Cellular Immune Therapy with Allogeneic Stem Cell Transplantation
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Transcript of Cellular Immune Therapy with Allogeneic Stem Cell Transplantation
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Cellular Immune Therapy with Allogeneic Stem Cell
Transplantation
Richard Champlin, M.D.
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HSCT
DRL
RRL
RR D
DD
D
D
D
D
D
Hematopoietic Stem Cell Transplantation
PreparativeRegimen
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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.
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HSCT +DLI
DTDNK
DRLRL
RRL R
R DB
DscDT
DNK
D
DD
Dsc
D
DT
DT
Dsc
D
Complete ChimeraRecipient Donor Mixed Chimera
Hematopoietic TransplantationPreparativeRegimen
R
Cellular Immune Therapy
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Relapse is main cause of treatment failure with Allogeneic HSCT for AML
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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
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Prevention of GVHD
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• 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)
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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
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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
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Questions with Tregs
• Production process, separation of Tregs from Tconv
• Cell Dose• Administration with calcineurin inhibitors
vs. sirolimus• Impact on GVL effects?
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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
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Di Stasi et al NEJM 2011
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Rapid Reversal of GVHD after Rx with AP1903.
Di Stasi A et al. N Engl J Med 2011;365:1673-1683
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Anti viral T-cells
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CTLMultimer
Multimer selection
IFN-
Gamma interferon selection
IFN-
Gamma Capture of Antigen Reactive T-cells
Feasible for high frequency T-cell responses: EBV, CMV
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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
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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
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Induction of Graft-vs-Malignancy Effects
Donor lymphocyte InfusionsAntigen specific CTLs
Chimeric Antigen Receptor T-cells
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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
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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)
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Shared ResourcesFlow Cytometry and Cellular Imaging Facility, Genetically Engineered Mouse Facility, Monoclonal Antibody Facility; Clinical Trials Support Resource
Antigen-Specific Immune Therapy for AML
Proteasom
eP3
NE
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
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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 b
TCR-complex
ge ed z z
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Production Methods
• Retroviral vectors• Letiviral vectors• Non viral systems, Sleeping Beauty
• Expansion using artificial APCs
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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
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2nd and 3rd Generation Chimeric Antigen Receptors
Propagation on Artificial APCs
Cooper et al
41BB
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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
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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
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Anti CD19 CAR T-cells for CLL
Porter DL et al. N Engl J Med 2011;365:725-733
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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
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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
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“Off-the-shelf” CD19-specific CAR+T Cells for Adoptive Immunotherapy
Cooper et al Blood 2010
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NK Cells
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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
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NK Cell Receptors
Murphy et al Biology of Blood and Marrow Transplantation 2012; 18:S2-S7
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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
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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
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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 ofCAR+Tcellswith integrated transposon on
g-irradiated K562-aAPC
IL-2 (50U/mL)
IL-21 (50U/mL)
Wave Bioreactor
Wave Bioreactor
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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
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42
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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.
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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