human immune response to monoclonal antibody-enzyme conjugates in adept pilot clinical trial

�9 1993 by Humana Press Inc. All rights of any nature whatsoever reserved. 0163 -4992/92/21 / 109-120/$ 3.40

Human Immune Response to Monoclonal

Antibody-Enzyme Conjugates in ADEPT Pilot Clinical Trial

SURINDER K. SHARMA, * ,1 KENNETH D. BAGSHAWE, 1

ROGER G. MELTON, 2'3 AND ROGER F. SHERWOOD 2

1CRC Labs, Department of Medical Oncology, Chafing Cross Hospital, Fulham Palace Road,

London, W6 UK, and 2pHLS, CAMR, Division of Biotechnology, Porton Down,

Salisbury, Wiltshire, UK

ABSTRACT

The human immune response to monoclonal antibody-enzyme conjugates has been studied in patients included in the pilot clinical trial of ADEPT. Each patient received murine monoclonal anti- CEA antibody fragments (A5B7-F(ab')2, conjugated to bacterial enzyme, carboxypeptidase G2 (CPG2) followed by a galactosylated monoclonal anti-CPG2 antibody (SB43), 36-48 h after the conjugate. Some patients were also given a dose of 131I-labeled conjugate

*Author to whom all correspondence and reprint requests should be addressed.

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110 Sharma et al.

(4-8 mg, 7-15 mCi) for blood clearance and gamma camera image studies. All patients studied developed human antimouse antibodies (HAMA) and anti-CPG2 antibodies within 10 d after a single course of treatment with the conjugate. In most cases, IgM response was detected at 7 d after the conjugate followed by the IgG response 14 d later. In one patient, HAMA and anti-CPG2 antibodies of the IgG type could still be detected at 10 mo after treatment. Anti-CPG2 antibodies in serum of one patient were found to inhibit CPG2 activity in vitro.

Generation of neutralizing antibodies limits the use of repeat cycles of ADEPT in patients. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying the appearance of HAMA and anti-CPG2 antibodies. Patients given cyclosporin A before and during ADEPT are currently being studied for HAMA and anti-CPG2 response.

Index Entries: ADEPT; antibody-enzyme conjugates; HAMA; anti-CPG2 antibodies.

INTRODUCTION

Anticancer agents linked directly to monoclonal antibodies achieve some selectivity, but have so far been of limited therapeutic value. To improve targeted cancer therapy, a novel approach has been developed (1-3) wherein a cytotoxic agent is generated at the tumor sites from a nontoxic precursor by a targeted enzyme. The enzyme is attached to a monoclonal antibody directed at a tumor-associated antigen. This approach has been termed "Antibody directed enzyme prodrug therapy" or "ADEPT."

Our studies in ADEPT have been carried out using a bacterial enzyme, carboxypeptidase G2 (CPG2), which has no known human analog, conjugated to monoclonal anti-hCG and anti-CEA antibody fragments. The feasibility of this approach has been demonstrated in a choriocarcinoma xenograft, CC3, and a colon carcinoma xenograft, LS174T. Using a two-phase approach, complete elimination of small tumors was achieved in the CC3 model (2,4), and using a three-phase system, growth delay in the LS174T model was achieved with only a single course of treatment (5,6).

Following these studies, a pilot clinical trial of ADEPT is being carried out in which murine monoclonal anti-CEA antibody, A5B7-

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Monoclonal Antibody-Enzyme Conjugates 111

F(ab')2 fragments conjugated to CPG2 are given in the first phase of treatment. As with other antibody-based systems, one of the major limi- tations in the use of murine monoclonal antibodies is the potential development of the immune response against the mouse antibody (7-9). Such immune responses can result in allergic reactions and may decrease effectiveness of therapy by removing the antibody from circulation before it reaches the target. In the ADEPT system, there may be an addi- tional immune response against the bacterial enzyme CPG2. One approach to the problem of HAMA is the use of genetically engineered chimeric monoclonal antibodies (10-13) or use of human monoclonal antibodies (14,15). The immune response against the enzyme may be avoided by using a human enzyme (3,16), but no studies on immune response against such enzymes have been reported so far and human- ization of monoclonal antibodies has reduced, but not so far eliminated, the problem of immunogenicity. Until data on the immunogenicity of humanized antibody-human enzyme conjugates are available, the need to study the nature of the human immune response to antibody-enzyme conjugates remains. The present studies were undertaken to study the development of immune response to murine monoclonal antibody- bacterial enzyme conjugates given to patients in ADEPT pilot clinical trial in order to plan effective therapy in the more immediate future with the currently available materials.

