Clinical Translation of Immunotherapy using WT1 and CMV specific Gene Transfer Dr Emma C Morris Reader, Dept of Immunology, UCL Consultant Haematologist (BMT), UCLH and RFH ISCT, 2/5/211
Gene Transfer Designer T cells with re-directed antigen specificity. Adoptively transfer T cells with known antigen specificity and known (high) functional avidity. Potential to manipulate homing ability, etc.
CD8 Retroviral gene transfer Antigen-specific CD8 T cell Insert into retro-viral vector CD3 CD3 CD3 Clone DNA of antigen-specific Before After Antigen-specific multimer
CMV Gene Transfer Shao-An Xue, Liquan Gao, Emma Nicholson
CMV Disease Post Allogeneic Haematopoietic Stem Cell Transplantation CMV Retinitis CMV Pneumonitis CMV Colitis CMV Inclusion Body
CMV Reactivation Post Allogeneic HSCT Recipient CMV seropositive HSC Donor CMV seropositive Infuse CMV-specific CTL Donor engraftment and T cell expansion Potential for expansion of CMV-specific T cells CMV reactivation conditioning Time Pre- and post transplant immune suppression Day Day 28 Recipient CMV seropositive HSC Donor CMV seronegative Donor engraftment and T cell expansion NO CMV-Specific T Cells conditioning CMV reactivation Pre- and post transplant immune suppression Time Day Day 28
mc CD8 pnlv-tetramer Generation of Cys1-hybrid CMV construct LTR hv 18 mc p2a hv 13 mc LTR SS Mock Td CMV- Td Wild-type Cys-1 Hybrid Cys-1 Hybrid % 41% ss ss ss ss ss ss pnlv-tetramer Mock Td CMV- Td.1% 18% 14%
Classical CD4+ T cell Class I restricted CD4+ T cell CD4 T cell CD4 T cell CD4 Class II restricted CD4 Class I restricted Class II MHC Class I MHC APC Virally infected cell or tumour cell Are MHC class I restricted CMV-specific CD4+ T cells functional? Do they function in the same way as a classical class II restricted CD4+ T cells?
IL2 TNF TNF Ag-specific function of CMV -Td T cells IL2 TNF TNF CD4+ CMV- Td CD8+ CMV- Td Irrelevant Peptide (CLG) Relevant Peptide (NLV) Irrelevant Peptide (CLG) Relevant Peptide (NLV) 5.8%.2% 14% 14.7%.6%.3% 2.7% 11.8% IFN IFN 5.7%.3% 15.4% 13.2%.9%.2% 7.6% 7.3% IL2 IL2.4%.1% 5.4% 7.5%.1%.4% 1.9% 7.7%.4% 8.4% 1.8% 19.9% IFN IFN
% Killing % Killing CMV -Td T cells recognise endogenously processed antigen A Bead sorted CD4+ CMV -Td T cells B Bead sorted CD8+ CMV -Td T cells 1 1 8 8 6 6 4 4 2 2 5 25 12 6 3 1.5 5 25 12 6 3 1.5 E:T Ratio E:T Ratio Target cells T2+NLV T2+CLG K562/A2/pp65 K562/A2/LMP2
