Evaluation of T Cell Products LMP-specific T cell therapies for Lymphoma CM The Long Winding Road to Successful T cell Therapy Tumor specific T cells in the clinic (CAR modified) Multi-virus specific T cells in the clinic Improved growth conditions rapid manufacture 2 Priming naïve T cells in vitro Low volume GMP grade culture systems Improved APC generation Target antigen identification Incorporating CD4 and CD8 responder Proof of principle Virus specific T cells Leukemia-specific T cells 1995 1
Bench to Bedside T cells EBV-specific T cells Can CTLs be used to treat real malignancies that occur in normal individuals? EBV+ Lymphomas post BMT EBV+ Hodgkin s Disease and NHL 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 4% of EBV antigens expressed by up to 4% of lymphomas are potential targets for T cell immunotherapy 2
Types of EBV Latency LMP 2 EBNA-3a EBNA-3b EBNA-3c EBNA-1 EBNA-2 LMP 1 EBNA 1 LMP 1 LMP 2 EBNA 1 EBNA-1 LP Type 3 EBV lymphoma post transplant Lymphoblastoid cell lines (LCL) Type 2 Hodgkin s disease Nasopharyngeal carcinoma Type 1 Burkitt s lymphoma EBV Specific Cytotoxic T Lymphocytes (CTL) Control EBV Infection in vivo EBV Infected B cells CTL Lytic EBNA 3a, b, c LMP 2 LP LMP 1 EBNA 2 EBNA1 PBMC LMP1 Inhibitory factors LMP2A EBV +ve Lymphoma Cell 3
EBV-Specific EBV-specific T CTL cell Generation PBMC EBV EBV-infected B cells (LCL) Step 1: LCL generation 4-6 weeks PBMC Step 2: CTL expansion 4-7 weeks IL-2 LMP2-CTL EBV-CTL Step 3: QA/QC Sterility HLA type Phenotype Cytotoxicity EBV Specific CTL in EBV+ve Hodgkin Lymphoma Gene Marked T-cells persisted for 12 months max EBV-CTL lines showed small populations of T cells reactive against LMP2 Some expansion of LMP2- specific T cells in PB post infusion. i Anti-tumor effects seen (2% CR/PR) Marked CTL by in situ PCR at tumor site et al, J Exp Med 24 Straathof et al, J Immunol 25 4
Bench to Bedside T cells EBV-specific T cells How can we improve and expand tumor specific T cells? EBV+ Lymphomas post BMT EBV+ Hodgkin s disease 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 5
Making LMP1 and LMP2 Immunodominant Antigens Adherent PBMC GM-CSF IL-4 IL-1b IL-6 TNF-a PGE-2 LCL LCL LCL PBMC mdc More recently Substituting pepmixes for ad vectors IL-15 IL-2 IL-2 LMP-specific Cytotoxic T Lymphocytes (CTL) et al, JIT 24, Straathof et al, JI 25 Phenotype of Autologous LMP-CTL Lines Expanded from Lymphoma Patients 12 45%+/-15% CD45RA- 62L- 34%+/- 5% CD45RA- 62L+ 1 8 % 6 4 2 CD19+ Total CD3 TCR(ab)+ TCR(gd)+ CD4+ CD8+ CD3+ CD56+ CD16+ CD19 CD3 TCR TCR CD4 CD8 CD3+ CD56+ CD16+ CD56+ CD3neg CD3neg CD56+ CD3neg CD3neg 6
LMP1 & LMP2 Specific Activity in LMP-CTL from a Patient with Hodgkin Lymphoma FLY-A2 LMP2 FC19Y8.131 tetramer CLG-A2 LMP2 FC19Y8.13 tetramer ILL-A29 LMP2 FC19Y8.133 tetramer YLL-A2 LMP1 FC19Y8.132 tetramer 1 4 1 4 1.2%.19% 4.17% 15.32%.3% 1.3% 1 4.1% 2.95% 15.32%.19% 1.3% 2.96% 1 3 1 3 1 3 1 3 FLY 1 2 CLG 1 2 ILL 1 2 YLL 1 2 1 1 1 1 1 1 1 1 19.48% 65.3% 16.83% 82.95% CD8 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 CD8 % Specific Lysis CD8 8% 6% 4% 2% % 18.96% 79.98% 1 1 1 1 2 1 3 1 4 CD8 16.53% 8.5% 1 1 1 1 2 1 3 1 4 CD8 Auto LCL Allo LCL PHA Blasts LMP1 target LMP2 target -2% Ratio 4:1 Ratio 2:1 Ratio 1:1 Ratio 5:1 LMP2-specific Cytotoxic T Lymphocytes (CTL) are CD4+ and CD8+ T cells+lmp2 T cells+cmv T cell alone LMP2 CMV CD8+ T cells CD4+ T cells media 2 4 6 8 1 SFC per 1x1 5 cells 7
