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Human anti-murine antibody responses in ovarian cancer patients undergoing radioimmunotherapy with the murine monoclonal antibody OC-125
GYNECOLOGIC
ONCOLOGY
38, 244-248 (1990)
Human Anti-murine Antibody Responses in Ovarian Cancer Patients Undergoing Radioimmunotherapy with the Murine Monoclonal Antibody OC-I 25 MICHAEL
G. MUTO, M.D. ,’ NEIL J. FINKLER, M.D., AMIN I.KASSIS, PH.D. ,* EVA MARIE LEPISTO, B.S., AND ROBERT C. KNAPP, M.D.
Divisions of Gynecologic Oncology and *Nuclear Medicine, Departments of Obstetrics and Gynecology and Radiology, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Harvard Medical School, 75 Francis Street, Boston, Massachusettes 02115
Received February 8, 1990
Human anti-murine antibody (HAMA) responses were monitored in 23 patients with recurrent or persistent epithelial ovarian carcinoma undergoing single-dose intraperitoneal radioimmunotherapy (RIT) with the murine monoclonal antibody OC-125. Sera of patients receiving escalating dosesof OC-125 F(ab’), (lo70 mg) radiolabeled with 18 to 141 mCi of iodine-13’ were assayed for HAMA by a protein A-based radioimmunoassay. Overall, 70% of patients (16/23) developed HAMA within 10 to 46 days (median = 29) postinfusion, with peak values (23 + 6 to 325 2 10 pg/ml) at 32 to 102 days (median = 38). HAMA was undetectable prior to infusion in all casesand persisted up to 76 weeks. Of patients receiving a dose of 123 mCi or less, 80% (16/20) developed HAMA, whereas in the 140-mCi group, none of the three patients had detectable levels. Two patients in the 140-mCi group demonstrated dose-limiting bone marrow toxicity (severe thrombocytopenia and neutropenia). It is concluded that a single intraperitoneal dose of monoclonal antibody leads to a high incidence of HAMA production. The results also suggest that the likelihood of HAMA formation in patients who either had undergone recent chemotherapy or had received the highest dose of the radioimmunoconjugate is reduced. These observations may be of significance in designing multiple-dose therapy trials as HAMA has been demonstrated to decrease antibody-to-tumor binding and may potentially increase renal, hepatic, and hematologic toxicity associated with radioimmunotherapy. Q 1990 Academic PRSS, I~C.
the radioimmunoscintigraphy (RIS) and the radioimmunotherapy (RIT) of cancer patients [l-5]. The development of human anti-murine antibody (HAMA) is common in patients receiving repetitive or single large doses of MoAb [6-101. HAMA has been demonstrated to affect the biodistribution of MoAb by forming antigen-antibody complexes which increase the blood clearance of MoAb and decrease their specific binding to tumor [6,7,11,12]. OC-125 is a murine MoAb raised against the antigenic determinant CA12.5, an ovarian cancer-associated antigen expressed in over 80% of nonmucinous epithelial carcinomas [ 131.We report HAMA responses in 23 ovarian cancer patients undergoing single-dose intraperitoneal RIT with F(ab’), fragments of OC-125 radiolabeled with iodine-13’.
INTRODUCTION Interest
continues
to grow in the potential
diagnostic
and therapeutic applications of murine monoclonal antibodies (MoAb). Radiolabeled MoAb directed against human tumor-associated antigens have been used in both I To whom reprint requests should be addressed at Department of Obstetrics and Gynecology. 244 two-8258/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS Patients. Between July 1987 and January 1989, 23 patients were enrolled in the radioimmunotherapy trial. Written informed consent was obtained through an approved protocol of the Human Subjects Committee of the Brigham and Women’s Hospital, Boston, Massachusetts. All patients had a confirmed diagnosis of persistent or recurrent epithelial ovarian carcinoma (20 serous, 3 endometrioid), with CA125 antigen levels >35 U/ml. The median age was 54 (range, 31 to 73 years). The majority of the patients presented with advanced, poorly differentiated lesions (Table 1). Exclusion criteria included prior pelvic or abdominal radiation therapy in excess of 3000 cGy; overt thyroid, cardiac, or renal disease; a Karnofsky score ~50; and cytotoxic chemotherapy
TABLE 1 Distribution of Stage and Grade Among Twenty-three RIT Patients Grade Stage
1
1 II III IV
-
2
3
1 (4%) 4 (18%) -
I (4%) 1 (4%) 14 (61%) 2 (9%)
1 month of enrollment. All patients had undergone cytoreductive surgery followed by multiple courses of postoperative chemotherapy, predominantly with cisplatinum-containing regimens. One patient received adjuvant whole-pelvis radiotherapy. In addition, patients with recurrences had undergone aggressive salvage therapy with either intravenous or intraperitoneal administration of a variety of cytotoxic agents. within
Radioimmunoconjugate preparation and RIA administration. OC-125 F(ab’), fragments (Centocor, Malvern,
PA) were radiolabeled with ‘?‘I (NEN DuPont, Billerica, MA) to a specific activity of 2 mCi/mg utilizing a singlevial Iodogen technique [ 141. The radioimmunoconjugate
