An engineered bifunctional recombinant molecule that regulates humoral and cellular effector functions of the immune system.
BACKGROUND
Humoral and cellular defense mechanisms mediate the rejection of transplanted cells, tissues, and organs after allogeneic or xenogeneic transplantation. Inhibition of complement and T-cell costimulation are strategies aimed at increasing transplant survival.


METHODS
Engineered novel fusion proteins that contain the functional domains of human CD152 (hCTLA4) or porcine CD152 (pCD152) and human CD59 (hCD152-hCD59, pCD152-hCD59) were developed to form bifunctional chimeric proteins that retain the effector functions of both moieties. Porcine aortic endothelial cells and murine Balb/3T3 cells were transduced or transfected to express the novel fusion proteins.


RESULTS
Fluorescence-activated cell sorter analysis of hCD152-hCD59 transduced primary porcine aortic endothelial cells or hCD152-hCD59 and pCD152-hCD59 transfected Balb/3T3 cells determined that the molecules were expressed on the cell surface, and that they retained conformational epitopes. We demonstrate that hCD152-hCD59 and pCD152-hCD59 chimeric proteins inhibit complement-mediated cell lysis. In addition, hCD152-hCD59 or pCD152-hCD59 expression resulted in a significant reduction in T-cell activation as the result of CD152 engagement of porcine CD86 or murine CD80 in when Jurkat cells were cocultured with the hCD152-hCD59 or pCD152-hCD59 expressing cells. Antibody-blocking experiments or phosphatidylinositol phospholipase C removal of the glycosyl-phosphatidylinositol-linked molecules resulted in increased serum-mediated cytolysis and eliminated the costimulatory blockade.


CONCLUSIONS
These data illustrate that a single molecule can confer resistance to humoral and cellular immune attack.
Pizzolato
Maryellen C
MC
Alexion Pharmaceuticals, Inc., Cheshire, CT, USA.
Fodor
William L
WL
D016475:3T3 Cells; D000069594:Abatacept; D000818:Animals; D000917:Antibody Formation / Q000235:genetics* / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000943:Antigens, Differentiation / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018122:B7-1 Antigen / Q000276:immunology / Q000378:metabolism; D051940:B7-2 Antigen; D018957:CD59 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D060908:CTLA-4 Antigen; D003165:Complement System Proteins / Q000276:immunology; D005816:Genetic Complementation Test; D006801:Humans; D007111:Immunity, Cellular / Q000235:genetics* / Q000276:immunology; D018796:Immunoconjugates; D007376:Interleukin-2 / Q000276:immunology; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013552:Swine; D013601:T-Lymphocytes / Q000276:immunology; D014181:Transplantation Immunology
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D051940:B7-2 Antigen; D018957:CD59 Antigens; C497964:CD86 protein, human; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C497965:Cd86 protein, mouse; C556707:Ctla4 protein, mouse; D018796:Immunoconjugates; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000069594:Abatacept; D003165:Complement System Proteins