Synthesis and evaluation of antibody-albumin-methotrexate ternary conjugates for cancer treatment.
Date
1994
Authors
Kondejewski, Leslie Henry.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
The anticancer drug methotrexate (MTX) was conjugated to the monoclonal antibody (mAb) K20 directed against a cell surface antigen on the human kidney cancer cell line, Caki-1, for the purpose of obtaining effective targeting agents. Linkage of MTX to mAb K20 was through the use of human serum albumin (HSA) as an intermediary, in which HSA containing between 28 and 35 mol MTX/mol HSA (HSA-MTX) was linked to the mAb. The hypothesis was that carrier-based conjugates would be more effective targeting agents than direct mAb K20-MTX conjugates on the basis of increased drug load and greater retention of antigen binding activity of the mAb. The conjugation of HSA-MTX to mAb K20 was carried out using a site-specific method in which HSA-MTX was linked to the carbohydrate moiety of the mAb. It was predicted that site-specific conjugates would retain greater antibody activity than non-site-specific conjugates in which HSA-MTX was linked to amino groups of the mAb since the carbohydrate moiety is located at a distance from the antigen binding site.
Two site-specific heterobifunctional spacers were synthesized to allow conjugation to the carbohydrate moiety of mAb K20. These were 3-(2-pyridyldithio)propionic acid hydrazide (HPDP) and 11- (3-(2-pyridyldithio)propionyl) aminoundecanoic acid hydrazide (AUPDP). Use of the longer AUPDP gave higher yields of ternary mAb K20-HSA-MTX conjugates, suggesting that steric factors limited conjugation of HSA to the carbohydrate of mAb K20 when using HPDP. Hydrazone-linked conjugates prepared using AUPDP were unstable and could not be isolated without prior stabilization by cyanoborohydride. Following stabilization, 1:1 site-specific mAb K20:HSA-MTX conjugates were isolated. Non-site-specific ternary conjugates were also synthesized using 3-(2-pyridyldithio)propionic acid succinimidyl ester as the cross-linker. The non-site-specific conjugates retained only one third the antibody activity of the corresponding site-specific conjugates. The site-specific mAb K20-HSA-MTX conjugate was more cytotoxic to target Caki-1 cells than either the non-site-specific mAb K20-HSA-MTX conjugates, or direct conjugates in which MTX was linked directly to mAb K20 amino groups. The site-specific conjugate also displayed the greatest selectivity for the target cell line Caki-1, compared to D10-1, a non-target cell line. It is likely that the greater cytotoxicity to Caki-1 cells as well as the increased selectivity was the result of the increased retention of antigen binding activity in the site-specific conjugate.
Thesis (Ph.D.)--Dalhousie University (Canada), 1994.
Two site-specific heterobifunctional spacers were synthesized to allow conjugation to the carbohydrate moiety of mAb K20. These were 3-(2-pyridyldithio)propionic acid hydrazide (HPDP) and 11- (3-(2-pyridyldithio)propionyl) aminoundecanoic acid hydrazide (AUPDP). Use of the longer AUPDP gave higher yields of ternary mAb K20-HSA-MTX conjugates, suggesting that steric factors limited conjugation of HSA to the carbohydrate of mAb K20 when using HPDP. Hydrazone-linked conjugates prepared using AUPDP were unstable and could not be isolated without prior stabilization by cyanoborohydride. Following stabilization, 1:1 site-specific mAb K20:HSA-MTX conjugates were isolated. Non-site-specific ternary conjugates were also synthesized using 3-(2-pyridyldithio)propionic acid succinimidyl ester as the cross-linker. The non-site-specific conjugates retained only one third the antibody activity of the corresponding site-specific conjugates. The site-specific mAb K20-HSA-MTX conjugate was more cytotoxic to target Caki-1 cells than either the non-site-specific mAb K20-HSA-MTX conjugates, or direct conjugates in which MTX was linked directly to mAb K20 amino groups. The site-specific conjugate also displayed the greatest selectivity for the target cell line Caki-1, compared to D10-1, a non-target cell line. It is likely that the greater cytotoxicity to Caki-1 cells as well as the increased selectivity was the result of the increased retention of antigen binding activity in the site-specific conjugate.
Thesis (Ph.D.)--Dalhousie University (Canada), 1994.
Keywords
Chemistry, Biochemistry., Health Sciences, Oncology.