Minor H Antigen-Specific T Cells -The Black Box of the GVL Effect

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1 Minor H Antigen-Specific T Cells -The Black Box of the GVL Effect All Allogeneic Stem Cell Transplants Are Blind Doctor Donor Patient Genetic polymorphism gives rise to unique peptides in donor and recipient proteins (most common nonsynonymous SNPs, gene deletion polymorphism)

2 Horowitz, Blood 199 HA-1 HA-2 LRH-1 Bcl2A1 Heatr1 Pane-1 Donor T Cells SMCY HA-8 UGT2B17 P2XR7 GVL GVHD CML, CLL > Myeloma > Lymphoma > AML > ALL Why Does The GVL Effect Work? Leukemic Cell Hematopoietic Epithelium derived APC Recipient Cells Multiple minor H antigens (+possibly TAA) are recognized on tumor cells Decreased potential for tumor escape by antigen or HLA loss Both CD8 + and CD4 + T cells participate in the immune response Minor H antigen-specific T cell responses are high avidity T cells able to recognize tumor cells with low levels of antigen

3 The GVL effect in action - Part 1 Nonmyeloablative HCT For CLL Fludarabine TBI PBSC Lymphocyte # (x1) UPN * >8% CLL CSP D D28 D6 D8 D1 Day post-hsct MMF % Lysis Day E:T ratio - Pt LCL - Pt CD4-CLL - Pt PBMC - Donor LCL

4 Analysis of the mechanisms of tumor eradication after NM-HCT for CLL (Nishida et al. Clinical Cancer Research 29) Pre-HCT Patient CD4 CLL - Stimulator - Post-HCT Patient PBMC (donor origin) - Responder - Co-culture T cell line Chromium-Release Assay Targets: - Recipient CD4CLL / B-LCL - Donor B-LCL IFN-γ capture assay and sorting of IFN-γ-secreting T-cells CD8+ T-cells Responder : post transplant PBMC Stimulator : CD4 activated CLL (B cell) 7 days R:S=4:1 IL-7 1ng/ml (day ) IL-2 2u/ml (day 3) anti-cd3 mab Cloning by limiting dilution Pt CD4 CLL 7 days R:S=4:1 IL-7 1ng/ml (day ) IL-2 1u/ml (day 1) IL-2 1u/ml (day 4) Pt / Do B-LCL CTL clone Screening + HLA-restriction assay Pt CLL + Pt / Do B-LCL Analysis minor H Ag cdna library screening minor H Ag / TAA

5 A. Responders % Specific Lysis CLL-reactive T cell responses are only detected in responding patients #22388 #28736 #1882 # R-LCL -R-CLL -D-LCL % Specific Lysis #24487 #21899 #2843 #9661 (post DLI) Day post transplant B. Non Responders % Specific Lysis #23134 #1976 #28196 # Day post transplant (Nishida et al. Clinical Cancer Research 29)

6 CLL-reactive T cells elicited after NMA-HCT are both CD4 + and CD8 + UPN UPN %.36% 2.4% 1.9% R-CD4-CLL.28%.%.9% 1.3% R-B-LCL IFN-γ IFN-γ IFN-γ IFN-γ.9%.11%.3%.7% D-B-LCL CD4 CD8 CD4 CD8 CD4 + T cells CD8 + T cells UPN UPN 9661 UPN UPN 9661 D-CD4-B D-CD4-B D-CD4-B D-CD4-B D-LCL D-LCL D-LCL D-LCL Primary CLL Primary CLL Primary CLL Primary CLL CD4-CLL CD4-CLL CD4-CLL CD4-CLL R-LCL R-LCL R-LCL R-LCL % Lysis % Lysis % Lysis % Lysis

7 Clonal evolution of a CD8 + GVL T cell response * * * [HLA A2, 31, B1, 4, Cw3] - B4 (broad), A2 (SMCY) -specific T cells remain 1A1 23H3 3B2 - CLL-specific T cell clones remain R-B-LCL R-CLL D-B-LCL GVHD Day ~16, circulating CLL cells NONE +GVHD +GVHD MRD CR CR DIED IN CR - Multiple T cell clones - All B4 restricted (1 specificity) - minor H antigen-specific, kill CLL - No recognition of skin fibroblasts - Multiple T cell clones - B4 (2 Ags),A2 (SMCY),A31(3 Ags),B1, Cw3 - minor H antigen-specific and kill CLL - At least 4 of these Ag expressed on skin fibroblasts CLL-specific T cell clones also isolated 2A2 D-LCL R-LCL 73 R-CLL 64 Fibro 14D1 17C R-B-LCL R-CLL D-B-LCL E 61

