cellular immune therapy with allogeneic stem cell transplantation
DESCRIPTION
Cellular Immune Therapy with Allogeneic Stem Cell Transplantation. Richard Champlin, M.D. Hematopoietic Stem Cell Transplantation. D. D. Preparative Regimen. D. D. HSCT. D. R. R. D. R. D. R L. D. R L. D. Cell Therapy Allogeneic SCT. - PowerPoint PPT PresentationTRANSCRIPT
Cellular Immune Therapy with Allogeneic Stem Cell
Transplantation
Richard Champlin, M.D.
HSCT
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Hematopoietic Stem Cell TransplantationPreparativeRegimen
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.
HSCT +DLI
DTDNK
DRLRL
RRL R
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DT
DNK
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DD
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Complete ChimeraRecipient Donor Mixed Chimera
Hematopoietic TransplantationPreparativeRegimen
R
Cellular Immune Therapy
Relapse is main cause of treatment failure with Allogeneic HSCT for AML
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
Prevention of GVHD
• 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)
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
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
Questions with Tregs• Production process, separation of Tregs
from Tconv• Cell Dose• Administration with calcineurin inhibitors
vs. sirolimus• Impact on GVL effects?
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
Di Stasi et al NEJM 2011
Rapid Reversal of GVHD after Rx with AP1903.
Di Stasi A et al. N Engl J Med 2011;365:1673-1683
Anti viral T-cells
CTLMultimer
Multimer selection
IFN-
Gamma interferon selection
IFN-
Gamma Capture of Antigen Reactive T-cells
Feasible for high frequency T-cell responses: EBV, CMV
T cell stimulation/ expansion
PBMC
CTLCytokines+IL4/7
EBV – EBNA1, LMP2, BZLF1CMV – IE1, pp65Adv – Hexon, PentonBK – LT and VP1HHV6 – U11, U14, U90
Cultured anti-viral CTLs
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
Induction of Graft-vs-Malignancy Effects
Donor lymphocyte InfusionsAntigen specific CTLs
Chimeric Antigen Receptor T-cells
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
Targets for Graft-vs.-Malignancy
Broadly expressed minorhistocompatibility antigen (GVHD)
Lineage restrictedminor histocompatibilityantigen (G-vs-hematopoietic),or Redirected CAR T-cells vs CD19
Aberrant overexpressed normal cellular constituent(Proteinase 3, WT1,telomerase)
Allo-Specific Malignancy SpecificIdiotype, Fusion peptide of translocation (bcr-abl)
Shared ResourcesFlow Cytometry and Cellular Imaging Facility, Genetically Engineered Mouse Facility, Monoclonal Antibody Facility; Clinical Trials Support Resource
Antigen-Specific Immune Therapy for AML
ProteasomeP3NE
TCR
Leukemia
PR1 peptide
HLA-
A2
PR1
PR1-CTL
PR1/HLA-A2
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
Redirect T-cell Specificity through the Introduction of Chimeric Antigen Receptors (CARs)
vL
vH
vL
vH
CH1
CL C L
CH1
Antibody
Fab
vH vL
Chimeric antigen receptor
a bTCR-complex
e e d z z
Production Methods
• Retroviral vectors• Letiviral vectors• Non viral systems, Sleeping Beauty
• Expansion using artificial APCs
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
2nd and 3rd Generation Chimeric Antigen Receptors
Propagation on Artificial APCs
Cooper et al
41BB
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
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
Anti CD19 CAR T-cells for CLL
Porter DL et al. N Engl J Med 2011;365:725-733
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
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
“Off-the-shelf” CD19-specific CAR+T Cells for Adoptive Immunotherapy
Cooper et al Blood 2010
NK Cells
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
NK Cell Receptors
Murphy et al Biology of Blood and Marrow Transplantation 2012; 18:S2-S7
Lysis
Lysisleukemia
DC
NK
DCDC
NK
NK
Donor alloreactiveNK cells
Lysis
T T T
Kill recipient APCs =protection from GvHD
Kill recipient T cells =improved engraftment
Kill leukemia =GvL effectT T T
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
4 Log expansion of NK cells using mbIL21 APCs
Cryopreserve in aliquots
Amaxa Nucleofector
scFv
Hinge
Stalk
TM
T-cellsignaling
Transposase Transposon
CARSB11
K562 aAPC
K562 aAPCMasterCell Bank/WorkingCell Bank
Antigen-specific proliferation ofCAR +Tcells
Cryopreservation
Infusion
T-75cm2 flasks
Cell Culture Bags
Cell Culture Bags
ApheresisProduct
PBMC separationBiosafeSepax PBMC
VolumeReductionBiosafeSepax
Numericexpansion of CAR+Tcellswith integrated transposon on
-irradiated K562-aAPC
IL-2 (50U/mL)
IL-21 (50U/mL)
Wave Bioreactor
Wave Bioreactor
IL-2 or IL-15
HaploidenticalAllo reactive NK Cells
Busulfan Fludarabine
Donor, Haploidenticalor Cord Blood NK Cells
IL-2
Allo matchPBPC
Melphalan Fludarabine
HaploidenticalAllo reactive NK Cells
HaploBMT Cy-tacro-MMF
Flag-ida
42
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.
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