(RIC) was tested for immunoreactivity by a cell binding assay with OVCAR 433 as described [15]. Pyrogen testing was performed using a chromogenic limulus amoebocyte assay, and sterility was evaluated by a 72 hour incubation in thyoglycolate medium. The content of free iodine was assessed by instant thin-layer chromatography (Gelman Sciences, Ann Arbor, MI). Pyrogen-free RIC with greater than 30% immunoreactivity and less than 10% free iodine was administered to the patient intraperitoneally in all cases by single-lumen catheters as described previously [I]. Serum collection and HAMA assay. Serum was obtained prior to infusion, on a daily basis during the hospital stay, and weekly for at least 6 weeks postinfusion. All samples were stored at -70°C until assayed. Human anti-murine IgG responses were detected by a radioimmunoassay (RIA) described by Mosely [ 161. Briefly, 96-well Remove-A-Well (TC) plates (Dynatech Corp., Chantilly, VA) were coated with protein A (Genzyme, Boston, MA) at a concentration of 10 pug/ml by a 24-hr incubation at 4°C. The plates were washed in 0.1 M phosphate-buffered saline (PBS) containing 10% fetal calf serum and incubated for an additional 24 hr in this buffer. Serum samples, diluted 1: 500 in Tris-HCl buffer, pH 8.0, were added to the precoated plates in a 50-,ul
TABLE 2 Dose of ‘3’I-OC-125 and HAMA Response Patient number
Antibody dose (md
1 2 3 4 5 6 7 8 9 10 11 12 13 14 I5 16 17 18 19 20 21 22 23 * Mean ? SD.
10 10 10 15 15 IS 20 20 30 30 30 40 40 40 50 50 50 60 60 60 70 70 70
‘j’I dose (mCi) 18 20 28 30 20 34 44 42 47 60 54 76 82 87 100 104 105 120 117 123 140 141 140
Follow-up (days) 677 285 51 190 170 536 51 60 41 150 32 56 47 35 60 52 46 53 63 59 54 93 51
Day HAMA first positive 17 10 21 nd 42 18 26 18 28 nd 32 29 40 nd 27 31 37 nd 30 25 nd nd nd
Peak HAMA Wml) 253 ? 27” 319 * 31 234 -t- 25 nd 13 5 4 308 k 34 221 + 25 77 -+ 17 107 + 16 nd 122 * 4 28 k I 23 + 6 nd 29 t 3 124 -+ 12 33 2 6 nd 325 ” 10 112 -c 15 nd nd nd
Interval from prior chemotherapy (months)
5 20 3 1 2 10 5 4 3 2 3 6 14 2 4 22 3 15 11 22 3 5 1
Grade of hematologic toxicity 2 0 0 0 1 0 0 0 2 0 0 2 0 2 2 2 3 2 0 0 1 3 4
246
MUTO ET AL.
23). Overall, 70% (16/23) of the patients developed detectable HAMA within 10 to 46 days (median = 29 days) of therapy (Table 2). Peak values ranged from 23 + 6 HAMA detected to 325 ? IO pg/ml and occurred from 32 to 102 days Interval (median = 38 days) following therapy. No patient had Yes (months) No preexisting detectable HAMA prior to infusion. No pa<3 1 4 tient had been previously treated with monoclonal an23 15 3 tibody. Patients were followed from 32 to 677 days (median = 60), with HAMA detectable at 536 days in one patient. volume. These plates were then incubated for 2 hr at Among the 20 patients who received RIT at doses room temperature, the wells were washed with PBS, and below 140 mCi, HAMA was detected in 80% (16/20), 50 ~1 of the tracer (OC-125 radiolabeled with 12’1)was whereas none of the 3 patients given 140 mCi developed added. The plates were then incubated an additional 2 detectable HAMA. When this latter group was compared hr at room temperature; the wells were washed and their to the other treated patients, there were no significant radioactive content was determined in a gamma counter. differences in age, pretreatment chemotherapy, or inA negative control was established from pooled sera terval from prior chemotherapy to RIT. of volunteers with no detectable HAMA IgG. A positive HAMA production was less likely in patients who had control was established from a previously studied patient received chemotherapy within 3 months of RIT adminwith a HAMA level of 200 pg/ml. Results are reported istration when compared with patients in which the inin micrograms based upon a standard curve established terval from prior therapy was 3 months or longer (Table by serial dilutions of a rabbit anti-OC-125. 3). There was no association between the number of courses of chemotherapy given prior to RIT and the agents used in antecedent therapy. RESULTS Various patterns of HAMA response were observed; Twenty-three patients received 10 to 70 mg of OC-125 two are illustrated in Fig. 1. Patient 1, followed for 677 F(ab’), radiolabeled with 18 to 141 mCi of 13’I. At the days, demonstrated a rapid rise in HAMA with a peak 140-mCi dose, 2 patients (out of 3) demonstrated at least at Day 38. HAMA was last detected on Day 205, and grade III toxicity (neutropenia with absolute neutrophil by Day 317, no further response was detectable. Patient count of 820 in patient 22, and severe thrombocytopenia 6, on the other hand, had a similar early rise, but perwith a platelet nadir of 20,000 and bleeding in patient sistently detectable levels up to 536 days postinjection. This response persisted despite four subsequent courses of intravenous c&platinum chemotherapy at a dose of 50 mg/m2. 300 HAMA
TABLE 3 versus Interval from Prior Chemotherapy
Formation
I
DISCUSSION
600
DAYS POS?- INJECTION
FIG. 1. Two types of HAMA response: (A) Patient 1, (B) patient 6. Asterisks for patient 6 indicate subsequent course of intravenous cis-platinum chemotherapy.
