Engineered T cells: Next-generation cancer
immunotherapy
Rahul Purwar, Assistant Professor
Department of Biosciences & Bioengineering
Indian Institute of Technology Bombay (IIT Bombay)
Mumbai, India, 400076
Outline
Introduction
Adoptive T cell therapy: Engineered CAR-T cells
Review of recent clinical trials
Challenges of CAR T cell therapy
Results
T cell subsets: Th17 and Th9 cells in cancer immunotherapy
Summary
Adoptive T cell therapy: what we learnt and what we should do next
Overview: Adoptive T cell therapy
Target therapy with Tumor specific T
cells
2. Expand and activate
T-cells ex vivo
Cancer: Melanoma
Autologous tumor infiltrating
lymphocytes (TILs); “Live drug”
3. Infuse the "boosted"
T-cells into the patient.
1. Isolation of TILs
or tumor specific
T-cells from blood
Advantages
High response rate (>50%),
Long-term remission,
Less toxic & gentler to the patient
Limitation:
Extraction of TILs,
Cell manufacturing
Possible alternate
T cell Engineering (CAR-T cells)
Rosenberg SA & Dudley ME 2009 Current Opinion of Immunology
Adoptive T cell therapy: CAR-T cells
CAR-T cells (Chimeric antigen receptor-T
cells0
Antigen specific
domain
T cells transduced with tumor-specific CAR
CAR: Single fusion molecule with antigen specificity
plus signaling domain
Three types of CAR: First/second/generations
Based on co-stimulatory receptors
Cancer: Solid tumor & hematological malignancies
Advantages of CAR T cells
“Live drug”
Tumor recognition
independent of HLA
(no HLA typing
needed)
Multiple antitumor immunomodulators can
be engineered
Target variety of
antigens (protein,
carbohydrate,
glycolipid)
Maus M V et al. Blood 2014;123:2625-2635
Clinical significance of CAR-T cells
Target
CAR
Cancer
Objective response
CD19
CAR:CD28-CD3z
Lymphoma and N=7: 1CR, 5 PR & 1SD
CLL
CAR:CD137-CD3z
ALL
2CR
CAR:CD28-CD3z
ALL
5CR
CD20
CAR:CD137-CD28CD3z
NHL
N=3: 1PR, 2NED
CEA
CAR-CD3z (1st gen)
Colorectal &
breast
N=7: minor responses
in two patients
GD2
CAR-CD3z (1st gen)
Neuroblastoma N=19: 3CR
ERBB2
CAR:CD28-CD137CD3z
Colorectal
cancer
N=1, patient died
Kershaw et. al. 2013 Nature Reviews cancer
Challenges of CAR-T cells
Toxicities
On target/off tumor toxicities
Metastatic colon cancer patient died after 5 days of infusion of
ERBB2+CAR-T cells
Low levels of ERBB2 express on lung epithelium (lung tox)
Renal cell carcinoma: 5/11 patients developed liver toxicity
Cytokine syndrome
Elevated levels of pro-inflammatory cytokines
Treatable by anti-IL-6mAb and steroids
Determinants of successful ACT: CAR-T cells
Tumor target
Efficacy & Long-term persistence
Target antigen is critical determinant for
efficacy & safety
Subtypes of CD4+T cells (Th1, Th2,
Th17, Th9 cells),
Ideal target uniquely express on tumor
cells or on cells which are not essential
for survival
CD8+T cells
Trafficking of CAR T cells to tumor
Expression of addressins
Route of CAR-T cell infusion
Intra-tumoral/intravenous
Optimal co-stimulation of T cells
naïve, central memory; long-term
effector; active but short lived
Patient conditioning before ACT
Reduced-intensity or nonmyeloablative
Increased intensity myelo ablative
Outline
Introduction
Adoptive T cell therapy: focus on engineered CAR-T cells
Overview of current & investigational CAR therapies
Challenges of CAR T cell therapy
Results
Roles of T-helper cell subsets in cancer immunotherapy
Summary
`
Adoptive T cell therapy: what we learnt and what we should do next
Adoptive T cell therapy: Right T cell population?
