Cellular Immune Therapy with Allogeneic Stem Cell Transplantation

1. Cellular Immune Therapy with Allogeneic Stem Cell Transplantation Richard Champlin, M.D.

2. Hematopoietic Stem Cell Transplantation D D Preparative Regimen D D HSCT D R R D R D RL D RL D

3. Cell Therapy Allogeneic SCT • High dose chemotherapy/radiation usually does not eradicate malignancy • Higher relapse rate with identical twin or with T-cell depletion • Reduced relapse with GVHD • Allogeneic GVL effect responsible for eradicating residual disease.

4. Hematopoietic Transplantation Dsc Preparative Regimen DT D Cellular Immune Therapy D D DT D +DLI HSCT DT DB Dsc R R DNK Dsc R DT DNK RL D R RL RL D Recipient Donor Mixed Chimera Complete Chimera

5. Relapse is main cause of treatment failure with Allogeneic HSCT for AML

6. Fundamental Problems with HSCT • Graft-vs.-malignancy which naturally occurs post transplant is relatively weak • Graft vs. Malignancy associated with GVHD • Relapse remains the major cause of treatment failure • Resistant infections can occur due to post transplant immune deficiency

7. Prevention of GVHD

8. Regulatory T-Cells (Tregs) • T-cells that down regulate immune responses termed regulatory T cells have been identified. • CD4+CD25+FoxP3+ • Challenge to separate from Tconv • Cord Blood vs. Peripheral Blood • Can suppress GVHD • Clinical Trials • Natural T regs • Inducible T regs

9. Anti-CD3/antiCD28-coated beads. Supplemented with IL-2 300 IU/mL Cord Blood Treg Expansion and Activation CD25 Selection Culture Reduced incidence of grade II-IV aGVHD (43% vs 61%) Brunsteinet al Blood 2011

10. Clinical outcomes of patients after nonmyeloablative umbilical cord blood transplantation who received Treg ≥ 30 × 105/kg (dotted line; n = 18) and historical controls (solid line; n = 108). Brunstein C G et al. Blood 2011;117:1061-1070

11. Questions with Tregs • Production process, separation of Tregs from Tconv • Cell Dose • Administration with calcineurin inhibitors vs. sirolimus • Impact on GVL effects?

12. Suicide Switch to Abrogate GVHD • Genetically modify T-cells to introduce gene to induce apoptosis upon treatment with an activating drug • Herpes virus tyrosine kinase – activated with ganciclovir • Modified Caspace 9

14. Di Stasi et al NEJM 2011

15. Rapid Reversal of GVHD after Rx with AP1903. Di Stasi A et al. N Engl J Med 2011;365:1673-1683

16. Anti viral T-cells

17. Multimer IFN- Gamma interferon selection Multimer selection Gamma Capture of Antigen Reactive T-cells CTL IFN- Feasible for high frequency T-cell responses: EBV, CMV

18. Cultured anti-viral CTLs Cytokines+IL4/7 CTL EBV – EBNA1, LMP2, BZLF1 CMV – IE1, pp65 Adv – Hexon, Penton BK – LT and VP1 HHV6 – U11, U14, U90 PBMC T cell stimulation/ expansion

19. Anti Viral T-cells • Initial studies indicate feasibility and suggest efficacy (CMV, EBV) • Effective for EBV-LPD • Rapid production techniques have been developed • Difficult to use in patients with GVHD-must avoid high dose steroids • Donor specific products • Off the shelf 3rd party CTLs under study

20. Induction of Graft-vs-Malignancy EffectsDonor lymphocyte InfusionsAntigen specific CTLsChimeric Antigen Receptor T-cells

21. Donor Lymphocyte Infusion • Effective treatment for EBV-LPD, relapsed CML, CLL, indolent NHL; less effective for relapsed AML and ALL • Planned DLI studied to enhance GVM effects • Frequently complicated by GVHD • Related to cell dose, time post transplant • Escalating cell dose

22. Targets for Graft-vs.-Malignancy Allo-Specific Malignancy Specific Broadly expressed minor histocompatibility antigen (GVHD) Idiotype, Fusion peptide of translocation (bcr-abl) Lineage restricted minor histocompatibility antigen (G-vs-hematopoietic), or Redirected CAR T-cells vs CD19 Aberrant overexpressed normal cellular constituent (Proteinase 3, WT1, telomerase)

24. Antigen-Specific Immune Therapy for AML PR1-CTL are naturally enriched (0.1-0.4%) in fetal cord blood PR1-CTL TCR PR1 peptide HLA-A2 NE PR1/HLA-A2 P3 Proteasome PR1 Leukemia No AML Clinical trials with cord blood-derived PR1-CTL are ongoing for transplant recipients (AML, CML) AML Shared Resources Flow Cytometry and Cellular Imaging Facility, Genetically Engineered Mouse Facility, Monoclonal Antibody Facility; Clinical Trials Support Resource Molldrem et al