The data presented in this paper demonstrate that 70% of patients with ovarian cancer undergoing RIT with a single intraperitoneal injection of ‘3’I-OC-125 will develop HAMA. Peak values occur at 4 to 6 weeks postinfusion and persist for up to 76 weeks. Our data are consistent with previously published reports of HAMA development in patients receiving murine MoAb. Among renal transplant patients receiving OKT3, the murine IgG,, for the immunotherapy of acute renal allograft rejection, 88% developed HAMA within 3 weeks of MoAb infusion [ 171. Patients with gastrointestinal malignancies who received anti-CEA MoAb also demonstrated an 89% HAMA formation rate [18]. Finally, in a series of eight patients with advanced carcinoma of the ovary, four (50%) developed HAMA after a second exposure to various antiovarian MoAbs [6]. It has been suggested that HAMA formation rates
HUMAN ANTI-MURINE
ANTIBODY
RESPONSES IN OVARIAN
CANCER
247
should approach 100% in immunocompetent patients and The formation of HAMA must be monitored in any prothat decreased HAMA response among cancer patients tocol involved in administering murine monoclonal anmay be related to generalized immunosuppression re- tibodies, particularly in multiple-dose therapy trials. lated to the malignancy or to cytotoxic chemotherapy [19]. As demonstrated by our data, RIC in doses suffiREFERENCES ciently high to cause bone marrow suppression may fur1. Finkler, N. J., Muto, M. G., Kassis, A. I., Weadock, K., Tumeh. ther reduce the ability of the patient to mount a HAMA S. S., Zurawski, V. R., Jr., and Knapp, R. C. Intraperitoneal raresponse. diolabeled OC 125 in patients with advanced ovarian cancer, GyWhile lower doses of MoAb are associated with high necol. Oncol. 34, 339-344 (1989). HAMA formation rates, the administration of very high 2. Stewart. J. S. W., Hird. V., Snook, D.. Sullivan, M., Hooker, G., doses of MoAb actually results in a reduced HAMA Coutenay-Luck, N. S., Siuolapenko, Cl., Griffiths, M. Myers, response rate. For example, Sears et al. have reported M. J., and Lambert, H. E. lntraperitoneal radioimmunotherapy for ovarian cancer: Pharmacokinetics, toxicity and efficacy of I-131 HAMA in eight of nine patients receiving less than 200 labelled monoclonal antibodies. Int. J. Radiat. Oncol. Biol. Phys. mg of murine MoAb. HAMA was detected in only one 16, 405-413 (1989). of nine patients when doses of infused MoAb ranged 3. Carrasquillo, J. A.. Bunn. P. A., Keenan, A. M., Reynolds, J. C., from 366 to 1000 mg [19]. This “tolerizing” effect of Schroff, R. W., Foonka, K. A., Su, M. H.. Gazdar. A. F., Mulhigh-dose immunotherapy has not been observed in the shine, J. L., and Oldham, R. K. Radioimmunodetection of cutadose ranges used in our study. It is therefore unlikely neous T-cell lymphoma with “‘In labeled TIOI monoclonal antibody, N. Engl. J. Mrd. 315, 613-678 (1986). that decreased HAMA formation among the highest dose 4. Epenetos, A. A., Munro, A. J., Stewart. S., Rampling, R., Lamgroup is related to the dose of MoAb alone. bert, R. E., Mckenzie, C. G., Soutter, P., Rahemtulla, A., Hooker. The phase I trial described in this report is based on G., and Siuolapenko, G. B. Antibody-guided irradiation of ada single intraperitoneal infusion of RIC. Hoever, effecvanced ovarian cancer with intraperitoneally administered radiotive RIT will probably require fractionation of dose or labeled monoclonal antibodies, J. C/in. Oncol. 5, 1890-1899 (1987). multiple treatment cycles. In some cases, RIT will be 5. Ward. B., Mather, S., Shepherd, J., Crowther, M., Hawkins, L., preceded by attempts at tumor detection or dosimetry Britton. K.. and Slevin, M. L. The treatment of intraperitoneal malignant disease with monoclonal antibody guided ‘2’1 radiotherestimation by radioimmunoscintigraphy utilizing the apy, Brir. J. Cancer 58, 658-662 (1988). same MoAb. The administration of serial doses of RIC 6. Courtenay-Luck, N. S.. Epenetos, A. A., Moore, R., Larche, M., in the presence of preexisting HAMA may have signifPectasodes. D., Dhokia, B., and Ritter, M. A. Development of icant effects upon the biodistribution and pharmacokiprimary and secondary immune responses to mouse monoclonal netics of the