Biodistribution Study of 188Re-Labeled Trisuccin-HuCC49 and Trisuccin-HuCC49ACa2 Conjugates in Athymic Nude Mice Bearing Intraperitoneal Colon Cancer Xenografts

The t r ihydroxamate bifunctional chelating agent (BCA), trisuccin, has been shown to be a potential ligand for radiolabeling of monoclonal antibodies (MAbs) with rhenium radioisotopes, through an indirect postconjugation approach. The use of this t r ihydroxamate BCA made it possible to prepare stable BCA-MAb conjugates in pure form that could be radiolabeled with carrier-free lSSRe. The antiTAG-72 murine MAb, CC49, and its humanized derivatives are promising agents in the t reatment of a number of malignancies with the CH2 domain-deleted MAb (HuCC49ACH2), which is of part icular interest due to its rapid blood clearance. The biodistribution of 188Re-labeled conjugates of trisuccin with both humanized CC49 (HuCC49) and HuCC49ACn2 in athymic nude mice implanted i.p. with LS174T human colon carcinoma was studied. Trisuccin-MAb conjugates were synthesized at different BCA:MAb ratios by the 6-oxoheptanoic acid method using trisuccin hydrazide. The conjugates were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy for the number of incorporated trisuccin molecules. The conjugates were radiolabeled with carrier-free, generator-produced lSSRe and purified by gel fil tration on Sephadex G-25. Labeling yields and homogeneity of the labeled conjugates were analyzed by high-pressure liquid chromatography and instant TLC. Athymic nude mice were injected i.p. with LS174T human colon carcinoma cells, 7 days prior to injection of the labeled antibodies, lSSRelabeled MAbs were injected i.p., and the mice were sacrificed 24 h postinjection. Matrix-assisted laser desorption/ ionization time-of-flight analyses showed stable incorporation of trisuccin into each MAb, with the measured l igand:MAb values positively correlating with the theoretical ratios. Labeling of the conjugates with lSSRe proceeded with high yields, producing homogeneous lSSRe-MAbs with good stabilities as shown by instant TLC and biodistrihution analyses. Biodistribution of the radiolabeled MAbs at 24 h after injection showed median tumor uptake values of 23.5 %ID/g and 17.6%ID/g for the 18SRe-HuCC49ACn2 and XSSReHuCC49, respectively. The blood clearance of the domaindeleted MAb was faster than that of the intact antibody. The blood values at 24 h after injection were 0.7%ID/g for 18SRe-HuCC49ACH2 and 3.2%ID/g for lSSRe-HuCC49. The results indicate that trisuccin is a promising agent for postconjugation labeling of antibodies with lSSRe. Additionally, these results illustrate the potential of ~8SRe-HuCC49ACrI2 in rad io immunodiagnos is and r ad io immuno the rapy of cancer. I n t r o d u c t i o n Radiolabeled MAbs 3 have shown promise for cancer therapy (1-8). Although utilization of this approach was started with isotopes of iodine as the source of radioactivity (9-15), it was realized later that the labeling of MAbs with radiometals might offer advantages (16-19). These include lack of dehalogenation, higher tumor uptake, and longer retention in tumor. The indirect labeling method, in which a BCA serves as a linker between the antibody and the radiometal, may offer chemical and biological advantages due to better-defined metal chelate structures and higher immunoreactivities (20, 21). In the postconjugation radiolabeling techniques, the BCA is first conjugated to the antibody and the resulting conjugate is then radiolabeled in a second step. Radioisotopes of the transition metal rhenium (i.e., 186Re and 188Re) have physical properties that may be useful in the radioimmunodiagnosis and RIT of cancer (22-25). ~S6Re is produced by neutron bombardment of the stable ~SSRe leading to a low specific activity product, whereas 188Re is available carrier-free from a lSSW/lSSRe generator (23). In addition to generating [3-particles with suitable energies for therapy, these radioisotopes also emit ",/-rays with energies suitable for imaging. Whereas lS6Re, with a half-life of 90 h, may be appropriate for antibodies with long circulation times, lSSRe with a half-life of 17 h, may be suitable for smaller molecules, such as antibody fragments and derivatives. Presented at the "Seventh Conference on Radioimmunodetection and Radioimmunotherapy of Cancer," October 15-17, 1998, Princeton, NJ. Supported by NIH Grant R01 CA62550 (to D. J. B.). 2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, University of Alabama at Birmingham, 1824 6th Avenue South, WTI 674, Birmingham, AL35294-6832. Phone: (205) 934-3681; Fax: (205) 975-7060; E-mail: [email protected]. 