Administration of Tumor-Specific Cytotoxic T Lymphocytes Engineered to Resist TGF-ß to Patients with EBV-Associated Lymphomas CM. Bollard, G Dotti, S Gottschalk, E Liu, A Sheehan, M Mims, H Liu, AP. Gee, MK. Brenner, HE. Heslop, CM. Rooney
Rationale of Immunotherapy for EBV-positive Lymphoma Significant failure rate of therapy for advanced stage or recurrent disease Long-term side effects of chemotherapy and radiation EBV antigens expressed by up to 40% of lymphomas are potential targets for T cell immunotherapy
EBV Specific Cytotoxic T Lymphocytes (CTL) Control EBV Infection in vivo PBMC TGFb EBV Infected B cells CTL LMP1 LMP2A Lytic EBNA 3 LMP 2 LP LMP 1 EBNA 2 EBNA1 EBV +ve Lymphoma Cell
LMP1 and LMP2A-specific CTL For Hodgkin and non-hodgkin Lymphoma LMP1 EBNA1 LMP1 and LMP2A are potential CTL targets LMP2A Hodgkin R-S Cell/NHL Cell
Making LMP1 and LMP2 Immunodominant Antigens Ad5f35 LMP2 OR Ad5f35 LMP1-I-LMP2 Dendritic Cells Lymphoblastoid cell line (LCL) PBMC IL-2 Bollard et al, JIT 2004 Gottschalk et al, Blood 2004 and Leen et al, JIT 2007 LMP-specific Cytotoxic T Lymphocytes (CTL)
% Specific Lysis FLY CLG ILL YLL LMP1 & LMP2 Specific Activity in LMP-CTL from a Hodgkin Disease Patient FLY-A2 LMP2 FC109Y08.131 tetramer CLG-A2 LMP2 FC109Y08.130 tetramer ILL-A29 LMP2 FC109Y08.133 tetramer YLL-A2 LMP1 FC109Y08.132 tetramer 10 4 10 4 10 4 0.17% 15.32% 0.02% 0.19% 0.03% 1.03% 10 3 10 3 10 3 10 3 10 4 0.01% 2.95% 15.32% 0.19% 1.03% 2.96% 10 2 10 2 10 2 10 2 10 1 10 1 10 1 10 1 19.48% 65.03% 16.83% 82.95% 18.96% 79.98% CD8 80% 70% 60% 50% 40% 30% 20% 10% 0% 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD8 CD8 CD8 16.53% 80.50% 10 0 10 1 10 2 10 3 10 4 CD8 Auto LCL Allo LCL PHA Blasts
Clinical Responses post LMP-CTL Relapsed Disease Arm (n=21) No toxicity 11 CR (1 also given Rituximab) (includes 1PR CR) 2 very good partial responses (up to 36 mths) 8 progressive disease (2-8 wks) Median clinical response: 1.5y (range: >6 to >40 mths) Patients with disease at CTL infusion PR CR Bollard et al, JCO 2013 in press n =21
Proportion disease-free Clinical Responses post LMP-CTL in Patients with Active Disease 50% Disease Free Survival at 2 years 1 0.8 0.6 0.4 0.2 0 LMP2-CTL Alascer study LMP1/2-CTL ALCI study P=0.744 0 1 2 3 X X X X X X X X PR NR PR CR CR Year n =21
Immune Reconstitution of LMP1 and LMP2- Figure 4. Immune Reconstitution specific T cells in Patients Treated with LMP1/2-CTL Responders Non-Responders LMP1 SFC per 2x10 5 LMP1 SFC per 2x10 5 LMP2 SFC per 2x10 5 LMP2 SFC per 2x10 5
SFC per 2x10 5 cells SFC per 2x10 5 cells Figure 4C. Immune Reconstitution Evidence of Epitope spreading in Responding Patients Treated with LMP1/2- CTL 120 100 80 60 40 20 0 60 40 MAGE A4 pre-infusion Survivin Responders post-infusion 120 100 80 60 40 20 0 60 40 MAGEA4 pre-infusion post-infusion Survivin Nonresponders 20 20 0 60 40 20 0 pre-infusion PRAME post-infusion pre-infusion post-infusion 0 70 60 50 40 30 20 10 0 pre-infusion post-infusion PRAME pre-infusion post-infusion
Can we make LMP-CTL resistant to the inhibitory effects of TGF-ß secreted by Lymphoma cells?
TGFb Effects on CTL Inhibits CTL proliferation Inhibits cytotoxicity - perforin Inhibits cytokine production - IFN
Creating a Mutant TGFb Receptor II Wild type Receptor Transmembrane domain Truncated TGFß Receptor II Dominant Negative Receptor (DNR) Stop codon 597 Retroviral vector SFG MoMLV Bollard et al, Blood 2002 NcoI/BamHI MoMLV U3 R U5 U3 R U5 SD PBSQ + SFG:DNR SA TM domain DNR
Rendering LMP-specific T cells Resistant to TGFb Ad5f35 LMP1-I-LMP2 EBV-LCL DC SFG:DNR PBMC IL-2 DNR-transduced LMP CTLs Bollard et al, JIT 2004, Bollard et al, Blood 2002, Foster et al, JIT 2008
Study Eligibility EBV + malignant cells EBER and/or LMP1 and/or LMP2 positive Lymphoma cells Patients with relapsed Hodgkin Disease or NHL including after allogeneic SCT
Transgene copy number in 100ng DNA TGFBRII PE CD4 and CD8 T cells are DNR-transduced NON Trans 10 4 10 3 0.03% 2.39% Trans 10 4 10 3 2.29% 10.19% 10 2 10 2 10 1 10 1 10 0 140000 120000 100000 16.26% 81.32% 10 0 10 1 10 2 10 3 10 4 CD8 FITC 80000 60000 40000 20000 0 n=6 CTL lines 10 0 10.68% 10 0 10 1 10 2 10 3 10 4 CD8 FITC 76.84% XX Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6
DNR-transduced CD4 and CD8 T cells are Predominantly Effector Memory 20% 18% 16% 35% 30% n=6 CTL lines 14% 12% 10% 17% 25% 20% 8% 6% 4% 11% 15% 10% 25% 28% 2% 0% 4% TGFβ+ CD3+ TGFβ+ CD3+ TGFβ+ CD3+ CD4+ CD8+ 5% 0% 1% CD45RA+ CD62L+ CD45RA- CD62L+ CD45RA-CD62L-
DNR-Transduced CTL are LMP-specific EBV-LCL
Patients Studied 5 females and 3 males EBV+ HL 7 relapsed post autologous SCT 1 relapsed post allogeneic SCT Two previously treated with LMP-CTL alone All refused additional chemotherapy
Copy numbers in 10 3 ng DNA DNR-transduced T-cells Persist in vivo for 5-23 months 1000 CTLs Patient 1 100 10 1 0.1 0 10000 CTLs Patient 3 1000 100 10 1 0.1 0
Conclusions No dose limiting toxicity TGFb-resistent LMP-CTL may beneficial in EBV+ Lymphoma DNR-trans LMP-CTL persist up to 3 years Now plan to explore the use of DNR- CTL in other TGFb-secreting cancers