25. Redirect T-cell Specificity through the Introduction of Chimeric Antigen Receptors (CARs) TCR-complex Antibody a b vL g e e d vL Fab z z vH vH CL CL CH1 CH1 vH vL Chimeric antigen receptor

26. Production Methods • Retroviral vectors • Letiviral vectors • Non viral systems, Sleeping Beauty • Expansion using artificial APCs

27. Sleeping Beauty Transposition Transposase Gene X Transposon Transposon (Donor) sequences flanked by inverted repeats are integrated into genome Transposase (Helper) expression is transient Cytoplasm Nucleus Cooper et al Cancer Res 2008

28. 2nd and 3rd Generation Chimeric Antigen ReceptorsPropagation on Artificial APCs 41BB Cooper et al

29. Chimeric Antigen Receptor T-cells • Can target nonimmunogenic targets, tissue/tumor specific antigens. Most experience targeting CD19 for B-cell lymphomas, CLL and ALL • First, second and third generation constructs including costimulatory molecules CD28, CD137 enhance survival of the cells in vivo and their proliferation • Optimal design of CAR not established • Affinity of antibody receptor, spacer, costimulatory molecules, coexpressed receptors, homing molecules

30. Clinical Trials of CAR T-cells • lymphodepleting chemotherapy and autologous CAR T-cells • some complete remissions, eradicating CD19+ cells (reported studies N=32; CR-3 PR-10) • Small number of HSCT patients treated with autologous or allogeneic CAR+ cells • Durable elimination of CD19+ normal B-cells

31. Anti CD19 CAR T-cells for CLL Porter DL et al. N Engl J Med 2011;365:725-733

32. Serum and Bone Marrow Cytokines before and after Chimeric Antigen Receptor T-Cell Infusion. Porter DL et al. N Engl J Med 2011;365:725-733

33. CAR Problem Areas • Autologous vs. Allogeneic • Survival, homing in vivo • In vivo expansion needed for activity • Toxicity, “cytokine storm” may occur, particularly with CD137 containing CARs- can produce respiratory failure • Time/ expense in producing patient specific products • Complex, regulatory considerations make multicenter studies difficult

34. “Off-the-shelf” CD19-specific CAR+T Cells for Adoptive Immunotherapy Cooper et al Blood 2010

35. NK Cells

36. NK Cells • Component of innate immune system • CD3- TCR-, CD16+, CD56+ • Mediates anti-tumor, anti-viral, BM rejection • Activating and inhibitory receptors (KIR) • Cytotoxicity governed by missing ligand hypothesis re: inhibitory receptors • Cw alleles that bind to KIR2DL1 have amino acid K at position 80. • Cw alleles that bind to KIR2DL2 or to KIR2DL3 have amino acid N at position 80 • Bw4 or Bw6, KIR 3DL1 amino acids at positions 82-83 • Missing ligand model has “not” predicted responses in most clinical trials

37. NK Cell Receptors Murphy et al Biology of Blood and Marrow Transplantation 2012; 18:S2-S7

38. NK NK NK Kill recipient APCs = protection from GvHD Donor alloreactive NK cells DC DC DC Lysis Lysis leukemia Lysis Kill leukemia = GvL effect T T T T T T Kill recipient T cells = improved engraftment

39. NK Cells- Clinical • NK reactivity reported to reduce relapse in AML following haploidentical transplants • Human studies infusing “selected” NK cells (CD3-depleted +/- CD56 selected) demonstrate safety, activity. • Limited by low and variable frequency (5-15%) in normal donors, cannot collect more than 106/kg by apheresis • NK cells already in PBPC, CB or BMT, adding low doses from donor unlikely to benefit • Ex vivo expansion feasible, entering human clinical studies

40. 4 Log expansion of NK cells using mbIL21 APCs Cryopreserve in aliquots

41. Donor, Haploidentical or Cord Blood NK Cells Allo match PBPC Busulfan Fludarabine IL-2 Haploidentical Allo reactive NK Cells Haplo BMT Cy-tacro-MMF Melphalan Fludarabine Haploidentical Allo reactive NK Cells Flag-ida IL-2 or IL-15

43. Conclusions • Adoptive cellular immunotherapy is a promising novel treatment modality for treatment of cancer. • Cellular immune therapy is a promising approach to control alloreactivity to prevent GVHD. Tregssuccessful to prevent GVHD in mice; improved approaches needed to achieve similar benefit in man. • Antigen specific CTLs and CAR T-cells can eradicate experimental tumors. Preliminary human clinical trials have been performed with autologous and allogeneic cells, demonstrating activity and feasibility in conjunction with HSCT.

44. Where do we go from here? • Rapidly evolving technology; optimal cellular designs and production methods need to be determined. • Need widely accepted products which can be taken into larger scale phsae II and III clinical trials. • The needed multicenter “gene therapy” trials will costly and complex to administer