Broad LMP2-specific Activity Present in CTL line 1x1 5 cells SFC per 25 2 15 1 5 9 8 7 6 5 4 3 2 1 18 17 16 15 14 13 12 11 1 Overlapping peptide pool number 19 2 21 22/23 CTL CTL+LCL SFC per 1x1 5 cells 88 11 HLA type: A2;29/B7;15(62) A2= FLYALALLL (4+5+2) 81 sa2 CLG 61 A29=ILLARLFLY (4+2) 41 21 1-19 CTL alone peptide peptide 28 29 ILL PYL Peptide 32 Peptide 33 Peptide 92 Peptide 93 Peptide Peptide 94 95 LLW CLG FLY Broad LMP1-specific Activity Present in CTL line 1x1e5 cells SFC per 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 1 CTL alone Overlapping peptide pool number LMP1 pepmix e5 cells SFC per 1x1e 1 9 8 7 6 5 4 3 2 1 LLIALWNLHGQALFL YLLEMLWRK HLA type: A2;29/B7;15(62) GLWIYLLEILWRLGATIWQLLAF #21 #22 #23 #24 #25 #26 #27 #28 #29 #3 #31 #32 #33 #34 #35 #36 #37 #38 #39 #4 #91 #92 #93 #94 #95 #96 #97 #98 #99 #1 CTL alone 15mer peptide number 8
CTL Product Autologous LMP2-CTL product (n=17) OR LMP1 and LMP2 CTL product (n=33) Dose escalation - Level 1: 4x1 7 /m 2, - Level 2: 1.2x1 8 /m 2 - Level 3: 3x1 8 /m 2 Types of Lymphoma Treated on LMP-CTL Studies Diagnosis LMP2 CTLs LMP1/2 CTLs Total Hodgkin s Lymphoma 9 14 22 NK/T lymphoma 3 8 11 Diffuse large B cell 3 4 7 lymphoma PTLD (lung, kidney, liver) 1 3 4 Other 1 3 5 Total 17 32 5 Age range 8-79years 9
FC per 1 5 cells S 15 1 5 pre 1wk 1 8 6 4 2 pre 1wk Immune Reconstitution of LMP1 and LMP2- specific T cells in Patients Treated with LMP1/2-CTL Pt1 15 4 Pt2 Pt3 2wk 4wk 6wk 8wk 3 2 1 pre 2wk 6wk 3mth 1 5 pre 1wk 35 3 25 2 15 1 5 2wk 4wk 8wk 3mth Pt4 Pt5 Pt6 Pt7 2wk 4wk 6wk 8wk 3mth 3 25 2 15 1 5 pre 1wk 2wk 4wk 6wk 8wk 3mth Time post infusion pre 1wk 2wk 4wk 6wk 8wk 3mth No difference between DL1 and DL3 patients 25 2 15 1 5 LMP2 LMP1 pre 1wk 2wk 4wk 6wk 8wk 3mth LMP2-tetramer Positive CTL Detected in LN Pre CTL Post CTL Supraclavicular Lymph Node FC12L4.19 FC12L4.2.19%.21% LMP2 CD8 LMP2 CD8 LMP2 tetram mers 1 1 1 1 2 1 3 1 4 CD8 FITC Peripheral Blood FC113L4.62 1 1 1 1 2 1 3 1 4 CD8 FITC CD8 1 1 1 1 2 1 3 1 4 CD8 FITC FC113L4.63.12%.% % 1 1 1 1 2 1 3 1 4 CD8 FITC 1
Rise in LMP-specific T cells Post Infusion and Accumulation in Lymph Node SFC per 2X1 5 cells 4 35 3 25 2 15 1 5 LMP1 LMP2 IIL LMP2_A2 29 pentamer ILL-PE IIL 1 4 1 3 1 2 1 1 PB.5% 1 1 1 1 2 1 3 1 4 CD8 1 4 LYMPH 1 NODE 3 1 2 1.31% 1 1 pre wk1 wk2 wk4 wk6 1 1 1 1 2 1 3 1 4 CD8 CD8-FITC Clinical Responses post LMP-CTL PRE PRE POST POST 11
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 >4 mths) Patients with disease at CTL infusion PR CR n =21 Clinical Responses post LMP-CTL in Patients with Active Disease 6% Disease Free Survival at 2 years ion disease-free Proport 1.8.6.4 P=.744.2 LMP2-CTL Alascer study LMP1/2-CTL ALCI study 1 2 3 Year X X X X X X X X PR NR PR CR CR n =21 12
