Cancer Immunol Immunother (1988) 26:187-201 ancer mmunology mmunotherapy © Springer-Verlag 1988 The intraperitoneal delivery of radiolabeled monoclonal antibodies: studies on the regional delivery advantage Richard L. Wahl l, Jeffrey Barrett 2, Onelio Geatti 1., Monica Liebert 1, Barry S. Wilson 3.*, Susan Fisher I, and John G. Wagner 2 ~University of Michigan Medical Center, Department of Internal Medicine, Division of Nuclear Medicine, Ann Arbor, Michigan, USA 2University of Michigan Medical Center, College of Pharmacy and Upjohn Center for Clinical Pharmacology, Ann Arbor, Michigan, USA 3University of Michigan Medical Center, Department of Pathology, Ann Arbor, Michigan, USA Summary. The i.p. delivery of murine monoclonal anti- those for i.v. delivery, though not beyond 48 h after i.p. in- body was compared with i.v. delivery in normal mice and jection. This study demonstrates the pharmacokinetic ra- rats, in normal nude mice and in those with i.p. human tionale for i.p. monoclonal antibody delivery, especially ovarian carcinoma xenografts. In normal rats, all classes for agents cleared rapidly from the blood, such as anti- of antibodies and antibody fragments evaluated were body fragments. In addition, definite i.p. delivery benefit cleared from the peritoneal cavity at comparable rates. for antibody specific to i.p. tumors in the i.p. ovarian can- The regional delivery (Rd 1) advantage to the peritoneal cer system was shown soon after injection. These data re- cavity following i.p. delivery was thus most dependent on garding i.p. antibody delivery should be useful in rational- the rate of clearance of the antibody or fragment from the ly planning diagnostic and therapeutic studies involving blood stream. Determining the exact i.p. delivery advan- the i.p. delivery of unmodified and immunoconjugated tage was problematic due to the difficulty in reliably ob- monoclonal antibodies. taining peritoneal fluid later than 9-10 h after i.p. injection in normal animals. During the first 9 h following i.p. injec- tion, the Rd(0-9/0-9) was, for a murine IgG2ak Fab > Introduction F(ab')2 > IgG (at 13.6 > l0 > 7.9). Two murine IgMs Regional delivery (Rd) of chemotherapeutic agents to the evaluated differed in Rd(0-9) at 27.1 and 9.2 respectively. peritoneal and other cavities has been used in the treat- When blood levels were extrapolated to infinity, these Rd ment of malignant diseases restricted to such spaces, such (0-9/oo) values were considerably lower with the Fab hav- as ovarian carcinoma [7, 11, 20]. With the development of ing the highest Rd at 4.67. The i.p. Rd advantage was al- monoclonal antibodies reactive with ovarian carcinoma, most solely due to the i.p. antibody levels seen in the first some of which when radiolabeled are capable of immuno- 24 h after injection, as after that time, blood levels become imaging such tumors, the i.v. and more recently the i.p. comparable to those seen following i.v. injection. Normal routes of antibody delivery to such regionally limited dis- tissues obtained at sacrifice 5-7 days after i.p. injection. eases have been explored [2, 4, 9, 10, 25, 27]. While there Normal tissues obtained at sacrifice 5-7 days after i.p. or has been preliminary clinical utilization of the i.p. delivery i.v. injection in rats showed comparable levels of radioan- route for ovarian cancer therapy in humans, relatively tibody activity, whether the injection was i.p. or i.v. (except little is known about the clearance rate of monoclonal an- for higher diaphragmatic levels following i.p. delivery). In tibodies from the peritoneal cavity. nude mice with i.p. human-derived ovarian tumors, intact It is clear that higher concentrations of radiolabeled IgG clearance from the peritoneal cavity to the blood was antibodies present in a given region will result in higher considerably slower than in normal animals, and early i.p. binding to target antigens in that region, due to binding ki- tumor uptake of specific antibody was significantly higher netic considerations [14]. We have recently shown for radio- than that following i.v. antibody delivery. With higher ear- iodinated intact murine IgG2ak, that increased antibody ly tumor uptake and lower systemic exposure, early concentration compared to blood levels exists in the peri- tumor/nontumor ratios were significantly greater than toneal cavity of rats following i.p. antibody administration [21, 24]. We also showed that this i.p. delivery advantage Rd is area under the curve (AUC) for peritoneal fluid activity / could be further enhanced by accelerating the clearance of AUC for blood radioactivity. Rd (0-9/0-9) is the Rd measured the intact IgG from the blood stream using a systemically from 0 to 9 h for both peritoneal fluid and blood. Rd (0-9/0o) is