High-Dose Methotrexate (MTX) as Part of a Novel Immunosuppressive Regimen in Canine MHC-Haploidentical Hematopoietic Cell Transplantation (HCT).

In a preclinical canine model using major histocompatibility (MHC)-haploidentical littermate peripheral blood stem cell (PBSC) grafts, initial donor chimerism could be achieved after a nonmyeloablative conditioning regimen consisting of monoclonal antibody S5 (anti-CD44) from days −7 to −2, 200 cGy TBI on day 0, and MMF and cyclosporine for postgrafting immunosuppression. However, sustainable donor chimerisms could not be achieved in many of these dogs, with 25 of 44 (57%) rejecting their grafts at a median of 7 weeks after transplant. In order to improve the engraftment rate, a novel strategy to overcome the immunological barrier created by this MHC-haploidentical setting was developed. By adding single, progressively higher doses of MTX on day+3 after transplantation to the above regimen, we hoped to control host T and NK cells, both of which contribute to graft rejection in MHC-haploidentical HCT, and to target alloreactive donor T cells which are responsible for graft-versus-host disease (GVHD). Once dose toxicity is established, this system will also allow the study of transplants using gene-modified, MTX-resistant CD34 selected PBSC, which can provide additional intrinsic graft protection on exposure to MTX. To date, 11 dogs have been transplanted using the above regimen and adding either 50 (n=5), 100 (n=3), or 200 (n=3) mg/m² MTX on day +3 followed by leucovorin rescue on days +4 and +5. Early results showed that 4 of 5 dogs experienced graft rejection in the MTX50 group, 0 of 3 dogs rejected in MTX100 (p=0.047, Monte Carlo), and all 3 dogs in MTX200 have continued to exhibit stable mixed chimerisms from +42 to +76 days after transplant. Dogs in all 3 cohorts had rapid engraftment within 1 week, with similar average peak mononuclear cell (MNC) chimerisms at comparable timepoints (83% at 5 wks, 88% at 6 wks, and 80% at 5 wks at MTX50, 100, and 200 respectively). None of the 11 dogs have shown evidence of GVHD or MTX toxicity, and all dogs cleared MTX within 48 hours. All dogs exhibited early donor tolerance on mixed lymphocyte culture assays within 2 weeks and preserved NK activity posttransplant. While these data are preliminary, adding single, progressively larger doses of MTX on day +3 to a novel, nonmyeloablative conditioning regimen appeared to improve engraftment among MHC-haploidentical canine recipients with no signs of GVHD. There was high initial donor chimerism without undue early toxicity. Further, this study will aid in our establishment of high-dose, posttransplant MTX as a way of promoting sustained donor chimerism after transplanting CD34 selected, MTX-resistant canine PBSC in the MHC-haploidentical setting. This preclinical data serve as a basis for developing nonmyeloablative transplant options for patients without HLA-identical donors.