Targeted Immunotherapeutic Strategies in the Management of Viral Infections and Caspase-9 Suicide Gene Strategies in Controlling Engineered T-cells

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1 Targeted Immunotherapeutic Strategies in the Management of Viral Infections and Caspase-9 Suicide Gene Strategies in Controlling Engineered T-cells Helen Heslop Disclosure Licensing agreement with Cell Medica for EBV-specific T cells in NHL and nasopharyngeal cancer Founder Viracyte - third party monovirus T cells BCM IP in Bellicum All trials use investigational IND therapies 1

2 Viral Infections Post Transplant Major cause morbidity and mortality Pharmacologic therapy not available for all viruses and expensive Recurrences when therapy stopped Clearly related to lack of virus specific T cell response 2

3 EB-VST Generation Lymphocyte s EBV LCL 1 LCL generation (4-6 weeks) Donor IL-2 CTL 2 VST expansion (4-6 weeks) 3 QC/QA (1-2 weeks) Smith et al J Hematother Apr;4(2):

4 EB-VSTs Post HSCT Small numbers ( / kg) Restore virus-specific immunity Reduce virus load Cure disease in over 8% Long-lasting protection Low toxicity Trivirus-Specific T Cells EBV, CMV and Adenoviruses 3 most common viral complications after HSCT Most donors immune Have detectable levels of T cells EBV CMV Adv 4

5 Generation Of Multivirus-specific VSTs Using Ad5f35 Vectors Clinical Outcome of Tri-VSTs In vitro expanded donor-derived VSTs targeting Adv, EBV, CMV Reconstituted antiviral immunity for EBV, CMV and Adv Effective in clearing disease Considerable expansion in vivo 3 SFC per 1 x 1 6 cells Pre CTL Adv T cell Adv DNA (trachea) 1,4 1,2 1, wk 4 wk 6 wk 8 wk Leen et al, Nat Med. 26 Leen et al, Nat Med 26 Adv copies/ml 5

6 Donor Virus Specific T cells First report of activity against CMV in 1992 (Riddell, Greenberg) Studies in multiple centers show Effective as prophylaxis Effective as therapy in over 8% patients But still boutique therapy * * NIH ARRA reviewer 29 Why are VSTs Not More Widely Used? Manufacture with original methods complicated lengthy and expensive Individualized products Impractical for widespread or urgent use Cannot make from seropositive donors 6

7 How Do We Extend Applicability? Use bank of allogeneic matched VSTs Simplify production patient specific product Donor-derived VSTs AdV Hexon, Penton EBV EBNA1, LMP2, BZLF1 CMV IE1, pp65 BKV LT, VP1 HHV6 U11, U14, U9 Ulrike Gerdemann Ann Leen +IL4/7 T cell stimulation/ expansion 1 days mvsts (multivirus VSTs) 7

8 Clinical Protocol Prophylaxis/treatment of EBV, CMV, Adv, BK and/or HHV6 Single infusion of mctls: 5x1 6-2x1 7 /m 2 Activity in EBV-PTLD Pre mvst Post mvst 1 6 EBV (copies/ug DNA) T cells Viral load SFC/5x

9 1 Activity Against Adenovirus VSTs T cells 25 Copies/ml Viral load SFC/2x1 5 Clinical Response - BKV 1 pvsts Blood 8 BKV copies/ml Viral load BKV T cells SFC/5x1 5 BKV copies/ml 1 1.E+1 1.E+8 1.E+6 1.E+4 1.E+2 1.E+ wk-3 wk-2 wk-1 Infusion wk1 wk2 wk3 wk4 wk6 wk8 wk1 wk12 Viral load pvsts Bladder-derived T cells Urine wk5 wk-3 wk-2 wk-1 Infusion wk1 wk2 wk3 wk4 wk6 wk8 wk1 wk12 SFC/5x1 5 BKV control 9