MATERIALS AND METHODS

Antibody-Enzyme Conjugates and Prodrug

The enzyme CPG2, a bacterial enzyme that catalyzes the hydro- lytic cleavage of reduced and nonreduced folates, was produced at PHLS, CAMR, Division of Biotechnology, Porton Down, UK (17) and conjugated to murine monoclonal anti-CEA antibody A5B7-F(ab')2 fragments via a stable thioether bond (18).

SB43, an IgG1 monoclonal antibody, was raised against CPG2 (19) and galactosylated according to a modification of the method of Mattes (20). The prodrug CMDA was developed as a substrate for CPG2 as described by Springer et al. (21). Alkaline-phosphatase-linked antihuman IgM and IgG antibodies used in ELISA assays were purchased from Sigma Chemicals (St. Louis, MO).

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Patients

Sharma et al.

In the first stage of the pilot clinical trial of ADEPT, six patients with advanced colorectal disease were entered for one course of ADEPT treatment. These patients tolerated escalating doses of the prodrug alone without toxicity. In the second stage of the trial, patients referred to here as patients 1-5 were given an infusion of murine monoclonal anti- CEA antibody fragments, A5B7-F(ab')2, conjugated to a bacterial enzyme, CPG2 (20,000 U/m) (22). In addition, these patients were given a dose of 131-iodine-labeled conjugate (4-8 mg, 7-15 mCi) for blood clearance and gamma camera imaging studies.

A galactosylated murine monoclonal anti-CPG2 antibody, SB43 (160-320 mg), was given 48 h after the conjugate infusion to inactivate and clear the residual CPG2 in plasma before prodrug injections. Blood samples were taken at intervals for 131-I-labeled conjugate clearance, counted in a gamma counter, and corrected for isotope decay. Serum samples for immune response assays were taken at intervals starting from d 7 after infusion of the conjugate.

ELISA

Human antimouse IgM and IgG were detected by standard ELISA procedures. Briefly, 96-well plates were coated with monoclonal antibody, A5B7, A5B7-F(ab')2 fragments, the enzyme CPG2, SB43, or the nonspecific anti-hCG antibody, SB10, at I ~tg/well and diluted in car- bonate/bicarbonate buffer, pH 9.6, by overnight incubation.

Nonspecific binding sites were blocked with phosphate-buffered saline containing 2% bovine serum albumin. Plates were washed with distilled water before use.

Patients' sera diluted in PBS from 1/10 dilution to 1/10,000 were added in duplicate. After incubation for I h, plates were washed three times, incubated for another hour with 50 ~tL of alkaline-phosphatase- linked goat antihuman IgM or IgG antibodies, and washed again.

One hundred microliters of substrate (p-nitrophenyl phosphate) were then added to each well and the reaction stopped after 45 min by addition of 3M NaOH solution. Absorbance was read at 405 nm in a Titertek multiscan Elisa Reader.

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Monoclonal Antibody-Enzyme Conjugates

RESULTS

113

Blood Clearance

The clearance of the radiolabeled conjugate in blood was similar in all five patients (Fig. 1). SB43gal was given 48 h after the conjugate, which inactivated CPG2 in plasma, but radioactivity was still detectable at 7 d after the conjugate.

HAMA and Anti-CPG2 Antibodies

Human antimouse and anti-CPG2 antibodies were detectable in all patients within 10 d after conjugate infusion. IgM type antibodies were present within 8 d, followed by IgG-type antibodies by day 20 (Fig. 2A,B) IgG-type antibodies to A5B7 and CPG2 were detected in the serum of all patients for a long period of time ranging from 20 d to 10 mo (Fig. 3A,B) after treatment.

