Treatment of B cell lymphomas with anti-idiotype antibody therapy in combination with alpha interferon

1989 73: 651-661

Merigan and R LevySL Brown, RA Miller, SJ Horning, D Czerwinski, SM Hart, R McElderry, T Basham, RA Warnke, TC combination with alpha interferonTreatment of B-cell lymphomas with anti-idiotype antibodies alone and in

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Blood, Vol 73, No 3 (February 15), 1989: pp 651-661 6�1

Treatment of B-Cell Lymphomas With Anti-idiotype Antibodies Alone and inCombination With Alpha Interferon

By Sherri L. Brown, Richard A. Miller, Sandra J. Horning, Debra Czerwinski, Sarah M. Hart, Roxena McElderry,

Teresa Basham, Roger A. Warnke, Thomas C. Merigan, and Ronald Levy

Idiotypes are distinct clonal markers for B-cell lymphomas.

Previously we reported the use of anti-idiotype antibodies

in the therapy of patients with B-cell malignancies.

Because synergy was demonstrated with the addition of

alpha interferon to anti-idiotype antibodies in a murine

lymphoma model, we performed a clinical trial combining

these two agents. Here we provide an update of the

original trial of anti-idiotype antibodies alone and report

the outcome of the new combination trial. In 16 treatment

courses of anti-idiotype antibodies alone there were seven

partial responses and one complete response. In 12

courses of combination anti-idiotype antibody and alpha

interferon there were two complete responses and seven

partial responses. Substantial tumor regressions occurred

J3-CELL LYMPHOMAS represent a special class of

human tumors because they express a cell-surface

immunoglobulin molecule unique for each patient that can

serve as a marker for the tumor.’ Antibodies can be produced

against the idiotype, or variable region of the immunoglobu-

lin molecule, and these anti-idiotype antibodies have been

shown to be exquisitely sensitive in discriminating tumor

cells from normal cells.24

Previously we reported the treatment of 1 1 patients with

B-cell malignancies with monoclonal anti-idiotype antibod-

ies.5 Seven of these patients had tumor regression, including

one complete response and four partial responses. An impor-

tant observation made in this trial was the emergence of

idiotype-negative variant tumor cells upon tumor pro-

gression.6 Subsequent studies showed that idiotype-negative

variant cells exist in the lymphoma cell populations prior to

treatment and that this heterogeneity is due to point muta-

tions in the expressed immunoglobulin-variable region

genes.7 This suggested that treatment with anti-idiotype

antibodies selectively reduced the idiotype-positive lym-

phoma cell population, allowing, in some cases, an idiotype-

negative variant cell to become dominant.

A murine lymphoma model has been developed that

allowed testing of the therapeutic effects of anti-idiotype

� Marked antitumor effects were seen after a

single administration of anti-idiotype antibodies, leading to

cure of up to 80% of mice previously injected with tumor

cells. In this murine model, anti-idiotype antibody therapy

resulted in the selection of idiotype-negative tumor cell

variants similar to those arising in human tumors.9 We have

subsequently used this model to examine the effects of

monoclonal anti-idiotype antibodies in combination with

other biological agents.’#{176}” When interferon, an agent that

has an independent antitumor action, was administered in

combination with monoclonal anti-idiotype antibodies, a

synergistic therapeutic effect was noted.”

In an attempt to address the problem of idiotype-negative

variant cells, we performed a clinical trial combining anti-

idiotype antibodies and alpha interferon, based on the inde-

pendent activities of anti-idiotype antibodies and interferon

in patients with B-cell lymphomas and their synergy when

with minimal toxicity in both trials even in patients refrac-

tory to conventional chemotherapy. Tumor specimens

obtained at the time of disease progression often con-

tamed a preponderance of idiotype-negative lymphoma

cells, suggesting that anti-idiotype antibody treatment

exerted a strong antitumor effect against antigen-positive

cells. Anti-idiotype antibodies have reproducible objective

antitumor activity in B-cell lymphoma. The addition of

alpha inteferon may improve the initial rate of response to

this treatment. Strategies that deal effectively with idio-

type-negative lymphoma cells should improve the extent

and duration of these responses.

S 1989 by Grune & Stratton. Inc.

combined in the murine lymphoma model. Here we provide

an update of the previous trial of anti-idiotype antibodies

alone and report the results of the trial combining anti-

idiotype antibodies and interferon. Both studies confirm the

antitumor effect of anti-idiotype antibodies. In both trials

patients had idiotype-negative cells that persisted after treat-

ment and contributed to regrowth of tumor.

MATERIALS AND METHODS

Patient selection. All patients included in these trials gave

informed consent in accordance with the guidelines established bythe Human Investigation Committee of Stanford University Medi-

cal Center. Eligibility criteria included an initial diagnosis of follicu-lar lymphoma, a peripheral lymph node accessible for biopsy ofgreater than 2 cm in diameter, a projected survival of greater than 1year, and an absence of other major medical problems. Immunophe-notyping of the tumor cells was required to confirm the presence of a

monoclonal surface immunoglobulin. Anti-idiotype antibodies wereproduced (see below) and then each patient was re-evaluated. Beforethe treatment phase patients were required to have measurabledisease and, in the antibody-plus interferon trial, a serum-idiotype

protein level less than SO �tg/mL. (The anti-idiotype antibody alonetrial demonstrated that above SO zg/mL the serum barrier ofidiotype protein makes it difficult to infuse enough antibody to

penetrate tumor tissue.) Prior to the initiation of treatment, a secondlymph-node biopsy was performed to confirm the continued reactiv-ity of the anti-idiotype antibody with the tumor cells. Pretherapy

From the Departments of Medicine and Pathology, Stanford

University School of Medicine, Stanford, CA and IDEC Pharma-

ceuticals Corporation, Mountain View, CA.

