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Evaluation of Ricin A Chain-containing Immunotoxins Directed against CD19 and CD22 Antigens on Normal and Malignant Human B-Cells as Potential Reagents for in Vivo Therapy¹

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 [M-A. G., R. D. M., M. T., J. W. U., V. G., E. S. V.]; Drug Targeting Laboratory, Imperial Cancer Research Fund Laboratories, P. O. No. 123, London WC-2A 3PX, United Kingdom [P. P. K., M. R., A. B., P. M. W., P. E. T.]; and Department of Immunology, Royal Free Hospital, Hampstead, London, United Kingdom [G. J., P. A.]

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')₂, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 x 10^-11 M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 x 10^-10 M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

¹ Supported in part by NIH Grants CA-218149 and CA-41081 and a grant from the Welch Foundation, I-0947.

² To whom requests for reprints should be addressed, at Department of Microbiology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235.

Received 9/21/87. Revised 12/23/87. Accepted 2/ 3/88.

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