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Cellular Immune Therapy with Allogeneic Stem Cell Transplantation
- 1. Cellular Immune Therapy withAllogeneic Stem Cell Transplantation Richard Champlin, M.D.
- 2. HSCT D RL R RL R R D D D D D D D D Hematopoietic Stem CellTransplantation Preparative Regimen
- 3. Cell Therapy AllogeneicSCT • 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. HSCT +DLI DT DNK D RLRL R RL R R DB Dsc DT DNK D DD Dsc D DT DT Dsc D CompleteChimeraRecipient Donor Mixed Chimera Hematopoietic Transplantation Preparative Regimen R Cellular Immune Therapy
- 5. Time(weeks) SurvivalProbability 0 20 4060 80 100 120 0.00.20.40.60.81.0 In remission, PB.blast=0 Active Disease, PB.blast=0 Active Disease, PB.blast>0 p<0.0001 Time(weeks) Event-freeprobability 0 20 40 60 80 100 120 0.00.20.40.60.81.0 In remission, PB.blast=0 Active Disease, PB.blast=0 Active Disease, PB.blast>0 p<0.0001 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.
- 8. • T-cells thatdown 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 Regulatory T-Cells (Tregs)
- 9. Cord Blood TregExpansion and Activation •Anti-CD3/antiCD28-coated beads. •Supplemented with IL-2 300 IU/mL Reduced incidence of grade II-IV aGVHD (43% vs 61%) Brunstein et al Blood 2011 CD25 Selection Culture
- 10. Clinical outcomes ofpatients 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 toAbrogate 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 etal NEJM 2011
- 15. Rapid Reversal ofGVHD after Rx with AP1903. Di Stasi A et al. N Engl J Med 2011;365:1673-1683
- 16.
- 17. CTLMultimer Multimer selection IFN- Gamma interferon selection IFN- GammaCapture of Antigen Reactive T-cells Feasible for high frequency T-cell responses: EBV, CMV
- 18. T cell stimulation/expansion PBMC CTL Cytokines+IL4/ 7 EBV – EBNA1, LMP2, BZLF1 CMV – IE1, pp65 Adv – Hexon, Penton BK – LT and VP1 HHV6 – U11, U14, U90 Cultured anti-viral CTLs
- 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- MalignancyEffects Donor lymphocyte Infusions Antigen specific CTLs Chimeric 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 Broadlyexpressed minor histocompatibility antigen (GVHD) Lineage restricted minor histocompatibility antigen (G-vs-hematopoietic), or Redirected CAR T-cells vs CD19 Aberrant overexpressed normal cellular constituent (Proteinase 3, WT1, telomerase) Allo-Specific Malignancy Specific Idiotype, Fusion peptide of translocation (bcr-abl)
- 24. Shared Resources Flow Cytometryand Cellular Imaging Facility, Genetically Engineered Mouse Facility, Monoclonal Antibody Facility; Clinical Trials Support Resource Antigen-Specific Immune Therapy for AML P3 NE Leukemia PR1 peptide PR1 PR1-CTL Clinical trials with cord blood-derived PR1-CTL are ongoing for transplant recipients (AML, CML) PR1-CTL are naturally enriched (0.1-0.4%) in fetal cord blood AML No AML Molldrem et al
- 25. Redirect T-cell Specificitythrough the Introduction of Chimeric Antigen Receptors (CARs) vL vH CH1 CL Antibody Fab vH vL Chimeric antigen receptor TCR-complex
- 26. Production Methods • Retroviralvectors • Letiviral vectors • Non viral systems, Sleeping Beauty • Expansion using artificial APCs
- 27. Sleeping Beauty Transposition Cytoplasm Nucleus Transposase Transposon GeneX Transposase (Helper) expression is transient Transposon (Donor) sequences flanked by inverted repeats are integrated into genome Cooper et al Cancer Res 2008
- 28. 2nd and 3rdGeneration Chimeric Antigen Receptors Propagation on Artificial APCs Cooper et al 41BB
- 29. Chimeric Antigen ReceptorT-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 ofCAR 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 CART-cells for CLL Porter DL et al. N Engl J Med 2011;365:725-733
- 32. Serum and BoneMarrow 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 Cellsfor Adoptive Immunotherapy Cooper et al Blood 2010
- 35.
- 36. NK Cells • Componentof 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 Murphyet al Biology of Blood and Marrow Transplantation 2012; 18:S2-S7
- 38. Lysis Lysis leukemia DC NK DC DC NK NK Donor alloreactive NK cells Lysis T TT Killrecipient APCs = protection from GvHD Kill recipient T cells = improved engraftment Kill leukemia = GvL effectTT
- 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 expansionof NK cells using mbIL21 APCs Cryopreserve in aliquots
- 41. IL-2 or IL-15 Haploidentical Alloreactive NK Cells Busulfan Fludarabine Donor, Haploidentical or Cord Blood NK Cells IL-2 Allo match PBPC Melphalan Fludarabine Haploidentical Allo reactive NK Cells Haplo BMT Cy-tacro-MMF Flag-ida
- 42.
- 43. Conclusions • Adoptive cellularimmunotherapy is a promising novel treatment modality for treatment of cancer. • Cellular immune therapy is a promising approach to control alloreactivity to prevent GVHD. Tregs successful 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 wego 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
Editor's Notes
- #12 Q6. retroviral vector issues.Adenoviral vector used for transient transfection situations Retroviral vector for stably transfected situations with low oncogenic risk (e.g. T cells)Lentivirus for stem cells (in development)