Cellular Immune Therapy with Allogeneic Stem Cell

Transplantation

Richard Champlin, M.D.

HSCT

DRL

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Hematopoietic Stem Cell Transplantation

PreparativeRegimen

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

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R DB

DscDT

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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

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

Redirect T-cell Specificity through the Introduction of Chimeric Antigen Receptors (CARs)

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CH1

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CH1

Antibody

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Chimeric antigen receptor

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TCR-complex

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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

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NK

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Donor alloreactiveNK cells

Lysis

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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 ofCAR+Tcellswith integrated transposon on

g-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

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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.

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