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4/11/2013
1
Cell Kinetics in Adoptive Cell
Therapy
April 11, 2013
David Stroncek, MD
Chief, Cellular Processing Section
DTM, CC, NIH
4/11/2013
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Cellular Therapies Cell suspensions used for therapeutic purposes
Examples
•Red Cells
•Platelets
•Granulocytes
•Hematopoietic stem cells
•T cells
•Dendritic cells
•Natural killer (NK) cells
•Bone marrow stromal cells (BMSCs)
•Embryonic stem cells (ESC)
•Induced pluripotent stem cells (iPSC)
What its not
•Tissue, bone, organs Maybe autologous or allogeneic
Blood Products
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Overview
RBCs – the simplest cell therapy
• Biologic variability
Granulocytes-more complex trafficking
• Cell phenotype effects kinetics
T cells – Adoptive Cell Therapy for Cancer
• Cell expansion effects kinetics
• Genetic engineering to improve kinetics
• Re-programming to change phenotype and improve kinetics
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Measuring Cell Kinetics Radionuclides
• Chromium-51 (27.7 days half-life)
• Technetium-99m (6.0 hrs)
• Indium-111 (2.8 days)
Applications
• Intravascular recovery
• Intravascular survival
• Tissue distribution
Issue = Radionuclides are Difficult Handle
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Molecular Biology for Cell Detection
Cell Clones – Same T cell receptor (same peptide and HLA restriction)
• HLA tetramers and peptide antigen
• T cell receptor PCR
• TCR b-chain V region sequencing
• TCR b-chain V region antibodies
Genetically Engineered Cells
• Antibodies
• PCR
Not
Quantitative
Good for Accessing Intravascular Recovery and Survival
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Iron Labeling – MRI Detection
Ferumoxytol Heparin Protamine
Ferumoxytol-Heparin-Protamine
Nanocomplexes
Cells
Cells
3 FDA Approved Drugs
Iron Labeled Cells
Infusion of Labeled Cells
MRI Detection
+ +
Self-Assembling Nanocomplexs
Thu MS et al. Nature Medicine Feb 2012
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Iron Labeling – MRI Detection
Ferumoxytol Heparin Protamine 3 FDA Approved Drugs
Iron Labeled BMSCs
Infusion of
Labeled BMSCs MRI Detection
+ +
Self-Assembling Nanocomplexes
Bone Marrow
Stromal Cells
(BMSCs)
Thu MS et al. Nature Medicine Feb 2012
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Measuring Cell Kinetics:
Summary
• Radionuclide labeling = gold standard
but limited availability
• Molecular methods can be useful for
cloned T cells or genetically engineered
cells
Alternative methods are needed
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Red Blood Cells: the simplest cell therapy
Function
• Transport oxygen
Production
• Bone Marrow
Clearance
• Spleen
Distribution
• Extracellular space
• 40 to 45% of blood volume
Life span
• 105-120 days
Well documented with radionuclides
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RBC Donation and Transfusion
Manufacturing RBCs for Transfusion •Remove plasma
•Add anticoagulant /storage solution
RBCs
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Recovery of Transfused RBCs Expected Recovery = 75% Expected Half-life = 30 days
AuBuchon JP et al. Blood 1988;71:448-452
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Variability in RBC Recovery:
Intra-donor and Inter-donor variability
Sources of Variability
Assay
Donor
•Intra-donor
•Inter-donor
Manufacturing
Storage
Recipient factors
•RBC-specific antibodies
•Enlarged spleen
Hess JR. Transfusion 2012
RBC Recovery in 27 healthy subjects
Concept: Cell Therapy Kinetics are Highly Variable
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Granulocytes
More Complex Trafficking
• Intravascular, organ and tissue distribution
• Marrow reserves
• Transfusion recipient factors affect the survival of transfused cells
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Circulating Leukocytes
Cell Type (%)
Lymphocytes 40 to 50
Granulocytes 50 to 80
Monocytes 5 to 10
Eosinophils 1 to 5
Basophils 0 to 1
Normal White Blood Cell Count = 4 to 5 x109 cell per L
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Granulocytes Function
• Antimicrobial
Production
• Bone Marrow
Clearance
• Spleen
• Tissue
• Sites of infection or inflammation
Distribution
• Intravascular
• Spleen, lungs
• Tissue
Life span
• 24 hours
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Life of Granulocytes
Bone Marrow
•7 to 10 days
•100x109 produced per
day
Spleen Lungs
Tissue
•24 hours
Circulation
•12 hour
•25x109 circulating cells
Marginal Pool
25x109 cells in the
lungs and spleen
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Collection of Granulocyte by
Apheresis
Donor Blood Cell
Separator
Granulocytes
Whole blood
Whole blood – Granulocytes