RIC. For example, Stewart et al. [2] have antibodies used in the diagnosis and therapy of malignant neoplasms, Cunc~v Res. 46, 6489-6493 (1986). compared the blood and urine clearance rates of five 7. Sakahara, H., Reynolds, J. C., Carrasquillo. J. A., Lora, M. E.. ovarian cancer patients who had developed HAMA from Maloney, P. J.. Lotze. M. T.. Larson, S. M., and Neumann, R. D. prior antiovarian MoAb infusions with a series of similar In vitro complex formation and biodistribution of mouse anti-tumor patients receiving primary therapy. Patients received 50 monoclonal antibody in cancer patients, J. NW/. Med. 30, 131lto 158 mCi of ‘3’1-labeled antiovarian MoAb intraperi1317 (1989). toneally as part of a phase I-II RIT trial. These authors 8. Schroff, R. W. Human anti-murine immunoglobulin responses in reported a significant increase in urinary clearance rates, patients receiving monoclonal antibody therapy, Cancer Res. 45, 879-885 (1985). a decrease in effective half-life and a pronounced de9 Shawler, D., Bartholomew, R., Smith. L., and Dillman, R. Human crease in dose to peritoneum in patients with preexisting immune rsponse to multiple injections of murine monoclonal IgG, HAMA. J. fmmunol. 135, 1530-1535 (1985). Sakahara et al. [7] studied 10 melanoma patients who IO. Herlyn, D., Lubek. M., Sears, H., and Koprowski. H. Specific also developed HAMA from prior exposures to ‘“I-ladetection of anti-idiotype immune responses in cancer patients beled monoclonal antibody 9.2.27, an antimelanoma antreated with murine monoclonal antibodies. J. Immunol. Methods tibody. Upon reinfusion, mean serum levels of RIC were 85, 27-38 (1986). reduced, serum clearance was increased, and hepatic 11. Zimmer, A. M.. Rosen, S. T., Spies, S. M., Goldman-Leikin, R., uptake of 13’1was significantly elevated due to the forKazikiewicz, J. M., Silverstein, E. A., and Kaplan, E. H. Radioimmunotherapy of patients with cutaneous T-cell lymphoma mation of antibody complexes in the serum. using an iodine-13 l-labeled monoclonal antibody: Analysis of reIn conclusion, our results indicate that patients retreatment following plasmapheresis, J. NucI. Med. 29, 174-180 ceiving single intraperitoneal doses of ‘3’I-OC-125 de(1988). velop HAMA frequently. HAMA formation seems to be 12. Pimm, M. V., Perkins, A. C., Armitage, N. C., and Baldwin, dependent upon the total dose of RIC, the interval from R. W. The characteristics of blood-borne radiolabels and the effect of anti-mouse IgG antibodies on localization of radiolabeled monoprior chemotherapy, and the patient’s immune status.
248
13.
14.
15.
16.
MUTO ET AL.
clonal antibody in cancer patients, J. NW/. Med. 26, 1011-1023 (1985). Bast, R. C., Klug, T. L., St. John, E., Jenison, E., Niloff, J. M., Lazarus, H., Berkowitz, R. S., Leavitt, T., Griffiths, C. T., Parker, L., Zurawski, V. R., Jr., and Knapp, R. C. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer, N. Engl. .I. Med. 309, 883-887 (1983). Haisma, H. J., Hilgers, J., and Zurawski, V. R. Jr. Iodination of monoclonal antibodies for diagnosis and radiotherapy using a convenient one vial method, J. Nucl. Med. 27, 1890-1895 (1986). Lindmo, T., Bowen, E., and Cultata, F. Determination of the immune reactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite excess, J. Immunol. Methods 72, 77-89 (1984). Mosely, K., Knapp, R. C., and Haisma, H. J. An assay for the
detection of human anti-murine immunoglobulins in the presence of CA125 antigen. J. Zmmunol. Methods, 106, l-6 (1988). 17. Goldstein, G., Fuccello, A., Norman, D., Shield, S., Colvin, R., and Cosimi, A. OKT, monoclonal antibody plasma levels during therapy and the subsequent development of host antibodies to OKT,, Transplantation 42, 507-511 (1986). 18. Sears, H., Herlyn, D., Steplewski, Z., and Koprowski, H. The effects of monoclonal antibody immunotherapy on patients with gastrointestinal adenocarcinoma, J. Biol. Response Modif. 3, 138150 (1984). 19. Dillman, R. O., Koziol, J. A., Zavanelli, M. I., Beauregard, J. C., Halliburton, B. L., Glassy, M. C., and Royston, I. Immunocompetence in cancer patients. Assessment by in vitro stimulation tests and quantitation of lymphocyte subpopulations, Cancer 53, 1489 (1984).