% IFN producing T cells Functional avidity of CMV- Td CD8+ and CD4+ T cells % of INFg+ T cells % of IFNg+ T cells % Killing % Killing % IL-2 producing T cells % of IL2+ T cells 5 of IL2+ T cells IFN secretion 6 IL-2 secretion CLG 1 8 6 4 2 1nm 1nM 1nM 1pM 1pM 1pM KA2 [peptide] 4. 3. 2. 1.. 1nm 1nM 1nM 1pM 1pM 1pM KA2 [peptide] CLG NLV CLG NLV 5 4 8 6 4 2 3 2 1 1nM 1nM 1nM 1pM 1pM 1pM KA2 [peptide] 25. 2. 15. 1. 5. 5. 1nM 1nM 1nM 1pM 1pM 1pM KA2 [peptide] NLV CLG NLV CMV- Td CD8 + T cells CMV- Td CD4 + T cells CMV- Td CD4+ T cells CMV- Td CD8+ T cells 7 12 6 5 4 3 2 1-1 1 2 3 4 5 6 1 8 6 4 2-2 1 2 3 4 5 6 T2+CLG T2+NLV KA2LMP2 KA2pp65 E:T from 4:1 E:T from 4:1
CMV- Td CD8+ and CD4+ T cells reject CMV pp65- expressing tumours in vivo CMV- Td CD8+ T cells CMV- Td CD4+ T cells EBV- Td CD8+ T cells d1 d5 d1 d15 d2 d25 2x1 6 K562A2-pp65-Luc tumor cells
Clinical GMP Grade CMV- Td CD8+ T cells SSC NLV-Tetramer CD3 FSC CD8 Mock transduction 1 round transduction 2 round transduction Anti Murine C
A Phase I Safety, Toxicity and Feasibility Study of Adoptive Immunotherapy with CMV -td donor-derived T cells for Recipients of Sibling Allo HSCT (CMV -1) TCD Myeloablative HSCT or Reduced Intensity HSCT Start anti-viral Rx if: Copy number at 1 wk CMV PCR pos at + 2 wks CMV disease Conditioning Weekly CMV PCR Follow Up D Exclusion criteria: agvhd grades II-IV Neuts <.5 x 1 9 /l +7 +14 +21 +28 +35 +42 CMV -td bulk donor-derived T cells 1 4-1 5 /kg recipient weight 1 st positive PCR (Pre-emptive Rx) Immunology Ix: V 13, tetramer staining Ag-specific proliferation Intracellular CK secretion Cytotoxicity repertoire PCR for V 13/ fragments Clinical Assessment for GVHD T cell subsets & phenotype
Class I restricted CD4+ T cell Class I restricted CD4+ T cell + additional CD8 CD4 T cell CD4 T cell CD4 Class I restricted CD4 Class I restricted Class I MHC CD8 Class I MHC Virally infected cell or tumour cell Virally infected cell or tumour cell
means of OD45 means of OD45 means of OD45 means of OD45 Co-transfer of CD8 co-receptor into CMV -Td CD4 + T cells improves function 3 CMV-CD4-INFg CLG NLV.5 CMV-CD4-IL2 CLG NLV 2.5.4 2.3 1.5.2 1.1.5 1nM 1nM 1nM 1pM 1pM 1pM KA2 1nM 1nM 1nM 1pM 1pM 1pM KA2 [peptide] 3 CMV-CD8-INFg CLG NLV 2.5 CMV-CD8-IL2 CLG NLV 2.5 2 2 1.5 1.5 1 1.5.5 1nM 1nM 1nM 1pM 1pM 1pM KA2 1nM 1nM 1nM 1pM 1pM 1pM KA2 [peptide] 3 2.5 2 1.5 1.5 CMV8-CD4-INFg 1nM 1nM 1nM 1pM 1pM 1pM KA2 CLG NLV 3 2.5 2 1.5 1.5 CMV8-CD4-IL2 CLG NLV 1nM 1nM 1nM 1pM 1pM 1pM KA2
Co-transduction with additional CD8 co-receptor into CMV- Td CD4+ T cells may improve in vivo function CMV- Td CD4+ T cells CMV- Td CD8+ T cells CMV- and CD8 Td CD4+ T cells d5 d1 d15 d2 d25
Improving function of -td T cells Maryam Ahmadi, Judy King
