title: Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation.
abstract: BACKGROUND
  It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function.
  
  
  METHODS
  The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb.
  
  
  RESULTS
  One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells.
  
  
  CONCLUSIONS
  These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.
authors.affiliation: Transplantation Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. sgraves@fhcrc.org
authors.forename: Scott S
authors.initials: SS
authors.lastname: Graves
authors.forename: Diane M
authors.initials: DM
authors.lastname: Stone
authors.forename: Carol
authors.initials: C
authors.lastname: Loretz
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Peterson
authors.forename: Marina
authors.initials: M
authors.lastname: Lesnikova
authors.forename: Billanna
authors.initials: B
authors.lastname: Hwang
authors.forename: George E
authors.initials: GE
authors.lastname: Georges
authors.forename: Richard
authors.initials: R
authors.lastname: Nash
authors.forename: Rainer
authors.initials: R
authors.lastname: Storb
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000494:pharmacology*; D015703:Antigens, CD / Q000276:immunology; D001666:Binding Sites, Antibody; D018106:CD28 Antigens / Q000276:immunology*; D060908:CTLA-4 Antigen; D049109:Cell Proliferation / Q000187:drug effects; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control*; D006825:Hybridomas; D008213:Lymphocyte Activation / Q000187:drug effects*; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D016377:Organ Transplantation / Q000009:adverse effects*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology; D013997:Time Factors; D014184:Transplantation, Homologous
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; C556707:Ctla4 protein, mouse; D011993:Recombinant Fusion Proteins