Clinical Responses post LMP-CTL- As Adjuvant Clinical Therapy Results Adjuvant Therapy Patients high risk for relapse n=29 at CTL infusion No toxicity 14 patients post BMT Relapse 15 post chemo alone 1 died of cardiac disease (at <8wks) 27 remain in remission Remain in remission - 1 relapsed 8 wks post CTL - CR median of 2.5 years Progression Free Survival Probability in LMP2-CTL vs LMP1/2-CTL groups Patients who received CTL as Adjuvant Therapy Propor rtion disease-free 1.8.6.4 Year P=.741.2 LMP2-CTL Alascer study LMP1/2-CTL ALCI study 1 2 3 13
Cause of death specific probability: All subjects XXXXXX Patients who received T cells Patients who received T cells as Treatment XXXXXXXXXX as Adjuvant Therapy Cumulative In ncidence Probability 1.8.6 4.4.2 XXXX Lymphoma Other 1 2 3 Year Deaths from Other causes Deaths from Other Causes In adjuvant Group 8/26 patients died 1 relapsed, died in CR after allo SCT 3 second cancers (2 MDS, 1 Sarcoma) 3 infection 1 cardiac disease Confirms need for targeted therapies 14
Conclusions-LMP1/2 data No toxicity especially when used as adjuvant therapy Accumulation of LMP-CTL at disease sites Anti-tumor effects seen (13/21 patients PR/CR) And what about the patients who relapse?? Immune Evasion Strategies in Hodgkin s Lymphoma - Do CTL have a chance? Reed-Sternberg cell LMP-1 LMP-2 EBNA-1 IL-1 APC IL-13 BARF TARC TGF-ß CD8+CTL CD4+ TH2 Cell IL-4 CD4+CD25+ Treg Cell 15
Bench to Bedside T cells EBV-specific T cells EBV+ Lymphomas post BMT EBV+ Hodgkin s Can Tumor-specific CTL Be Disease Genetically Modified to Become Resistant to Inhibitory Effects of Lymphoma Cells? TGF Effects on CTL Inhibits CTL proliferation Inhibits cytotoxicity - perforin Inhibits cytokine production - IFN 16
Creating a Mutant TGF Receptor II Wild type Receptor Transmembrane domain Truncated TGFß Receptor II Dominant Negative Receptor (DNR) Stop codon 597 Retroviral vector SFG MoMLV NcoI/BamHI MoMLV U3 R U5 + U3 R U5 SD SA PBSQ et al, Blood 22 SFG:DNR TM domain DNR Rendering LMP-specific T cells Resistant to TGF Ad5f35 LMP1-I-LMP2 EBV-LCL DC SFG:DNR PBMC IL-2 et al, JIT 24, et al, Blood 22, Foster et al, JIT 28 DNR-transduced LMP CTLs 17
CD4 and CD8 T cells are DNR-transduced NON Trans TGFBR RII PE 1 4.3% 2.39% 1 3 1 2 1 1 Trans TGFBR RII PE 1 4 2.29% 1.19% 1 3 1 2 1 1 Transgene copy number in 1ng DN NA 1 14 12 1 8 6 4 2 16.26% 1 1 1 1 2 1 3 1 4 CD8 FITC n=6 CTL lines 81.32% 1 1.68% 1 1 1 1 2 1 3 1 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 2% 18% 16% 35% 3% n=6 CTL lines 14% 12% 1% 17% 25% 2% 8% 6% 4% 2% % 4% 11% TGFβ+ CD3+TGFβ+ CD3+ TGFβ+ CD3+ CD4+ CD8+ 15% 1% 5% % 1% CD45RA+ CD62L+ 25% CD45RA- CD62L+ 28% CD45RA-CD62L- 18
DNR-Transduced CTL are LMP-specific EBV-LCL DNR-Transduced LMP-CTL are Functional in vitro 8 7 Proliferation CTL 6 5 4 3 2 1 +TGF +TGF NT Non trans NT+TGFb CTL Transduced Trans+TGFb CTL 19
