clinical and immunological effects of treatment with ...vol. 1.481-491, may 1995 clinical cancer...

Vol. 1. 481-491, May 1995 Clinical Cancer Research 481

Clinical and Immunological Effects of Treatment with Murine

Anti-CD3 Monoclonal Antibody along with Interleukin 2 in

Patients with Cancer’

Jacquelyn A. Hank,2 Mark Albertini, Osvaldo H.

Wesly, Joan H. Schiller, Aggie Borchert, Karen

Moore, Robin Bechhofer, Barry Storer, Jacek

Gan, Carlo Gambacorti, Jeffrey Sosman, and

Paul M. Sondel

Departments of Human Oncology [J. A. H., M. A., J. H. 5, A. B.,K. M., R. B., B. S., J. G., P. M. S.], Pediatrics [P. M. S.], and

Medical Genetics [P. M. S.], University of Wisconsin-Madison,

Madison, Wisconsin 53792; Memorial Regional Cancer Center/SIUMedical School, Springfield, Illinois 62794-9248 [0. H. W.]; IstitutoNazionale Tumori, Milan, Italy 20133 [C. G.]; and Loyola University

Medical Center, Maywood, Illinois 60153 [J. S.]

ABSTRACT

Anti-CD3 mAb and interleukin 2 (IL-2) were used in aPhase I study to treat 29 patients with cancer. The anti-CD3was given as an i.v. bolus infusion over 10 mm followed bytwo i.v. 96-h continuous infusions of IL-2 at 3 x 106 units/

m2/day with a 3-day rest between the IL-2 infusions. Fourpatients were treated with 6, 18, 60, and 300 �g/m2 anti-

CD3. One patient received 3000 �.ag/m2 anti-CD3. This pa-tient developed profound hypotension and the IL-2 infusions

were delayed for 2 weeks. Two patients were treated at anintermediate dose of 600 �iWm2. These patients developeddose-limiting toxicities including hypotension, dyspnea and

increased blood urea nitrogen, creatinine, and bilirubin.They were unable to complete their first course of therapy.

In an effort to achieve a dose of anti-CD3 which wouldactivate T cells in vivo, pentoxifylline was given to blunt thetoxicities seen with anti-CD3 thought to be due predomi-

nantly to the cytokine syndrome and tumor necrosis factorrelease. Four patients received p.o. pentoxifyffine to cover ananti-CD3 dose of 600 pig/m2. The IL-2 infusion was initiated1 week after the mAb. While there was an anti-CD3 dose-dependent increase in serum tumor necrosis factor level 1 hafter mAb infusion, pentoxifylline did not reduce the serumtumor necrosis factor level. There was also an anti-CD3dose-dependent increase in the serum soluble IL-2 receptorlevels. Other immune parameters monitored, including in

vitro cytotoxic and proliferative responses and lymphocytecount, were similar to treatment courses with IL-2 alone.Fourteen of 26 patients examined developed human anti-

murine antibodies following a single dose of anti-CD3. There

were no objective antitumor responses. We conclude that in

vivo treatment with anti-CD3 did not enhance T cell activityor expansion with subsequent IL-2 infusion and that thecombination of anti-CD3 followed by IL-2 did not improve

upon the antitumor activity previously seen with IL-2 alone.

INTRODUCTION

IL-2,3 a polypeptide produced by activated T lymphocytes,

was first described as a T cell growth factor and utilized in the

propagation and long-term in vitro culture of T lymphocytes (1).

IL-2 can enhance the proliferative and cytotoxic activity of both

T lymphocytes and NK cells in vivo and in vitro (2-4). Most

fresh, unprimed T lymphocytes do not respond well to IL-2,

since a prior encounter with specific antigen or exposure to

mitogen is necessary for a proliferative response (5). However,

some lymphocytes in fresh peripheral blood specimens can

respond directly to IL-2, and these IL-2-activated cells are able

to recognize and lyse tumor cells (6, 7). In addition, clinical

trials using IL-2 as anticancer therapy have demonstrated that

the systemic administration of IL-2 can activate and preferen-

tially expand NK cells in vivo (8-12). Although systemically

administered IL-2 increases the absolute number of CD3� lym-

phocytes, the relative frequency of these cells as a fraction of the

circulating leukocyte population and their immunological func-

tion is suppressed (13, 14).

The CD3 complex of the T cell antigen receptor complex is

able to be targeted by mAbs (15). Depending on the adminis-

tration dose, anti-CD3 mAb is able to function as either an

immunostimulating or an immunosuppressive agent. At very

high doses, anti-CD3 mAb depletes CD3-bearing cells and has

been successfully used to inhibit antigen-specific T cell-medi-

ated graft rejection in the transplant setting (16, 17). Treatment

with low doses of anti-CD3 (approximately 1/100th of the

immunosuppressive dose) can trigger T cell activation through

the CD3 T cell receptor complex and induce expression of the a

chain of the IL-2 receptor on these T cells (18). Thus, admin-

istration of low doses of anti-CD3 have demonstrated immuno-

stimulatory activity, both in vitro and in vivo (19, 20). Animal

models have shown that an immunostimulatory dose of anti-

CD3 can inhibit growth of immunogenic malignant tumors (21,

22). As T cell activation with anti-CD3 mAb induces IL-2

receptor expression, the combination of anti-CD3 mAb and

Received 11/16/94; accepted 2/17/95.

1 This research was supported by NIH Grants and Contracts CA-53441,

CA-05436, CA-32685, CM-87290, CA-14520, CA-13539, CM-47669,HL-02143, RR-03186 and by American Cancer Society Grant CH-237.2 To whom requests for reprints should be addressed, at K4/454 CSC,600 Highland Avenue, Madison, WI 53792.

3 The abbreviations used are: IL-2, interleukin 2; NK, natural killer;

LAK, lymphokine-activated killer; MTh, maximum tolerated dose;l’TX, pentoxifylline; ADCC, antibody-dependent cellular cytotoxicity;PBMC, peripheral blood mononuclear cells; HS, human serum; HAMA,human anti-murine antibody; sIL-2R, serum-soluble IL-2 receptor;TNF, tumor necrosis factor.