10 # of pts 4 viruses 1 3 viruses 2 2 viruses 3 1 virus 2 Summary Virus EBV, CMV, BK, HHV6 EBV, CMV, BK EBV, BK, HHV6 CMV, BK BK, EBV BK, EBV Adv BK Best outcome at wk12 CR, CR, PR, CR CRs CR, NR, CR PR, CR CRs PRs CR CR CR: complete response; PR: partial response; NR: No response Papadopolou et al Science Trans Med 214 Rooney, et al, Lancet EBV Heslop, et al, Nat Med EBV EBV Rooney, et al, Blood EBV, CMV, AdV Leen et al, Nat. Med. EBV, AdV Leen, et al, Blood Cytomegalovirus Adenovirus EBV, CMV, AdV et al, Mol. Ther. Papadopoulou, et al, STM Epstein-Barr Virus VST timeline EBV, CMV, AdV, HHV6 BK virus HHV6 3 months 1 virus Safety and efficacy maintained 1 days 5 viruses 1

11 Broader Implementation Of VSTs Problem 1: Seronegative donors Use of cord blood Use of younger donors Problem 2: Production (1 days) and release (7-1 days) time does not allow urgent use Can we use VSTs as an off the shelf product? 3 rd Party VST Therapy Bank of VSTs Infected Patients Cryopreservation HLA - A HLA - B HLA - A HLA - B HLA - DR HLA - DR HLA - A 11

12 Most Closely HLA Matched Allogeneic VSTs to Treat Persistent Reactivation or Infection with Adenovirus, CMV and EBV after Hemopoietic Stem Cell Transplantation CAGT Helen Heslop Ann Leen Clio Rooney Cath Bollard Malcolm Brenner Adrian Gee Other Sites MDACC EJ Shpall Harvard Joe Antin, B Dey Duke Paul Szabolcs CHLA Neena Kapoor Children s Boston Sun Yun Pai Miami Gary Kleiner Hackensack Scott Rowley SCCT Multicenter Study of Multivirus CTLs Cumulative Incidence CR/PR 1..8 Cumulative Incidence of First CR/PR N= Cumulative Incidence of First CR/PR by Infection Probability.6.4 Probability Days Post VST Infusion At day 42: Overall 74. (95% CI: ) Days Post VST Infusion At day 42: CMV 73.9 (95% CI: ) EBV 66.7 (95% CI: ) AdV 77.8 (95% CI: ) 71% CMV (N=23) EBV (N=9) Adenovirus (N=18) Leen et al Blood

13 Multivirus-directed T Cell Lines Targeting Adv, CMV, EBV, BK, HHV6 AdV Hexon, Penton EBV EBNA1, LMP2, BZLF1 CMV IE1, pp65 BKV LT, VP1 HHV6 U11, U14, U9 Ifigineia Tzannou Bilal Omer +cytokines T cell stimulation/ expansion 1 days mvsts (multivirus VSTs) Clinical Protocol Single center, phase II study Treatment of refractory EBV and/or CMV and/or AdV and/or BKV and/or HHV-6 2 part process Screen for a suitable line Matched at least 1 HLA allele Activity against infecting virus through shared allele Treatment 13

14 Treated 28 Patients With 31 Infections Viruses CMV EBV Adv BKV HHV6 1 virus 2 viruses VST Safety Acute GVHD within 45 days of infusion 1 patient developed grade I skin GVHD Resolved with topical steroids 1 chronic skin GVHD flare (discontinued immunosuppression) 2 patients with transient fevers 14

15 Clinical Response CMV copies/ml VSTs T cells Viral load SFC/5x1 5 Month -2 Month -1 pre-infusion Week1 Week 2 Week 4 Week 6 Clinical Response BKV viral load plasma 12 VSTs viral load urine 9 copies/ml copies/ml pre-infusion Week2 Week 3 Week 4 15

16 Outcomes: 28 Patients with 31 Infections Viruses CMV EBV Adv BKV HHV6 1 virus 2 viruses 9% Response Rate Viruses CMV EBV Adv BKV HHV6 1 virus 2 viruses PR PR PR PR PR PR PR PR PR PR PR PR NE 16

