Abstract
A new method has been devised to eliminate T cells from murine bone marrow grafts across major histocompatibility barriers and thus prevent graft-vs.-host disease (GVHD). The method utilizes a monoclonal antibody directed at the Thy-1.2 antigen but is complement independent. To make anti-Thy-1.2 toxic, the antibody is covalently linked to the toxin ricin. Ricin ordinarily binds, enters, and kills cells through receptors containing galactose. The hybrid protein, anti-Thy-1.2-ricin, can enter and kill cells via the Thy-1.2 receptor. In the presence of lactose the usual entry route for ricin is largely blocked and the hybrid is shown to be a highly selective reagent that is T cell specific in its inhibition of mitogen-stimulated splenocytes. We have used a model of severe and fatal GVHD where BALB/c splenocytes and bone marrow cells are given to irradiated C57BL/6 recipients. Over 90% of these mice die by day 70, exhibiting signs of GVHD. When donor cells are pretreated with 0.5 microgram/ml of anti-Thy-1.2-ricin plus 200 mM lactose before injection, 10 of 11 animals survive through day 70 without signs of GVHD. These studies demonstrate that ricin linked to monoclonal antibodies may have utility related to the prevention of GVHD in human bone marrow transplantation.
Highlights
A new method has been devised to eliminate T cells from murine bone marrow grafts across major histocompatibility barriers and prevent graft-vs.-host disease (GVHD)
In the presence of lactose the usual entry route for riein is largely blocked and the hybrid is shown to be a highly selective reagent that is T cell specific in its inhibition of mitogen-stimulated splenocytes
Summary
A description of synthesis of the anti-Thy-l.2-ricin molecule has been reported [5]. Ricin is reacted with m-maleimidobenzoyl-N-hydroxysuecinimide ester to link maleimide residues to ricin. A standardized monoelonal IgG against Thy-l.2 (clone 30-H12 derived by Ledbetter and Herzenberg [6]) was prepared for cross-linking by partial reduction of disulfide bonds. The maleimide-linked ricin was mixed with the sulfhydryl containing antibody, forming a nonreducible thioether linkage between the two species. C57BL/6 and BALB/c strains used in these experiments were bred and housed at the Minnesota Mouse Colony, University of Minnesota. Adult males (26-31 g) were housed in conventional cages with filter lids. Transplanted animals were fed a fat-supplemented diet and antibiotic supplemented water ad libitum
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