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482 Anti-CD3 mAb and IL-2 Therapy in Cancer Patients

Table 1 Anti-CD3 treatment schema”

Course 1 Course 2 Course 3

Group Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10

Ab” Ab

1-6 � IL-2 � � IL-2 J I IL-2 � � IL-2 � I IL-2 � � IL-2

Ab Ab Ab Ab Ab Ab Ab Ab Ab Ab

7 I lL-2 � � IL-2 � I IL-2 � � IL-2 � � IL-2 � � IL-2Ab

8 � IL-2 � � IL-2 � I IL-2 � [ IL-2 � � IL-2 � � IL-2

PTX PTX PTX

9 Ab � IL-2 � [ IL-2 � I lL-2 � � IL-2

a IL-2 was given as a 96-h continuous infusion of 3 X 106 units/m2/day each week for courses 1, 2, and 3. For courses 1 and 3, the IL-2 was

initiated I h after the anti-CD3 infusion. For group 9, IL-2 was initiated 1 week after the mAb. Anti-CD3 was given as an iv. bolus infusion over

10 mm. PTX, 2 g/day (400 mg p.o. 5 times per day for 3 days), was given the day before, the day of, and the day after each dose of the anti-CD3infusion. In this group, IL-2 was not initiated until 1 week after anti-CD3, and was given as a 96-h continuous infusion for 2 weeks of each treatmentcourse.

b Ab, antibody.

IL-2 in vitro leads to an augmentation of the T cell response to

IL-2 and increased activation and expansion of peripheral blood

lymphocytes over that seen following IL-2 treatment alone (23,

24). We thus performed the clinical trial described here, which

combined treatment with anti-CD3 mAb and IL-2 in vivo. The

goal of this treatment protocol was to augment LAK cell func-

tion via activation of CD3 bearing T cells through the CD3

molecule itself. Furthermore, the in vivo administration of anti-

CD3 could potentially expand putative endogenous T cells able

to selectively recognize autologous tumor.

MATERIALS AND METHODS

Patients. Twenty-nine patients were enrolled in this

Phase I trial. All patients had refractory cancers for which other

proven effective treatments were not available, and all patients

signed approved informed consent forms. Patients had an East-

ern Cooperative Oncology Group performance status of 0-1

(Karnofsky, 80-100) and a life expectancy of >4 months.

Eligibility criteria included normal hematological parameters

(hemoglobin, � 1 1.5 g/dl; WBC, �3.5 K/pA; granulocyte count,

>2,0004al; and platelets � 100,000), adequate liver function

(total serum bilirubin, �2.0 mg/dl; serum glutamic oxaloacetic

transaminase, less than two times the upper limits of normal),

and adequate renal function (serum creatinine, <2.0 mg/dl, or a

creatinine clearance, >60 ml/min). Criteria for exclusion in-

cluded patients who had major surgery within the last 3 weeks

or treatment with cytotoxic chemotherapy in the prior 3 weeks,

radiation therapy, or IFN therapy 2 weeks prior to the study.

Patients who had previously received murine mAb therapy or

IL-2, or with clinically significant cardiac abnormalities, or

significant pleural effusions were ineligible. Patients with sig-

nificant central nervous system disease or central nervous sys-

tern metastases, psychiatric or seizure disorders, or serious re-

cent infections, or those who required continued therapy with

corticosteroids, aspirin, or nonsteroidal antiinflammatory agents

were also ineligible. In addition, patients must not have received

more than two chemotherapeutic regimens. Patients with a his-

tory of chronic obstructive pulmonary disease were required to

have adequate ventilatory function (forced expiratory volume �

60% of predicted by pulmonary function testing).

Treatment Schema. The purpose ofthis Phase I protocol

was to determine what single dose of anti-CD3 mAb was

tolerated when followed by two sequential 96-h infusions of

IL-2 at 3 X 10� units/m2/day. All patients were scheduled to

receive 96-h IL-2 infusions in weeks 1 and 2 (course 1), weeks

5 and 6 (course 2), and weeks 9 and 10 (course 3) (Tables 1 and

2). The patients in the initial six groups received a single dose

of anti-CD3 mAb on day 1 of course 1 and course 3 as shown

in Table 1. In course 1, patients received an initial bolus (10

mm) injection of anti-CD3 mAb i.v. followed 1 h later by the

first 96-h continuous infusion of IL-2 at 3 X 106 units/m2/day.

Patients then rested for 72 h and the 96-h IL-2 infusion was

repeated. Following a 2-week rest, patients received course 2

which consisted of 2 weekly 96-h continuous infusions of IL-2

without the mAb. Patients were treated in groups of four,

stratified according to the dose of anti-CD3 mAb. These in-

cluded 6, 18, 60, 300, 3000, and 600 �.Wm2. An additional

group of four patients was scheduled to receive the MTh of

anti-CD3 daily for 5 days during week 1 of their treatment

(group 7). An additional group of four patients was treated

without the anti-CD3 in course 1, and received the mAb in the

second course (week 5), 1 h prior to initiating the 96-h IL-2

infusion (group 8). These patients served as a control group for

the data analyses of course 1 because they received treatment

with IL-2 alone in contrast to the others, who received the

combination of anti-CD3 plus IL-2. A final group of four

patients received PTX p.o. 2 g/day for 3 days, the day preceding,

of, and following the anti-CD3 infusion. In this group, the IL-2

was initiated 1 week after the anti-CD3 and was given as 2

weekly 96-h continuous infusions as indicated above (Tables 1

and 2).

Anti-CD3 mAb, IL-2, and PTX. Anti-CD3 (OKT3; Or-tho) mAb and IL-2 (Hoffmann LaRoche) were supplied by the

National Cancer Institute, Division of Biological Response



Clinical Cancer Research 483

Table 2 Treatment schema

Patients (completing/initiating) indicated course

GroupAnti-CD3 dose

(p.g/m2)Anti-CD3

schedule”

Course I :

weeks 1-2

Course 2:

weeks 5-6

Course 3:

weeks 9-10

1

2

3

4

5

6

7

8

9

6

18

60

3003000

600

MiD(300)MTD

(300)w/PTX”

(600)

Day 1 of courses 1 and 3

Day 1 of courses 1 and 3Day 1 of courses 1 and 3

Day 1 of courses 1 and 3Day 1 of courses 1 and 3Day 1 of courses 1 and 3Days 1-5 of courses 1 and 3

Day 1 of course 2

Day 2 of courses 1 and 3

4/4

4/44/4

4/40/10/20/2

4/4

4/4

3/3

4/44/4

4/40

0/12/2

3/4

3/3

2/2

2/2

2/2

1/100

1/1

2/2

2/2

a See foot note a to Table 1 for t reatment schema details.

