Effects of specific anti-B and/or anti-plasma cell immunotherapy on antibody production in baboons:: depletion of CD20-and CD22-positive B cells does not result in significantly decreased production of anti-αGal antibody

Anti-Gal alpha1-3Gal antibodies (anti alpha Gal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alpha Gal oligosaccharide columns temporarily depletes anti-alpha Gal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of anti alpha Gal Ab following EIA. The effects of the mouse anti-human IT anti-CD22-ricin A (proportional to CD22-IT, directed against a B cell determinant) and anti-CD38-ricin A (proportional to CD38-IT, B and plasma cell determinant) and the mouse anti-human anti-CD38 mAb (proportional to CD38 mAb) and mouse/human chimeric anti-human anti-CD20 (alpha CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and anti alpha Gal Ab production were assessed both in vitro and in vivo in baboons (n=9) that had previously undergone splenectomy. For comparison, two baboons received nonmyeloablative whole body irradiation (WBI) (300 cGy), and one received myeloablative WBI (900 cGy). Depletion of B cells was monitored by flow cytometry of blood, bone marrow (BM) and lymph nodes (LNG, staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and anti alpha Gal Ab levels were measured daily. In vitro proportional to CD22-IT inhibited protein synthesis in the human Daudi B cell line more effectively than proportional to CD38-IT. Upon differentiation of B cells into plasma cells, however, less inhibition of protein synthesis after proportional to CD22-IT treatment was observed. Depleting CD20-positive cells in vitro from a baboon spleen cell population already depleted of granulocytes, monocytes, and T cells led to a relative enrichment of CD20-negative cells, that is plasma cells, and consequently resulted in a significant increase in anti alpha Gal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in anti alpha Gal Ab production. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood and BM,> 80% depletion in LN, with substantial recovery of B cells after 21 days and only transient reduction in anti alpha Gal Ab after EIA. proportional to CD22-IT depleted B cells by > 97% in blood and BM, and by 60% in LN, but a rebound of B cells was observed after 14 and 62 days in LN and blood, respectively. At 7 days, serum anti alpha Gal IgG and IgM Ab levels were reduced by a maximum of 40-45% followed by a rebound to levels up to 12-fold that of baseline anti alpha Gal Ab by day 83 in one baboon. The results obtained with proportional to CD38-IT were inconclusive. This may have been, in part, due to inadequate conjugation of the toxin. Cell coating was 100% with proportional to CD38 mAb, but no changes in anti alpha Gal Ab production were observed. proportional to CD20 mAb resulted in 100% depletion of B cells in blood and BM and 80% in LN, with recovery of B cells starting at day 42. Adding 150cGy WBI at this time led to 100% depletion of B cells in the BM and LN. Although B cell depletion in blood and BM persisted for >3 months, the reduction of serum anti alpha Gal IgG or IgM Ab levels was not sustained beyond 2 days. proportional to CD20 mAb+WBI totally and efficiently depleted CD20- and CD22-positive B cells in blood, BM, and LN for >3 months in vivo, but there was no sustained clinically significant reduction in serum anti alpha Gal Ab. The majority of antibody secretors are CD38-positive cells, but targeting these cells in vitro or in vivo with alpha CD38-IT was not very effective. These observations suggest that CD20-and CD22-positive B cells are not the major source of anti alpha Gal Ab production. Future efforts will be directed towards suppression of plasma cell function.