Submitted August 2, 1988; accepted October 17, 1988.

Supported by National Institutes of Health Grants CA 33399

and CA 34233.

Address reprint requests to Ronald Levy, MD, Division of

Oncology. M207, Stanford University Medical Center, Stanford,

CA 94305.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. section /734 solely to

indicate this fact.© I 989 by Grune & Stratton, Inc.

0006-4971/89/7303-0008$3.00/0




652 BROWN ET AL

evaluation also included physical examination; CBC; platelet count;general chemistry survey; urinalysis; chest x-ray; lymphangiogram;computed tomography (CT) scan of the chest, abdomen, and pelvis;

and bone marrow biopsy. Patients had not received antitumortherapy for at least 4 weeks before beginning the experimental

treatment. Here we provide an update on the initial group of nineevaluable lymphoma patients treated with anti-idiotype antibodies

alone, along with the results of this trial in five subsequent patients.Additionally the outcomes are reported for 1 1 patients who com-

pleted combined treatment with anti-idiotype antibody and interfer-

on.Anti-idiotype antibodies. Mouse or rat monoclonal anti-idio-

type antibodies were produced by methods previously described indetail.5 Large-scale production of antibodies was accomplishedeither by growing hybridomas as ascites in mice or by in vitrocell-culture techniques.’2”3 In the anti-idiotype antibody alone trial,antibodies for the first nine patients were purified by ammonium

sulfate precipitation. The antibodies for all other patients were

additionally purified by ion exchange chromatography (IDEC Phar-maceutical Corporation, Mountain View, CA). These later prepara-tions were greater than 90% pure IgG as determined by sodiumdodecyl sulfate-palyacrylamide gel electrophoresis (SDS-PAGE).Therapeutic preparations passed general safety, sterility, and endo-

toxin testing and were found to be free of adventitious viruses.’4”5Trial design. Patients received therapy in a hospital setting.

Acetaminophen and diphenhydramine were given every four hoursuntil the end of each antibody infusion. Anti-idiotype antibody wasadministered over four hours by intravenous (IV) infusion using a0.22-sm in-line filter. Treatment was given three times per weekuntil all the available antibody had been delivered. This was usuallya duration of three to four weeks. In both trials it was assumed that

the anti-idiotype antibody needed to bind to the tumor cells to havean antitumor effect. In our originial trial with anti-idiotype antibod-ies alone, biopsies of tumor tissue were performed 24 hours afterantibody infusion to document the presence of mouse protein binding

to the target cells.5 Based on this previous experience, antibody was

infused in doses sufficient to overcome circulating serum-idiotypeprotein and to permit penetration of solid-tumor tissue. Because the

antibody treatment, serum-idiotype level, and amount of tumor bulk

were different for each patient, the antibody doses were individual-ized. Recombinant alpha interferon (Roferon, Hoffmann LaRoche,

Nutley, NJ) was given two hours before each antibody infusion at a

dose of 12 x 106 jz/m2 intramuscularly (IM) in the combinationtherapy trial. After the first 3 to 4 weeks of the treatment theinterferon was continued alone at the same dose three times weeklyfor a total duration of eight weeks.

In both trials a general chemistry survey, CBC, and platelet countwere performed prior to each antibody infusion. Serum samples were

collected immediately before, immediately after, two hours after,and 24 hours after each antibody dose. Chest x-ray, lymphography,and CT scans (as appropriate) were performed prior to initiation of

therapy on both protocols. These studies were obtained at the end ofthe three to four weeks of antibody, at the end of the eight weeks ofinterferon in the combination therapy trial, and in follow-up monthlyor bimonthly thereafter in both trials. Bone marrow aspirate andbiopsies were performed at study entry and were repeated if all othermeasurable disease sites responded completely. Repeat lymph-nodebiopsies were performed if the patient had progressive disease

following treatment.Clinical responses were scored according to objective measure-

ment of disease. Since all patients had multiple disease sites, in eachcase several key lesions were designated. A complete response wasdefined as the disappearance of all sites of disease. A partial response

was defined as greater than 50% reduction of the product of the

aggregate perpendicular diameters of the measured key lesions. Aminor response was defined as a reduction of less than 50% of the

product of the aggregate perpendicular diameters of the measuredkey lesions. Responses were required to last at least 1 month with the

observation of response noted on two separate occasions. Freedomfrom progression was scored from the time treatment was initiated

until disease progression occurred.

