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
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)
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
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
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
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
T cells From CB Recognize Adenovirus, CMV, and EBV Spots per 1x10e5 cells 1000 100 10 1 n=9 CTL lines Irrelevant peptide Adv CMV pp65 EBV 0 1 2 3 4 Hanley et al, Blood 2009
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
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
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
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+
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 (28-1278) 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
Are Virus-specific CTL safe?
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
Do we further delay engraftment in Cord Blood Recipients Receiving only 80%?
Cumulative Incidence Probability 0.0 0.2 0.4 0.6 0.8 1.0 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)) 0 10 20 30 40 50 Days post transplant
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
Adenovirus, EBV, and CMV infection after CTL administration
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 350 300 EBV DNA EBV T cell 70,000 60,000 SFC per 2x10 5 cells 250 200 150 100 50,000 40,000 30,000 20,000 EBV copies/ug DNA 50 10,000 0 0 Pre CTL wk1 wk2 wk4 wk5 wk6 wk8
EBV Copies/ug DNA Spots per 100,000 cells EBV-specific T cell Responses in CB P3923 2500 2000 EBV Load EBV-T cells 20 15 1500 1000 10 500 5 0 1 2 3 Month Post CTL Infusion 0
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
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
CMV copies/ml CMV copies/ml CMV specific T cell Responses in Patient with CMV Reactivation Spots/2x10e5 cells T Cells 2000 2000 1500 1500 CMV DNA/mL pp65-specific CTL CMV DNA/mL 200 150 1000 1000 T Cells 100 500 500 50 0 0 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
Do Infused T cells Persist?
Duration of CTL Persistence Depends on Viral Reactivation % CTL TCRs No Reactivation (P3275) 10 10 10 0 0 0 10 10 10 CMV, adeno Reactivation (P2891) 0 0 0 Pre Infusion Month 1 Month 6 % PBMC TCRs
Infused CTL Clones Persist and Expand in vivo in P2891 % of all TCRs 16 12 CASSIKGNNNSP 8 4 Pre Month 1 Month 3 Month 6 Month 12
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%
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