Cellular Immune Therapy with Allogeneic Stem Cell Transplantation


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  • 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)
  • Cellular Immune Therapy with Allogeneic Stem Cell Transplantation

    1. 1. Cellular Immune Therapy with Allogeneic Stem Cell Transplantation Richard Champlin, M.D.
    2. 2. HSCT D RL R RL R R D D D D D D D D Hematopoietic Stem Cell Transplantation Preparative Regimen
    3. 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. 4. HSCT +DLI DT DNK D RLRL R RL R R DB Dsc DT DNK D DD Dsc D DT DT Dsc D Complete ChimeraRecipient Donor Mixed Chimera Hematopoietic Transplantation Preparative Regimen R Cellular Immune Therapy
    5. 5. Time(weeks) SurvivalProbability 0 20 40 60 80 100 120 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 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. 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. 7. Prevention of GVHD
    8. 8. • 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 Regulatory T-Cells (Tregs)
    9. 9. Cord Blood Treg Expansion 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. 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. 11. Questions with Tregs • Production process, separation of Tregs from Tconv • Cell Dose • Administration with calcineurin inhibitors vs. sirolimus • Impact on GVL effects?
    12. 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
    13. 13. Di Stasi et al NEJM 2011
    14. 14. Rapid Reversal of GVHD after Rx with AP1903. Di Stasi A et al. N Engl J Med 2011;365:1673-1683
    15. 15. Anti viral T-cells
    16. 16. CTLMultimer Multimer selection IFN- Gamma interferon selection IFN- Gamma Capture of Antigen Reactive T-cells Feasible for high frequency T-cell responses: EBV, CMV
    17. 17. 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
    18. 18. 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
    19. 19. Induction of Graft-vs- Malignancy Effects Donor lymphocyte Infusions Antigen specific CTLs Chimeric Antigen Receptor T- cells
    20. 20. 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
    21. 21. Targets for Graft-vs.-Malignancy Broadly expressed 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)
    22. 22. Shared Resources Flow Cytometry and 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
    23. 23. Redirect T-cell Specificity through the Introduction of Chimeric Antigen Receptors (CARs) vL vH CH1 CL Antibody Fab vH vL Chimeric antigen receptor TCR-complex
    24. 24. Production Methods • Retroviral vectors • Letiviral vectors • Non viral systems, Sleeping Beauty • Expansion using artificial APCs
    25. 25. Sleeping Beauty Transposition Cytoplasm Nucleus Transposase Transposon Gene X Transposase (Helper) expression is transient Transposon (Donor) sequences flanked by inverted repeats are integrated into genome Cooper et al Cancer Res 2008
    26. 26. 2nd and 3rd Generation Chimeric Antigen Receptors Propagation on Artificial APCs Cooper et al 41BB
    27. 27. 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
    28. 28. 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
    29. 29. Anti CD19 CAR T-cells for CLL Porter DL et al. N Engl J Med 2011;365:725-733
    30. 30. 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
    31. 31. 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
    32. 32. “Off-the-shelf” CD19-specific CAR+T Cells for Adoptive Immunotherapy Cooper et al Blood 2010
    33. 33. NK Cells
    34. 34. 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
    35. 35. NK Cell Receptors Murphy et al Biology of Blood and Marrow Transplantation 2012; 18:S2-S7
    36. 36. Lysis Lysis leukemia DC NK DC DC NK NK Donor alloreactive NK cells Lysis T TT Kill recipient APCs = protection from GvHD Kill recipient T cells = improved engraftment Kill leukemia = GvL effectTT
    37. 37. 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
    38. 38. 4 Log expansion of NK cells using mbIL21 APCs Cryopreserve in aliquots
    39. 39. IL-2 or IL-15 Haploidentical Allo reactive 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
    40. 40. 42
    41. 41. 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. 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.
    42. 42. 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