Establishment of Long-Term Tolerance to SRBC in Dogs by Recombinant Canine CTLA4-Ig

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. 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. 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. 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.