Modifiers (Frederick, MD). PTX is a commercially available

medication manufactured by Hoechst-Roussel Pharmaceutical

Incorporated (Somerville, NJ).

ADCC. All ADCC assays were performed in RPMI 1640

supplemented with 10% HS (Pel-Freez, Rogers, AR), 25 msi

HEPES, 100 units/mI penicillin, and 100 p.g/ml streptomycin

sulfate (RPMI-HS) and in RPMI-HS supplemented with IL-2 at

100 IU/rnl. Effector cells in a total volume of 50 �i.l were plated

in quadruplicate into 96-well U-bottomed microtiter plates at

E:T ratios of 50, 16.7, and 5.6:1. Fifty pA of media or IL-2 at a

final concentration of 100 units/ml were added to effector cells

and incubated for 30-45 mm at 37#{176}C+ 5% CO2. Just prior to

the addition of target cells, 50 �i.l of antibody were added to the

effectors. These included: (a) human mouse chimeric antibody

ING-1 (25), a gift from R. Robinson of Xoma Corp.; (b) the

bispecific anti-CD3-113F1, a gift from Dr. D. Segal ofNIH and

Dr. D. Ring of Cetus (Emoryville, CA), in which murine anti-

CD3 mAb was chemically conjugated to the murine 1 13F1 mAb

reactive with certain breast and colon cancer cell lines (26); (c)

mouse monoclonal anti-CD3 (Ortho). The final concentrations

per well were 0.25 �ig/ml for ING-1, 100 ng/ml for anti-CD3-

1 13F1 conjugate, and 0.25 pg/ml for anti-CD3. Effector cells in

media and IL-2 were also plated to determine their ability to

mediate lysis of target cells in the absence of antibody. Target

cells were labeled with 250 pCi 51Cr in 0.2 ml RPMI-HS.

Target cells were mixed every 15-30 mm during labeling to

keep the cells in suspension. After washing twice with RPMI

1640, 5 x i03 target cells were plated with effector cells and

centrifuged at 200 X g for 5 mm. The plates were incubated at

37#{176}C+ 5% CO2 for 4 h, and the supernatants were harvested

using the Skatron Harvesting System (Skatron, McLean, VA).

Maximum 5tCr release was measured by lysing target cells with

the detergent cetrimide (Sigma). Spontaneous 51Cr release was

measured by incubating target cells in RPMI-HS media alone.

Percentage of cytotoxicity values were calculated for each E:T

ratio as follows:

% cytotoxicity =

experimental release - spontaneous releasebox

maximum release - spontaneous release

Results are expressed as percentage cytotoxicity for the inter-

polated percentage of cytotoxicity expected for an E:T ratio of

17: 1 or as lytic units, where 1 lytic unit is the number of effector

cells necessary to achieve 20% lysis of 5 X i0’ targets.

LAK Cell Functional Assays. PBMCs were assayed for

their ability to kill the NK-resistant, LAK-sensitive, Daudi Bur-

kitt’s lymphoma line. PBMCs in 50 p.1 RPMI-HS were plated in

quadruplicate U-bottomed microtiter wells at E:T ratios of 50,

16.7, and 5.6:1. RPMI-HS or RPMI-HS supplemented with IL-2

at a final concentration of 100 units/mI were added to quadru-

plicate wells. Target and effector cells were handled as de-

scribed above.

Surface Marker Analysis. T cell markers CD3, CD5,

and y-& T cell receptor, the Fc receptor (CD16), the IL-2

receptor (CD25), the NK marker (CD56), and the MHC class II

(HLA-DR) were examined on PBMCs gated for lymphocytes.

Double marker analysis was used to determine the percentage of

cells expressing both CDS6 and CD16, CD3 and CD2S, and

CD3 and HLA-DR. All fluorescence labeling was performed at

4#{176}Cin the dark for 30 mm. Patient PBMC populations were

characterized by incubating 2 X i0� Ficoll-Hypaque-isolated

PBMCs in 100 p.1 PBS with FITC-conjugated or phycoerythrin-

conjugated antibodies (Becton Dickinson) at the recommended

concentrations according to the manufacturer’s procedure. Pro-

pidium iodide at a final concentration of 1 p.g/ml was added just

prior to analysis on fluorescence-activated cell sorting to sepa-

rate live from dead cell populations.

Detection of Anti-CD3 Antibody on the Surface of Cir-

culating T Cells. Anti-CD3 antibody binding to circulating T

cells in vivo was detected by staining patient PBMCs directly

with goat anti-mouse antibody and control goat anti-rabbit an-

tibody using the method described above.

Proliferation Assays. The proliferative responses of pa-

tient PBMCs were determined using a [3H]thymidine incorpo-

ration assay. Briefly, 1 X i05 patient cells in 100 p.1 RPMI-HS

were cultured with 100 p.1 of the following stimulators:

RPMI-HS alone, 1% phytohemagglutinin/ml, 100 units/mI IL-2

(3-day assay), 100 units/ml IL-2, 0.5 p.g/ml anti-CD3, 0.5 p.g/ml

anti-CD3 + 100 units/ml IL-2, or 1 X 10� irradiated normal

donor cells Xx and Yx (6-day assay) in 96-well U-bottomed




microtiter plates. The plates were incubated for 3 or 6 days at

37#{176}C+5% CO,. On either day 2 or day 5 each well was pulsed

with 50 �i.l of a 20 liCi/ml solution of [3H]thymidine for 16-

18 h at 37#{176}C+ 5% CO2 and then MASH harvested and cpm

quantitated by scintillation counting.