17 Published Studies with Third Party VSTs Virus Responses GVHD Study EBV 17/33 None Haque et al 27 EBV 4/5 None Doubrovina et al 212 EBV 8/1 None Vickers et al 214 EBV,CMV,ADV 37/5 8/5 Leen et al 213 EBV,CMV,ADC 4/6 None Uhlin et al 212 EBV,CMV,ADV 2/2 1/2 Withers et al Tandem BMT 216 EBV,CMV,ADV, BK,HHV6 2/22 2/22 Tzannou et al Tandem BMT 216 What Are Requirements For Banked Cells? Donor evaluation Level of testing of banked lines Edinburgh group manufactured new bank with optimal donors 17

18 Screening Additional Donors for Third Party VST Studies Normal adult donors Health care providers Age and occupation result in more virus exposure 4 viruses 3 5 viruses Rayne Rouce Ifigenia Tzannou What Type of VST is Best for Third Party Setting? Multivirus for broader coverage Monovirus as less risk competition may result in broader activity against infecting virus One stimulation versus two stimulations 18

19 VSTs After HSCT Low attributable toxicity Confirmed activity in multiple centers Sustained response rates 8-9% in donor specific studies 6-7% with third party CTLs Rapid manufacturing methodologies facilitate definitive clinical trials Important to include comparative effectiveness analyses Can we provide a T cell product with antiviral and anti tumor activity without alloreactivity? 19

20 24 Improving Efficacy And Safety Of T-cell Add Back Allo- depleted T-cells Ex-vivo allodepletion of T-cells using anti-cd25 immunotoxin allows for safer T-cell add back At cells/kg decreases rate of death from viral disease CD34+ selected Haplo transplants However, rate of relapse remains high possibly due to lower frequency of tumor-specific precursors in infused cells Amrolia et al, Blood 26 Improving Efficacy And Safety Of T-cell Add Back Allo- depleted T-cells Allo-depleted T-cells with suicide gene Increase number of allodepleted T cells by dose escalation from 1 6 cells/kg to 1 7 cells/kg CD34+ selected Haplo transplants To further ensure safety incorporate an inducible suicide gene in allodepleted T-cells DiStasi et al, NEJM 211 2

21 Improving Efficacy And Safety Of T-cell Add Back AP193 icasp 9 suicide gene relies on mitochondrial apoptotic pathway FKBP12 domain Caspase9 domain Upon activation induces apoptosis within minutes Caspase 3 activation Cell apoptosis DiStasi et al, NEJM 211 Inducible Caspase 9 (ic9).2a.dcd19 Transgene Construct Inducible Casp9 Suicide Gene Selectable Marker 5 LTR FKBP12-F36V Caspase 9 2A CD19 3 LTR SGGGS Tey SK et al. Biol Blood Marrow Transplant 13 (27) 21

22 CASPALLO Trial Overview Product generation time : 1 weeks LCL generation Donor PBMC + recipient LCL Allodepletion Expansion CD19 selection Haplo SCT -8 wks OKT3 activation icasp9.cd19 transduction Cryopreservation ic9 T-cell Infusion CASPALLO Trial Outcome Demonstrated safety and efficacy of the ic9 suicide gene system Infused ic-9 T cells Expand and persist long-term Enhance antimicrobial immunity Accelerate endogenous immune recovery Activation of suicide gene system Rapidly and safely abrogates GVHD No effect on antimicrobial responses DiStasi et al, NEJM 21 Zhou et al, Blood

23 Can We Simplify The Process By Using In Vivo Allodepletion Alone? Allo- depleted T-cells Allo-depleted T-cells with suicide gene Allo-replete T-cells with suicide gene CD34+ selected Haplo transplants Shorten the manufacture time Simplify process and increase scalability Potentially preserve tumor specific precursors by excluding ex-vivo allodepletion DOTTI Trial Overview Product generation time : 11 days Donor T-cell activation Expansion dcd19 selection Haplo SCT Day icasp9.dcd19 transduction Cryopreservation ic9 T-cell Infusion 23

