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Monoclonal antibody (MAb) CC49 reacts with tumor-associated glycoprotein (TAG)-72, a human pancarcinoma antigen. In clinical trials, radiolabeled CC49 has shown excellent tumor localization; however, many of the patients receiving MAb CC49 develop a human antimouse antibody response. In an attempt to prevent this antiimmunoglobulin response, we have developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto the variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28' CL, respectively, while retaining those murine framework residues that may be required for the integrity of the antigen combining-site structure. The HuCC49 MAb was compared with native murine CC49 (nCC49) and chimeric CC49 (cCC49), using a variety of assays. SDS-PAGE analysis under nonreducing conditions showed that the HuCC49 MAb has virtually identical mobility to that of cCC49. Under reducing conditions, the HuCC49 yielded two bands of approximately 25-28 and approximately 50-55 kDa, characteristic of heavy and light immunoglobulin chains. In competition radioimmunoassays, HuCC49 completely inhibited the binding of 125I-labeled nCC49 to TAG-72, although 23- to 30-fold more HuCC49 was required to achieve a level of competition similar to those of cCC49 and nCC49. The relative affinity of HuCC49 was 2- to 3-fold less than those of the cCC49 and nCC49 MAbs, respectively. The plasma clearance in mice of HuCC49 was virtually identical to that of cCC49. Biodistribution studies demonstrated equivalent tumor-targeting of HuCC49 and cCC49 to human colon carcinoma xenografts. These studies thus suggest that HuCC49 and genetically modified molecules, such as sFv and domain-deleted immunoglobulins developed by using the HuCC49 variable region as a cassette, may be potentially useful in both diagnostic and therapeutic clinical trials in patients with TAG-72-positive tumors.

Eight different human breast carcinoma cell lines, as well as clonally derived cell lines, were used to study the expression of four different monoclonal antibody (MAb)-defined tumor-associated antigens (TAAs) and the ability of recombinant human (rHu)-interferon (IFN)-alpha A and rHu-IFN-gamma to increase tumor-associated and/or normal cell-surface antigen expression. The different breast tumor cell lines expressed a wide range of antigenic phenotypes with respect to four different cell-surface TAAs. The cell lines could be divided into high and low antigen-expressing groups based on their constitutive levels of the four TAAs. In general, those breast tumor cell lines that expressed high levels of carcinoembryonic antigen, the MAb DF-3-defined 290-kD antigen, and the 90-kD antigen reactive with MAb B6.2 were poor candidates for antigen augmentation by rHu-IFN-alpha A or rHu-IFN-gamma. In contrast, breast cell lines that constitutively express low levels of these TAAs were found to be highly responsive to the ability of either of the rHu-IFNs to enhance MAb binding to the cell surface. Treatment with either of the rHu-IFNs increased the level of surface binding of MAbs as much as fourfold. The relative abilities of the IFNs to increase MAb binding to the surface of human breast tumor cells thus appeared to depend on the degree of constitutive surface antigen expression of the breast tumor cells. The high-molecular-weight TAA, TAG-72, is known not to be expressed on most cell lines but is expressed on the majority of human carcinoma biopsies. Most breast tumor cell lines employed in this study did not express the TAG-72 high-molecular-weight TAA. However, rHu-IFN-alpha A did substantially increase the level of expression of TAG-72 on the surface of breast tumor cells that were isolated from a pleural effusion. These studies may thus provide an important insight into the criteria used in the selection of carcinoma patients for IFN-mediated antigen augmentation when employing MAb-guided radioimmunolocalization or MAb-guided therapy.