8 HA-1 tetramer+ T cells in peripheral blood in relation to clinical effect of DLI Marijt W A E et al. PNAS 23;1:

9 In vivo priming T cells from recipient post transplant The GVL effect in action - Part 3 Adoptive T Cell Transfer CD8 + T cell clones specific for minor H antigens inhibit human AML and ALL engraftment in NOD/SCID/γc-/- mice Recipient APC (PBMC or leukemia) AML Alone Assay BM Engraftment at >6 weeks IL-2 AML + Control CTL Cloning by Limiting Dilution AML + Specific CTL Bonnet et al. PNAS, 1999; Bleakley et al. Blood 211

10 Specific T cell therapy for relapsed acute leukemia (Warren et al Blood, 21) Patients with AML or ALL > 2nd CR undergoing allogeneic stem cell transplant Isolate minor histocompatibility antigen-specific T cell clones early post SCT Select clones with preferential killing of recipient hematopoietic cells and leukemia (exclude clones that recognized recipient skin fibroblasts) 3 7 days Cryopreserve At Relapse Expand T Cells T Cell Infusions CTX * Day IL-2 Patient Monitoring Safety In vivo persistence Migration Cell doses - 3x1 7 /m 2-3x1 9 /m 2

11 Transferred T cell clones have antitumor activity in advanced relapsed leukemia UPN primary refractory ALL - Relapse 7 months post SCT (>9% blasts) - Persistent leukemia after Mitoxantrone/VP16 CTL Clone 11C6 Donor Fibroblasts Donor LCL Leukemic Blasts Recipient Fibroblasts Recipient LCL Post Chemotherapy - Relapse >1% Blasts % Specific Lysis Post 6 T cell infusions - Remission 1% donor chimerism

12 UPN: Post chemotherapy 8% Blasts Post 6 T cell infusions <% Blasts (CR) Transferred T cells were detected in vivo but did not persist long term Clone-specific TcR V beta PCR % Total PBMC T1/2 < 7 days Day After Infusion? Clones Lack of cell intrinsic properies Activation induced cell death

13 Safety of adoptively transferring CD8+ minor H antigen-specific T cell clones Seven patients treated with a total of 42 T cell infusions, /7 achieved CR Safety: Fever and chills were common, no acute onset GVHD No evidence of transferred T cells at the site of GVHD 3 patients developed pulmonary toxicity within 2 hours of cell infusions at doses of >1 9 cells. Toxicity was serious (1 patient) but reversible. Subsequent studies showed the antigen (P2RX7) recognized by the T cell clone was expressed on alveolar epithelium. (On-target toxicity) P2RX7 expression (lung alveoli)

14 Lost in Translation 1. No current strategy allows us to completely separate GVL from GVHD 2. The number of CHARACTERIZED minor H antigens that are relevant targets for GVL therapy is small and limits efforts to transfer T cells specific for single targets 3. Efforts at high throughput discovery using gene expression/sequencing data have so far yielded less than anticipated 4. The isolation of minor H antigen-specific T cells for adoptive therapy is arduous. Other strategies (TCR gene transfer or vaccination) hold promise but #2 is limiting Should we continue discovery efforts and pilot studies to employ minor H antigens as therapeutic targets? YES THE GVL EFFECT WORKS

15 Human minor H antigen discovery is accelerating HA-2 V HA-1 H HB-1 H HB-1 Y HA-8 R HA-1 H B*6 B*42 UGT2B17 ACC-1 Y ACC-2 D HA-3 LRH-1 LB-EGFR 1H AAC-4 R ACC- R PANE-1 SP11 R LB-ADIR 1F ACC-6 V LB-P14K2B 1S ACC-1 C CD19 L LB-MTHFD1 1Q LB-PTK2B 1T LB-MR1 1R LB-LY7 1K UGTB17 A*26 SLC1A A SLC19A1 R LB-WNK1 1L LB-SSR1 1S LB-PRCP 1D LB-ERAP 1R LB-BCAT2 1R LB-ARHGDIB 1R LB-PDCD11 1F LB-GEMIN4 1V LB-EBI3 1L LB-APOBEC3B 1L FLJ871.2 H FLJ871.1 R P2RX7 H HEATR1 E DPH1 V HPLC & mass spectroscopy cdna library Genetic linkage analysis GWAS Bacterial cdna library

16 Scientists prediction of a home computer in

17 Acknowledgements Fellows/Trainees Marie Bleakley Tetsuya Nishida Rebecca Gardner Michael Hudecek Suzanne McGoldrick Seitaro Terakura Cameron Turtle FHCRC Faculty Edus Warren David Maloney Brenda Sandmaier Rainer Storb Aichi Cancer Center/ Fujita Health University Yoshiki Akatsuka National Institutes of Health; Leukemia and Lymphoma Society