Serum assays. Enzyme-linked immunosorbent assays were used

to measure serum levels of idiotype protein, mouse immunoglobulin,and human antibody to mouse immunoglobulin as previously

described.5

Immunophenotyping. Single-cell suspensions of tissue samples

were stained with fluorescent antibodies, F(ab’)2 fragments of goat

antihuman immunoglobulin chains and F(ab’)2 fragments of goat

antimouse IgG (Tago, Burlingame, CA) by methods previously

described5 and analyzed by flow cytometry (FACS 440, Becton

Dickinson, Mountain View, CA). An estimate of the total number oftumor cells in each sample was obtained by staining with anti-lambda and anti-kappa antibodies. The number of cells staining with

the light chain not expressed by the tumor was doubled andsubtracted from the total number of cells expressing the tumor’s

heavy chain. In this way small numbers of normal B cells in theanalyzed samples were excluded. Frozen sections of biopsy speci-

mens were stained by the immunoperoxidase method, as previously

Cytocentrifuge preparations were made from lymphocytes from

whole blood or bone marrow following isolation by Ficoll-Hypaque

(FH) sedimentation. Slides were fixed and stained with murinemonoclonal antibodies (MoAbs), followed by enzyme-labeled anti-

bodies. Color was generated with APAAP (Dako, Santa Barbara,

CA) and developed with a naphthol AS-MX phosphate.

RESULTS

Therapeutic trial of anti-idiotype antibody alone. Atotal of 14 evaluable patients with lymphoma have been

treated with anti-idiotype antibodies alone. Nine of these

patients have been reported previously. Two of these have

been treated a second time, and five additional patients are

now evaluated.

Table 1 summarizes the clinical characteristics of the

patients treated with anti-idiotype antibodies alone. Thirteen

of 14 patients had follicular lymphoma at the time of initial

biopsy. Progression to a diffuse histology occurred in the

tumors of three patients prior to antibody treatment. Twelve

patients had previously been treated with chemotherapy.

Eight patients had tumor masses greater than 5 cm.

A summary of the clinical responses is presented in Table

2. In 16 treatment courses there were eight complete or

partial responses with four of these responses lasting 6

months or longer, including one complete response lasting 6

years. The cumulative antibody doses ranged from 500 mg in

patient PK to 1 5,500 mg in patient KC. The importance of

the pretherapy serum-idiotype protein level was demon-

strated by the treatment courses of patients EL and JC. Both

patients were initial nonresponders who had major clinical

responses once the serum-idiotype level was reduced. In five

patients more than one anti-idiotype antibody was used to

achieve comprehensive reactivity with the tumor-cell popula-

tions. Three of these five patients had partial responses

lasting 6 months or greater. Two patients had progressive

disease at 6 and 1 2 months, while patient CC has had




CVP

Bleomycin

Interferon

CC/M/71 FSC

Pretherapy

Id Level

�ig!mL

5.0

.01

Patient

PK

CC

Humana Mouse

Total

Antibody

Dose (mg)

400

6,908

15,500

Tumor

Response

CR

PR

Freedom

fromProgression

6Y

25M+

11,700 -

+ RDPE

CJ

EL(2)

CG

BJ

+ EL(1)CP

TG

JC (1)

PR 12M

PR 6M

- PR 5M

- PR 4M

+ PR 1M

- PR -�

- MR 3M

+ MR 1M

- NR -

+ NR -

+ NR -

- NR -

1.993

3,183

3,079

3,106

3,173

2,492

2,101

3,080

1,775

9,630

Disease Sites

Nodes, liver,

spleen, scalp

nodules

Nodes, bone

marrow

Nodes, bone

marrow

Nodes, femoral

mass, skin

nodules

Supraclavicular

mass, lung

mass, abdomi-

nal mass

Abdominal mass

nodes, bone

marrow

Nodes, liver

Nodes, spleen.

bone marrow

Nodes, liver,

bone marrow

Nodes, liver

Nodes, bone

marrow

Nodes, bone

marrow

Nodes, bone

marrow

Nodes, bone

marrow

9,600 - NR -

3.060 NR

TG/M/36 FSC

EW/M/33 FSC

ANTI-IDIOTYPE ANTIBODY THERAPY 653

Table 2. Anti-idiotype Antibody Therapy: Clinical Responses

Table 1 . Anti-idioty pe Antibody Therapy: Pati ent Characteristics

Patient!Sex/Age Dx Prior Therapy

Tumor

Masses�5 cm

PK/M/67 FSC

DSC

+

KC/M/27 FSC CVP

Chlorambucil

JC/M/44 FSC CVP

DLC Chlorambucil

High-dose

cyclophosphamide

AdriamycinSplenectomy

RD/M/44 DSC CVP

DLC Chlorambucil

MTX

VM 26

Adriamycin

Bleomycin

XRT to abd mass

Interferon

PE/M/38 FSC CVP

Chlorambucil

CJ/M/29 FSC CVP

CMOPP

CHOP

ChlorambucilSplenectomy

EL/F/36 FSC CVP

BVP

Chlorambucil

Adriamycin

Vinblastine

CG/F/53 FM CVP

Interferon

Splenectomy

BJ/F/4O FM CHOP

BVP

CCNU

Splenectomy

CP/M/42 FSC Interferon

CVP

Splenectomy

CVP

BL/F/49 FSC Splenectomy

Abbreviations: FSC, follicular small-cleaved cell lymphoma; DSC,

diffuse small-cleaved cell lymphoma; FM, follicular mixed lymphoma;

DLC. diffuse large-cell lymphoma, CVP, cyclophosphamide, vincristine,

prednisone; BVP, bleomycin, vincristine, prednisone; CHOP, cyclophos-

phamide, Adriamycin, vincristine, prednisone; MTX, methotrexate; XRT,

radiation therapy.