Human antimouse response to A5B7-F(ab')2 fragments was stron- ger than that to the intact A5B7 antibody alone in one patient as judged by the higher OD reading at the same dilution of the sample. This may be owing to the presence of antiidiotypic antibodies in the serum. There was a weaker response to SB43 antibody and only a slight response (lower OD readings) to an isotype matched nonspecific antibody (SB10) in the same patient (Fig. 4) for the same dilution of serum sample (1/100).

CONCLUSIONS

Human immune response to murine monoclonal anti-CEA antibody, A5B7, and the bacterial enzyme carboxypeptidase, G2 (CPG2), has been studied in the first five patients included in the ADEPT pilot clinical trial. Although the number of patients studied is small, several trends in the development of human immune response to antibody- enzyme conjugates can be seen from this preliminary study. All patients developed antimouse (HAMA) and anti-CPG2 antibodies within 10 d of a single course of treatment with the conjugate. Antibodies of the IgM type were detectable within 8 d after treatment. The titers of these


114 Sharma et al.

Io

1

,01 0 24 48 72 96 120 144 168

Time (hrs)

Fig. 1. Blood clearance of 131-iodine-labeled A5B7-F(ab')2-CPG2 in five patients.

antibodies decreased after 14 d, when IgG-type antibody titers began to rise. This is consistent with studies carried out by other workers (7,23).

The IgG-type antibodies persisted in circulation for a longer period of time. HAMA and anti-CPG2 antibodies could still be detected in one patient at 10 mo after treatment. Anti-CPG2 antibodies in the serum of one patient were able to inactivate CPG2 in vitro. Although use of the bacterial enzyme confers specificity to the prodrug activation, it also enhances the problem of immunogenicity in humans. Anti- bodies developed against CPG2 in patients have the potential to neutralize CPG2 at the tumor sites as well as clear it from plasma. This probably limits the ADEPT treatment to one cycle only. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying appearance of HAMA and anti- CPG2 antibodies. Our preliminary studies in Balb/C mice showed that Cyclosporin A treatment suppressed immune response to CPG2 for 3 wk. Use of immunosuppressive agents such as Cyclosporin A (25) FK506


Monoclonal AnUbody-Enzyme Conjugates 115

~21 A

1.0.

0.8.

d 0.6. d

0.4

0.2-

0.0 i i i

0 10 20 30 40 Days after conjugate infusion

IgM Response �9

= i 50 60

tgG Response

a d

1.2.

1.0.

0 , 8 -

0.6.

0,4

0.2

0.0 0

B

J

10 20 30 40 Day== attm" conjugate infusion

50 60

Fig. 2. Development of immune response to (A) A5B7 and (B) CPG2 in patient 3. Serum dilutions at 1/1000.

(26), or anti-T-cell antibodies (27) may allow ADEPT cycles to be repeated in patients to achieve a better therapeutic effect. Patients given cyclosporin A during ADEPT are currently being studied for HAMA and anti-CPG2 responses.


116 Sharrna et al.

A 2,0

15

[ o~ 1.0

r< d

05

O0 I0 100 1000 10000

Serum dilution

B 20

15

I.C,

d d

05

,30 10 100 I000 10000

Serum dilution

Fig. 3. IgG immune response in five patients to (A) A5B7 and (B) CPG2 at various intervals of time. [] 1 day 8; 1 2 day 17; ~ 3 day 26; [] 4 day 60; and U 5 at 10 mo.


Monoclonal Antibody-Enzyme Conjugates 117

A

E r -

z n o v

ci

6

1.0

0,5 �84

0.@ 25 50 75 100 125

Days after conjugate infusion

Fig. 4. IgG immune response in patient 1. - -@-- anti-A5B7; I A - - anti-CPG2; - -O I anfi-A5B7-F(ab')2; -MZ]--- anti-SB43; and - -O I anti- SB10. (Serum dilution 1/100)

ACKNOWLEDGMENTS

This work is supported by the Cancer Research Campaign and the Post-natal Chorionepithelioma Trust.

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human immune response to monoclonal antibody-enzyme conjugates in adept pilot clinical trial

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