Elimination of anti-Gal B cells by ??-Gal ricin1

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Abstract
Background. A major barrier in pig to human organ transplantation is the binding of human anti-Gal to α-gal epitopes (Galα1-3Galβ1-4GlcNAc-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 α1-acid glycoprotein modified to carry α-gal epitopes. This complex, designated α-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. Methods. Carbohydrate chains on α1-acid glycoprotein were converted to carry α-gal epitopes by enzymatic treatment with recombinant α1,3 galactosyltransferase (α1,3GT). This molecule and ricin A were biotinylated and coupled by avidin to generate α-gal ricin. The efficacy of α-gal ricin in eliminating anti-Gal B cells was studied in the experimental model of α1,3GT knockout (KO) mice. These mice produce large amounts of anti-Gal immunoglobulin G when immunized with pig kidney membranes, as measured by ELISA with α-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. Results. Repeated administration of α-gal ricin into α1,3GT 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 α-gal ricin that were not toxic to the mice and did not affect production of antibodies with other specificities. Conclusions. Administration of α-gal ricin results in specific elimination of anti-Gal B cells in α1,3GT KO mice. The elimination of these B cells may prove to be helpful in attempts to achieve immune tolerance to α-gal epitopes in primates.