Antibody

Isotype�

IgG2b

lgG2a

IgG2b

lgG 1

KC 12.8 lgG2b

lgG 1

JC (2) 0.8 lgG2a

IgG1

lgG 1

0.10 lgGl

14.50 IgG2b

2.20 IgGi

20.0 IgGi

0.01 IgGi

0.02 lgG2b

243.0 IgGi

0.01 IgGl

3.26 lgG2a

49.4 IgG2a

lgG 1

lgG 1

EW 34.0 IgGi

IgG 1

+ BL 3.0 lgG2blgGl

+

Number in parentheses refers to the fact that the patient has been

treated more than once on this antibody protocol.

tAll antibodies are mouse monoclonals.

�This patient was entered on another treatment protocol after receiv-

ing antibody therapy, and the duration of the PR could not be

determined.

continued tumor regression for over 25 months. Therapy in

patient BL was aborted after five antibody infusions because

of an immune response against horse immunoglobulin, a

minor contaminant, in the antibody preparation. Toxicity

+ was limited to urticaria. Patient EW never achieved circulat-

ing levels of free anti-idiotype antibody in serum because of a

+ relatively high serum-idiotype level and large tumor bulk.Patient PK, the first patient treated with anti-idiotype

antibody, achieved a complete remission. He remained free

ofdisease for 6 years with no additional therapy. At that time

- an erythematous macular skin lesion appeared on the dorsum

of one foot distal to a site of cellulitis. Biopsy of this lesion

- revealed diffuse small-cleaved cell lymphoma. CT scans of

the chest, abdomen, and pelvis; lymphangiogram; bone mar-

- row biopsy; and immunostaining of the bone marrow aspirate

- and peripheral blood lymphocytes were all negative for

disease. Immunoperoxidase staining of the skin lesion

showed this recurrent disease to be idiotype positive.

In the original report of nine patients with B-cell malig-

nancies treated with anti-idiotype antibodies, four patients

developed human antimouse immunoglobulin responses

necessitating cessation of therapy with the murine proteins.

Subsequent patients were treated with purer antibody prepa-

rations, and no further immune responses of this type were

seen in the antibody-alone trial. The only toxicity that was




Table 3. Combination Anti-idiotype Antibody and Interferon

BR/M/45 FSC CVP

Chlorambucil

Vinblastine

Chlorambucil Nodes, bone

marrow, mes-

enteric mass

Stomach ulcer

and associated

mass

Nodes, skin nod-

ules, bone

marrow

Nodes, bone

marrow, mes-

enteric mass

Nodes, bone

marrow

Nodes, neck

mass, skin

nodules

+

+

+

+

RT/M/46 FSC

KB/F/43 FSC

RV/M/55 FSC

Chlorambucil

CVP

654 BROWN ET AL

consistently noted was fever and chills on the first day of

antibody administration. Once circulating idiotype-positive

cells and idiotype protein in the serum were cleared, patients

were able to receive multiple antibody infusions without side

effects.

Therapeutic trial of anti-idiotype antibody and interfer-

on. A summary of the clinical features of the patients

treated with anti-idiotype antibodies and interferon is shown

in Table 3. Eleven patients were entered on this protocol,

including six males and five females. Ages ranged from 32 to

Therapy: Patient Characteristics

TumorPatient! MassesSex/Age Dx Prior Therapy Disease Sites �5 cm

BL/F/49 FSC Splenectomy -

DT/F/44 FSC

MW/F/5 1 FM CVP-B

DLC CMOPP

CHOP

RI

XRT to T spine

mass

XRT to flank

mass

BE/M/32 FSC CVP

FM Chlorambucil

RW/M/47 FM

F + DM

w/DLC

PC/F/46 FSC

Chlorambucil

CVP

BCEPP

CVP

Chlorambucil

JC/M/44 FSC CVP

DLC Chlorambucil

High-dose

cyclophosphamide

Adriamycin

Splenectomy

Nodes, bone

marrow

Nodes, bone

marrow

Nodes, bone

marrow

Nodes, bone

marrow

Nodes, bone

marrow, mes-

enteric mass,

spleen

Abbreviations: FSC, follicular small-cleaved cell lymphoma; FM, fol-

licular mixed lymphoma; DLC. diffuse large-cell lymphoma; F + DM

w/DLC, follicular and diffuse mixed lymphoma with focal areas of diffuse

large-cell lymphoma; CVP, cyclophosphamide, vincristine, prednisone;

C-MOPP, cyclophosphamide. vincristine, prednisone, procarbazine;

CHOP, cyclophosphamide, Adriamycin, vincristine, prednisone; �CEPP,

bleomycin, cyclophosphamide, VP-i 6, prednisone, procarbazine; RI,

total lymphoid radiation; XRT, radiation therapy; T spine, thoracic spine.