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Changes in Donor Granulocyte Trafficking Increases the Concentration of Granulocytes in
the Blood and Apheresis Collection yields
Granulocyte in a unit of whole blood = 2.5x109 cells
Donors given Dexamethasone
• Granulocytes in an apheresis concentrate =
20x109 cells
Donor given Dexamethasone plus G-CSF
• Granulocyte in an apheresis concentrate = 40 to
80x109 cells
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Survival of Transfused Granulocytes
Explanation
•Granulocytes are trapped in the lungs
•Likely due to normal trafficking and activation during
collection
Concept
Kinetics of even minimally processed cells may be
much different than naïve cells
Issue
•Recovery of transfused granulocytes is less than
expected
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Method of Collection (Mobilization) Effects
Granulocyte Kinetics
Classical Granulocyte Products
•Donors given Dexamethasone
•Collection yield = 20 x 109 cells
•Expected increase in counts = 5 x109/L
•Actual increase in counts = none
•Can find transfused cells in buccal swabs
G-CSF Granulocyte Products
•Donors given G-CSF + Dexamethasone
•Collection yield = 40 to 80 x 109 cells
•Expected increase in counts = 15 x109/L
•Actual increase in counts = 1 to 3 x109/L
Effects of G-CSF
•Improved recovery is due to mild activation and down
regulation of adhesion receptor CD62L
•Improved survival is due to the release of slightly less mature
granulocytes
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Sequential CT scans of the thorax and response to granulocyte transfusions
O'Donghaile D et al. Blood 2012;119:1353-1355
Granuolcyte Transfusions in a 16 year female with Severe Aplastic Anemia
Dose of G-CSF+Dex
mobilized Granulocytes
(x1010 cells)
•Weak relationship between and dose and
count increment
•Count increments are reduced as more
transfusions are given
Inflammation increases after initial (4) transfusions
due to granulocyte trafficking to the site of infection
Pre -transf Pre-transf
Post 4 transf Post 4 transf
Post 9 transf Post 9 transf
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(A) Relationship between ANC increment and granulocyte dose transfused for the first
five non-alloimmunized patients who received 69 granulocyte concentrates.
Quillen K et al. Haematologica 2009;94:1661-1668
©2009 by Ferrata Storti Foundation
(B) ANC over time for one patient who developed de novo HLA antibodies during the
course of granulocyte therapy. Arrows denote granulocyte transfusions.
Effects of Granulocyte Transfusion Cell Dose and the Presence
of HLA antibodies on Neutrophil Count Increments
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Effect of splenomegaly and positive HLA antibody screen on
absolute WBC increments 0–4 h post-transfusion
Quillen K et al. Haematologica 2009;94:1661-1668
©2009 by Ferrata Storti Foundation
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T cells – Most Common Cell Used For Adoptive
Immune Therapy of Cancer and viral infections
Function
•Adoptive cytoxicity
Production
•Bone marrow
•Lymph nodes
Distribution
•Blood
•Lymph nodes
•Thymus
•Tissue
Life span
•Short
•Long – months to years
Bone
Marrow Blood Peripheral
Lymphatic Tissue
Thymus
Tissue
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Adoptive Immune Therapy: Antigen Specific T Cell
Antigen specific T cells recognize tumors and viral
infected cells using T cell receptor to recognize
peptide (antigen)-loaded HLA antigen (MHC)
Tumor
T Cell
Clinical Applications: Cancer therapy (melanoma) and viral infections (after marrow transplantation)
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T Cell Therapy for Melanoma Tumor Infiltrating Lymphocyte (TIL) Therapy
Tumor Tumor Lysate
Bulk TIL Isolation Patient with Metastatic Melanoma
Expanded TIL Transfuse 30 to 60 x 109 cells and administer IL-2
Clinical Response Rate: 10 to 70%
+ IL-2
IL-2 + Anti-CD3 +
Feeder Cells
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Typical TIL Treatment Protocol
Collect Cells
Process and Expand Cells
Infuse Cells High dose IL-2
Time
Clinical responses may not occur for several weeks
post-therapy
Post infusion IL-2 enhances T cell survival
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Adoptive Cell Therapy for Metastatic Melanoma:
Kinetics and Outcome
Patients: 23 with melanoma
TIL : Clones produced from tumors or peripheral blood
leukocytes
Treatment Schedule: Treated with 2 or more times
Average Cell Dose: 10 x 109 cells
Measurement of Cell Kinetics: T-Cell receptor PCR
Survival: Concentration of cells in the blood reached a
maximum after 1 hour and rapidly declined to
undetectable levels by 2 weeks
Clinical outcome: One partial response (PR)
Issues
•Peripheral blood counts should have been much higher
•Cells should have persisted much longer
Dudley M, et al. Journal of Immunotherapy 2001;24:363-373.