CD4+ T cells
IL23R
Zou W & Restifo NP Nature Reviews Immunology 2010
Role of Th17 cells in tumor immunity is
controversial
Pro-tumor:
- enhances vascularization/angiogenesis
- promotes metastasis
- promotes growth
Langowski et al, Nature 2006; Murugayen 2011 J.Immunol; Wang et al. J.Exp.Med 2009
Anti-tumor:
- enhances tumor immunity by promoting CD8+T cell and DC
function
Martin-Orcazzo et al Immunity 2010
Tumor growth suppression in ROR-/- mice (Th17 cell
deficient)
Survival of tumor bearing host
Melanoma tumor growth
RORg+/+ chimeric mice
RORg-/- chimeric mice
100
3000
2500
2000
1500
1000
500
0
**
***
0 10 20 30 40
Days after tumor induction
% of mice (survival)
Tumor volume (mm3)
3500
ROR+/+chimeric mice
ROR-/-chimeric mice
75
50
25
0
5 10 15 20 25 30 35
Days after tumor induction
Abrogation of Th17 pathways promotes anti-tumor immune responses
Slc15a2
Robo3
Robo3
Gzmc
Il9
Abp1
Gzmb
Foxn4
Ak1
Il28ra
Zfp423
S100a4
Rhod
Ckmt1
Lgr4
Apobec2
Cckbr
Galntl4
Phlda3
Pgr
Slc2a4
Ms4a4b
Il1f9
Ly6d
Mal2
Id4
Gpr81
Penk
Ptger3
Psrc1
Penk
Ms4a4b
Il12b
Ebf4
Gzmd
Spib
Mmp13
Serpinb9
Serpinb6b
Il10
Ifng
Il4
Eomes
Tbx21 (Tbet)
Il17re
Il17f
Ccr2
Malat1
Serpinb1a
Pkib
Ccl24
Col16a1
Ramp1
Itga7
Serpinb2
Il17a
Col4a2
Serpinb2
Cysltr1
Selenbp2
Selenbp1
Il23r
Numbers of IL9+ve cells (%)
Gene-expression analysis: ROR-/- CD4+T cells
IL-9
6
5
4
**
Increased IL-9
expression in
RORγ-/- CD4+T
cells
3
2
1
0
WT
ROR-/IL-17f
Th17 genes
IL-17a
IL-23r
-100-95-90-85-80-75-70-65-60-55-50-45-40-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65
CD4+T cells differentiated under Th17 conditions (ROR-/- / ROR +/+Fold change)
Day 0
Subq
B16F10 (1x105/100 ml)
LLC (1x105/100 ml)
Day 0 onwards
Follow tumor
development
(3x/week)
rIL-9 100
mg/mouse
(i.p.),
Every alternate
day
Results
Delayed tumor growth in IL-9 treated group
IL-9 mediated anti-tumor effects are not
limited to melanoma tumor model
Melanoma (B16F10)
Control
2000
rIL-9 treated
1500
1000
*
500
0
5
Tumor volume (mm3)
Efficacy study plan
Tumor volume (mm3)
Treatment with exogenous rIL-9 suppresses tumor growth
10
15
control
Lung carcinoma
(LLC)
rIL-9 treated
1200
1000
*
800
600
400
200
0
5
10
15
20
Days after tumor induction
Effects of rIL-9 on melanoma tumor growth in Rag1-/- mice
(T cell and B cell deficient host)
Tumor volume (mm3)
1,500
Control
rIL-9 treated
1,000
**
500
0
5
10 15 20 25
Time after tumor induction (d)
IL-9 mediated tumor growth suppression is independent of T cells and
B cells
Effects of rIL-9 on tumor tumor growth in mast cell-/- mice
1500
Control
rIL-9 treated
1000
ns
500
0
10
Tumor volume (mm3)
Tumor volume (mm3)
Melanoma (B16F10)
Lung carcinoma (LLC)
control
rIL-9 treated
800
600
400
200
0
5
15 20
Days after tumor induction
10
15
IL-9 mediated tumor growth suppression is dependent of mast cells
Engineering Th9 cells: TAA specific Tumor model
Engineer tumor specific Th9 cells
TGFβ + IL-4
Th9
OT2 TCR transgenic mice
(CD4 cells recognize ova)
Naïve
OT2
CD4+CD25CD62L+
Normal WT or Rag1-/TGFβ + IL-6
Th17