Serum TNF and sIL-2R. Serum TNF kits were obtained

from Biosourse International (Camarillo, CA) and sIL-2R kits

were obtained from T cell Diagnostics (Cambridge, MA). These

sandwich enzyme immunoassays were used to determine TNF

and sIL-2R levels in serum according to the specifications of the

manufacturer.

HAMA Levels in Patient Serum Determined by ELISA.A 96-well flat-bottomed polystyrene plate (Immulon-2; Dyna-

tech) was coated with 150 p.1/well of mouse monoclonal IgGi

(Sigma MOPC-21) in a concentration of 2 �igJml in 0.05 M

sodium carbonate buffer (pH 9.6) overnight at +4#{176}C.The plate

was washed three times with PBS (pH 7.2) containing 0.05%

Tween 20 (Sigma). Serum test samples and controls (100 gil)

were added in 10-fold dilutions in sample buffer (PBS + Tween

20 + gelatin + milk) and incubated overnight at +4#{176}Cin wet

chambers. After washing three times with PBSI1’ween 20, the

plate was incubated with 100 �il/well biotin-labeled mouse

monoclonal IgG1 in a 1:300 dilution in sample buffer for 3 h at

room temperature before washing as described above. Bound

biotinylated antibody was detected by adding extravidin conju-

gated to alkaline phosphatase (Sigma) diluted to 1:5000 in

TrisiTween 20 buffer. After a 1-h incubation at room tempera-

ture and washing as described above, staining was performed

using p-nitrophenyl phosphate (Sigma) in a concentration of 2

mg/mI in diethanolamine buffer. After 1-h incubation at room

temperature in the dark, substrate conversion to colored product

was determined at 405 nm versus 492 nm with an ELISA reader

(Dynatech MR 580). The absorbances were calculated to ng/ml

according to a standard curve, which was generated using a

defined concentration of goat anti-mouse immunoglobulin (Sig-

ma). The range of the standard curve for this assay is 2-100

ng/ml based on the binding of goat anti-mouse IgG antibody.

The sensitivity is approximately 2 ng/ml.

Statistical Analysis. Treatment effects were assessed as

the change in parameter values from baseline to various times

during treatment. Except for parameters relating to cell surface

phenotype, the data were generally log transformed prior to

analysis. Paired t tests were used to test for treatment effects

within groups of patients; two-sample t tests were used to test

for differences in treatment effects between groups. Linear

regression of treatment effects on log dose of anti-CD3 dose

response was lacking, and data were pooled over the anti-CD3

dose for subsequent analyses. Logistic regression on the log of

the anti-CD3 dose was used to assess whether the probability of

developing HAMA was dose related.

RESULTS

Patient Characteristics. Twenty-nine patients were en-

tered into this Phase I/lb clinical trial. Twelve patients had renal

cell carcinoma, 15 had malignant melanoma, and 1 each had

breast and ovarian carcinoma. Two patients had no prior ther-

apy, 14 had surgery alone, and the remaining 13 had surgical

resection of their primary tumors followed by other therapies

which included radiotherapy (9), chemotherapy (2), immuno-

therapy (4), and hormonal therapy (3). The median age was 54

years and all patients had an Eastern Cooperative Oncology

Group performance status of 0 or 1.

Clinical Laboratory Changes and Significant ClinicalToxicities. The laboratory parameters and clinical toxicities

observed with this anti-CD3 and IL-2 treatment regimen are

presented in Table 3. Data are presented for the courses with

mAb and IL-2 (course 1, groups 1-7 and course 2, group 8); for

courses with only IL-2 (course 2, groups 1-7 and course 1,

group 8); and for courses with PTX, mAb, and IL-2 (group 9,

courses 1 and 3). Treatment with IL-2 plus anti-CD3 induced a

significant lymphocytosis, although the rise in lymphocytes was

comparable in the patients receiving IL-2 alone (4). Hepatic

studies showed greater changes in patients receiving IL-2 plus

anti-CD3 (n = 34 courses) compared to those receiving IL-2

alone (n = 29 courses) for aspartate aminotransferase (P =

0.007) and bilirubin (P = 0.02). Most clinical toxicities encoun-

tered were similar in incidence and severity for combined mAb

plus IL-2 courses and IL-2 courses alone.

Patient entry was planned to proceed consecutively from

group 1 through group 5 (Table 2). While the 300-p.g/m2 anti-

CD3 dose was tolerated well (i.e., group 4), severe and pro-

longed hypotension was seen in the first patient in group 5

treated with a 3000-p.g/m2 dose of anti-CD3. This patient was a

54-year-old man with metastatic renal cell carcinoma who pre-

sented with a right hilar lung mass 6 years after nephrectomy.

The patient received his scheduled anti-CD3 treatment with

subsequent facial flushing and mild tachycardia. He developed

subsequent chilling, nausea, and vomiting for which he received

parenteral Demerol and Haldol. One h following anti-CD3 ad-

ministration he became hypotensive, with a decline in blood

pressure to 70/40. This hypotension was poorly responsive to

i.v. fluid challenge with normal saline, and required pressor

support for approximately 36 h with a dopamine drip and a

phenylephrine drip. His blood pressure subsequently stabilized,

allowing discontinuation of both the phenylephrine and dopa-

mine. Because his hypotensive response was quite profound, the

scheduled IL-2 infusion was delayed for 2 weeks. He was

initially treated with 3 X 10� units/m2/day of IL-2 and received

this dose for each of his initial 96-h continuous infusion treat-

ments. The IL-2 was started at this dose for course 2 following

the 2-week rest, but was discontinued following 3 days of

therapy due to constitutive symptoms. The second 96-h IL-2

infusion was then started at 1.5 X 10� units/m2/day, but was

discontinued following 4 h of therapy due to profound hypoten-

sion with a decrease in blood pressure to 70/40. This hypoten-

sion resolved with i.v. fluid support, but no further IL-2 therapy

was administered. This patient has since been followed clini-

cally and has remained without evidence of progressive disease

for 3.5 years since completing protocol therapy. The patient is

inevaluable regarding disease response in that his only site of

pretreatment disease was his right hilar mass and this site had

received local radiotherapy 1 month prior to initiation of IL-2

and anti-CD3 therapy. No other patients received this dose.