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2
T-Cell Receptor Specific PCR to Detect TIL Clones in the Blood
Adoptive Transfer of Cloned Melanoma-Reactive T Lymphocytes for the Treatment of Patients with Metastatic Melanoma. Dudley, Mark; Wunderlich, John; Nishimura, Michael; Yu, David; Yang, James; Topalian, Suzanne; Schwartzentruber, Douglas; Hwu, Patrick; Marincola, Francesco; Sherry, Richard; Leitman, Susan; Rosenberg, Steven Journal of Immunotherapy. 24(4):363-373, July/August 2001.
BV7 Clone
Control
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2
Circulating TIL were Detected 1-day post infusion in
approximately 50% of Patients/Cycles
Adoptive Transfer of Cloned Melanoma-Reactive T Lymphocytes for the Treatment of Patients with Metastatic Melanoma. Dudley, Mark; Wunderlich, John; Nishimura, Michael; Yu, David; Yang, James; Topalian, Suzanne; Schwartzentruber, Douglas; Hwu, Patrick; Marincola, Francesco; Sherry, Richard; Leitman, Susan; Rosenberg, Steven Journal of Immunotherapy. 24(4):363-373, July/August 2001.
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Why is T cell Survival Limited?
1. Unfavorable recipient environment
2. Inadequate cell dose
3. Prolonged expansion leads to apoptosis
4. Phenotype changes to a short-lived
effector T cells
TN TCM TEM TEFF
Naïve
CD62L+ CD45RO-
Central Memory
CD62L+ CD45RO+
Effector Memory
CD62L- CD45RO+ Effector
T Cell Phenotypes
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Improving the Survival of Infused
Adoptive Cell Therapies
Modify the Infused Cells
•Different cell subset/type
•Shorter Duration of Expansion
Modify the Recipient
•Growth Factors/cytokines
•Leukocyte Reduction
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New TIL Treatment Protocol:
Recipient leukoduction
Collect Cells
Process and Expand Cells
Infuse Cells High dose IL-2
Time
Chemotherapy
Total Body Irradiation
Autologous CD34+
cells
Potential Benefits of Leukocyte Reduction
•Increased cytokine levels
•Elimination of inhibitory cells
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TIL Therapy and Lymphodepleting Chemotherapy
Treatment Protocol
•Nonmyeloablative lymphodepletion: cyclophosphamide and
fludarabine, no autologous CD34+ cells
•TIL: approximately 7 x109 cells
•Analysis of TIL persistence: by TCR b-chain V region
sequencing
Clinical Outcomes
•Objective clinical responses in 13 of 25 patients
TIL Kinetics
•Post-infusion lymphocytosis in 2 patients
•Persistence of cells more for 23 to 62 days
•Greater degree of persistence of TIL in patients with
clinical responses
Robbins PF et al. Journal of Immunology 2004;173:7125-7130
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Persistence of T Cell Clones in the Blood Following TIL Therapy
Robbins PF et al. Journal of Immunology 2004;173:7125-7130
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T Cell Clones in the Blood Following TIL Therapy: Comparison of
Responder and Non-Responders (23-63 days post infusion)
Robbins PF et al. Journal of Immunology 2004;173:7125-7130
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Leukocyte Depletion Enhances TIL Therapy Outcomes
SA Rosenberg, ME Dudley Curr Opin Immunol. 2009;21:233-40
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Mean telomere length, the number of CD27+CD8+ cells, and the percentage persistence of the
infused cells in peripheral blood at 1 month after cell infusion are significantly different in objective
responders (CR + PR) compared with nonresponders (all P2 < 0.001)
Rosenberg S A et al. Clin Cancer Res 2011;17:4550-4557
Young TIL Telomere Length, CD27 expression and
Persistence affect Patient Outcome
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Genetically Engineered T cells: Improved
Kinetics and Improve efficacy
Why Engineer T cells?
