Establishment of long-term tolerance to SRBC in dogs by recombinant canine CTLA4-Ig.
BACKGROUND
Blockade of the CD28 costimulatory molecule by recombinant human cytotoxic T lymphocyte (CTL)-associated antigen (CTLA4)-Ig or CD40-CD154 interaction with the monoclonal antibody 5C8 together with donor-specific transfusion led to enhanced engraftment in the canine model of dog leukocyte antigen (DLA)-identical marrow transplantation after 1 Gy total body irradiation. To reduce or eliminate total body irradiation conditioning regimens, we have sought to develop canine specific reagents.


METHODS
We have created a fusion protein of the extracellular domain of canine (c) CTLA-4 linked to the hinge-CH2-CH3 domains of canine IgG1 in a pcDNA3.1+ vector. Chinese hamster ovarian cells were cotransfected with CTLA4-Ig vector and a dihydrofolate reductase-containing vector. Stable, high producing clones were generated.


RESULTS
Cell binding and mixed leukocyte reactions indicated no significant differences in activity between cCTLA4-Ig and human CTLA4-Ig. Mixed leukocyte reaction data indicated that combinations of cCTLA4-Ig and the monoclonal antibody 5C8 were superior in blocking H-thymidine uptake compared to either reagent alone. In dogs, the circulating half-life of cCTLA4-Ig was approximately 7 days with no immune response against the fusion protein. Finally, two injections of cCTLA4-Ig effectively tolerized two dogs against eight consecutive challenges with sheep red blood cells, given over 330 days as indicated by a complete block of IgG antibody production. Tolerance was broken in one of the two dogs when a ninth injection of sheep red blood cell was given subcutaneously in incomplete Freund's adjuvant.


CONCLUSIONS
cCTLA4-Ig is an effective nonimmunogenic blocking reagent of the CD28 costimulatory pathway in dogs and is a promising reagent for studies of tolerance induction in hematopoietic cell transplantation in the canine model.
Graves
Scott S
SS
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109-1024, USA. sgraves@fhcrc.org
Stone
Diane
D
Loretz
Carol
C
Peterson
Laura
L
McCune
Jeannine S
JS
Mielcarek
Marco
M
Storb
Rainer
R
D000069594:Abatacept; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D018106:CD28 Antigens / Q000276:immunology; D016466:CHO Cells; D060908:CTLA-4 Antigen; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D004912:Erythrocytes / Q000187:drug effects* / Q000276:immunology; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects*; D018796:Immunoconjugates / Q000493:pharmacokinetics / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D007959:Lymphocyte Culture Test, Mixed; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012756:Sheep; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D015703:Antigens, CD; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D011993:Recombinant Fusion Proteins; C442103:hu5C8 antibody; D000069594:Abatacept