Pretreatment of CD138− B Lymphocytes with a Covalent Reactive Antigen Analog to Factor VIII Reduces Antibody Production in response to FVIII (88.14)

Abstract

Abstract B lymphocytes producing high titer antibodies to factor VIII (FVIII) are of major concern in the treatment of bleeding episodes in a subpopulation of patients with hemophilia A. We hypothesize that an electrophilic, covalent reactive analog of FVIII (FVIII-CRA) can induce antigen-specific tolerance by permanent engagement of B cell receptors. FVIII-CRA was prepared by derivitizing lysine side chains with phosphonate diester groups. These groups are predicted to bind enzyme-like nucleophilic residues located within the BCR. To test the effects of FVIII-CRA binding on B lymphocytes, we isolated CD138− splenocytes from FVIII-deficient mice (B6;129S4-F8tm1Kaz/J) immunized with FVIII. These putative memory B lymphocytes were then re-stimulated for 6 days with identical doses of FVIII or FVIII-CRA. The number of antibody secreting cells (ASC) produced in response to FVIII-CRA re-stimulation was significantly lower than FVIII re-stimulation (avg of 36 ASC vs 111 ASC, p = 0.03). In addition, cell viability following low dose FVIII-CRA treatment was decreased by ~ 22% when compared with identical doses of FVIII. These results suggested that FVIII-CRA may tolerize memory B lymphocytes. To confirm this, we pretreated CD138− splenocytes with FVIII-CRA and then added a stimulatory dose of FVIII to the cultures. Pretreatment with FVIII-CRA resulted in a significant decrease in the number of ASC produced in response to FVIII (by 17-fold compared to control pretreatment). These results suggest that FVIII-CRA may represent a viable means to induce antigen-specific B cell tolerance in hemophilia.