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CAR-T cell therapy
Usanarat Anurathapan, MD
Department of Pediatrics
Faculty of Medicine Ramathibodi Hospital
Outline
• Related basic immunology
• Cellular immunotherapy
• CAR-T cell therapy
Basic immunology
• T-cell receptor
• T-cell activation and costimulation
• Effector T cells and cytotoxic T cells
T-cell receptor
• Self-MHC restriction
– Specific to a combination of an antigen and a major
histocompatibility complex (MHC), also known as a
human leukocyte antigen (HLA)
αβ and γδ T-cell receptors
Heterodimer composed of either α and β chains or
γ and δ chains
Comparison of αβ and γδ T cells
Feature αβ T cells γδ T cells
Proportion of CD3+ cells 90-99% 1-10%
TCR V gene germline repertoire Large Small
CD4/CD8 phenotype
CD4+
CD8+
CD4+ CD8+
CD4- CD8-
60%
30%
< 1%
< 1%
< 1%
30%
< 1%
60%
MHC restriction CD4+ : HLA class II
CD8+ : HLA class I No MHC restriction
Ligands MHC + peptide
antigen
Phospholipid, intact
protein
TCR multigene families
Gene Chromosome
location
Number of gene segments
V D J C
α chain 14 54 - 61 1
β chain 7 67 2 14 2
γ chain 14 14 - 5 2
δ chain 7 3 3 3 1
TCR rearrangement
TCR complex: TCR-CD3
Associated on its membrane with CD3,
multicomponent signal-transducing complex
T-cell activation & costimulation
T-cell activation
• The central event in the generation of both
humeral & cell-mediated immune response
• Signal 1 = Initiated by interaction of TCR-
CD3 complex with a processed antigenic
peptide bound to either HLA class I
(CD8+ cells) or class II (CD4+ cells) on
the surface of an antigen-presenting cell
TCR & CD4 or CD8 coreceptor
Affinity of TCR for peptide-MHC complexes is enhanced
by CD4 or CD8 coreceptors
TCR-CD3 mediating signaling
T-cell costimulation
Signal 2 = costimulatory signals are required for
full T-cell activation
T-cell activation & differentiation
Effector T cells
CTLs kill cells in two ways
Necrosis vs Apoptosis
Cytotoxic T cells
Cancer immunotherapy
• Monoclonal antibodies: -mab
• Cytokines & T cell subsets
• T cell activation: CTLA-4 Ig, anti-CD3
• B-cell based therapy: anti-CD20
• Cellular therapeutics
Cellular immunotherapy
• Active immunization
– Vaccines: DNA, protein, whole-cell, dendritic cell
• Adoptive immunotherapy
Adoptive immunotherapy:
T cells to induce tumor regression
1
Blood draw
Adapted from Ronald Levy, MD
*Antigen
Specificity*
2
T-cell product
generation
3
Infusion 4
T cells
Adoptive immunotherapy
in allogeneic setting
1
Blood draw
Adapted from Ronald Levy, MD
*Antigen
Specificity*
2
T-cell product
generation
3
Infusion 4
T cells Donor
Recipient
Types of adoptive T cell therapy
• Natural tumor-specific T cells
– Natural-killer cell (NK cell)
– Tumor infiltrated lymphocyte (TIL)
– Cytotoxic T lymphocyte (CTL)
• Genetic-modified tumor-specific T cells
NK cell
TIL
• Solid tumor
– Melanoma
– Pancreatic cancer
– Prostate cancer
CTL specifically kill tumor cells
HLA-restricted cytotoxicity effect
Types of adoptive T cell therapy
• Natural tumor-specific T cells
• Genetic-modified tumor-specific T cells
Principles of gene & cell therapy for cancer
• To exert cytotoxicity on cells in neoplastic
tissue
• To stimulate anti-tumor immune responses
• To interfere with cellular signaling pathways
conferring malignant properties
Singh, HM et al. Expert Opin Biol Ther 2014:15;1-12.
Modalities of gene & cell therapy for
cancer Modality Gene delivery systems Transduced cell types
Gene addition or editing
(in vivo or ex vivo gene therapy)
Lentivirus
Gammaretrovirus
Adenovirus Adeno-associated virus
Tumor cell
Stromal cell Immune cell
Genetically engineered cell
therapy (ex vivo gene transfer)
Transposon system
DNA/RNA transfection Nanoparticle
T lymphocyte
Antigen-presenting cell
Isolated tumor cell
(whole cell vaccine)
Mesenchymal stem cell Bacterial cell
Oncolytic virotherapy
(infection +/- in vivo gene transfer)
Selectively replicating
virus, unmodified or recombinant
Target tumor cell Cellular delivery vehicle
Singh, HM et al. Expert Opin Biol Ther 2014:15;1-12.