As 300 �i.g/m2 anti-CD3 were well tolerated, an interme-

diate dose of 600 p�g/m2 anti-CD3 was given to a sixth group

scheduled to treat four patients. Dose-limiting toxicities were

observed in the first two patients at this 600-�i.g/m2 dose, in-




Table 3 Laboratory c hanges and clinical toxicity associated with IL-2 + anti-CD3 treatment

No. of treatment courses associated with the indicated lab changes

(treatment of 29 patients)

IL-2 alone: IL-2 + anti-CD3:course 2, groups 1-7; courses 1 and 3, groups 1-7; IL-2 + anti-CD3 with PTX:

course 1, group 8 course 2, group 8 courses 1 and 3, group 7

(29 courses) (34 courses) (6 courses)

Hematology

Hemoglobin <10 g/100 ml 19 14 3WBC <2000/pJ 0 5 0Neutrophil count

500-1000/pA 2 4 1<500/p.l 1 0 0

Eosinophil count >10,000/pA 4 1 2Lymphocyte count >6,000/pA 24 26 6Platelet count <50,000/pA 0 3 0

Renal creatinine

2.0-2.9 mg/dl 3 5 0>3.0 mg/dl 1 2 0

HepaticAspartate aminotransferase

2-3 times normal 1 6 0>3 times normal 1 6 2

Alkaline phosphatase2-3 times normal 9 8 0

>3 times normal 5 12 2

Total bilirubin2-3 times normal 0 6 0

Clinical toxicityFever �40#{176}C 1 4 0

Hypotension >40 mm Hg reduction in 7 7 1

systolic blood pressureRequiring pressors 0 1 1

Decline in ECOG” performance statusof3(0-*3) 1 2 0

Weight gain �10% of total body weight 4 3 3Central venous catheter thrombosis 1 1 1Dyspnea or hypoxemia requiring oxygen 2 5 0Central nervous system (moderate to 3 4 0

severe transient headache)

a ECOG, Eastern Cooperative Oncology Group.

cluding hypotension, dyspnea or hypoxemia requiring oxygen,

as well as elevations in blood urea nitrogen, serum creatinine,

and bilirubin. One patient did not receive any of the scheduled

IL-2, while the second patient received 54 h ofthe planned 96-h

IL-2 infusion for week 1 and no IL-2 was given in the second

week of course 1 . This patient started the second course of

treatment at week 5 with an IL-2 dose reduction of 50%.

Following a completed 96-h IL-2 infusion, the second week of

course 2 was not given due to disease progression. The MiD

was thus defined as 300 p.g/m2.

PTX Treatment. Since T cell activation and expansion

had not been noted in patients treated thus far, an additional

group of four patients received p.o. PTX to blunt the cytokine

syndrome (26, 27-31). Common toxicities associated with the

cytokine syndrome include elevations in serum creatinine, Se-

rum bilirubin, blood urea nitrogen, and aspartate aminotransfer-

ase and changes in blood pressure (32, 33). The change between

the baseline value and the maximum or minimum post-anti-CD3

value for these parameters were compared for patients treated

with anti-CD3 at 300 and 600 �i.Wm2 (n 6) and anti-CD3 at

600 �.Wm2 with PTX (n = 4). The differences observed between

these two groups were not significant.

Immunological Monitoring. Statistical analyses of lab-

oratory data analyzed by anti-CD3 dose using Student’s t test

revealed that there was no significant anti-CD3 mAb dose effect

detected on the variables of cell surface phenotype, in vitro

proliferative and in vitro cytotoxic responses (data not shown).

Therefore, these data are pooled from the 16 patients receiving

the 6-, 18-, 60-, and 300-p.g/m2 doses anti-CD3 (groups 1-4).

At the higher doses of anti-CD3 mAb, many of the clinical

blood samples were not available at the appropriate sampling

times due to modifications in the treatment schedule resulting

from anti-CD3-induced toxicities; thus, paired comparisons

were not possible for laboratory data from these patients.

Cell Surface Phenotype. The cell surface phenotypepresented in Table 4 shows that in the 16 patients receiving the

combined IL-2 plus anti-CD3 mAb treatment, there was a sig-

nificant drop in the percentage of CD3� T lymphocytes with a

corresponding increase in CD16�/CD56� NK cells. A signifi-

cant increase in the percentage of cells expressing the CD25 �




Table 4 Cell s urface phenotype

% of PBMCs positivea

Sample time CD3 CD5 CD3 + CD5 CD16 CD2S CD56 MHC class II

Groups 1-4 (anti-CD3 + IL-2), n = 16On study 67Week 1 65

7065

7259

1622

616”

2034”

18351)

Week 2 55C 55C 52d 31h 20” 44” 26�

Group 8 (IL-2 alone), n = 4

On study 68 70 NA� 20 13 22 20Week 1 56 56 NA 31 18 44 41Week 2 45 44 NA 42 18 56 44

Group 9 (anti-CD3 + PTX), n = 4

On study 62 22 59 12 16 18 16

Week 2 35 40 27 35 13 58 40

Week 3 37 42 24 41 18 62 32

(I PBMCs were isolated by density gradient centrifugation and directly labeled with FITC or phycoerythrin-conjugated antibodies. The percentageof cells positive was based on PBMCs gated for lymphocytes.

I) p < 0.001 based on change from on study.

‘. P < 0.01.dp < 005�

.. NA, not applicable.

Table 5 in vitro proli ferative respo nses in patients receiving IL-2 and anti-CD3”

[3H]thymidine incorporation (cpm)

On study Week 1 Week 2

CD3 + IL-2 IL-2 CD3 + IL-2 IL-2 CD3 + IL-2 IL-2

(groups 1-4) (group 8) (groups 1-4) (group 8) (groups 1-4) (group 8)

3-DayMedium 763 424 520 386 449 644

Phytohemagglutinin

IL-2

36,927

4,333

30,451

4,157

34,094 34,077

27,037” 27,173

30,31 1

24,660”

14,251

17,759

CD3 21,996 20,124 2,040” 1 1,452 5,566k 2,490CD3 + IL-2 30,31 1 28,084 33,109 30,728 37,900 17,672

6-DayMedium 1,035 933 927 823 1,004 1,085

Xx 10,597 10,634 3,037 4,985 2,392’� 2,181Yx 10,129 10,566 4637d 1,497 4,532d

1,452

IL-2 20,937 24,836 42,607� 27,678 37,462” 15,778

CD3 22,109 6,102 2,021” 2,294 1,257” 1,257CD3 + IL-2 77,981 24,854 51,157k 49,239 52,834 28,301

a PBMCs were isolated from patients prior to (on study) and 24 h after each 96-h continuous infusion of IL-2 in course 1. Quadruplicate cells

(1 X i0� cells/well) were stimulated with the indicated stimuli including irradiated PBMC from allogeneic control donors X and Y (Xx, Yx) andcultured for 54 h, labeled with [3H]thymidine for 18 h, and harvested. Values represent the mean of 16 patients treated in groups 1-4. n = 16, groups1-4; n = 4, group 8.