ONCOLOGY
38, 244-248 (1990)
Human Anti-murine Antibody Responses in Ovarian Cancer Patients Undergoing Radioimmunotherapy with the Murine Monoclonal Antibody OC-I 25 MICHAEL
G. MUTO, M.D. ,’ NEIL J. FINKLER, M.D., AMIN I.KASSIS, PH.D. ,* EVA MARIE LEPISTO, B.S., AND ROBERT C. KNAPP, M.D.
Divisions of Gynecologic Oncology and *Nuclear Medicine, Departments of Obstetrics and Gynecology and Radiology, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Harvard Medical School, 75 Francis Street, Boston, Massachusettes 02115
Received February 8, 1990
Human anti-murine antibody (HAMA) responses were monitored in 23 patients with recurrent or persistent epithelial ovarian carcinoma undergoing single-dose intraperitoneal radioimmunotherapy (RIT) with the murine monoclonal antibody OC-125. Sera of patients receiving escalating dosesof OC-125 F(ab’), (lo70 mg) radiolabeled with 18 to 141 mCi of iodine-13’ were assayed for HAMA by a protein A-based radioimmunoassay. Overall, 70% of patients (16/23) developed HAMA within 10 to 46 days (median = 29) postinfusion, with peak values (23 + 6 to 325 2 10 pg/ml) at 32 to 102 days (median = 38). HAMA was undetectable prior to infusion in all casesand persisted up to 76 weeks. Of patients receiving a dose of 123 mCi or less, 80% (16/20) developed HAMA, whereas in the 140-mCi group, none of the three patients had detectable levels. Two patients in the 140-mCi group demonstrated dose-limiting bone marrow toxicity (severe thrombocytopenia and neutropenia). It is concluded that a single intraperitoneal dose of monoclonal antibody leads to a high incidence of HAMA production. The results also suggest that the likelihood of HAMA formation in patients who either had undergone recent chemotherapy or had received the highest dose of the radioimmunoconjugate is reduced. These observations may be of significance in designing multiple-dose therapy trials as HAMA has been demonstrated to decrease antibody-to-tumor binding and may potentially increase renal, hepatic, and hematologic toxicity associated with radioimmunotherapy. Q 1990 Academic PRSS, I~C.
the radioimmunoscintigraphy (RIS) and the radioimmunotherapy (RIT) of cancer patients [l-5]. The development of human anti-murine antibody (HAMA) is common in patients receiving repetitive or single large doses of MoAb [6-101. HAMA has been demonstrated to affect the biodistribution of MoAb by forming antigen-antibody complexes which increase the blood clearance of MoAb and decrease their specific binding to tumor [6,7,11,12]. OC-125 is a murine MoAb raised against the antigenic determinant CA12.5, an ovarian cancer-associated antigen expressed in over 80% of nonmucinous epithelial carcinomas [ 131.We report HAMA responses in 23 ovarian cancer patients undergoing single-dose intraperitoneal RIT with F(ab’), fragments of OC-125 radiolabeled with iodine-13’.
