As before, both MORF and its complement cMORF were obtained from GeneTools (Philomath, OR) with a primary amine on the 3′-equivalent end. The base sequences were 5′-TCTTCTACTTCACAACTA-linker-amine (MORF) and 5′-TAGTTGTGAAGTAGAAGA-linker-amine (cMORF). The murine anti-CEA antibody MN14 was a gift from Immunomedics (Morris Plains, NJ). The murine antiTAG-72 antibody CC49 was prepared by Strategic Biosolutions (Ramona, CA) from the CC49 murine hybridoma cell line, a gift from Dr Jeff Schlom (Laboratory of Tumor Immunology and Biology, Center for Cancer Research, NCI, NIH, Bethesda, MD). The p-SCN-Benzyl-DTPA was from Macrocyclics (Dallas, TX). The Hydralink kit for the conjugation of MORF to antibody was from Solulink (San Diego, California). The Sephadex G100 resin was from Pharmacia Biotech (Uppsala, Sweden). The 99Mo-99mTc generator and the 111InCl3 were from Perkin Elmer Life Science Inc (Boston, MA). All other chemicals were reagent grade and used without purification.
Preparation of 99mTc-cMORF, MORF-antibodies, and 111In-antibodies
In labeling, the CC49 and MN14 were first conjugated with the bifunctional agent p-SCN-Benzyl-DTPA and, after purification, mixed with 111
in an acetate buffer (21
The cMORF was conjugated with MAG3
as previously described (22
) and stored at −20 °C. For labeling, a vial was returned to room temperature, 10 μL of 99m
Tc generator eluate was added to a combined solution of 10 μL (0.3 μg/μL) MAG3
-cMORF, 4 μL (50 μg/μL) sodium tartrate 2H2
O in a pH 9.2 buffer (0.5 M Na2
, 0.25 M NH4
OAc, 0.175 M NH3
), and 1.5 μL (4 μg/μL) SnCl2
O in 10 mM HCl-sodium ascorbate. After heating at boiling water temperature for 20 min, the labeling efficiency by size-exclusion HPLC was over 95%.
The antibody was conjugated with MORF using a commercial Hydralink approach (23
) in which both CC49 and MN14 were conjugated with succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) and the MORF was conjugated with succinimidyl 4-formylbenzoate (SFB). After purification, the hydrazine-modified antibody and the benzaldehyde-modified MORF were combined to form a hydrazone link between the antibody and MORF. Purification of the MORF-antibodies from the free MORF was achieved on a 1.0×50 cm Sephadex G-100 glass Econo-column and the average number of MORFs per antibody (gpm) was determined as described previously (3
Biodistributions of 111In labeled antibodies
All animal studies were approved by the UMMS Institutional Animal Care and Use Committee. For tumor implantation, 106 LS174T cells were injected into the left thigh of each Swiss NIH nude mouse (Taconic Farms, Germantown, NY). The animals were used 12–13 days later when the tumors were about 0.5 g. Four tumored mice each received intravenously 20 μg (20–30 μCi) of 111In-labeled DTPA-benzyl-CC49 or DTPA-benzyl-MN14. After 48 h, the mice were killed by exsanguination via heart puncture under halothane anesthesia. At necropsy, samples of blood and other organs were removed, weighed, and counted in a NaI(Tl) well counter (Cobra II automatic gamma counter, Packard Instrument Company, CT) along with a standard of the injectate. Blood and muscle were assumed to constitute 7 % and 40 % of body weight respectively. Since the LS174T tumor is difficult to separate from normal tissues, the entire tumor thigh was first excised and as much skin, muscle and bone as possible were removed before counting. Since the radioactivity levels in the normal tissues were negligible, the counts within the tumor samples were attributed to the tumor. After counting, the residual skin, muscle and bone were surgically removed for each tumor sample weighed and the tumor weight determined by subtraction.
Internalization of MORF-conjugated antibodies
The selection of the CC49 antibody for this investigation was partially based on reports that antiTAG-72 antibodies are unlikely to internalize (8
). After conjugation with MORF, the internalization property of the CC49 and the MN14 antibodies was examined in LS174T cells using a method described previously (24
Specifically, about 0.5 ×106
LS174T cells were seeded in each well of 7 twelve-well plates and were used after 1.5–2 days when the cells reached a confluency of greater than 75%. As described previously (24
), 150 μL (about 2 μCi) of the 99m
Tc-cMORF/MORF-antibody complex and 1 mL of PBS/0.5%BSA at 37 °C was added to each well in the direct-targeting group, while 150 μL of MORF-antibody and 1 mL of PBS/0.5%BSA at 37 °C was added to each well of the pretargeting group. After a designated time of incubation at 37 °C, the same amount of 99m
Tc-cMORF in 10 μL was added to each well of the pretargeting group. Five min later, the cell-associated radioactivity and the radioactivity in the media for both groups were separated and counted. Samples were measured in triplicate for each time-point of each group. The cell-associated radioactivity in the direct-targeting group measured the total antibody accumulation (both internalized and on the surface), while the cell-associated radioactivity in the pretargeting group measured only the antibody on the surface.
Determination of the accessible level of MORF in tumor
Seventeen tumored mice each received intravenously 30 μg of MORF-CC49 (gpm = 0.68) on the 11th day and a dosage of 99mTc-cMORF varying between 1 and 6 μg on the 13th day. The mice were killed for biodistribution 3 h thereafter. At this time, the tumors weights averaged 0.36 ± 0.10 g.
As detailed in Appendix
, the above measurements were used to estimate the optimum dosage of cMORF effector, the dosage that just saturates the accessible MORF in tumor. A timing schedule identical to that used previously with MN14 could again be used since the CC49 is also an IgG and therefore with similar pharmacokinetics and the since the same cMORF effector was to be used. A single mouse weighing 27 g with a 0.85 g LS174T tumor was administered 53.9 μg MORF-CC49 (gpm = 1.12) 48 h before the injection of 6.3 μg (3.8 mCi) of 99m
Tc-cMORF and was imaged 3 h later.
Images were obtained on a pinhole HiSPECT system (Bioscan, Washington DC) installed on a three-head PRISM-3000 (25
). The mouse was anesthetized by intraperitoneal injection of ketamine-xylazine and placed on a flat Lucite extension clamped to the headrest of the gamma camera bed. The mouse was in the axis of a circular orbit formed by the rotation of three six-pinhole apertures with a radius of 3.7 cm. The radius choice was dictated by our experience in positioning the mouse for convenient data requisition. The acquisition process required 20 min for 36 intervals through 360 degrees resulting in 30 projections. The projection data were reconstructed using commercial software also from Bioscan.