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Analysis of gp74 Expression by Transformed Rat Fibroblasts from Experimental Pulmonary Metastases following Specific Ricin A-Chain Immunotoxin Therapy¹

Departments of Thoracic Surgery [J. A. R., K. D. F.], Tumor Biology [J. A. R.], General Surgery [S. T.], Pathology [S. T.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and Xoma Corporation [P. S., C. C.], Berkeley, California 94710

Ricin A chain immunotoxin (IT) 45-2D9-RTA mediates regression of spontaneous pulmonary metastases and lung colonies from K-ras trans-formed rat fibroblasts (TRF cells). However, residual metastases are frequently noted after IT therapy, and therefore, possible mechanisms mediating tumor cell escape were investigated. Individual lung colonies were dissected from lungs of BALB/c mice, and single-cell suspensions of fresh cells from short-term cultures (eight passages) were tested. Immunoperoxidase staining with 45-2D9 monoclonal antibody showed that stable loss of surface antigen by cells cultured from IT-treated mice did not occur after four injections of specific IT. Sensitivity to specific IT in vitro was equal for metastatic tumor cells from mice treated with either two or four doses of specific IT compared to cells from nonspecific IT-treated mice and to parental cells. Clones derived from metastases of IT-treated mice were not resistant to IT. Clones derived from metastases of specific IT-treated mice internalized bound antibody or IT at the same rate as untreated cells. Freshly disaggregated cells from specific IT-treated mice were as sensitive to specific IT as were cells from nonspecific IT-treated or untreated mice. Specific IT successfully mediated reduction of lung colonies derived from fresh suspensions of lung colony TRF cells from IT-treated mice. This reduction was equivalent to that seen for cells not previously exposed to specific IT. Immunoperoxidase stains of lung sections with 45-2D9 showed that colonies consisting entirely of unstained TRF cells were present in both specific IT and phosphate buffered salinetreated mice. There was a trend toward a higher percentage of antigennegative colonies in mice treated with IT, although 9 days following specific IT therapy, greater than 80% of lung colonies expressed gp74 antigen. When TRF cells were grown on agar plugs, which promoted three-dimensional growth, groups of cells showing absence of immunoperoxidase staining with antibody to gp74 were identified during 2 weeks of growth. Thus, stability of antigen-negative variants is favored by three-dimensional growth conditions and the selective pressure of IT administration. Our results also suggest that impaired trafficking of IT to antigenpositive cells may also contribute to escape from IT therapy.

¹ This work was supported by NIH Grant Ca45187 from the National Cancer Institute, USPHS, Department of Health and Human Services.

² To whom requests for reprints should be addressed, at Department of Thoracic Surgery, P. O. Box 109, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030.

Received 2/ 2/89. Revised 4/ 5/89. Revised 7/ 6/89. Accepted 7/20/89.