3 The abbreviations used are: MAb, monoclonal antibody; BCA, bifunctional chelating agent; L:MAb ratio, ligand:MAb ratio; OHA, 6-oxoheptanoic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; HPLC, high-pressure liquid chromatography; RIT, radioimmunotherapy; DPBS, Dulbecco's PBS; MW, molecular weight; SEC, size-exclusion chromatography; %ID/g, percentage of injected dose per gram of tissue; RY, radiolabeling yield; ITLC, instant TLC. Research. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Clinical Cancer Research 2995s Despite their attractive features, the chelation chemistries of rhenium isotopes have hampered their full utilization in radioimmunodiagnosis and RIT. In contrast to 99mTc, 186/lS8Re isotopes are difficult to reduce to lower oxidation states, a process necessary for their ligation by organic BCAs (26). Higher concentrations of the reducing agent (e.g., SnC12) at elevated temperatures and longer reaction times are required for radiolabeling with 186/18SRe isotopes. A rhenium-specific BCA must also have appropriate features to satisfy the requirements of stable antibody conjugation. Most importantly, the metalchelating groups of this compound must not interfere chemically with the antibody binding terminus. Of different BCAs examined for lS6/188Re and 99myclabeling of MAbs, only the amido-thiol system has been in frequent use (27-29). These molecules consist of one or two sulfur atoms in its sulfide form and two or three nitrogen atoms, usually as amide groups. These produce stable ligands for both 186/laSRe and 99myc radionuclides. However, because of the reactivity of the thiol groups, the standard procedure of postconjugation radiolabeling used for other radiometals is not feasible or, at best, not efficient. Interference from the thiol functions necessitates masking the BCA prior to conjugation to the MAb. Because removal of the thiol protecting groups is not chemically compatible with maintaining the biological integrity of the antibody, the thiol masking and radiometal chelation have been combined in a procedure referred to as the "preformed chelate approach" (27, 30). In this protocol, the activated BCA is first labeled at high temperature with 186/188Re or 99mTc isotopes, in the presence of stannous ion. In a second step, the complex is purified on a reversed-phase cartridge, and the pH of the medium is adjusted to a pH suitable for antibody conjugation. Next, the purified activated chelate is conjugated to the antibody. A second purification affords the final labeled antibody. Low to medium RYs are achieved. In an effort to design rhenium-binding BCAs without the shortcomings discussed above, we introduced hydroxamic acids as a new class of BCAs and reported on the synthesis of the first member of this family, trisuccin (31). In this design, we were interested in a chelator that allows the postconjugation radiolabeling of antibodies with isotopes of ~s6/~SSRe at high yields and good in vivo stability. The rationale behind the design of hydroxamate BCAs was 3-fold: (a) the oxyphilic nature of rhenium nuclides, which favors the oxygen-metal bonding between the hydroxamate ligand and rhenium isotopes and eliminates the need for a sulfur chelator; (b) the stability of unprotected hydroxamate functions (in contrast to free thiols); and (c) the incorporation of chemically noninterfering groups for both antibody conjugation and radiometal chelation. We developed and optimized a suitable conjugation protocol (31-33) and recently reported the hydrazone bond protocol as our standard and optimized procedure for antibody conjugation of unprotected hydroxamates (33). This chemistry has resulted in efficient postconjugation radiolabeling of MAbs with ~SSRe through a quite simple protocol. Murine CC49 has a high binding affinity for TAG-72, and this antigen is plentiful in selected human adenocarcinomas (34). Humanization of this antibody (35) would reduce immunogenicity and allow more than one treatment course to be given. However, construction of chimeric or CDR grafted molecules results in a substantial increase in circulating plasma half-life (36), and this prolonged radiation exposure of the marrow reduces the maximum tolerated dose and limits dose administration (37). This is true for i.p. as well as i.v. administration because the majority of the dose administered into the peritoneal cavity eventually enters the plasma compartment. Schlom and co-workers (38) have shown that deletion of the CH2 region of the molecule results in dramatic shortening of plasma half-life with retention of binding affinity and tumor