1) P < 0.001 based on change from on study.

�P < 0.01.dp < 005�

(IL-2 receptor) and MHC class II activation antigens was ob-

served. These values are comparable to those seen in previous

studies of patients receiving IL-2 alone (4, 8) and also are not

statistically different from comparable values obtained from the

four patients in group 8, who received only IL-2 in their first

course of treatment and the four patients in group 9 who re-

ceived PTX with the anti-CD3 infusion (Table 4). In addition,

there was no change in the -1’-S T cells, which remained stable at

a mean of 2% throughout the study.

In Vitro Proliferative Responses. The in vitro prolifer-

ative responses are shown for patients in groups 1-4 receiving

anti-CD3 and IL-2 and group 8 receiving only IL-2 in course 1

(Table 5). While there was not a significant change in the

response to the mitogen phytohemagglutinin, there was a sig-

nificant enhancement of the proliferative response to IL-2. This

was noted for the patients in groups 1-4 following the first and

second week of IL-2 and was also significant in both the 3- and

6-day proliferative assays. The in vitro proliferative response to

anti-CD3 mAb stimulation was depressed following both 1 and

2 weeks of the combined therapy and was noted in both the 3-

and 6-day assays. These enhanced proliferative responses to

IL-2 and depressed responses to irradiated allogeneic lympho-

cytes are similar to the responses seen in patients in group 8,

course 1, receiving only IL-2 (Table 5). In addition, they are




Table 6 Fresh cytotoxic ass ays in patien ts receiving IL-2 and anti-CDY’

Lytic units/107 effectors

On study Week I Week 2

CD3 + IL-2 IL-2 CD3 + IL-2 IL-2 CD3 + IL-2 IL-2(groups 1-6) (group 8) (groups 1-6) (group 8) (groups 1-4) (group 8)

NKJLAK on Daudi in the presence of

Medium 3 17 12 98 16” 74

IL-2 7 171 285k 852 422k 616

CD3 4 21 32� 148 22� 1(XJ

CD3 + IL-2 8 95 328k 976 512” 830

ADCC on BT2() in the presence of

Medium 24 10 6tY 143 65’ 95

IL-2 31 25 348” 643 405k 624

ING-l 232 83 552k 1354 761” 959

ING-1 + lL-2 304 151 96O� 2059 1190” 1866

ll3Fl-CD3” 28 18 94” 119 74� 96

113F1-CD3 + IL-2 40 46 410” 914 412” 537

‘, Fresh PBMCs obtained prior to (on study) and 24 h after each of the 96-h IL-2 infusions of course 1 were tested in a 4-h 51Cr release assay

at three E:T ratios. Values represent the mean lytic units obtained from 16 patients treated in groups 1-4. n = 16, groups 1-4; n = 4, group 8.

I, p < o.ooi based on change from on study.

(. P < 0.05.

“P < 0.01.

(S l3Fl-CD3 antibody conjugated to mAb anti-CD3.

similar to responses noted in previous studies with continuous

infusion IL-2 (4, 8). The proliferative responses of patients

receiving PTX was not significantly different from the response

of patients in groups 1-4 and group 8 (data not shown).

In Vitro Cytotoxic Response. The in vitro cytotoxic

responses are shown in Table 6 for the 16 patients treated with

anti-CD3 and IL-2 (groups 1-4) and the 4 patients receiving

only IL-2 in course 1 (group 8). PBMCs obtained prior to

initiation oftreatment (day 1) did not mediate significant lysis of

the NK-resistant Daudi cell line, while PBMCs obtained from

patients following week 1 of treatment with IL-2 plus anti-CD3

mAb demonstrated a significant increase in LAK activity

against Daudi in vitro. This increased lytic activity was further

boosted following a second week of IL-2. This activity was also

markedly enhanced when IL-2 was included in the cytotoxicity

assay medium. Inclusion of anti-CD3 antibody in the assay

enables antibody-coated CD3� effectors to bind the Fc receptor-

positive Daudi target. A low, yet significant, amount of this

‘ ‘reverse’ ‘ ADCC was observed. The lytic activity mediated by

PBMCs obtained from patients in group 8 receiving only IL-2

during the first course was not significantly different from the

lytic activity mediated by PBMCs from patients in groups 1-4

(Table 6).

LAK Activity and ADCC. LAK activity and ADCC

were also measured against the BT2O human breast carcinoma

line (Table 6). As with Daudi, significant LAK activity was

induced in vivo against BT2O with the combined systemic IL-2

plus anti-CD3 therapy. ADCC activity was measured with in-

clusion of the ING-1 human mouse chimeric IgGi antibody

(25), which recognizes a carcinoma-specific surface antigen on

BT2O. Prior to the start of therapy, there was significant ADCC

activity when ING-1 was included in the medium. Following

weeks 1 and 2 of IL-2 plus anti-CD3 therapy, this ADCC

activity was significantly enhanced and inclusion of IL-2 in the

assay medium further augmented this cytotoxicity. To determine

if CD3� effectors activated in vivo with anti-CD3-mediated

enhanced cytotoxicity, a bifunctional heteroconjugate antibody,

consisting of anti-CD3 cross-linked to the I 13F1 antibody,

which recognizes an antigenic determinant expressed on BT2O

target cells (26), was included in the assay. While the hetero-

conjugate did facilitate a slight increase in the destruction of the

BT2O target, this increase was minimal compared to the IL-2 or

ING-1 antibody effects. The PBMC samples from patients in

group 8 receiving only IL-2 in their first course of therapy

(Table 6) and from patients receiving PTX with anti-CD3 (data

not shown) responded in a similar fashion to those from patients

receiving the combined therapy with anti-CD3 and IL-2 in the in

vitro cytotoxic LAK, NK, and ADCC assays.