administered polyclonal anti-mouse antibody [21]. the conservative estimate of Rd with i.p. fluid AUC measured to Through such a manipulation, the Rd advantage following 9 h, with blood levels extrapolated to infinity. Rd 2 is Rd/(AUC i.p. fluid (0-9)/AUC blood (0-9)) after i.v. injection. i.p. administration could be increased by approximately * Present address: Istituto di Medicina Nuclear, Ospedale Civile, 50% in the first 10 h after injection. Udine, Italy Since quite marked differences exist among the blood ** Present address: Hybritech Incorporated, Department of Cell clearance rates and tumor imaging properties of intact Biology, San Diego, CA, USA monoclonal antibodies and monoclonal antibody frag- Offprint requests to: Richard L. Wahl, University of Michigan ments following intravascular delivery, it is possible that Medical Center, Division of Nuclear Medicine, Box 0028, 1500 similar differences in clearance may exist for i.p. delivered East Medical Center Drive, Ann Arbor, MI 48109-0028, USA monoclonal antibodies [3, 5, 23] and that there might be 188 large differences in the Rd advantage when given i.p. The neum exposed. The i.p. injections of antibody were per- present study was conducted to determine the extent of the formed with a 27-gauge needle attached to a 20 ml syringe Rd advantage to the peritoneal space for monoclonal an- under direct visualization. Preliminary studies using India tibodies, as well as the influence of antibody class and ink in saline demonstrated that this approach resulted in fragmentation on the delivery advantage. This report also i.p. fluid delivery without significant leakage. Peritoneal evaluates the influence of i.p. human tumor xenografts on fluid was subsequently sampled (50-100 gl aliquots) us- antibody clearance from the peritoneal space and ex- ing a tuberculin syringe fitted with a 28-gauge needle (un- amines the extent of localization improvement of specific der direct visualization). The i.v. injections were carried antibody to i.p. human ovarian carcinoma xenografts fol- out by direct cannulation of the femoral vein with a lowing i.p. versus i.v. delivery. 28-gauge needle after an incision had been made in the skin overlying the vein. Material and methods In studies using i.p. tumor-bearing nude mice (approxi- mately 8-12-week-old tumors), the 1251 5G6.4 antibody was Monoclonal antibodies. The 225.28S is an IgG2ak murine given either i. p. or i.v. with animal sacrifice 4, 24, 48, or monoclonal antibody reactive with the high molecular 120 h later. Uptake in i.p. tumor foci (HTB77 IP3 line of weight antigen of melanoma [33]. Hybridoma cells produc- human ovarian cancer) [31] and blood was compared be- ing this reagent were grown in pristane-primed (Aldrich) tween delivery routes (i.p. or i.v.). In addition, a dual-label Balb/c mice as ascites, and then purified by staphylococ- experiment comparing simultaneously i.p. administered cal protein A chromatography [8]. Then 225.28S F(ab')2 5G6.4 and UPC-10 was conducted to confirm the specifici- fragments were prepared by 2% pepsin digestion in pH 4.2 ty of tumor uptake. The rate of i.p. clearance of the acetate buffer, with subsequent purification by dialysis nonspecific IgG2ak, UPC-10, was also studied in normal and on a staphylococcal protein A column [8, 15, 23]. The nude mice, nude mice with i.p. ovarian tumors, and in 225.28S Fab fragment was prepared by 2% papain diges- healthy BALB/c mice without such tumors. Gamma imag- tion with purification of the Fab from the Fc on a staphy- ing following i.p. administration was also performed using lococcal protein A column, followed by additional purifi- a large field of view gamma camera equipped with a high- cation using TSK sizing HPLC [16]. FTI66 is an IgM energy parallel hole or a pinhole collimator in selected in- of murine origin, which was purified by DEAE chroma- stances. The images were digitally recorded in a dedicated tography from mouse ascites (Liebert et al, unpublished computer for subsequent display and analysis. All images data) [32]. UPC-10 is an IgG2a monoclonal antibody pur- were performed using a 20% window centered at 364 KeV. chased in purified form (Bionetics Research, Charleston, SC). BA-1 is a murine IgM, (generously provided by Hy- Time course experiments. In experiments evaluating i.p. an- britech, LaJolla, Calif.) [1], reactive with human B-cells tibody delivery in normal rats, groups of 3-7 adult female which was purified from carrier human albumin protein rats were injected i.p. with approximately 5-20 p.Ci of la- using TSK sizing HPLC. 5G6.4 is an IgG2a murine mono- beled antibody (0.2-1 p~g) in a total volume of 20 ml of clonal antibody with preferential reactivity with ovarian warm, 0.01 M phophate-buffered saline, pH 7.0.