INTRODUCTION Interest
continues
to grow in the potential
diagnostic
and therapeutic applications of murine monoclonal antibodies (MoAb). Radiolabeled MoAb directed against human tumor-associated antigens have been used in both I To whom reprint requests should be addressed at Department of Obstetrics and Gynecology. 244 two-8258/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS Patients. Between July 1987 and January 1989, 23 patients were enrolled in the radioimmunotherapy trial. Written informed consent was obtained through an approved protocol of the Human Subjects Committee of the Brigham and Women’s Hospital, Boston, Massachusetts. All patients had a confirmed diagnosis of persistent or recurrent epithelial ovarian carcinoma (20 serous, 3 endometrioid), with CA125 antigen levels >35 U/ml. The median age was 54 (range, 31 to 73 years). The majority of the patients presented with advanced, poorly differentiated lesions (Table 1). Exclusion criteria included prior pelvic or abdominal radiation therapy in excess of 3000 cGy; overt thyroid, cardiac, or renal disease; a Karnofsky score ~50; and cytotoxic chemotherapy
TABLE 1 Distribution of Stage and Grade Among Twenty-three RIT Patients Grade Stage
1
1 II III IV
-
2
3
1 (4%) 4 (18%) -
I (4%) 1 (4%) 14 (61%) 2 (9%)
1 month of enrollment. All patients had undergone cytoreductive surgery followed by multiple courses of postoperative chemotherapy, predominantly with cisplatinum-containing regimens. One patient received adjuvant whole-pelvis radiotherapy. In addition, patients with recurrences had undergone aggressive salvage therapy with either intravenous or intraperitoneal administration of a variety of cytotoxic agents. within
Radioimmunoconjugate preparation and RIA administration. OC-125 F(ab’), fragments (Centocor, Malvern,
PA) were radiolabeled with ‘?‘I (NEN DuPont, Billerica, MA) to a specific activity of 2 mCi/mg utilizing a singlevial Iodogen technique [ 141. The radioimmunoconjugate
(RIC) was tested for immunoreactivity by a cell binding assay with OVCAR 433 as described [15]. Pyrogen testing was performed using a chromogenic limulus amoebocyte assay, and sterility was evaluated by a 72 hour incubation in thyoglycolate medium. The content of free iodine was assessed by instant thin-layer chromatography (Gelman Sciences, Ann Arbor, MI). Pyrogen-free RIC with greater than 30% immunoreactivity and less than 10% free iodine was administered to the patient intraperitoneally in all cases by single-lumen catheters as described previously [I]. Serum collection and HAMA assay. Serum was obtained prior to infusion, on a daily basis during the hospital stay, and weekly for at least 6 weeks postinfusion. All samples were stored at -70°C until assayed. Human anti-murine IgG responses were detected by a radioimmunoassay (RIA) described by Mosely [ 161. Briefly, 96-well Remove-A-Well (TC) plates (Dynatech Corp., Chantilly, VA) were coated with protein A (Genzyme, Boston, MA) at a concentration of 10 pug/ml by a 24-hr incubation at 4°C. The plates were washed in 0.1 M phosphate-buffered saline (PBS) containing 10% fetal calf serum and incubated for an additional 24 hr in this buffer. Serum samples, diluted 1: 500 in Tris-HCl buffer, pH 8.0, were added to the precoated plates in a 50-,ul
TABLE 2 Dose of ‘3’I-OC-125 and HAMA Response Patient number
Antibody dose (md
1 2 3 4 5 6 7 8 9 10 11 12 13 14 I5 16 17 18 19 20 21 22 23 * Mean ? SD.
10 10 10 15 15 IS 20 20 30 30 30 40 40 40 50 50 50 60 60 60 70 70 70
‘j’I dose (mCi) 18 20 28 30 20 34 44 42 47 60 54 76 82 87 100 104 105 120 117 123 140 141 140
Follow-up (days) 677 285 51 190 170 536 51 60 41 150 32 56 47 35 60 52 46 53 63 59 54 93 51
Day HAMA first positive 17 10 21 nd 42 18 26 18 28 nd 32 29 40 nd 27 31 37 nd 30 25 nd nd nd
Peak HAMA Wml) 253 ? 27” 319 * 31 234 -t- 25 nd 13 5 4 308 k 34 221 + 25 77 -+ 17 107 + 16 nd 122 * 4 28 k I 23 + 6 nd 29 t 3 124 -+ 12 33 2 6 nd 325 ” 10 112 -c 15 nd nd nd
Interval from prior chemotherapy (months)
5 20 3 1 2 10 5 4 3 2 3 6 14 2 4 22 3 15 11 22 3 5 1
Grade of hematologic toxicity 2 0 0 0 1 0 0 0 2 0 0 2 0 2 2 2 3 2 0 0 1 3 4
246
MUTO ET AL.
23). Overall, 70% (16/23) of the patients developed detectable HAMA within 10 to 46 days (median = 29 days) of therapy (Table 2). Peak values ranged from 23 + 6 HAMA detected to 325 ? IO pg/ml and occurred from 32 to 102 days Interval (median = 38 days) following therapy. No patient had Yes (months) No preexisting detectable HAMA prior to infusion. No pa<3 1 4 tient had been previously treated with monoclonal an23 15 3 tibody. Patients were followed from 32 to 677 days (median = 60), with HAMA detectable at 536 days in one patient. volume. These plates were then incubated for 2 hr at Among the 20 patients who received RIT at doses room temperature, the wells were washed with PBS, and below 140 mCi, HAMA was detected in 80% (16/20), 50 ~1 of the tracer (OC-125 radiolabeled with 12’1)was whereas none of the 3 patients given 140 mCi developed added. The plates were then incubated an additional 2 detectable HAMA. When this latter group was compared hr at room temperature; the wells were washed and their to the other treated patients, there were no significant radioactive content was determined in a gamma counter. differences in age, pretreatment chemotherapy, or inA negative control was established from pooled sera terval from prior chemotherapy to RIT. of volunteers with no detectable HAMA IgG. A positive HAMA production was less likely in patients who had control was established from a previously studied patient received chemotherapy within 3 months of RIT adminwith a HAMA level of 200 pg/ml. Results are reported istration when compared with patients in which the inin micrograms based upon a standard curve established terval from prior therapy was 3 months or longer (Table by serial dilutions of a rabbit anti-OC-125. 3). There was no association between the number of courses of chemotherapy given prior to RIT and the agents used in antecedent therapy. RESULTS Various patterns of HAMA response were observed; Twenty-three patients received 10 to 70 mg of OC-125 two are illustrated in Fig. 1. Patient 1, followed for 677 F(ab’), radiolabeled with 18 to 141 mCi of 13’I. At the days, demonstrated a rapid rise in HAMA with a peak 140-mCi dose, 2 patients (out of 3) demonstrated at least at Day 38. HAMA was last detected on Day 205, and grade III toxicity (neutropenia with absolute neutrophil by Day 317, no further response was detectable. Patient count of 820 in patient 22, and severe thrombocytopenia 6, on the other hand, had a similar early rise, but perwith a platelet nadir of 20,000 and bleeding in patient sistently detectable levels up to 536 days postinjection. This response persisted despite four subsequent courses of intravenous c&platinum chemotherapy at a dose of 50 mg/m2. 300 HAMA
TABLE 3 versus Interval from Prior Chemotherapy
Formation
I
DISCUSSION
600
DAYS POS?- INJECTION
FIG. 1. Two types of HAMA response: (A) Patient 1, (B) patient 6. Asterisks for patient 6 indicate subsequent course of intravenous cis-platinum chemotherapy.