1. Higher affinity T cell receptors
2. Develop therapies that are not dependent
on HLA type and T cell receptor restrictions
3. Improve kinetics
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Kershaw MH et al. Nature Reviews Immunology. 2005:5;928-40.
Chimeric Antigen Receptor (CAR): T Cell + Antibody = T Body
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AA Chekmosova RJ Brentjens Discov Med 2010;9:62-70
Chimeric Antigen Receptor (CAR) T cells
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Production of Autologous Anti-CD19 CAR T Cells
Autologous PBMCs
T cell Stimulation Anti-CD3/CD28 beads + IL-2
Transduction + Anti-CD19/CD28/CD3 zeta vector
Expansion
Continue culture with anti-CD3/CD28 beads + IL-2
Treat Acute Lymphocytic Leukemia
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AA Chekmosova RJ Brentjens Discov Med 2010;9:62-70
Chimeric Antigen Receptor (CAR) T cells Clinical Trial with first generation CAR T cells
•Receptor Directed to an ovarian cancer-associated antigen: a-
folate receptor (FR)
•Treated 14 patients with metastatic ovarian cancer
Anti-FR
No CD8
or
CD28
CD3z chain
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Anti-Folate Receptor CAR T Cells For Ovarian Cancer
Kinetics
•PCR analysis of gene modified cells
•111In-labeling
Clinical Outcomes
•No responses
Treatment Protocol
•Phase I/II
•1 or 2 cycles of Anti-FR CAR
Phase I
Phase II
Cell dose = 4 to 7x109
Kershaw MH et al Clin Cancer Res 2006;12:6106-6115
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Biodistribution of radiolabeled T cells.
Kershaw M H et al. Clin Cancer Res 2006;12:6106-6115
©2006 by American Association for Cancer Research
Anti-Folate Receptor CAR T Cells For Ovarian Cancer
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Anti-Folate Receptor CAR T Cells For Ovarian Cancer:
Little Persistence Beyond 5 days
Kershaw MH et al Clin Cancer Res 2006;12:6106-6115
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T Cell Receptor Co-Stimulatory Molecules
Addition of co-stimulatory molecules to CARs
enhances the persistence of infused cells
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Song D et al. Cancer Res 2011;71:4617-4627
Anti-Folate Receptor CAR T Cells: Adding Co-Stimulatory Domain
Improves Anti-Tumor Response
CD137 = 4-1BB
co-stimulatory
domain
Control Vectors
Only Anti-FRa CAR with 4-1BB Reduce Tumor Volume
Anti-FRa CAR Vectors
Anti-FRa CAR with 4-1BB
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Song D et al. Cancer Res 2011;71:4617-4627
Anti-Folate Receptor CAR T Cells: Adding Co-Stimulatory Domain
Improves Anti-Tumor Response
CD137 = 4-1BB
co-stimulatory
domain
Control Vectors
Cell persistence with Anti-FRa
CAR plus 4-1BB
Anti-FRa CAR Vectors
Anti-FRa CAR with 4-1BB
Effectiveness of Anti-FRa CAR plus 4-
1BB is not effected by route of
injection
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AA Chekmosova RJ Brentjens Discov Med 2010;9:62-70
Clinical Trial with Second Generation CAR T cells •CAR Directed to the B cell antigen CD19
•CAR contained a co-stimulator molecule domain
•Treated 3 patients with Chronic Lymphocytic Leukemia (CLL)
Anti-CD19
4-1BB
CD3z chain
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Kalos M et al. Sci Transl Med 2011;3:95ra73-95ra73
Published by AAAS
Anti-CD19 CAR Treatment Protocol: Chemotherapy followed by 3 daily doses
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65 year old male,
CR (11+ months)
77 year old male,
PR (7+ months)
65 year old male,
CR (11+ months)
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Kalos M et al. Sci Transl Med 2011;3:95ra73-95ra73
Published by AAAS
Prolonged Persistence of CD19 CAR Cell in the Blood
and Marrow
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Kalos M et al. Sci Transl Med 2011;3:95ra73-95ra73
Published by AAAS
Clinical Reponses After CD19 CAR Therapy
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Improving T Cell Adoptive
Cellular Therapy by Starting with
T Cells with Better Survival
Kinetics
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Genetically Engineered T cell Receptors
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TN TCM TEM TEFF
Naïve
CD62L+ CD45RO-
Central Memory
CD62L+ CD45RO+
Effector Memory
CD62L- CD45RO+
What Type of T Cells are Best for Adoptive Cell Therapy
Hinrichs C S et al. Blood 2011;117:808-814
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J. Clin. Invest. 118:294–305 (2008). doi:10.1172/JCI32103.