55 years. All 1 1 patients had follicular lymphoma at the time

of initial biopsy. However, in three cases a progression in

tumor histology occurred during the period between initial

biopsy and entry into the treatment phase of this study. The

tumors of two patients transformed to diffuse large-cell

lymphoma, and one transformed from a follicular mixed to a

follicular and diffuse mixed with focal areas of diffuse

large-cell lymphoma. Nine patients had been previously

treated with chemotherapy and/or radiotherapy. Two

patients, JC and BL, had previously been treated with

anti-idiotype antibodies. One patient, RT, had no prior

therapy. Nine patients had disease that was progressing prior

to the initiation of therapy, and two patients, RT and BL,

had stable, measurable disease. Ten patients had involve-

ment of five or more lymphoid anatomic sites. The remaining

patient, MW, had diffuse involvement of the gastrointestinal

(GI) tract. Five patients had individual tumor masses of S cm

or greater.

+ Prior to the initiation of therapy, peripheral-blood mono-

nuclear cells were examined as cytocentrifuge preparations.

- Immunostaining with anti-idiotype antibodies allowed the

detection of circulating lymphoma cells in every patient

(manuscript in preparation). These circulating lymphoma

- cells could only rarely be identified by morphology alone

with routine hematologic stains.

Table 4 summarizes the treatments with anti-idiotype

antibodies and interferon. Mouse MoAbs, predominantly of

the IgG 1 class, were used in nine patients, and rat MoAbs

were used in two patients. There were two patients, JC and

BR, who were treated with more than one monoclonal

- anti-idiotype antibody. Patient BR received two separate

courses of treatment. Individual doses of antibody ranged

Table 4. Combination Anti-idiotype Antibody and Inte

Therapy: Antibody and Interferon Treatments

rferon

No. of RXPretherapy Total After

Id Level Antibody Antibody No. of AB Sustained IFN DosePatient �zg!mL lsotype� Dose (mgI RX AB Levels Delivered

BL 7.0 IgGi 4,308 ii 10 80%

BR(i) 4.0 rIgGi 3,800 7 3 75%

(2) 1.0 rIgGi 7,530 11 10

rlgG2a

75%

DT 12.3 IgG1 6.840 12 10 80%

MW 19.8 rlgG2a 4,740 9 8 100%

BE 4.6 IgGi 8,400 12 11 100%

RW 0.1 IgGi 2,930 11 O� 88%

PC 1.7 IgGi 4,440 9 5 76%

JC 1.0 lgG2a 2.500 6 5

lgG 1

lgG 1

46%

RT 4.7 IgGi 2,880 6 4 100%

KB 2.5 IgGi 1,680 6 5 80%

RV 3.5 IgGi 7,440 9 3 45%

‘Number in parentheses refers to the fact that the patient has been


tAll antibodies are mouse monoclonals with the exception of those

with the prefix ‘r,” which are rat monoclonals.

�This patient was treated on a different dosing schedule than other

patients and did not achieve the same peak serum levels.




900 mg

0 2 4 6 8 10 12 14 16 18

DAYS

Fig 1 . Pharmacokinetics of anti-idiotype antibody. The anti-body treatments from the first course of therapy in patient BR areshown. A total of seven treatments were given. The first four

treatments were at a dose of 500 mg. The last three treatmentswere at a dose of 900 mg. Only with the last treatments were peak

serum levels greater than 200 �g/mL and trough levels of freeanti-idiotype antibody in serum sustained.


from 240 mg to 900 mg. The cumulative antibody dose over

the treatment courses in these patients ranged from 2,500 mg

to 8,400 mg, depending on the availability of antibody.

The initial dose of anti-idiotype antibody was approxi-

mately 500 mg and was adjusted upward in an attempt to

achieve peak serum levels of at least 200 sg/mL and trough

levels that rose with successive treatments. In previous

studies we have demonstrated that tissue penetration occurs

at these serum levels.5 Not all treatment doses resulted in

sustained levels of antibody in serum. For that reason the

number of treatments delivered after levels of free anti-

idiotype were achieved are designated separately in Table 4.

As an example of how dosage adjustments were made, Fig 1

shows the pharmacokinetics of MoAb in patient BR during

the first course of treatment. Four infusions were given at the

500-mg dose level. When the dose was then raised to 900 mg,

peak serum levels greater than 200 �g/mL were achieved

and trough levels of free anti-idiotype rose progressively.

All patients experienced some toxicity attributable to the

interferon. Fever and rigors were invariably noted on the first

day of interferon administration. Fever was less frequent on

subsequent days. Seven patients developed neutropenia

(WBC <2,500), four patients developed thrombocytopenia

(platelet count < 100,000), and/or three patients developed

GI distress. Because of these side effects, only three of 1 1

patients could tolerate the full interferon dose. No toxicity

could be related to the antibody administration, although it

could have contributed to the fever and chills observed on the

first day of each patient’s therapy. Two patients (RT and

KB) in this trial made an immune response against mouse

immunoglobulin. Interestingly this included the single

patient who had no prior therapy for lymphoma (RT) and a

patient treated only in the past with limited amounts of pulse

chlorambucil (KB). Symptomatic arthralgias necessitated

discontinuation of antibody therapy in patient RT on day 10.

400

350

300

.� 250

� 200

� 150

100

50

0

500mg

Q Q ,c� v�

In patient KB difficulty swallowing without respiratory

compromise necessitated discontinuation of antibody ther-

apyonday 14.