Patients Studied 5 females and 1 male EBV+ HL 5 relapsed post autologous SCT 1 relapsed post allogeneic SCT Two previously treated with LMP-CTL alone All refused additional chemotherapy DNR-transduced T-cells Persist in vivo for 5-23 months 1 CTLs Patient 1 1 Copy num mbers in 1 3 ng DNA 1 1.1 1 CTLs 1 1 1 1.1 Patient 3 2
DNR-transduced T-cells Persist in vivo for 5-23 months Copy num mbers in 1 3 ng DNA 1 1 1 1.1 1 1 1 1 1.1 CTLs Patient 4 Patient 6 Patient 5 Partial Response Pre Copy nu umbers in 1 3 ng DNA 1 1 1 1.1 sion 3hr ek1 ek 2 ek3 ek4 ek6 th 2 th 3 th 4 th 5 th 6 th 7 eek Pre-infus We Wee Wee We We Mont Mont Mont Mont Mont Mont Month 7+ 1 W Post 21
Patient 2 Complete Response PRE CR 8 wk Copy numbers in 1 3 ng DNA 5 45 4 35 3 25 2 15 1 5 LMP2-CTL DNR Specific Assay Response durable for over 34mths SFC per 2x1 5 cells pre week 2 CTL #2 week 4 week 8 month 3 Patient 1 Mixed Response to CR Copy numbe ers in 1 3 ng DN NA 16 CTL EBV T-cells 14 12 1 8 6 4 2 DNR-trans T-cells Mixed Response Anti-CD25 RIT CR SFC per 2x1 5 cells pre wk3 wk6 wk8 mth3 mth 6 mth 9 mth 12 Tumor Cells LMP2pos PreCTL Residual Tumor Cells LMP2neg Post CTL 22
Patient 3 Partial Response in Copy numbers 1 3 ng DNA 3 DNR-T-cells 25 LMP1-CTL 2 LMP2-CTL 15 1 5 pre 2wks 4wks 8wks SFC per 2x1 5 cells PRE PRE POST POST Partial Response (8mths) LMP-specific CTL Accumulate in Tumor Sites CD3 T-cells infiltrating tumor sites post CTL Patient 3 SFC pe er 2x1 5 ce lls 4 12 1 Tumor infiltrating T-cells are LMP-specific 8 6 4 2 LMP1 LMP2 Adhexon EBV SFC per 2x1 5 cells Residual LMP2+ Tumor Cells Present post CTL 9 8 7 6 5 4 3 2 1 Infused CTL line Tumor infiltrating CTL Pool1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 Pool 7 Pool 8 Pool 9 Pool 1 LMP1 peptide pools 23
Clinical Outcomes after DNR-transduced LMP-CTL Pt ID Age Dose # Doses 1 27 2x1 7 /m 2 2 2 47 2x1 7 /m 2 2 3 43 6x1 7 /m 2 8 4 47 6x1 7 /m 2 6 5 32 2x1 7 /m 2 6 6 28 6x1 7 /m 2 4 Response post CTL Mixed response CR Complete response Partial response Stable Disease Very good PR Stable Disease Duration of Response CTL Persistence 14 months 12months 34 months+ 2 months 18 months 23 months 12 months 18 months 1 months + 1months+ 7 months 5months Conclusions No dose limiting toxicity TGF -resistant LMP-CTL may beneficial in EBV+ Lymphoma DNR-trans LMP-CTL persist up to 12 months Enrolment continues 24
Conclusions-LMP1/2 data Evaluation of T cell products important to perform detailed immune reconstitution analysis and persistence Accumulation of LMP-specific T cells at disease sites even when peripheral blood persistence low No toxicity i especially when used as adjuvant therapy Anti-tumor effects seen (PFS 6% at 2 years) CAGT Laboratory Malcolm Brenner Cliona Rooney Helen Heslop Ann Leen Barbara Savoldo Gianpietro Dotti Steve Gottschalk Juan Vera Adrian Gee Nabil Ahmed Carlos Ramos Carl Allen Collaborators EJ Shpall John Barrett Acknowledgments Cath Lab Stephanie Ku, Sharon Lam, Russell Cruz, Patrick Hanley, Alana Kennedy-Nasser Yasmin Hazrat, Mo Baki, Javier El-Bietar, JW Blaney, Gerrit Weber, Francesco Saglio 25