Stem Cell Sources 2/22/13. Cellular Therapy Today and Tomorrow. Cellular Therapy in HCT. Bone Marrow

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1 2/22/13 Cellular Therapy Today and Tomorrow Robert S. Negrin, MD Division Chief, Stanford Bone and Marrow Transplant Program Professor of Medicine Cellular Therapy in Clinical Medicine Established Hematopoietic cell or bone marrow transplantation Under investigation Immune cell therapy for cancer and infectious diseases Stem cell therapy for heart attacks and stroke Mesenchymal stromal cell therapy in orthopedics Islet cell transplantation for diabetes Future prospects Stem cell therapies for broad range of disorders Cellular Therapy in HCT Hematopoietic cell transplantation Disease control Cytotoxic T cells NK cells CIK cells Memory T cells Dendritic cells Treatment of infections CTLs Acceleration of immune reconstitution CLP, CMP Treatment and prevention of GVHD MSCs Treg Stem Cell Sources Bone Marrow Mobilized Peripheral Blood G-CSF mobilized Chemotherapy plus G-CSF Other cytokines Cord Blood 1

2 Theoretical Considerations Challenges Donor Hematopoiesis HSCs, T Rejection T, NK GVHD, GVT T, NK Recipient Biological Do the principles apply? How different are the preclinical models from the clinical situation? Technical Cell separation techniques (columns, FACS, cell expansion) Availability of reagents Laboratory requirements Regulatory Imaging Do the cells traffic to the appropriate locations? How long do they survive? Cost Response to Donor Leukocyte Infusions in CML Patients Generation of LMP2-specific CTL with LMP2-transduced DC & LCL Stimulations Adf3 LMP2 DC LCL IL2 IL2 IL2 Dazzi et al. Blood :212, 2 PBMC LMP2- specific CTL Pre CTL Post CTL Complete Radiological Response EBV+ve NK-T NHL Pre CTL 1 Pre CTL 2 Pre CTL 3 Probability of EBV lymphoma

3 Expansion of CTLs Potential Targets Day Polyclonal Activation of T Cells with OKT3 Day 3 Electroporation of OKT3- Activated PBMC Day Addition of Selection Drug Day 14 Cloning of Surviving T Cells Day 2 Expansion of Drug- Resistant Clones Day 42 Large Scale Expansion of scfvfc:ζ+ Clones for Re-Infusion Tumor specific Bcr/abl Wt-1 Lineage specific Minor HAG CD1, CD2 PR1 Idiotype Viral EBV Virus Specific T Cells Products Donor Specific Direct-selection strategies in licensing trials in Europe companies will likely lead US studies Rapid donor specific T cells using peptides and - day culture in studies in several centers Third Party Studies with EBV, CMV and multivirus Greatest need probably CMV Multimer Direct Selection T Cells Multimer selection CTL IFN-γ IFN-γ Gamma interferon selection Rapid CTLs Administration of Rapidly Generated Multivirus-Specific Cytotoxic T-Lymphocytes for the Prophylaxis and Treatment of EBV, CMV, Adenovirus, HHV, and BK virus Infections post Allogeneic Stem Cell Transplant Pepmix mastermix EBV EBNA1, LMP2, BZLF1 CMV IE1, pp Adv Hexon, Penton BK LT and VP1 HHV U11, U14, U pepmix +IL4/ PBMC +IL4/IL CTL T cell stimulation/ expansion days Gerdemann et al, 212, Mol Ther; Gerdemann et al, 213, Blood Natural Killer Cells -% of peripheral blood lymphocytes. Large granulated cells morphologically. Capable of killing virally infected and tumor cells without prior activation. Can be further activated with cytokines such as IL-2, IL-12 and IL-1. 3

4 2/22/13 Haploidentical NK cell therapy in a non-transplant setting Collect donor cells Activate T-cell deplete (CliniMACS) Allogeneic NK cells 2 x /kg Rest with IL-2 In vivo expansion Cyclophosphamide mg/kg Fludarabine 2 mg/m2 14 IL-2 MU 3x/week x doses JS Miller et al, Blood :31, 2 Cytokine Induced Killer Cells (CIK Cells) Day : IFN-g Day 1: a-cd3 and IL-2 Day 21: Expansion of T cells which co-express NK cell markers In vitro and in vivo antitumor cytotoxicity PBMC (human)! Splenocytes (mouse)! 12 d 1 h. h Dual Viral-Cell Combination EffectsTherapy In Vivo CIK! (NK T Cells) IL2! αcd3! Day 1! A2 Lymphoma 3 d Dual Biotherapy IFNγ! CIK Cell Trafficking and Tumor Infiltration 2 UCI-1 12 IL2! Day! Day! CD3+ NK1.1+ (or DX +) i.v. injection! Monitor tumor! burden, viral distribution! or CIK cell distribution! C! CIK+vvDD Time (Days) Days post treatment PBS CIK+vvDD CIK vvdd 2 Time (Days) Photons/mouse/second Broad Specificity! Not MHC restricted! Not TCR mediated! CD1, ICAM, NKG2D! Perforin-mediated! 2 PBS CIK vvdd CIK/vvDD 12 vvdd! SKOV Time (Days) Thorne et al. Science 2 4