During the first week of antibody therapy, several patients

developed swelling in one or more lymph node sites that

subsided within 2 weeks. All tumor sites that showed this

reaction had major regressions following treatment.

Eleven patients and 12 treatment courses were evaluable

for tumor response (Table 5). Ten of the 1 1 patients had

objective evidence of tumor regression. In 1 2 treatment

courses there were two complete responses, seven partial, and

two minor responses. Response durations ranged from 2

months for patient JC to greater than 24 months for patient

BL. The median response time was 7 months, with a mean

that is yet to be determined since two patients are still

responding at 21 and 24 months. Tumor regressions were

first noted between ten days to 3 weeks after initiation of

treatment. In many cases tumors continued to regress slowly

for weeks to months after the discontinuation of therapy.

Details ofseveral cases are shown in Figs 2 through S. The

course of Patient BR was particularly interesting. Immedi-

ately prior to inclusion on this trial he had rapidly progressive

disease after failing chemotherapy. All nodal sites and bone

marrow were involved, including large tumor masses in the

mediastinum and iliac nodes. Two weeks after the initiation

of anti-idiotype antibody and interferon treatment an objec-

tive tumor response was noted. With each evaluation over the

next 3 months the tumor mass continued to decrease (Fig 2).

In the pelvis, however, the initial tumor response occurred

mostly in the right iliac lymph nodes, while the nodes on the

left responded to a lesser degree (Fig 3A and B). Three and

one-half months after the initiation of treatment, the left

iliac lymph nodes began to regrow (Fig 3C). The patient

received a second course of treatment, this time with two

anti-idiotype antibodies (anti-BR 1 and anti-BR 2). Ten days

after the reinitiation of treatment a second tumor response

was detected. The left iliac lymph nodes responded (Fig 3D).

The tumor continued to regress over the subsequent 8 months

until no radiographic evidence of disease was apparent. Bone

Table 5. Combination Anti-idiotype Antibody and Interferon

Therapy: Clinical Responses

Freedom

PatientHuman

a MouseTumor

Responsefrom

Progression

BL - CR 24M+

BR(2) - CR 21M+

DT - PR i3M

MW - PR 9M

BE - PR 7M

RW - PR SM

BR(i) - PR 3M

PC - PR 3M

Jc - PR 2M

RT + MR 6M

KB + MR SM

RV - NR -

#{149}Numberin parentheses refers to the fact that the patient has been





656 BROWN ET AL

Fig 2. Chest CT scans of

patient BR. A and C prior totreatment and B and D follow-ing the first course of combina-tion anti-idiotype and inter-

feron treatment. Shown is areduction in the prevascular.

paratracheal, and pericardial

adenopathy.

marrow biopsy and aspirate at that time showed no lympho- treatment was initiated. In each case a pretherapy biopsy

matous involvement, and the patient remains in a complete confirmed that the lymphoma cells continued to express

remission. idiotype. All three of these patients achieved objective tumor

Three patients, JC, MW, and RW, had histologic trans- responses. Patient JC had regression of a neck mass that

formation to a predominantly large-cell pattern by the time extended from the mandible to the thoracic inlet; MW had

Fig 3. Pelvic CT scans ofpatient BR. Prior to treatment

(A) enlarged lymph nodes areevident bilaterally in the iliacchains, but only the lymphnodes on the left were opaci-fled by lymphangiogram dye.After the first course of ther-apy (B) the lymph nodes in the

right iliac chain underwent amarked regression. Lymphnodes in the left iliac chain alsoregressed but not completely.Prior to the second course oftreatment (C) lymph nodes inthe left iliac chain grew. Lymphnodes in the right iliac chain re-

mained stably regressed. Afterthe second course of treatment(D) the lymph nodes in the leftiliac chain completely re-

greased.





Fig 4. Lymphangiogram of patient BE before and after treat-ment with anti-idiotype antibody and interferon. Patient BE hadslowly progressive disease despite chemotherapy. Sites ofinvolvement included bone marrow. multiple skin nodules scat-

tered across the trunk and face. and enlarged para-aortic and leftiliac nodes. Tumor regression was noted 3 weeks after theinitiation of treatment and continued for 5 months before stabiliz-ing. Opacified nodes (see left iliac region) on lymphangiogramwere decreased in size. and multiple skin lesions resolved. Someskin lesions became smaller. less indurated. and less erythema-tous without disappearing completely. These skin lesions pro-greased at 71/2 months. and several new peripheral nodesdeveloped. The regression in the nodal disease visualized onlymphangiogram was maintained.

endoscopically proven regression of an ulcerating gastric

mass; and RW had regression of bulky peripheral disease

and a mesenteric mass.

Analysis oftumor escape. In both trials repeat biopsies

were performed at the time of disease progression. Tumor-

cell populations were analyzed by immunoperoxidase stain-

ing and flow cytometry using panels of anti-idiotype antibod-

ies including those used for therapy. Results are summarized

in Table 6. Biopsies in five of ten patients treated with

anti-idiotype antibodies alone and four of eight patients

treated with anti-idiotype antibody and interferon demon-

strated an increase of more than 50% in the numbers of

idiotype-negative variant cells compared to pretreatment

biopsy specimens. In all of these cases the idiotype-negative

cells were derived from the original malignant clone. In every

case except one (patient MW) the idiotype-negative tumor

cells continued to express surface immunoglobulin. Although

phenotypic analysis of patient MW’s escaping tumor cells

showed they were surface-immunoglobulin negative, gene

rearrangements revealed clonal fidelity.