Detection of Anti-CD3 on Circulating Lymphocytes.

Lymphocytes were examined for in vivo binding of anti-CD3 by

staining directly with a goat anti-mouse FITC-conjugated mAb.

Lymphocytes obtained 1 week following the injection of anti-

CD3 were consistently negative for in vivo bound anti-CD3. The

eight patients receiving the 300- and 600-pg doses of anti-CD3

had blood samples examined at 10 and 60 mm and at 2 and 6

days following the antibody dose. As noted in prior studies,

there was a dramatic decrease in the number of circulating

CD3� cells within 1 h of the antibody treatment. Of the circu-

lating lymphocytes found at 10 mm and 1 h after the anti-CD3

injection, 95% of the circulating CD3� cells showed evidence

of anti-CD3 mAb binding in vivo as detected by flow cytometry.

Over the next 2 days the detectable number of CD3� circulating

peripheral blood lymphocytes retaining in vivo bound anti-CD3

mAb decreased to 63% and by day 6 was no longer detected.

sIL-2Ra Chain Levels Were Examined. Fig. 1 demon-

strates a significant dose-dependent increase (P = 0.01) in the

sIL-2Ra chain for patients receiving 6-300 �ag/m2 anti-CD3.



A

A

A

A

1�

10000

1000

Fig. 1 Anti-CD3 dose-dependent increase in sIL-2Ra level noted in serum samples obtained on day 6 of course 1 for the 16 patients (groups 1-4)receiving 6-300 p.g/m2 anti-CD3. Regression slope is significant at P < 0.005.

A

A

A

10 100 1000

anti -CD3 DOSE (ug/m sq)


E

This increase was noted in serum obtained on day 6. The rise in

the sIL-2R is thought to reflect the mass of systemic lymphoid

activation in patients treated with IL-2 alone and is due to

secretion of the receptor, primarily by T cells in IL-2-treated

patients (34, 35). The increased activation, determined by higher

sIL-2Ra levels with greater doses of anti-CD3, may well reflect

augmented activation of IL-2Rcs release by anti-CD3-activated

T cells, as noted in vitro (36) and in mice (20, 21). The four

patients receiving PTX in conjunction with anti-CD3 showed a

similar increase in the sIL-2Ra levels, demonstrating that the

PTX dose and administration schedule did not inhibit the im-

mune stimulation in these patients.

Serum TNF Induction. An increase was noted in serum

TNF in samples obtained 1 h following the mAb infusion for the

25 patients treated in groups 1-8. There was a highly significant

dose-dependent relationship between the dose of anti-CD3 and

serum TNF levels (P < 0.001) (Fig. 2). Evaluation of serum

TNF levels in four patients receiving PTX (group 9) indicated

that oral PTX did not decrease TNF levels, as compared with

four patients receiving 300 p.g/m2 and two patients receiving

600 p.g/m2 anti-CD3 without PTX (groups 4 and 6). Further-

more, there was not a statistically significant relationship be-

tween clinical toxicity and TNF-a levels within these small

groups of patients.

HAMA Response. Fourteen of the 26 patients examined

generated an antibody response against the mouse monoclonal

following one dose of anti-CD3 antibody. The patients receiving

greater doses of antibody were more likely to mount a HAMA

response (P 0.016 by logistic regression). However, if

FIAMA developed, the level of serum anti-murine antibody was

not related to the dose of anti-CD3.

Antitumor Activity. While we had no partial or com-

plete antitumor responses noted in this Phase lB trial, four

patients demonstrated signs of antitumor activity. These in-

cluded one melanoma patient with multiple pulmonary metas-

tases and a large axillary node. Following two courses of ther-

apy, the patient had a 45% shrinkage of the axillary node and

stable disease in the lung. This level of response was maintained

for 4 months. A second melanoma patient who had previously

resected local and regional recurrences was treated with no

evidence of disease. This patient currently demonstrates no

evidence of disease over 48 months following treatment. A

patient with renal carcinoma demonstrated a 34% shrinkage of

the kidney mass and pulmonary disease. This response was

maintained for 2 months. A fourth patient with renal cell carci-

noma with pulmonary and lymph node involvement demon-

strated shrinkage of some lung lesions, while many remained

stable. This response was maintained for 2 months.

DISCUSSION

This clinical trial combined anti-CD3 with IL-2 with the

intent of activating and expanding in vivo the T cell population

in addition to the NK population as is seen in vitro with the

combination of anti-CD3 plus IL-2 (23, 24). While the current

standard clinical use of anti-CD3 is limited to its immunosup-

pressive effects at high doses (5 mg/dose; Refs. 37 and 38), it is

clear that this antibody can function as an immunostimulant

both in vitro and in animal models in vivo (19-22, 39). Fur-



A A

A

A

A

A

A A

10 100 1000 10000


100000

�.10000E

� 1000

I

� 100

Cl)10

1

I

antl-CD3 DOSE (ug/m sq.)

Fig. 2 Anti-CD3 dose-dependent increase in serum TNF measured by ELISA in serum samples obtained 1 h following the anti-CD3 infusion for

the 25 patients in groups 1-8. Regression slope is significant at P < 0.001. TNF levels of < 10 were recoded to 10.

thermore, in animals bearing transplanted tumors, low-dose

anti-CD3 treatment induced an immune stimulation associated

with specific antitumor immunity (19, 22).