The data presented in this paper demonstrate that 70% of patients with ovarian cancer undergoing RIT with a single intraperitoneal injection of ‘3’I-OC-125 will develop HAMA. Peak values occur at 4 to 6 weeks postinfusion and persist for up to 76 weeks. Our data are consistent with previously published reports of HAMA development in patients receiving murine MoAb. Among renal transplant patients receiving OKT3, the murine IgG,, for the immunotherapy of acute renal allograft rejection, 88% developed HAMA within 3 weeks of MoAb infusion [ 171. Patients with gastrointestinal malignancies who received anti-CEA MoAb also demonstrated an 89% HAMA formation rate [18]. Finally, in a series of eight patients with advanced carcinoma of the ovary, four (50%) developed HAMA after a second exposure to various antiovarian MoAbs [6]. It has been suggested that HAMA formation rates
HUMAN ANTI-MURINE
ANTIBODY
RESPONSES IN OVARIAN
CANCER
247
should approach 100% in immunocompetent patients and The formation of HAMA must be monitored in any prothat decreased HAMA response among cancer patients tocol involved in administering murine monoclonal anmay be related to generalized immunosuppression re- tibodies, particularly in multiple-dose therapy trials. lated to the malignancy or to cytotoxic chemotherapy [19]. As demonstrated by our data, RIC in doses suffiREFERENCES ciently high to cause bone marrow suppression may fur1. Finkler, N. J., Muto, M. G., Kassis, A. I., Weadock, K., Tumeh. ther reduce the ability of the patient to mount a HAMA S. S., Zurawski, V. R., Jr., and Knapp, R. C. Intraperitoneal raresponse. diolabeled OC 125 in patients with advanced ovarian cancer, GyWhile lower doses of MoAb are associated with high necol. Oncol. 34, 339-344 (1989). HAMA formation rates, the administration of very high 2. Stewart. J. S. W., Hird. V., Snook, D.. Sullivan, M., Hooker, G., doses of MoAb actually results in a reduced HAMA Coutenay-Luck, N. S., Siuolapenko, Cl., Griffiths, M. Myers, response rate. For example, Sears et al. have reported M. J., and Lambert, H. E. lntraperitoneal radioimmunotherapy for ovarian cancer: Pharmacokinetics, toxicity and efficacy of I-131 HAMA in eight of nine patients receiving less than 200 labelled monoclonal antibodies. Int. J. Radiat. Oncol. Biol. Phys. mg of murine MoAb. HAMA was detected in only one 16, 405-413 (1989). of nine patients when doses of infused MoAb ranged 3. Carrasquillo, J. A.. Bunn. P. A., Keenan, A. M., Reynolds, J. C., from 366 to 1000 mg [19]. This “tolerizing” effect of Schroff, R. W., Foonka, K. A., Su, M. H.. Gazdar. A. F., Mulhigh-dose immunotherapy has not been observed in the shine, J. L., and Oldham, R. K. Radioimmunodetection of cutadose ranges used in our study. It is therefore unlikely neous T-cell lymphoma with “‘In labeled TIOI monoclonal antibody, N. Engl. J. Mrd. 315, 613-678 (1986). that decreased HAMA formation among the highest dose 4. Epenetos, A. A., Munro, A. J., Stewart. S., Rampling, R., Lamgroup is related to the dose of MoAb alone. bert, R. E., Mckenzie, C. G., Soutter, P., Rahemtulla, A., Hooker. The phase I trial described in this report is based on G., and Siuolapenko, G. B. Antibody-guided irradiation of ada single intraperitoneal infusion of RIC. Hoever, effecvanced ovarian cancer with intraperitoneally administered radiotive RIT will probably require fractionation of dose or labeled monoclonal antibodies, J. C/in. Oncol. 