Elimination of anti-Gal B cells by alpha-Gal ricin1.

Abstract

A major barrier in pig to human organ transplantation is the binding of human anti-Gal to alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on pig cells, resulting in hyperacute and acute vascular rejection of pig xenografts. Moreover, the immune system in xenograft recipients is activated by these epitopes to produce high affinity anti-Gal, which is also detrimental to xenografts. Production of anti-Gal can be prevented by specific elimination of anti-Gal B cells. This was achieved with the toxin ricin A, coupled to human alpha1-acid glycoprotein modified to carry alpha-gal epitopes. This complex, designated alpha-gal ricin, is targeted in vivo to anti-Gal B cells by interaction with the immunoglobulin molecules (i.e., B cell receptors) on these cells. Carbohydrate chains on alpha 1-acid glycoprotein were converted to carry alpha-gal epitopes by enzymatic treatment with recombinant alpha 1,3 galactosyltransferase (alpha 1,3GT). This molecule and ricin A were biotinylated and coupled by avidin to generate alpha-gal ricin. The efficacy of alpha-gal ricin in eliminating anti-Gal B cells was studied in the experimental model of alpha 1,3GT knockout (KO) mice. These mice produce large amounts of anti-Gal immunoglobulin G when immunized with pig kidney membranes, as measured by ELISA with alpha-gal epitopes linked to bovine serum albumin (BSA). In the absence of anti-Gal B cells, these mice lack the ability to produce anti-Gal. Repeated administration of alpha-gal ricin into alpha1,3GT KO mice resulted in elimination of anti-Gal B cells, thereby preventing production of anti-Gal immunoglobulin G after immunization with pig kidney membranes. This prevention of anti-Gal production occurred with doses of alpha-gal ricin that were not toxic to the mice and did not affect production of antibodies with other specificities. Administration of alpha-gal ricin results in specific elimination of anti-Gal B cells in alpha 1,3GT KO mice. The elimination of these B cells may prove to be helpful in attempts to achieve immune tolerance to alpha-gal epitopes in primates.