Co-transduction of and CD3 into T cells Increased surface expression of leads to increased functional avidity of the δ α β Υ CD3 rate limiting for expression of introduced ζ ζ Can surface expression of be increased by introduction of additional CD3? Does this lead to increased functional avidity of the T cell expressing a class I restricted? 5 LTR CD3 2A CD3 2A CD3 2A CD3 IRES GFP 3 LTR
CD3 is rate limiting for expression WT1 F5 CD3 Vβ2.1 Tet Vβ11 Tet Mock x1 x1 x1 x1 x1 +GFP + CD3-GFP x26 x16 x2 x5 x4 GFP BW cells
CD3 is rate limiting in primary T cells F5- WT1- CD3 Vβ11 Tet CD3 Vβ2.1 Tet x1 x1 x1 x1 x1 +GFP + CD3-GFP x3 x4 x1 x3 x5 GFP Murine splenocytes
CPM IL-2 (pg/ml) Vβ11 CD3 improves antigen-specific IL2 production 16 4 12 3 8 2 4 2 15 1 5 F5- F5-+CD3 F5 F5+CD3 GFP 1 1pM 1-6 1pM 1nM 1nM pnp (μm) 1-5 1-4 1nM 1-3 1uM 1uM 1-2 1-1 [NP conc, um] 1 1 pwt SV9 Sorted CD8+ murine T cells
IFNg CPM pg/ml CPM CPM/1 Vβ11 CD3 improves antigen-specific proliferation 4 6 4 3 5 4 2 3 2 1 F5 F5- F5+CD3 F5-+CD3 F5+CD3 GFP 1 1-6 1-5 1-4 1-3 1-2 1-1 1-6 1-5 1-4 1-3 1-6 1-5 1-4 1-3 1 1-2 1-1 [NP conc, um] [NP conc, um] -2 1-1 [NP conc, um] 1 1 pwt1 1 1 pwt1 1 1 pwt1 pnp peptide conc (um)
In vivo function of +CD3 transduced T cells d d1 > 8 weeks EL4-NP tumour cells (luciferase) + CD3 td CD8 + T cells (luciferase) Monitor tumour clearance and T cell memory
CD3 limits tumour growth in vivo 1 9 8 4 Tumour volume Mock +CD3 D5 D8 D11 D13 D17 D2 D33 D5 3 2 1 8 13 15 2 24 35 42 Days post tumor injection Photons./sec x 1 1 Mock Mock Mock 4. 4.11 4. 4. 1 1 Mock 1 1 1 +CD3 +CD3 +CD3 3. 3.11 3. 3. 1 1 1 1 1 2. 2.11 2. 2. 1 1 1 1 1 + CD3 1. 1.11 1. 1. 1 1 1 1 1.... 5 5 8 8 5 5 11 13 11 13 8 8 11 13 11 13 17 2 23 17 2 23 Days post tumor injection 17 2 23 17 2 23 33 5 33 5 33 33
+CD3 enhances T cell accumulation in tumours +CD3 D 3 D 5 D 6 D 8 D 11 D 16
+CD3 enhances T cell accumulation in tumours 5 5 75. 6. 4 4 45. 3 3 Photon flux x 1 8 3. 2 2 15. 1 1. 3 3 75. Mock 75. Mock Mock F5 6. +CD3 F5+CD3 6. +CD3 F5+CD3 +CD3 45. F5+CD3 45. 45. 3. 3. 3. 15. 15. 15.... 3 5 6 8 11 16 22 3 5 6 8 11 16 22 5 5 6 6 8 8 11 11 Days post tumor injection 16 16 22 22 T cell no x 1 5 3 3 3 5 5 5 6 6 6 8 8 8 11 11 11 Days post tumor injection 16 16 16 22 22 22
Vβ11 +CD3 T cell persist in larger numbers than -only T cells Spleen Lymph nodes Pre-injection 56 44 Day 35 Post-injection 35 65 Absolute no/1 3.2 15.15 *.15 1.1.1 5.5.5 * Absolute no/1 3.2.15.1.5 5 *.. +CD3. +CD3 +CD3 GFP +CD3 +CD3 F5- F5-+CD3
% Transduced % cells Cells % Transduced Cells % cells +CD3 T cells dominate central and effector memory development 1 8 8 ns Spleen 1 ns *** *** 1 1 8 8 Lymph nodes *** *** *** *** 6 6 6 6 4 4 4 4 2 2 2 2 +CD3 CD44 + /CD62L + CD F5+CD3 CD62L+ F5 +CD3 CD44 + /CD62L - F5+CD3 F5 F5+CD3 F5 CD62L- F5+CD3 CD62L- +CD3 +CD3 CD44 + /CD62L + CD44 + /CD62L -