24 Study Design Patients: Recipients of CD34+ selected haploidentical transplants Dose escalation schedule: Dose level 1: 1x1 4 cells/kg Dose level 2: 1x1 5 cells/kg Dose level 3: 5x1 5 cells/kg Dose level 4: 1x1 6 cells/kg Dose level 5: 5x1 6 cells/kg Measurement: Immune reconstitution Functional studies GvHD No GVHD GVHD grade I GVHD grade II/> GVHD management Monitor AP193 Day (2) (4) (6) AP193 Day (2) (4) (6) Steroids Patient Characteristics Pt Age (yrs) Diagnosis Disease status at HSCT Infused T-cells / kg (number of infusions) #1 49 T-cell ALL Active 1x1 4 (1) #2 4 ALL (CNS) CR2 1x1 4 (1) #3 2 JMML Active 1x1 5 (1) #4 1 ALL CR3 1x1 5 (1) #5 1 MDS CR3 1x1 5 (1) #6 5 T-cell ALL CR3 5x1 5 (1) #7 4 EBV-LPD Active 5x1 5 (2) #8 15 MDS CR3 1x1 6 (1) #9 8 HLH (CNS) Extensive prior therapy 1x1 6 (1) #1 9 AML CR2 5x1 6 (1) #11 17 ALL CR1 5x1 6 (1) #12 19 AML CR2 5x1 6 (1) 24

25 DOTTI Results Can haploidentical alloreplete ic9 T-cells engraft and persist? What is the effect on endogenous immune reconstitution? Does the use of AP193 eliminate alloreactive ic9 T-cells? Do the ic9 T cells contribute to anti viral activity in vivo? Can toxicities be managed effectively? Can The ic9 Alloreplete Haploidentical Cells Engraft And Persist? CD3 + cells/µl CD3 + CD19 + T cells Pt #1 Pt #2 Pt #3 Pt #4 Pt #5 Pt #6 Pt #7 Pt #8 Pt #9 Pt #1 Pt #11 Pt # Months after T-cell infusion 12 25

26 What Is The Effect On Endogenous Immune Recovery? CD3+ cells / µl CD3 + CD19 T cells Pt #1 Pt #2 Pt #3 Pt #4 Pt #5 Pt #6 Pt #7 Pt #8 Pt #9 Pt #1 Pt #11 Pt # Months after T-cell infusion 12 Does AP193 Deplete Alloreactive T Cells In-vivo? Pt # 9 developed skin GVHD, not responsive to topical therapy. 6 AP193 CD3+19+ CD4+19+ CD8+19+ CD3+ cells / µl Months after T-cell infusion Despite re-proliferation of IC-9 T-cells there was no recurrence of GVHD 26

27 Effect Of AP193 On CNS T-cell Infiltration Pt. # 6 had disseminated 55 days after T cell VZV infusion at time of ic9 T-cell infusion with 9 days recovery before AP193 1 week after infusion A Detection of ic9-t cells in CSF 25% C 79 days after T cell infusion 14 days after AP193.6% 75% 99% B D Detection of ic9-t cells in PB CD19 33% 66% 2.5% 97.5% CD3 Do ic9 T Cells Contribute To Antiviral Activity In-vivo? CMV Pt. #12 5 without AP193 with AP193 (in vitro) CMV viral load 4 SFC / 5x1 5 PBMCs CMV copies/ ml Weeks after T-cell infusion 27

28 Do Ic9 T Cells Contribute To Antiviral Activity In-vivo? HHV6 Pt. # 11 SFC / 5x1 5 PBMC without AP193 with AP193 (in vitro) HHV6 viral load HHV6 Copies/ ml Months after T-cell infusion Similar responses were noted in patients with other viruses including EBV, Adeno, BK and VZV Can The Safety Switch Be Turned On In The Event Of A Cytokine Release Syndrome? Pt. # 8 presented with high fever, 3 weeks post infusion Temp (F ) AP193 Counts / µl CD3+19+ CD4+19+ CD8+19+ AP Times to AP193 (day) Weeks after T-cell infusion 28