5, 1890-1899 (1987). multiple treatment cycles. In some cases, RIT will be 5. Ward. B., Mather, S., Shepherd, J., Crowther, M., Hawkins, L., preceded by attempts at tumor detection or dosimetry Britton. K.. and Slevin, M. L. The treatment of intraperitoneal malignant disease with monoclonal antibody guided ‘2’1 radiotherestimation by radioimmunoscintigraphy utilizing the apy, Brir. J. Cancer 58, 658-662 (1988). same MoAb. The administration of serial doses of RIC 6. Courtenay-Luck, N. S.. Epenetos, A. A., Moore, R., Larche, M., in the presence of preexisting HAMA may have signifPectasodes. D., Dhokia, B., and Ritter, M. A. Development of icant effects upon the biodistribution and pharmacokiprimary and secondary immune responses to mouse monoclonal netics of the RIC. For example, Stewart et al. [2] have antibodies used in the diagnosis and therapy of malignant neoplasms, Cunc~v Res. 46, 6489-6493 (1986). compared the blood and urine clearance rates of five 7. Sakahara, H., Reynolds, J. C., Carrasquillo. J. A., Lora, M. E.. ovarian cancer patients who had developed HAMA from Maloney, P. J.. Lotze. M. T.. Larson, S. M., and Neumann, R. D. prior antiovarian MoAb infusions with a series of similar In vitro complex formation and biodistribution of mouse anti-tumor patients receiving primary therapy. Patients received 50 monoclonal antibody in cancer patients, J. NW/. Med. 30, 131lto 158 mCi of ‘3’1-labeled antiovarian MoAb intraperi1317 (1989). toneally as part of a phase I-II RIT trial. These authors 8. Schroff, R. W. Human anti-murine immunoglobulin responses in reported a significant increase in urinary clearance rates, patients receiving monoclonal antibody therapy, Cancer Res. 45, 879-885 (1985). a decrease in effective half-life and a pronounced de9 Shawler, D., Bartholomew, R., Smith. L., and Dillman, R. Human crease in dose to peritoneum in patients with preexisting immune rsponse to multiple injections of murine monoclonal IgG, HAMA. J. fmmunol. 135, 1530-1535 (1985). Sakahara et al. [7] studied 10 melanoma patients who IO. Herlyn, D., Lubek. M., Sears, H., and Koprowski. H. Specific also developed HAMA from prior exposures to ‘“I-ladetection of anti-idiotype immune responses in cancer patients beled monoclonal antibody 9.2.27, an antimelanoma antreated with murine monoclonal antibodies. J. Immunol. Methods tibody. Upon reinfusion, mean serum levels of RIC were 85, 27-38 (1986). reduced, serum clearance was increased, and hepatic 11. Zimmer, A. M.. Rosen, S. T., Spies, S. M., Goldman-Leikin, R., uptake of 13’1was significantly elevated due to the forKazikiewicz, J. M., Silverstein, E. A., and Kaplan, E. H. Radioimmunotherapy of patients with cutaneous T-cell lymphoma mation of antibody complexes in the serum. using an iodine-13 l-labeled monoclonal antibody: Analysis of reIn conclusion, our results indicate that patients retreatment following plasmapheresis, J. NucI. Med. 29, 174-180 ceiving single intraperitoneal doses of ‘3’I-OC-125 de(1988). velop HAMA frequently. HAMA formation seems to be 12. Pimm, M. V., Perkins, A. C., Armitage, N. C., and Baldwin, dependent upon the total dose of RIC, the interval from R. W. The characteristics of blood-borne radiolabels and the effect of anti-mouse IgG antibodies on localization of radiolabeled monoprior chemotherapy, and the patient’s immune status.
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detection of human anti-murine immunoglobulins in the presence of CA125 antigen. J. Zmmunol. Methods, 106, l-6 (1988). 17. Goldstein, G., Fuccello, A., Norman, D., Shield, S., Colvin, R., and Cosimi, A. OKT, monoclonal antibody plasma levels during therapy and the subsequent development of host antibodies to OKT,, Transplantation 42, 507-511 (1986). 18. Sears, H., Herlyn, D., Steplewski, Z., and Koprowski, H. The effects of monoclonal antibody immunotherapy on patients with gastrointestinal adenocarcinoma, J. Biol. Response Modif. 3, 138150 (1984). 19. Dillman, R. O., Koziol, J. A., Zavanelli, M. I., Beauregard, J. C., Halliburton, B. L., Glassy, M. C., and Royston, I. Immunocompetence in cancer patients. Assessment by in vitro stimulation tests and quantitation of lymphocyte subpopulations, Cancer 53, 1489 (1984).