Several of the biopsies from the combination antibody and

interferon trial were analyzed with anti-idiotype antibodies

Fig 5. Lymphangiogram of patient DT before and after treat-ment with anti-idiotype antibody and interferon. Note the

decrease in para-aortic and iliac adenopathy. Patient DT had bulkylymph nodes in the cervical and supraclavicular areas. a mesen-teric mass, and a positive bone marrow. Tumor regression wasdetected 21/2 weeks after the initiation of treatment and continuedfor 1 1 months. At 1 3 months following treatment the patientdeveloped a single, new, peripheral lymph node. The regressionnoted at other disease sites was maintained.

Table 6. Analysis of Escaping Tumor Populations

Anti-idiotype Antibody andAnti-idiotype Antibody Therapy Interferon Therapy

Increased IncreasedNumbersof Numbers of

Idiotype ldiotype

Negative Site NegativePatient Cells Seen Biopsied Patient Cells Seen Site Biopsied

PK - Skin JC + Skin

EL(2) - Node BR(1) - Node

RD + Node MW + Stomach, node

BJ - Node BE + Node, skin

CJ + Node DT - Node

CG + Node RW - Node

PE + Node PC + Node

KC - Node RT - Node

Jc (2) - Node 4/8

CC + Node

5/10

Pretreatment and post-treatment biopsies were compared by immu-

noperoxidase staining of tissue sections and/or by flow cytometry. A

greater than 50% increase in the number of idiotype-negative cells in the

post-treatment biopsy specimen is indicated by a “ +“.




658 BROWN ET AL

Table 7. Analy sis of Escaping Tumor-C elI Populations: Patient BE

AnalysisPretreatment Biopsy

% ld+ CellsPost-treatment Biopsy

% ld+ Cells

a-BE 1

a-BE 2

a-BE 3

85

90

88

0.5

25

84

C Numbers represent the percent of the tumor population that reacted

with each anti-idiotype antibody. Total tumor population was estimated

by staining with antikappa and antilambda antibodies.

specific for the patient’s idiotype that were not used for

treatment. An example of this is shown in Table 7. Patient

BE had a biopsy of a progressing skin lesion that showed the

tumor cells retained reactivity with anti-BE 1, the antibody

used for therapy. A lymph-node biopsy at the same time,

however, contained almost exclusively anti-BE 1 idiotype-

negative cells. Analysis with other anti-idiotype antibodies,

anti-BE 2 and anti-BE 3, was revealing. (These antibodies

were not available in amounts large enough for inclusion in

the treatment program.) The determinant (idiotope) recog-

nized by anti-BE 2 was expressed by 90% of the tumor cells

in a pretreatment biopsy but by only 25% of the tumor cells

in the biopsy at the time of progressive disease. This example

demonstrates the strong selective pressure anti-idiotype anti-

bodies exert against antigen-positive cells and also shows that

coselection against a related idiotope can occur. An idiotope

defined by the third anti-idiotype, antibody anti-BE 3, was

expressed by 88% of the tumor cells pretreatment and 84% of

the tumor cells post-treatment and was not selected against

by the treatment antibody.

A more subtle selective pressure was revealed by analysis

of tissue samples from patient BR. In those samples flow

cytometry analysis with the two anti-idiotype antibodies

showed three distinct populations of lymphoma cells (Fig 6).

One population reacted with anti-BR 1 alone, one population

reacted with anti-BR 1 and anti-BR 2, and one population

reacted with anti-BR 2 alone. During’the time between the

intake and pretreatment biopsies the total number of tumor

cells staining with anti-BR 1 (the number of cells that stain

with anti-BR 1 alone plus the number of cells that stain with

both antibodies) remained fairly constant and was 96% or

greater. However, heterogeneity that can exist in lymphoma

cell populations from site to site and in different points in

time was demonstrated by the change in the number of cells

reacting with both anti-idiotype antibodies. The pretreat-

ment lymph node showed comprehensive reactivity with

anti-BR 1 and was biopsied from a chain of lymph nodes that

underwent a major tumor regression in response to treatment

with that antibody (Fig 3B). At the time of disease

progression a biopsy from a left pelvic lymph node chain that

responded incompletely to therapy showed a population of

cells (approximately 9.5%) reacting with anti-BR 2 but not

with anti-BR 1. These cells were not effectively eliminated

until the patient received infusions of both anti-BR 1 and

anti-BR 2 (Fig 3D).

DISCUSSION

The current results confirm that treatment with anti-

idiotype antibodies produce significant tumor regressions in

B-cell lymphoma. In 16 courses of therapy there were eight

complete or partial responses with anti-idiotype antibodies

alone. This compares to nine complete or partial responses in

12 courses of therapy with combination anti-idiotype anti-

bodies and interferon. In both trials tumor responses

occurred in patients who had failed multiple conventional

forms of treatment and in patients who had histologic

conversion to intermediate-grade lymphoma. Because of its

lack of toxicity, therapy with anti-idiotype antibodies was

well tolerated, even in patients who had been heavily

pretreated with other therapies.