localization. We describe the conjugation of trisuccin to the humanized MAb CC49 (HuCC49) and its CH2 domain-deleted derivative HuCC49ACn2, labeling of the resulting conjugates with ~88Re, and a biodistribution study of the labeled conjugates in athymic nude mice bearing i.p. human colon cancer xenografts. Materials and Methods General. All solvents and reagents were purchased from commercial suppliers and were used as received. Humanized and CH2 domain-deleted CC49 MAbs were provided by the National Cancer Institute (Dr. Jeffrey Schlom). The benzylprotected trisuccin was prepared as described previously (31). HPLC analyses were carried out on a Bio-Rad model 5000 Titanium system (Richmond, CA) equipped with a model 1806 UV/Vis detector and a Beckman model 170 radioisotope detector, operated by ValueChrome software (Bio-Rad). For analytical SEC, a 7.5 mm • 25 cm G3000SW column (TosoHaas, Montgomeryville, PA) was used. 10 mM PBS containing 10 mM Na2SO 4 at pH 6.7 was used as a solvent. Preparative SEC was carried out using Sephadex G-25 columns (PD-10, Amersham Pharmacia Biotech AB, Uppsala, Sweden) eluted with DPBS. Mass spectra were run on an API VI triple quadruple mass spectrometer (PE-Sciex, Toronto, Ontario, Canada) in electrospray mode and PerSeptive Biosystems (Framingham, MA) Voyager Elite MALDI instruments. N-Succinimidyl oxoheptanoate was provided by Biolinx, Inc. ([email protected], Birmingham, AL). Metal-free purified water was obtained from a Milli-QF system (Millipore, Bedford, MA). Sodium [188Re]perrhenate was eluted with normal saline from a ~8SW/lSSRe generator (Oak Ridge National Laboratory, Oak Ridge, TN). ITLCs were carried out on silica gel-impregnated glass fiber slides (Gelman Sciences, Ann Arbor, MI). Synthesis of Trisuccin Hydrazide and Antibody Conjugation. This compound was synthesized as described earlier (33). Briefly, benzyl-protected trisuccin carboxylic acid (500 rag, 0.75 mmol; Ref. 31) was condensed with tert-butyl carbazate (Aldrich Chemical Co., Milwaukee, WI) at a 1:1 molar ratio, with dicyclohexylcarbodiimide (171 rag, 0.83 retool) and 1-hydroxylbenzotriazol (102 rag, 0.75 retool) in tetrahydrofuran. The resulting fully protected product was catalytically hydrogenated to remove the benzyl protecting groups, and the semiprotected intermediate was treated with trifluoroacetic acid to afford the fully deprotected final product ligand at an overall yield of 72%. Structure identification was carried out through 1H NMR and electrospray mass spectroscopic analyses. Antibody Conjugation. Step 1: OHA-MAb. The OHA protocol reported previously (33) was used. In this procedure, N-succinimidyl OHA was conjugated to the MAb at an OHA: Research. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 2996s t88Re-labeled HuCC49 MAb Biodistribution Table l Measured ligand:MAb values for trisuccin-CC49 conjugates" MAb OHA : MAb b Trisuccin : MAb ;~ HuCC49 5.1/7.5 2.7/100 3.0/300 4.1/500 HuCC49ACH2 5.5/7.5 2.9/100 2.7/300 3.0/500 Experimental values measured by MALDI-TOF. b Experimental/theoretical. MAb ratio of 7.5, in 50 mM PBS buffer at pH 8. l and at 0~ for 1 h. The conjugate was purified and buffer-exchanged in a Centricon concentrator (Amicon, Beverly, MA) with 50 mM acetate buffer at pH 5.5. For each antibody, this solution was divided into three 300-ml portions for the trisuccin conjugation (Step 2). Antibody Conjugation. Step 2: Trisueein-MAb. To the solution of the OHA-MAb from Step 1, and at 0~ a solution of the trisuccin hydrazide was added followed by NaCNBH 3 after 1 h and at an overall concentration of 50 raM. Three trisuccinto-MAb molar ratios (100:1, 300:1, and 500:1) were used for each MAb. The mixture was stirred at 4~ for 18 h and purified as above. M A L D I T O F Analyses. Samples were analyzed in the positive mode on a Voyager Elite mass spectrometer with delayed extraction technology (PerSeptive Biosystems. The acceleration voltage was set at 25 kV, and 50-100 laser shots were summed. Sinapinic acid (Aldrich), dissolved in a 1:1 (v/v) mixture of acetonitrile and 0.1% trifluoroacetic acid, was used as the matrix. A 1 pmol/>l solution of BSA was added as an internal standard. Equal volumes of sample and matrix were mixed on a smooth plate. For each reaction, the intact MAb was scanned as the control for evaluation of the conjugation. The average of three measurements was used. For the OHA method, the MW increase of the OHA-MAb intermediate was calculated using the unconjugated antibody. The M W of this intermediate then served as the control to measure the increase in the MW of the final conjugate product. Each