PREPARATION AND PROPERTIES OF THE IMMUNOCONJUGATE COMPOSED OF ANTI-HUMAN COLON CANCER MONOCLONAL-ANTIBODY AND MITOMYCIN C-DEXTRAN CONJUGATE

Monoclonal antibody (mAb) A7, produced against human colon cancer, was conjugated with a polymeric prodrug of mitomycin C (MMC), the MMC-dextran conjugate with an anionic charge (MMCD(an)) and a molecular weight of 70 000. The amino groups were introduced into the MMCD(an) by reacting ethylenediamine with the carboxyl group in the spacer arm of the dextran bridge by a carbodiimide-catalyzed reaction. The coupling to mAb A7 was performed using SPDP. A 15 M excess of ethylenediamine produced an optimal MMCD(an) with amino groups, which resulted in a homogenous conjugate (A7-MMCD) with minimal formation of high-molecular-weight aggregates in about a 30% yield of both IgG and MMC. The molar binding ratio of IgG:dextran:MMC in A7-MMCD was estimated to be 1:1.2:40. A7-MMCD, having MMC prodrug properties, released active MMC with a half-life of 29.1 h and had an almost neutral electric charge under physiological conditions. A competitive binding assay using I-125-labeled A7 revealed that the A7-MMCD almost fully retained its antibody-binding activity. The cytotoxicity of A7-MMCD was assayed by determining the degree of inhibition of [H-3]-thymidine in corporation in two different ways using the human colon cancer cell line SW1116. A 48-h continuous exposure test revealed that the pharmacological activity of MMC in A7-MMCD was completely preserved. In addition, A7-MMCD exhibited about a 14-fold greater cytotoxicity than MMCD(an) when the IC50 values determined using a 2-h pretreatment exposure system were compared. These results suggest that A7-MMCD could be useful in immunotargeting chemotherapy for colorectal cancer.