How does it work?
http://stemcells.nih.gov/StaticResources/info/scireport/images/4_3.jpg
Types of adoptive T cell therapy
• Natural tumor-specific T cells
• Genetic-modified tumor-specific T cells
– Transgenic TCR-modified T lymphocyte
– Chimeric antigen receptor-modified T lymphocyte
Transgenic TCR T cells
Chhabra A. The Scientific world J. 2011; 11: 121-9
What are the disadvantages?
Chhabra A. The Scientific world J. 2011; 11: 121-9
Genetic modification that
enhanced T cell targeting
Anurathapan U, et al. Cytotherapy. 2013; Epub ahead of print
What is CAR?
Chimeric Antigen Receptor
What is CAR?
Chimeric Antigen Receptor
What is CAR?
Chimeric Antigen Receptor
How can CAR work?
T cell
CAR
Anurathapan U, et al. Cytotherapy. 2013; Epub ahead of print
CAR detect specific-TAA on tumor cells
Tumor-associated antigen (TAA)
Tumor cell
T cell
CAR
Anurathapan U, et al. Cytotherapy. 2013; Epub ahead of print
Dimerization of CAR produce signal
T cell
ZAP70
Signal transduction
CD3ζ
membrane
Cytotoxicity
Genetically modified T cells redirect the
immune response against cancer cells
VL
CL
VH CH
CH2
CH3
Tumor specific
Antibody
CH2
CH3
TM
TAA
scFv VH
VL
T cell
Signaling
domains
Clone into
Expression vector
Gene transfer
Into T cells
Cancer cells
(TAA+)
CAR-modified T cell
Ex vivo expansion
of modified T cells
Cytotoxicity
Membrane
Evolution of CAR
First
Exodomain
Hinge
Endodomain
Second Third Generation
Costimulatory molecules
CD28, 41BB, OX40
Signaling domain
CD3ζ
Survival
Proliferation/cytokine production
Fourth
Chimeric cytokine
receptor
IL4/IL2, IL4/IL7
CAR T cells for clinical application
• Be produced and expanded according to GMP
guidelines and in larger scale
• GMP-graded materials and reagents will be used
during all processes
• All cell preparation will also be conducted in GMP
facilities
• Be tested for viability, expression, efficacy and
sterility prior to administration
Acute lymphoblastic leukemia
Zeller JL, et al. JAMA. 2007;297:1278.
• The most common
cancer in children
• Fever, bleeding,
pallor, bone pain
• Chemotherapy for
3 years
CAR-T against CD19+ leukemic cells
Bridget M, et al. JAMA. 2017;318:2167.
Phase I clinical trials: meta-analysis
• 133 pts with B-cell malignancies
• 119 pts with eligible for response rate evaluation Zhang T, et al. Oncotarget. 2015. 33961.
14 Clinical trials
Zhang T, et al. Oncotarget. 2015. 33961.
Forest plot for overall response rates
Zhang T, et al. Oncotarget. 2015. 33961.
Better response rate in ALL: 93 (65-100)%
ALL
CLL
Lymphoma
Overall
Zhang T, et al. Oncotarget. 2015. 33961.
Better response rate with lymphodepletion: 88 (65-100)%
Lymphodepletion
No lymphodepletion
Zhang T, et al. Oncotarget. 2015. 33961.
Better progression-free survival with
>108 infused CAR T cells
Zhang T, et al. Oncotarget. 2015. 33961.
Conclusion
• High clinical response rate of CAR-CD19 T
cell-based immunotherapy
• Lymphodepletion & increasing no. of infused
CAR-CD19 T cell have positive correlation
with the clinical efficacy
Zhang T, et al. Oncotarget. 2015. 33961.