29 Can The Safety Switch Be Turned On In The Event Of A Cytokine Release Syndrome? Concentration (pg / ml) Pre-AP hrs after 48 hrs after IL-6 IL-13 IFN TNF IL-1RA IL-8 IL-5 IL-1 Can The Safety Switch Be Turned On In The Event Of A Cytokine Release Syndrome? Before AP min after initiation of AP193 administration 29

30 Clinical Outcome CASPALLO DOTTI Number of patients 1 12 GVHD 4 3 Response to AP Recurrence of GVHD Relapse 4 4 Viral reactivations Viral disease 2 Deaths 5 5 Viral disease Relapse 4 3 Other 1 2 Relapse remains an ongoing concern Changing The Platform To Reduce Relapse Rates Allo- depleted T-cells Allo-depleted T-cells with suicide gene Allo-replete T-cells with suicide gene Allo-replete T-cells with suicide gene CD34+ selected Haplo transplants TCR αβ depleted Haplo transplants 3

31 Effect of BP-51 On Immune Reconstitution CD3+ T Cell Recovery in All BP-4 Pts CD3+ T Cell Recovery in Non-Malignant Pts Cells / ul Cells / ul Time from HSCT days Time from HSCT days In non-malignant patients who received BP-51, a mean improvement of 4 days in achieving a T cell count of 5 cells/ul was seen when compared to this site s historical controls In all BP-4 patients, a mean improvement was detected of approximately 25 days in achieving a T cell count of 5 cells/ul when compared to historical controls Bertaina/Locatelli 215 ASH Conclusions ic9 safety system can rapidly, effectively and sustainably remove alloreactive T cells causing GvHD after haplo-hsct Infusion of ic9-t cells leads to rapid immune reconstitution with effective anti-viral immunity that persisted after in vivo depletion of alloreactive cells. Activation of the ic9 transgene also rapidly controlled symptoms and signs related to cytokine release syndrome and depleted ic9-t cells not only in the PB but also in the CNS. 31

32 TRL Lab PIs Cliona Rooney Malcolm Brenner Ann Leen Stephen Gottschalk Nabil Ahmed Juan Vera Carlos Ramos Caroline Arber Transplant Service Bob Krance Kathy Leung Caridad Martinez George Carrum Ram Kamble Premal Lulla Swati Naik Cath Bollard Acknowledgements TRL Laboratory Lisa Rollins Olga Dakova Clinical Research Bambi Grilley Bridget Medina Milica Stojavic Kristal Black Yu-Feng Lin Vicky Torrano Amy Reyna GMP Laboratory Adrian Gee Natasha Lapteva Debbie Lyon Zhuyong Mei TRL Junior Faculty/ Postdocs/PhD students Bilal Omer Robin Parihar Rayne Rouce Meena Hegde Andras Hegzey Paulina Velasquez Chris Derenzo Max Mamonkin Serena Perna Ulrike Gerdemann Anastasia Papadopolou Ifigeneia Tzannou Sandhya Sharma Minhtran Ngo T cell Laboratory Huimin Zhang Tamara Trpic Pallavi Mohpatra Birju Mehta SCCT CHALLAH STUDY Joe Antin B Dey David Avigan Paul Szabolcs EJ Shpall Neena Kapoor EMMES Adam Mendizabal NMDP Dennis Confer Icasp 9 Antonio Distasi iaoou Zhou David Spencer Siok Tey Gianpietro Dotti Barbara Savoldo Funding: NCI Program Project Grant, NHLBI Somatic Cell Therapy Center, Lymphoma SPORE, Leukemia and Lymphoma Society Specialized Center of Research, Doris Duke Distinguished Clinical Scientist Award, PACT 32