Vaccinating Female Donors with Y Chromosome-Encoded Disparities in the Pre-Clinical Canine Model of Allogeneic Hematopoietic Cell Transplantation

Abstract 4123

Adoptive transfer of tumor antigen-sensitized lymphocytes has been a long-studied approach for the treatment of solid malignancies. Transfer of lymphocytes sensitized against minor histocompatibility (H) antigens, disparate between the marrow donor and recipient, may be a similar approach to enable improved host versus tumor responses in the treatment of hematological malignancies. We have developed a pre-clinical canine transplant model that establishes a state of stable mixed donor-recipient hematopoietic chimerism after a non-myeloablative conditioning regimen, dog leukocyte antigen (DLA)-identical sibling marrow graft, and a short course of post-grafting immunosuppression. Residual recipient hematopoiesis represents a surrogate for relapse from the underlying hematologic malignancy. The only way to eliminate the residual recipient hematopoiesis is by a donor lymphocyte infusion (DLI) from a donor sensitized by minor H antigens though either an infusion of cryopreserved recipient peripheral blood mononuclear cells, a kidney graft, or a skin graft. A DLI from an unsensitized donor does not alter chimerism in the recipient. This model lends itself to testing of vaccines to minor H antigens.

We hypothesized that vaccinating donor dogs with autologous antigen presenting cells (APC) expressing recipient minor H antigens followed by DLI will result in conversion to full donor hematopoiesis in the mixed chimerism recipient, providing the experimental evidence in a large animal model of the potential therapeutic benefit for a minor H antigen vaccine to treat relapsed disease. To test our hypothesis, we sensitized two donor dogs with autologous APC's transfected with poly(A)-selected mRNA from their respective recipients followed by DLI into these mixed chimeric recipients. We performed subcutaneous injections into the donor of 5 to 12 x 10^⋀6 CD40-ligand stimulated donor B-cells electroporated with mRNA isolated from PHA blasts from the chimeric recipient every three weeks for a total of three vaccinations. This was followed by a DLI infusion into the chimeric recipient. There was no change in chimerism in the recipient dogs, and we were unable to demonstrate an Elispot response in the donor dogs to the transfected recipient mRNA. Due to the difficulty in isolating sufficient quantities of mRNA and a lack of an ELISpot response, we changed the source of antigen.

Utilizing the canine genome sequence database we identified non-synonymous disparities between the × and Y chromosome genes of SMCY, UTY, and SRY. We created ‘minigenes’ encoding these disparities flanked by a T7 promoter and a 3' beta globin untranslated region with a poly(A) tail. These minigenes encoded 80% of the Y chromosome-associated disparities between canine SMCY, UTY, and their × chromosome homologs. The entire SRY gene was included as there is no × homolog for SRY. Minigenes were transcribed in vitro with a cap analogue to produce fully translatable mRNA. These Y chromosome-specific minigenes produced a strongly positive ELISpot in a female dog that had been sensitized with cells from a DLA-identical male sibling. We then performed three consecutive intravenous vaccinations at least one week apart with a combination of 25 to 40 × 106̂ CD34-derived donor dendritic cells and CD40 ligand-stimulated B cells transfected with these Y chromosome-specific minigenes. Following the third vaccination, we demonstrated a weak positive ELISpot reaction to the Y chromosome-specific minigenes. However, a DLI into a mixed chimeric recipient failed to convert the recipient to full donor hematopoiesis and did not elicit any graft versus host disease.

These studies suggest that in vivo sensitization of the marrow donor against recipient minor H antigens presented by autologous dendritic cells will require additional immune-modulation to accomplish improved graft versus tumor effects in the treatment of hematological diseases.