5 Apheresed Peripheral Blood (steady state) CIK Process Reinfuse into Patient Chimeric Antigen Receptors Tumor Antigen Monoclonal Antibody TCR complex α β γ ε ε δ ζ ζ Culture overnight with IFNγ (Aim V + % autologous plasma) QC testing Tumor CAR Linker v H v L Add OKT3 and IL-2 Add Media & IL-2 every 3-4 days Harvest (day 21) T T Cell cell CD2 Or 41BB Spacer Intracytoplasmic CD1 as a Target Present on B lineage cells from the pro-b cell stage to mature B cells High expression most B lineage lymphoma and leukemia NOT expressed on hematopoietic stem cells (or other tissues) Should not be myelosuppressive effects Should not be other organ toxicities Will cause depletion normal B cells CD1 CARs Strengths Multiple studies Promising results from some published results in CLL and NHL Recent reports of activity in relapsed ALL pre or post transplant - area where unmet need Reference! Brentjens et al." Targeted disease! CLL " Jensen et al." DLCL or NHL" Construct! Cell Activation! Retrovirus OKT3/ CD3ζ,CD2ζ" CD2" Plasmid CD3ζ" OKT3" Kalos et al." CLL" Lentivirus OKT3/ CD3ζ,4-1BB" CD2" Kochenderfer et al." Savoldo et al." CLL or Follicular Lymphoma" NHL" Total treated = 32 CR 3 PR SD Published Studies Retrovirus OKT3" CD3ζ,CD2ζ Retrovirus CD3ζ, CD2ζ" OKT3/ CD2" Auxiliary Therapy! n! Responses! CTX" " 3 SD " Fludarabine IL-2" Bendamustine or Pentostatin/CTX" CTX/Fludarabine IL-2" None or post auto SCT" 4" 3" 2 CR, 1 PR " " 1CR, PR, 1SD," " 4 SD" Mesenchymal Stromal Cells (MSCs) Adherent cells that can differentiate into a variety of cell types, secrete cytokines and have immunomodulatory activities Treatment of Acute GVHD Organ damage (liver, pulmonary) Poor marrow function 3 rd party banks can be used

6 Clinical Dose Bank with sufficient doses for many individuals Master Cell Bank MSC Banks Harvest from eligible donor Adherent cells cultured Expansion of MSCs Regulatory T Cells Several populations of T cells that regulate immune responses termed regulatory T cells have been identified. Phenotype CD4+CD2+FoxP3+ Can suppress GVHD Many investigators testing these cells in the clinic Natural T regs Expanded T regs Enhanced T regs Inducible T regs Phase I Treatment Plan for Umbilical Cord Bloodderived Regulatory T Cells ntreg Unit Expansion and Activation Culture in Minnesota Separation and start culture of UCB-derived UCB-derived Production Cyclophosphamide Fludarabine Monitori ng -1 (±1) Phase I semi-log fast-track dose escalation: Level 1-1 x /kg Level 2-3 x /kg Level 3 - X /kg Level 4-3 x /kg Level - 3 x /kg (X2) Level ª - 3 x /kg (X2) rapa 1/ MMF Csa CSA or Rapa Single or Double UCBT 2cGy TBI G-CSF UCB-derived UCB-derived Monitori ng CD2+ selection Flask culture Culture in X-VIVO 1 Human AB serum %, Anti-CD3/antiCD2-coated beads. Supplemented with IL-2 3 IU/mL Lot Release Gram stain neg Endotoxin < EU/kg Viability % CD4+/CD2+ % CD3+/CD+ % Sterility neg Mycoplasma neg Bead count < /3 x Isolation of High Purity T reg on a Clinical Scale at Stanford CD34 flow through CD2 selected CD4+/CD12dim sorted Purified nt reg + T con Trial 4.1%.% SSC CD4- FITC SSC CD12- PE FOXP3-APC FoxP3- APC % 2.% CD34 + cell selected graft CD4 + CD2 + CD12 + T CD4 + /CD + T reg con Endpoints: Chimerism FTBI/VP1/Cy or Immune reconstitution BCNU/VP1/Cy or Bu/VP1/Cy Acute and chronic GVHD EFS, OS Percent CD4 + FoxP3 + 4 (1) Yield 1 x (.1. x ) Recovery % (1%) Day - to - +2

7 Clinical Trials of Adoptive Transfer of Regulatory T Cells T reg + T con in Haploidentical transplantation (U. of Perugia) Di Ianni et al. Blood 211 Expanded UBC T reg for GVHD prevention (U. Minnesota) Brunstein et al. Blood 211 T reg + T con in MRD transplantation (Stanford) T reg for treatment of GVHD (planned) Conclusions Cellular Therapy is a reality in clinical medicine Many different concepts Dissemination beyond small single institution clinical trials a major challenge Potential in many different fields beyond HCT

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