difference in MW, divided by the MW of the corresponding linker, measured the linker:MAb ratio. Radiolabeling of Antibody Conjugates. Rhenium-188, in the form of Na [~SSRe]ReO4 (5 mCi), was eluted from a 188W/188Re generator (Oak Ridge National Laboratory) with metal-free normal saline (23, 39, 40). The perrhenate was reduced at 90~ with 250 txl of a reducing solution containing 1.1 M 2-hydroxy-2-methyl propionic acid and 26 mM stannous ion, for 1 h. The mixture was cooled in an ice bath, and the pH was adjusted to pH 8 with 1 M sodium hydroxide, at which time, the excess stannous chloride precipitated in the form of a heavy insoluble stannic hydroxide precipitate. The mixture was then added to the solution of the antibody (200 pA at a concentration of 1 mg/ml) followed by incubation at 45~ for 45 min. The radiolabeled antibody was purified on a PD-10 column with 0.1% human serum albumin in DPBS. A 3.2 mg/ml solution of the unconjugated HuCC49ACH2 (85 txl) was labeled by the same procedure as the conjugates. ITLC and SEC were used for determination of RYs and purity evaluations. ITLCs were run in 12% trichloroacetic acid. In this solvent, the radiolabeled MAb remains at the origin, Table 2 Radiolabeling of trisuccin-HuCC49 and trisuccinCC49ACH2 conjugates with different L:MAb ratios SA;' L:MAb %RY a (txCi/txg) %IR c HuCC49 100 80 2.0 71 300 84 2.1 69 500 92 2.2 69 HuCC49ACH2 100 83 2.0 74 300 94 4.0 77 500 98 4.8 69 a Percentage of radiolabeling yield by SEC-HPLC. ;' Specific activity. c Percentage of immunoreactivity. whereas free radioisotope and other by-products move with the solvent front. The SECs were carried out on a HPLC system as described above. In this system, the retention times for HuCC49 and HuCC49ACH2 were 9.1 and 9.5 min, respectively, for the UV traces followed, within about 0.4 min, by the radioactive traces. Imnmnoreaetivity Assay. A modified Lindmo assay was used to evaluate the immunoreactivity of the radiolabeled conjugates (41). Briefly, polystyrene beads (6.4 mm, specular finish) were coated with TAG-72 positive bovine submaxillary mucin (Sigma Chemical Co., St. Louis, MO) in PBS (4.4 rag/100 ml) and blocked with 0.1% human serum albumin. A control test tube containing no bead, as a counting standard for added l SSRe, and a nonspecific binding tube containing the bead and 5 ~l of a 5 mg/ml solution of the unlabeled MAb, as a competitor of the labeled MAb, were used. Five IxL of decreasing concentrations (1:2 to 1:200) of the same unlabeled MAb were added to other tubes. To all tubes were added 100 ix1 of the labeled MAb (12,000-20,000 cpm, approximately l0 mg/ml), which were vortexed at room temperature for 1 h. All tubes, excluding the counting standard, were washed with DPBS, and the beads were transferred into clean test tubes and counted in a gamma counter. The percentage of radioactivity bound to the mucin coated beads was calculated. The inverse of the percentage of binding values was plotted against the cold MAb concentrations. Extrapolation to infinite MAb concentration determined the percentage of immunoreactivity of the labeled MAbs. Animal Model . Athymic nude female nu/nu mice with a BALB/c background, were obtained from the National Cancer Institute-Frederick Cancer Research & Development Center (Frederick, MD) and were kept under sterile conditions. Procedures to minimize discomfort, pain, and distress were in accord with the Animal Resource Program at the University of Alabama at Birmingham, accredited by the American Association for Accreditation of Laboratory Animal Care. The LS174T human colon cancer cells (American Type Culture Collection, Manassas, VA) were harvested and suspended in sterile PBS at a concentration of 7.5 • 108 viable cells/ml. Cell viability was determined by trypan blue dye exclusion. Viable cells (10 s) in sterile PBS were injected i.p. into the mice. Biodistribution of 188Re-MAb Conjugates. Groups of mice with established 7-day i.p. tumors received 5 IxCi of the ss Re-labeled antibodies as an i.p. bolus injection. At 24 h Research. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Clinical Cancer Research 2997s Fig. 1 Biodistribution of lSSRe-HuCC49 (n = 4) and lS8Re-HuCC49ACH2 (n = 5) conjugates at 24 h after i.p. injection in athymic nude mice bearing LS174T i.p. tumors. Columns, median; bars, interquartile range. TU, tumor; *BL, blood; KI, kidney; LI, liver; SP, spleen; HT, heart; LU, tung; ST, stomach; SI, small intestine; SK, skin; *BO, bone; MU, muscle; *AL, abdominal lining; UT, uterus; PA, pancreas. *, significant by the Wilcoxon rank-sum test at P < 0.05. [~] 188Re-HuCC49