Relationship between Mixed Chimerism and Clinical Tolerance after Combined Kidney and Hematopoietic Cell Transplantation using Total Lymphoid Irradiation and Antithymocyte Globulin Conditioning Stephan Busque, John Scandling, Judith Shizuru, Robert Lowsky, Asha Shori, Kent Jensen, Edgar Engleman and Sam Strober

TTS 2010 Congress, Vancouver Immune Tolerance Definition The specific absence of a destructive immune response to a transplanted tissue without immunosuppression Operational Criteria 1) Complete withdrawal of immunosuppression followed by, 2) No evidence for rejection of transplanted organ for > 1 year Specificity 1) In animals: Normal rejection of third-party graft 2) in humans: Specific in vitro non-responsiveness to donor Strober S and Sachs D TTS 2010 Congress, Vancouver 2

The host’s chimeric immune system sees the donor graft as self Pathway to Tolerance Immune chimerism has been thought to be the way to tolerance since the work of Medawar and colleagues in neonatal mice six decades ago* The host’s chimeric immune system sees the donor graft as self * Billingham RE, et al. Nature 172: 603, 1953

Two Types of Chimerism Complete Mixed  Partial Nature of replacement of recipient immune and blood-forming cells with donor cells Partial Used to treat cancer  No recipient immune cells present Both donor and recipient immune cells present Host vs donor tolerance can be tested Donor vs host tolerance can be tested Significant risk of GvHD Significant risk of infection Goal of Stanford protocol

Stanford Tolerance Induction HLA-Matched and -Mismatched Protocols 2005-2016 Goals No immune graft loss Achieved in all 45 patients enrolled over the last 11 years No severe or chronic infection, or GvHD Achieved in all 45 No maintenance immunosuppressive drugs Achieved in 80% of HLA-matched Not yet achieved in mismatched

Stanford Tolerance Induction

Stanford Tolerance Induction Combined Kidney and Hematopoietic Cell Transplantation rATG (1.5 mg/kg x 5) TLI (120 cGy x 10) CD34+ cells CD3+ cells Day 0 Kidney Transplant MMF > 1 mo Corticosteroid Calcineurin Inhibitor > 6 mo Withdraw immunosuppression if: - Mixed chimerism > 6 months - No evidence of rejection - No GvHD

HLA-Matched Protocol Immunosuppression-Independent Chimerism Days After Kidney Transplantation

HLA-Matched Protocol Immunosuppression-Dependent Chimerism Days After Kidney Transplantation Days After Kidney Transplantation

HLA-Matched Protocol 2005-2016 Current Status 28 transplanted 21 mixed chimeras withdrawn from immunoRx drug 20 without subsequent rejection Up to 8 years off drug 7 of the 21 have not lost mixed chimerism 14 of the 21 lost mixed chimerism after year 1 1 developed acute rejection at 4 years off drug 5 did not achieve mixed chimerism 2 are in drug taper

HLA Haplotype-Matched Protocol 2010-2016 Goals and Current Status Step 1 T-cell dose escalation study to achieve mixed chimerism for at least 1 year 17 transplanted; 5 achieved goal Step 2 In persistent chimeras: Withdraw MMF during months 9-12 Taper tacrolimus to minimum effective dose (MED), 3-5 ng/mL, during months 12-15 5 achieved goal Step 3 Withdraw tacrolimus during year 2 Return to MED if chimerism lost In progress

HLA Haplotype-Matched Protocol Loss of Chimerism after Drug Withdrawal 10 % Donor Cells CNI/MMF dose (mg/day) Serum Creatinine Days After Kidney Transplantation

Stanford Tolerance Induction 2005-2016 Summary HLA-Matched Protocol No graft loss 80% developed mixed chimerism and came off drug HLA Haplotype-Matched Protocol Persistent mixed chimerism achieved Drug withdrawal in chimeras in year 2 to determine fraction on MED tacrolimus versus off drug No GvHD No severe or chronic infection

Conclusion What of mixed chimerism as a biomarker? HLA-Matched Loss after drug withdrawal may be a biomarker of risk of losing tolerance HLA-Mismatched Biomarker of host vs donor tolerance Guide to withdrawal of immunoRx drug

Stanford Tolerance Induction Core Clinical and Translational Investigative Team Medicine Robert Lowsky, MD John Scandling, MD Judy Shizuru, PhD, MD Sam Strober, MD Radiation Therapy Richard Hoppe, MD Surgery Stephan Busque, MD Asha Shori, MD Pathology Ed Engleman, MD