CAR T cells for solid tumors
• Currently, around 100 CAR T cell trials
registered at the ClinicalTrial.gov
• Most of studied targets
– EGFRvIII for glioblastoma
– GD2 for neuroblastoma
– Mesothelin for various epithelial cancers
Major obstacles to the efficacy
Selected CAR T cell trials
Antigen Indication Lymphodepletion Route of administration
CD70 Pancreatic/Renal Cell/Breast/ Ovarian CY/FLU Systemic
CD171 Neuroblastoma N/S Systemic
EGFRvIII Recurrent Glioblastoma/-sarcoma - Intracerebral
ErbB Head and Neck Cancer - Intratumoral
FAP Malignant Pleural mesothelioma - Intrapleural
GD2 Neuroblastoma CY/FLU Systemic
GPC3 Hepatocellular Carcinoma Cy/FLU Systemic
HER2 (ErbB2) Glioblastoma - Intracerebral
IL13Rα2 Glioblastoma, Brain Tumors - Intracerebral
MET Melanoma, Breast Cancer - Systemic
Mesothelin Breast/Cervical/Pancreatic/Ovarian/Lung Cancer CY/FLU Systemic
MUC-16 Ovarian/Fallopian tube Carcinoma CY/FLU Systemic
PSCA Prostate/Pancreatic Cancer - Systemic
ROR1 Triple Negative Breast Cancer/NSCLC CY/FLU Systemic
Combinatorial antigen targeting for
solid tumors
Somsak Prasongtanakij, Amornrat Tungprasitipap,
Maytawan Thanunchai, Thitinee Vanichapol,
Jiraporn Jirakkakul, Korakot Atjanasuppat,
Sassawat Lertrit-anan, Bunyada jittorntrum,
Vichaya Suttisunhakul, Suradej Hongeng,
Usanarat Anurathapan
Faculty of Medicine Ramathibodi Hospital, Mahidol University
Development of cellular therapy
for the treatment of cancers
Phase1/2 clinical trial
• Relapsed/refractory B cell leukemia patients
• Age 1-18 y/o
• No co-existing diseases
• Available donors for stem cell transplantation
• Priming with 1.5-3g/m2 of cyclophosphamide
• Start with 5x105 cell/kg in the first 3 patients
Protocol flow chart
Maximum tolerated dose
total T cells (cells/kg)
Tier 1 5 x 105
Tier 2 1 x 106
Tier 3 2 x 106
Tier 4 4 x 106
Enrolled subjects Pts with
complications Plan
Upto 3 pts 0 Increase CAR-CD19 T cell dosage to the next tier
upto 3 pts 1 Enrolled 3 more pts receive CAR-CD19 T cell
dosage at the same tier
4 – 6 pts 1 Increase CAR-CD19 T cell dosage to the next tier
4 – 6 pts 2
Assume the CAR-CD19 T cell dose is jmaximum
tolerated dose, the next enrolled 3 pts receive the
previous CAR-CD19 T cell dose to assure that is no
complications
CAR-CD19 T cells release criteria
Test Specimen Specification RESULT
Viability by trypan blue Cell product >70% viable 97.30% (Total live cell = 252-295 x 106)
Phenotyping Cell product >10% CAR-CD19 T cells 16-21%
Potency Cells on day 7
of culture
> 20% lysis at 20:1 of effector to target ratio
32.92-38.97%
Mycoplasma test Final product Negative Negative
Presence of bacteria by light microscope
Final product Negative Negative
Sterility Bactec Final product Negative Negative
N=2
GD2-expressing neuroblastoma
• Disialoganglioside antigen that is expressed on
neuroblastoma cells
• Patients with neuroblastoma were found to have
significantly elevated free GD2 levels in serum
• In normal tissues, GD2 expression is largely
limited to neurons, skin, melanocytes, &
peripheral pain fibers suitable for targeted
antitumor therapy
CAR-GD2 (1st Gen) CAR-GD2 (2nd Gen)
GD2-binding domain
“A kindly gift from Baylor College of Medicine”
CAR-GD2 second generation
62
0
10
20
30
40
50
60
70
80
90
100
CD3 CD4 CD8 CD25 CD27 CD62L CD45RA CD45RO
Day0
Day4
The effector cell numbers increase after CD3/CD28 activation
Immunophenotyping
63
CAR expression
• Using qRT-PCR
• Extract total RNA from CAR T cells
PCR product
CAR mRNA expression by RQ-PCR
100 bp
200 bp
300 bp
Ct value Normalized ct
CAR GAPDH CAR/GAPDH
Mock - 16.27 -
GD2 Full 24.43 15.97 1.53
GD2 Int 24.5 16.1 1.52
GD2 Short 24.05 16.31 1.47
Mock Full Int Short
Product size 281 bp
•Effector: Mock or CAR-GD2 T cells
•Target: SH-SY5Y (GD2 positive NB), SK-N-SH
(GD2 negative NB)
•Effector : Target ratio 20:1
•Labeling effector cells with 0.25 µM CFSE (FITC)
•4 hours co-cultures
•All cells were stained with 7-AAD
Cytotoxicity test using flow cytometry
GD2 expression of NB cell lines
18.76%
96.15%
GD2 positive
SH-SY5Y cell line
GD2 Negative
SK-N-SH cell line
0
10
20
30
40
50
60
70
80
90
100
SH-SY5Y SK-N-SH HEK
% s
pe
cifi
c ly
sis
Mock
Full
Int
Short
SH-SY5Y: GD2 positive NB cell line SK-N-SH: GD2 negative NB cell line HEK: human embryonic kidney cell line
Cytotoxicity test: 4h
Effector to target ratio 20:1
Questions?