The dose and scheduling of anti-idiotype antibodies are

both important issues. In the current trials the dose of

antibody was individualized for each patient with the goal of

penetrating tumor tissue. This pharmacologic concept led to

cumulative doses greater than 2 g for most patients. It is

possible that lower doses would be equally effective. For

instance, the patient with the longest remission, PK, received

only 500 mg of antibody. By analogy to chemotherapy

models, antibody treatment may also be improved by

changes in the schedule of administration. For instance,

multiple cycles of antibody may be preferable. The timing of

such cycles would need to be chosen according to a better

understanding of the way in which antibodies act.

The mechanism of action of anti-idiotype antibodies has

not been elucidated in man, although antibody-dependent

cellular cytotoxicity (ADCC) plays a role in the murine

lymphoma model.’ In the clinical trials, antibody affinity

and isotype do not seem to have discernible impact on

treatment results.’9 The presence of a tumor response, or the

magnitude of that response, could not be correlated in either

trial with the subclass of the antibody, since major clinical

responses were induced by antibodies of IgG 1 as well as

IgG2a and IgG2b subclasses. Anti-idiotype antibodies are

more active in B-cell malignancies than antibodies that react

with nonimmunoglobulin differentiation antigens.2#{176}’23 This

could be related to the greater specificity of the anti-idiotype

antibodies or to their inherent biological activity, since

anti-idiotypes have regulatory functions in the immune sys-

tem.2�26 In both trials it was common to see tumor regression

continuing months after the treatment phase was complete,

suggesting that passive infusion of anti-idiotype antibodies

may have an antitumor effect by altering the idiotype-

anti-idiotype network that plays a role in the regulation of

B-cell clones.

The addition of interferon to the treatment program was

based on both its known independent activity in follicular

non-Hodgkin’s lymphoma and its synergistic activity with

anti-idiotype antibodies in a murine lymphoma model. The

interferon dose chosen in this study, 12 x 106 z/m2 three

times weekly for 8 weeks, was based on the results of an

Eastern Cooperative Oncology Group pilot trial.27 In that

study seven of 16 patients with previously untreated follicu-

lar lymphoma had objective tumor responses, including one

complete response and six partial responses. Responding

patients on that study continued to receive maintenance

doses of interferon; however, no additional responses were

noted after the first 8-week treatment block. For this reason

we chose to limit the time of treatment with interferon to 8




BIOPSY

98%�

BIOPSY

2.6% 12%� 75.5%

�-1

L �

I-z

0.1% 9.5%


There was a trend toward a higher rate of tumor response

INTAKE PRE TREATMENT POST TREATMENT

BIOPSY

ANTI-BR 2

ANTI-HUMAN KAPPA

Fig 6. Distinct tumor cell populations in patient BR. Two-color flow cytometry analysis was performed on cell suspensions from biopsyspecimens. The percentages of the total tumor population staining with each antibody alone and with both antibodies are indicated. Thetotal tumor population. as measured by those cells staining with antihuman kappa, remains constant. Intake biopsies from right and leftinguinal lymph nodes showed a population of cells staining with anti-BR 1 and a population doubly stained with anti-BR 1 and anti-BR 2.Biopsy immediately prior to treatment. a right iliac lymph node. showed that anti-BR 1 stained virtually all of the B cells. Biopsy from a leftiliac lymph node after the first course of combination anti-idiotype antibody and interferon treatment showed an increasing population ofcells staining exclusively with anti-BR 2. Additionally the population of cells doubly stained with anti-BR I and anti-BR 2 had increased.

weeks. In other studies response rates to alpha interferon

have ranged from 1 6% to 54% in low-grade lymphoma.2834

The dose of interferon was chosen for its antitumor effect,

but this may not have been the optimal dose and schedule to

provide a synergistic biological effect with the anti-idiotype

antibodies. The addition of interferon did not prevent the

emergence of idiotype-negative clones in this study. Indeed,

in recent animal experiments the addition of interferon” or

interleukin-2 (IL-2)’#{176}to MoAb therapy has enhanced the

action of antibody against idiotype-positive disease, resulting

in more efficient selection for antigen-negative variant tumor

cells.

in patients receiving the combination of antibody and inter-

feron. However, the antitumor contribution of interferon was

difficult to assess because of the small number of patients on

both trials. Furthermore, the trials were not identical in

several respects. Although an attempt was made to select

patients with similar clinical characteristics in both trials,

this was difficult to assure. The purity of the antibody

preparations was improved after the first nine patients were

treated on the antibody-alone trial and for the combination

trial. There was a lower rate of antimouse immunoglobulin

responses in these later patients. When the patients who

made antimouse immune responses are excluded from analy-

sis, the response rates are more comparable between the two




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660 BROWN ET AL

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negative variants still occurred. Thus the current results

suggest a stronger antitumor effect by the antibodies than by

the interferon.

In previous clinical trials with MoAbs, the development of

a human antimouse immunoglobulin response has been a

significant obstacle to therapy. In our first report of nine

patients with B-cell lymphoma treated with murine anti-

idiotype antibodies, four immune responses to mouse immu-

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Documents

Treatment of B cell lymphomas with anti-idiotype antibody therapy in combination with alpha interferon