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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ADJUVANT IMMUNOTOXIN THERAPY WITH ANTI-B4-bR zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA227 1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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Tiêu đề	Adjuvant Immunotoxin Therapy With Anti-B4-Blocked Ricin After Autologous Bone Marrow Transplantation for Patients With B-Cell Non-Hodgkin’s Lymphoma
Tác giả	Michael L. Grossbard, John G. Gribben, Arnold S. Freedman, John M. Lambert, Jeanne Kinsella, Susan N. Rabinowe, Laura Eliseo, James A. Taylor, Walter A. Blattler, Carol L. Epstein, Lee M. Nadler
Trường học	American Society of Hematology
Chuyên ngành	Hematology/Oncology
Thể loại	Research Article
Năm xuất bản	1993

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Số trang	9
Dung lượng	1,03 MB