The current protocol design was to give an initial bolus

injection of anti-CD3 to initially activate T cells followed by

continuous infusion IL-2 therapy to expand both the activated T

cells and the NK cells. The MiD of anti-CD3 given in this

manner is less than that achievable in immunosuppressed recip-

ients of solid organ allografts. However, our MiD of 300 p.g/m2

was 10-fold higher than the maximum tolerable dose recently

reported (40) when anti-CD3 was given as single agent i.v.

therapy over 3 h, administered four times over 2 weeks. In that

study, the MiD was 30 �ig and the dose-limiting toxicity was

noted to be headache associated with aseptic meningitis. While

our most clinically significant dose-limiting toxicity was hypo-

tension, central nervous system toxicity of a moderate to severe

transient headache, evaluated as a grade II toxicity, was noted in

only 4 of 34 courses of combined treatment using IL-2 plus

anti-CD3 mAb and was not seen in the 6 courses with PTX

(Table 3). It is uncertain whether the single dose of anti-CD3 or

its combination with IL-2 in the current study helped blunt the

central nervous system toxicity noted by Urba et al. (40). No

patient in the PTX group (group 7, six courses) developed

significant neurological toxicities.

The immunological monitoring of cytotoxic and prolifera-

tive responses as well as cell surface phenotypic data indicated

that there was significant immune activation occurring for pa-

tients treated in this study. However, all of these parameters are

also altered by treatment with IL-2 alone, and these treatment-

induced changes were similar for these patients during courses

of IL-2 compared with courses of anti-CD3 antibody combined

with IL-2. Included in the cytotoxic monitoring was the hetero-

conjugate facilitated lysis of BT2O tumor targets. The 1 13F1

antibody, which recognizes an antigenic determinant expressed

on BT2O, was chemically cross-linked to anti-CD3 (26). This

bifunctional antibody was used to facilitate ADCC with T

lymphocytes as effectors. While a slight increase in ADCC was

noted following in vivo therapy with anti-CD3, the magnitude of

the response was markedly less than that ofthe ADCC seen with

the IL-2-activated NK cells.

Two serum markers clearly indicated that the in vivo use of

the anti-CD3 mAb produced immunomodulation. These were

the sIL-2R and TNF-a levels, both of which were noted to have

significant anti-CD3 dose-dependent increases. In addition, the

dose of anti-CD3 impacted on the development of antimouse

antibodies. Eleven of the 23 patients examined developed de-

tectable circulating antimouse antibody. Seven of the 1 1 were

treated with doses of anti-CD3 of 300 p.g/m2 or more. One

patient receiving three courses of anti-CD3 at 18 p.g/m2 and two

patients receiving two courses at 60 �ig/m2 did not develop

detectable HAMA. In the study of Urba et a!. (40), 8 of 16

patients examined developed HAMA.

Anti-CD3 induced a marked drop in the lymphocyte count

as is also noted with IL-2 (4, 8). Urba et a!. (40) also noted an

anti-CD3 dose-dependent decrease in the lymphocyte count. It

has been proposed that the activated lymphocytes leave the

peripheral circulation and home to the lymphoid organs. The

anti-CD3 may lead to a massive release of lymphokines (39) and

the mechanisms responsible for the loss of lymphocytes in the




circulation may be similar; the more activated lymphocytes are

likely not in the peripheral circulation (8, 41). Furthermore, the

few residual CD3� cells in the circulation did show surface

murine antibody 10 and 60 mm after mAb treatment, indicating

that the murine antibody effectively reached CD3� cells in vivo.

Since T cell activation and expansion were not seen at the

lower doses of anti-CD3, and the highest dose given was quite

toxic, an intermediate dose of anti-CD3 was combined with

PTX administration. PTh is a methylxanthine which reduces

TNF secretion seen with administration of anti-CD3 (27, 28)

and the capillary leak syndrome seen with administration of

IL-2 (29-31). Four patients were treated with PTX the days

before, of, and after anti-CD3 infusion. Unlike the severe tox-

icity in both patients treated with 600 p.g/m2 of anti-CD3

without PTX, only one of the four patients in the PTX-treated

group experienced marked side effects requiring intensive care

monitoring (Table 3). This single patient developed nausea and

vomiting during the 3 days of p.o PTX treatment, and as a result

may not have been able to achieve the same systemic level of

PTX. The other three patients tolerated this dose of anti-CD3

well. Thus, the inclusion of PTX appeared to provide a subtle

degree of protection from the toxicities of anti-CD3, based

strictly on subjective clinical impression. However, neither the

serum TNF levels nor other objective toxicity parameters mea-

sured for the patients receiving the FiX were significantly

different from those seen in the patients receiving the 300- and

600-�j.g/m2 dose of anti-CD3 without the PTX. The levels of

sIL-2Ra measured in patients receiving PTX were not de-

pressed compared to values from patients not receiving PTX,

suggesting that the PTX did not inhibit the ability of the mAb to

exert an immunostimulatory effect (42).

While it is clear from animal studies that anti-CD3 can be

effective as an antitumor agent in immunogenic and weakly

immunogenic tumor models (19-22), our current study com-

bining anti-CD3 with IL-2 in a small number of patients and a

previously reported study (43) did not demonstrate any addi-

tional clinical benefit over that previously reported for IL-2

alone. Increases in two immunological parameters, the sIL-2R

and TNF, indicated that anti-CD3 induced a dose-dependent

immunological response, although it is difficult to determine if

these parameters are relevant to the induction of an antitumor

response. Furthermore, laboratory data from these patients do

not support any selective in vivo T cell expansion using anti-

CD3 mAb as a broad, antigen nonspecific activation of T cells.

It is possible that repeated anti-CD3 infusions may activate T

cells in vivo; however, we were not able to examine this possi-

blity, since the first two patients who were to receive the MiD

for 5 days did not tolerate more than 2 days of anti-CD3.

Whether this agent, in combination with other immunomodula-

tors, could help induce the generation of specific antitumor

immunity in humans remains unknown. As other more direct

approaches are now being pursued attempting to directly acti-

vate specific T cell populations with tumor-related vaccines, we

feel that the immunological and clinical data obtained from this

study are not significantly encouraging to continue evaluation of

soluble anti-CD3 antibody as an immunostimulatory agent. The

potential use of anti-CD3 in bifunctional antibodies or as means

to expand (in vivo or in vitro) antigen-reactive T cells for

adoptive transfer requires further clinical analyses.

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1995;1:481-491. Clin Cancer Res J A Hank, M Albertini, O H Wesly, et al. patients with cancer.anti-CD3 monoclonal antibody along with interleukin 2 in Clinical and immunological effects of treatment with murine

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