Isolation of and labeling of TCM and TEM cells
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TCM cells
TEM cells
TCM cells
TEM cells
Macaque 1
Macaque 2
Better Persistence and Migration of TCM-derived CD8+ TE clones
J. Clin. Invest. 118:294–305 (2008)
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TN TCM TEM TEFF
Naïve
CD62L+ CD45RO-
Central Memory
CD62L+ CD45RO+
Effector Memory
CD62L- CD45RO+
Memory Stem T Cells (TSCM) Which Have
Characteristics of Naïve and Memory Cells are Present
in the Peripheral Blood
TSCM
Stem Memory T Cells Stem cell characteristics:
•CD62L+, CD45RO+, CD45RA+, CD27+, CD28+ and IL-7Ra+
Memory Cell Characteristics
•CD95+, IL-2Rb+, CXCR3+ and LFA-1+
TSCM Function
•Increased proliferative capacity
•Superior anti-tumor cell responses
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More Adoptively Transferred Mesothelin-Specific Human
TSCM cells are Recovered from NSG Mice
Gattinoni L et at. Nature Medicine 2011; 17: 1290-7
106 cells of each type were transferred to each mouse
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Adoptively Transferred Mesothelin-Specific TSCM
Cells Have Greater Antitumor Activity
Gattinoni L et at. Nature Medicine 2011; 17: 1290-7
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Production of Tscm cells
•Tscm cells are found in the blood but their
quantities are limited
•Cell re-programming methods are being
tested for the expansion of Tscm cells
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Wnt/β-catenin Signaling is Involved with T-cell Differentiation,
Polarization, and Survival
Gattinoni L et al. Clin Cancer Res 2010;16:4695-4701
Wnt/b-catenin signaling
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Wnt Signaling
Yao H, Ashihara E and Mackawa T. Expert Opin Ther Targets. 2011;15:873-887
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TWS119 Activates Wnt Signaling by Inhibiting GSK-3b
TWS119
Yao H, Ashihara E and Mackawa T. Expert Opin Ther Targets. 2011;15:873-887
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Production of Memory Stem T Cells (TSCM) From
Peripheral Blood Leukocytes
Anti-CD45RO
Depletion
Anti-CD62L
Selection
Peripheral Blood
Lymphocytes CD45RO-
Lymphocytes
CD62L+ CD45RO-
Lymphocytes
Anti-CD3/CD28 Beads
+
TWS119
TSCM Cells
TSCM Cells
•CD45RO- CD62L+ CCR7+ CD28+
•Both Naïve and Memory characteristics
•Increased proliferative capacity
•Superior anti-tumor cell responses
Anti-CD8
Selection
CD8+
Lymphocytes
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Conclusions
• Cellular Kinetics are important for the licensure of RBCs and platelets.
• Improvements in cell persistence have improved adoptive T cell therapy for cancer.
• Gene engineering is being used to increase the survival and clinical effectiveness of adoptively transferred T cells.
• T Cell reprogramming may be a useful tool for improving the persistence and effectiveness of adoptive T cell therapy.
