Multi-Virus-Specific T cell Therapy for Patients after HSC and CB Transplant

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1 Multi-Virus-Specific T cell Therapy for Patients after HSC and CB Transplant Hanley PJ, Krance BR, Brenner MK, Leen AM, Rooney CM, Heslop HE, Shpall EJ, Bollard CM

2 Hematopoietic Stem Cell Transplantation Treatment for relapsed and high risk malignancies Genetic disorders Involves necessary myeloblative regimens Chemotherapy Total body irradiation T cell depletion High incidence of viral infections post-transplant Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (Ad) Highest incidence when donor seronegative (cord blood)

3 Expanding T cells targeting CMV, EBV and adenovirus (Multivirus-specific CTL) Ad5f35-pp65 EBV-Lymphoblastoid cell lines (LCL) p p 6 5 LMP1 LMP2 EBNA-2 EBNA 3a,3b,3c EBNA-1 LP

4 Generating Multi-Virus-Specific T cells From Peripheral Blood (PB) of Seropositive Donors Ad5f35CMVpp65 vector EBV LCL Antigen Stimulation IL-2 IL-2 PBMC Multivirusspecific CTL

5 Generating Multi-Virus-Specific T cells From Cord Blood (CB) Using Same Methodology Ad5f35CMVpp65 vector EBV LCL Antigen Stimulation IL-2 IL-2 CBMC Multivirusspecific CTL

6 Successfully Generating Multi-Virus-Specific T cells From Cord Blood Ad5f35CMVpp65 vector EBV LCL Xxx Antigen Stimulation IL-15 Xxx xxx CB DCs CBMC IL-7 IL-12 IL-15 Hanley et al, J. Vis. Exp, 2012 Virus-specific CB-derived T cells

7 T cells From CB Recognize Adenovirus, CMV, and EBV Spots per 1x10e5 cells n=9 CTL lines Irrelevant peptide Adv CMV pp65 EBV Hanley et al, Blood 2009

8 Eligibility Criteria Prophylaxis and Treatment Day +30 post HSCT or CBT GvHD <grade III at enrollment In addition for Cord Blood: 2.5x10 7 TNC/kg Fractionated cord blood unit

9 Cell Dosing Phase 1 dose escalation study Peripheral Blood CTL: 1x10 7 /m 2 5x10 7 /m 2 1x10 8 /m 2 Cord Blood CTL: 5x10 6 /m 2 1x10 7 /m 2 1.5x10 7 /m 2 2.5x10 7 /m 2

10 Analysis of CTL Lines 34 CTL Lines analyzed 25 generated from peripheral blood of donors 9 generated from cord blood Phenotypic Analysis Specificity for Viruses Cytotoxicity assay ELISPOT assay for IFN-γ release Pentamer analysis

11 Percentage of Live Cells CTL Express Effector and Central Memory Markers 100% 75% Mean CB CTL Mean PB CTL CB CTL PB CTL 50% 25% 0% CD4 CD8 CD45RA- CD62L- CD45RA- CD62L+ CD3- CD56+ CD3- CD19+

12 CTL Lines Recognize Multiple Viruses IFN-γ ELISPOT: Spots per 1x10 5 cells CTL Line Adeno EBV CMV Control Peripheral Blood CTL 86 (9-542) 183 (0-351) 648 ( ) 14.5 (3-65) Cord Blood CTL 83 (1-504) 117 (4-339) 36 (1-110) 8 (1-28) 27/34 CTL were Ad-specific 32/34 CTL Lines were EBV-specific 29/34 CTL Lines were CMV-specific

13 Are Virus-specific CTL safe?

14 Minimal Related Toxicity Study Peripheral Blood CTL Donor/Recipient Matching # of Patients Acute GvHD Haplo 9 None MUD 6 1 Grade I MRD 10 1 Grade I Cord Blood CTL CBT: 5/6 6 None CBT: 6/6 3 None

15 Do we further delay engraftment in Cord Blood Recipients Receiving only 80%?

16 Cumulative Incidence Probability Similar time to neutrophil engraftment in patients who received only 80% fraction of the CB unit P=0.023 Median: 20 days Median: 25 days 100% fraction (n=19) 80% fraction (n=12)) Days post transplant

17 Patient Characteristics Group Median Age (range) Alternative Donors Campath or ATG in vivo Median day CTL infused Off Immune Suppression Peripheral blood CTL 10 years (1-62) 84% (21/25) 90% (23/25) +84 (35-164) 63% (14/25) Cord Blood CTL 17 months (5-60) 100% 0 92 (84-146) 0 Most patients received transplant for malignant disease Haploidentical donors included (n = 9) 7 patients had Adv infection 11 patients had CMV infection/reactivation 8 patients had EBV reactivation

18 Adenovirus, EBV, and CMV infection after CTL administration

19 Multi-virus CTL Protect Against EBV 8/34 patients had EBV reactivation 8/8 patients had decrease in EBV viral load with coinciding elevation in EBV-specific CTL in PB No additional antiviral therapy required EBV DNA EBV T cell 70,000 60,000 SFC per 2x10 5 cells ,000 40,000 30,000 20,000 EBV copies/ug DNA 50 10, Pre CTL wk1 wk2 wk4 wk5 wk6 wk8

20 EBV Copies/ug DNA Spots per 100,000 cells EBV-specific T cell Responses in CB P EBV Load EBV-T cells Month Post CTL Infusion 0

21 Multi-virus CTL Protect Against Adenovirus 7/7 patients with adenoviral infection (stool/blood) cleared virus after 1-2 doses of CTL 1/1 patient with adenovirus disease treated for progressive Adv pneumonia Cleared the infection from lungs post CTL

22 Multi-virus CTL Protect Against CMV 11/34 patients developed CMV reactivation immediately pre or post CTL infusion 9/11 cleared virus long term (>12 months) without additional antivirals 1 patient decreased viral load before Foscarnet 1 patient died of CMV

23 CMV copies/ml CMV copies/ml CMV specific T cell Responses in Patient with CMV Reactivation Spots/2x10e5 cells T Cells CMV DNA/mL pp65-specific CTL CMV DNA/mL T Cells Pre Pre 1st 1st infusion week 11 week 2 Pre 2nd infusion week 1 week 88 Pre 1 2 Pre 1 8 Weeks post CTL infusion 0

24 Do Infused T cells Persist?

25 Duration of CTL Persistence Depends on Viral Reactivation % CTL TCRs No Reactivation (P3275) CMV, adeno Reactivation (P2891) Pre Infusion Month 1 Month 6 % PBMC TCRs

26 Infused CTL Clones Persist and Expand in vivo in P2891 % of all TCRs CASSIKGNNNSP 8 4 Pre Month 1 Month 3 Month 6 Month 12

27 Summary: PB Multi-virus CTL Protective and Efficacious in vivo Routinely generate multi-virus CTL from Cord Blood and peripheral blood of healthy donors Virus-specific CTL appear to be safe and protective in vivo T cell clones derived from CTL product are detected in the peripheral blood for up to 12 months in CBT Recipients Overall response rate is 93%

28 Conclusions and Significance: Virus-specific T cells after stem cell transplant We can now expand virus-specific CTL from 2 donor sources: cord blood and peripheral blood (BMT) Safe to infuse to patients (minimal toxicity) Persistence of virus-specific T cells in presence of antigen Regardless of source of virus-specific T cells (naïve/memory), both populations appear protective Need to determine whether naïve-derived CTL as efficacious as memory-derived CTL