+CD3 T cell mount an effective memory recall response D 8 16 12 9 12 F5- F5-+CD3 6 months Absolute no x 1 3 8 6 4 3 TDLN LND Non-TDLN non-lnd
WT1 Gene Transfer Shao-An Xue, Liquan Gao, Sharyn Thomas, Irma Martinez-Davila
IFN-g (ng/ml) CD19 % of survival WT1 -td T cells display in vitro function and eliminate A autologous 1 leukaemia cells in NOD/SCID mice. 1 25 T2+pWT235 T2+pWT126 BV173 8 6 Cys1 WT1 -td T cells 2 4 2 Control -td T cells 15 B 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 Weeks 1 Control 1 Control 2 Control 3 Control 4 Control 5 5 Cys1 WT1 1 Cys1 WT1 2 Cys1 WT1 3 Cys1 WT1 4 Cys1 WT1 5 Normal AML CML T cells T cells T cells Human Class I Xue SA, et al. Blood. 25; 16(9):362-7 Xue SA, et al. Haematologica. 21;95(1):126-34.
V 2 Ladder 4 4 4 PBMC C(-) Clinical Scale Production of GMP grade WT1 -td T cells Lymphocyte Gate Isotype Control.56% qrt-pcr of PBMC to determine persistence of GMP grade WT1 -td T cells Diluted in 1 6 PBMC V 2 Mock-Transduced Post 1 Round.4x1 6 4x1 4 4x1 3 7.61% 26.13%
Trial Outline UK Multi Centre, non-randomised, dose-escalation, Phase I/II pilot study Infusion of WT1 -td Autologous T cells into HLA-A2 + adult AML or CML patients Ex-vivo Transduction (5-7 days) QC on transduced cells: expression i.c. CK secretion Microbiology Leucapheresis D-14 Start infection Prophylaxis *Provision to escalate conditioning intensity if required Lymphodepleting Conditioning* Flu/MP or Flu/Cy D-7 to D-2 Cryopreserved D Re-infusion of WT1 -td Bulk T cells Cohort 1 (n=6): Cohort 2 (n=12): + Low dose IL2 (D to D+5) 2 x 1 7 /kg 1 x 1 8 /kg Monitoring & Follow Up Immunology Ix: V 2.1, tetramer staining Ag-specific proliferation Intracellular CK secretion Cytotoxicity repertoire PCR for V 2.1/ fragments Clinical Assessment for toxicity T cell subsets & phenotype Integration site analysis & archives
21 22 23 24 25 26 27 28 29 WT1 Gene Therapy: Time Lines Pre-clinical development: Ongoing Including vector development 1 st expression in murine model Vector development (ongoing) In vitro expts with Td-T cells NOD/SCID In vivo expts (ongoing) CRUK/LRF funding LRF funding GTAC applic Vector production commenced DOH funding Clin scale tests EU ATMP Regs MHRA CTA/EAG Legal issues Stauss/Morris In vivo data Blood/PNAS Prov GTAC approval Rosenberg et al Science Phase 1 Trial Full GTAC approval? Opening End 21
Acknowledgments UCL Immunology Hans Stauss Shao-an Xue Liquan Gao Sharyn Thomas Emma Nicholson Judy King Irma Martinez-Davila GOSH/ICH Adrian Thrasher, Waseem Qasim, Sue Swift, Kate Parsley, Hong Zhan