title: 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-alphaGal antibody.
abstract: Anti-Galalpha1-3Gal antibodies (antialphaGal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alphaGal oligosaccharide columns temporarily depletes antialphaGal 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 antialphaGal 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 mAb (proportional to CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and antialphaGal 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 (LN), staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and antialphaGal 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 antialphaGal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in antialphaGal 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 antialphaGal 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 antialphaGal 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 antialphaGal 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 antialphaGal 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 antialphaGal 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 antialphaGal Ab. The majority of antibody secretors are CD38-positive cells, but targeting these cells in vitro or in vivo with proportional to CD38-IT was not very effective. These observations suggest that CD20-and CD22-positive B cells are not the major source of antialphaGal Ab production. Future efforts will be directed towards suppression of plasma cell function.
authors.affiliation: Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, BioTransplant, Inc., Boston 02129, USA.
authors.forename: I P
authors.initials: IP
authors.lastname: Alwayn
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: M
authors.initials: M
authors.lastname: Basker
authors.forename: C
authors.initials: C
authors.lastname: Wu
authors.forename: L
authors.initials: L
authors.lastname: Buhler
authors.forename: D
authors.initials: D
authors.lastname: Lambrigts
authors.forename: S
authors.initials: S
authors.lastname: Treter
authors.forename: D
authors.initials: D
authors.lastname: Harper
authors.forename: H
authors.initials: H
authors.lastname: Kitamura
authors.forename: E S
authors.initials: ES
authors.lastname: Vitetta
authors.forename: S
authors.initials: S
authors.lastname: Abraham
authors.forename: M
authors.initials: M
authors.lastname: Awwad
authors.forename: M E
authors.initials: ME
authors.lastname: White-Scharf
authors.forename: D H
authors.initials: DH
authors.lastname: Sachs
authors.forename: A
authors.initials: A
authors.lastname: Thall
authors.forename: D K
authors.initials: DK
authors.lastname: Cooper
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000917:Antibody Formation; D015703:Antigens, CD / Q000276:immunology*; D018951:Antigens, CD20 / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D015815:Cell Adhesion Molecules; D004187:Disaccharides / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control; D006801:Humans; D007167:Immunotherapy; D037102:Lectins; D008212:Lymphocyte Depletion; D051379:Mice; D010215:Papio; D010950:Plasma Cells / Q000276:immunology*; D012276:Ricin / Q000276:immunology; D051918:Sialic Acid Binding Ig-like Lectin 2; D014183:Transplantation, Heterologous / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; C497868:Cd22 protein, mouse; D015815:Cell Adhesion Molecules; D004187:Disaccharides; D000939:Epitopes; D037102:Lectins; D051918:Sialic Acid Binding Ig-like Lectin 2; C055075:galactosyl-(1-3)galactose; D012276:Ricin