B16F10-Ova cells
Generation of Ovalbumin expressing B16 tumor cells
(Lentiviral method)
Follow tumor
development
Treatment with Th9 cells suppresses tumor growth
Immunocompetent host (Wild type)
no T cells
Tumor volume (mm3)
2000
No T cells
Th17
Th9
1500
Th17 cells
1000
***
500
Th9 cells
0
0
5
10
15
20
Days after tumor induction
Th9 cell therapy: efficacy studies in immunodeficient host
Rag1-/- (T cells and B cells deficient)
no T cells
Tumor volume (mm3)
Th0 cells
Th9
5000
Th9 plus a-IL-9
4000
3000
**
2000
1000
0
10
*
15
20
Days after tumor induction
Th9 cells suppresses tumor growth independent of T cell and B cell
presence
Mechanism of anti-tumor effects of Th9 cells
Effects of Th9 cells on CD8+T cells (OT-1 cells) proliferation
90%
No Th cells
CFSE labeled
OT1-CD8-T cells
+
irradiated B16Ova cells
±
OT2-Th0 or OT2Th9 cells
3 days
45%
+Th0 cells
Presence of Th9 cells promotes the OT1CD8-T cells proliferation.
OT1 cells: ovalbumin specific CD8+T cells
CFSE: Carboxyfluorescein succinimidyl ester
counts
Evaluation of OT1-CD8-T cells by flow
cytometry
5%
+ Th9 cells
CFSE
Mechanism of anti-tumor effects of Th9 cells
Examining Cytotoxic activity of Th9 cells
EL-4 B16-Ova
5mM CFSE labeled B16F10-ova cells + 0.5 mM CFSE labeled EL-4
cells
±
No Th cells
69
31
+ Th0 cells
(25:1)
71
29
+ Th9 cells
(25:1)
89
10
OT2-Th0 or OT2-Th9
Tumor cell lysis was measured by flow cytometry.
Th9 cells lyse tumor cells in antigen/
target dependent manner
Target cells: B16-Ova
B16 ova specific Th cells: OT2-Th cells
counts
36 h.
CFSE
What is the relevance of these findings in human?
Translational research
Th9 cells are present in human “skin” and “blood”
Gate on CD4+IFN-IL-4CD4+IL-9+IFNg-IL-4-IL-17- (%)
IL-9
Th9 cells PU.1
IL-17
Skin
Blood
IL-9
*
*
8
6
4
2
0
Blood
Skin
Healthy subjects
T cells from
metastasis
Melanoma
Th9 cells are not just a murine phenomenon
Summary
CAR-T cells
Limitation of CAR-T cells
T cells transduced with tumor-specific
Chimeric Antigen Receptor (CAR)
Tumor recognition independent of HLA (no
HLA typing needed)
Target: variety of tumor antigens (protein,
carbohydrate, glycolipid)
High response rate (up to 88%): pre-clinical
and clinical findings
Toxicities

On target/off tumor toxicities

Cytokine syndrome
Tumor microenvironment

Presence of MDSCs & Treg in
tumor

Immunosuppressive agents
Results & Conclusion
IL-9 is a novel anti-tumor cytokine and anti-tumor effects are mediated via mast cells
Th9 cells are the most superior anti-tumor Th cells
Th9 cells exists in human: not just murine phenomenon
Strategies that promotes IL-9 production will be a critical for the development of
robust treatment for melanoma and lung carcinoma.
Acknowledgement
Laboratory members at IIT Bombay
Richa Agarwal
Mukti Vats
Farha Memon
Sathya
Atish
Department of Dermatology, Brigham &
Women’s Hospital (BWH)
Thomas S Kupper
Department of Neurology, BWH
Vijay K Kuchroo
Wassim Elymann
Sheng Xiao
National Institute of Health (NIH/NIEHS)
Anton J Jetten
Hong Soon Kang
!!Thanks a lot!!