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

3. Infuse the "boosted"

T-cells into the patient.

Overview: Adoptive T cell therapy

2. Expand and activate

T-cells ex vivo

Target therapy with Tumor specific T

cells

Cancer: Melanoma

Autologous tumor infiltrating

lymphocytes (TILs); “Live drug”

Advantages

High response rate (>50%),

Long-term remission, 1. Isolation of TILs

or tumor specific

T-cells from blood

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

Antigen specific domain

CAR-T cells (Chimeric antigen receptor-T

cells0

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 malignanciesCancer: Solid tumor & hematological malignancies

Maus M V et al. Blood 2014;123:2625-2635

“Live drug”

Tumor recognition

independent of HLA

(no HLA typing

needed)

Multiple anti-

tumor immuno-

modulators can

be engineered

Target variety of

antigens (protein,

carbohydrate,

glycolipid)

Advantages of CAR T cellsAdvantages of CAR T cells

Clinical significance of CAR-T cells

Target CAR Cancer Objective response

CD19 CAR:CD28-CD3ζ Lymphoma and

CLL

N=7: 1CR, 5 PR & 1SD

CAR:CD137-CD3ζ ALL 2CR

CAR:CD28-CD3ζ ALL 5CR

CD20 CAR:CD137-CD28- NHL N=3: 1PR, 2NEDCD20 CAR:CD137-CD28-

CD3ζNHL N=3: 1PR, 2NED

CEA CAR-CD3ζ (1st gen) Colorectal &

breast

N=7: minor responses

in two patients

GD2 CAR-CD3ζ (1st gen) Neuroblastoma N=19: 3CR

ERBB2 CAR:CD28-CD137-

CD3ζ Colorectal

cancer

N=1, patient died

Kershaw et. al. 2013 Nature Reviews cancer

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

Challenges of CAR-T cells

Cytokine syndrome

Elevated levels of pro-inflammatory cytokines

Treatable by anti-IL-6mAb and steroids

Efficacy & Long-term persistence

Subtypes of CD4+T cells (Th1, Th2,

Th17, Th9 cells),

CD8+T cells

naïve, central memory; long-term

effector; active but short lived

Tumor target

Target antigen is critical determinant for

efficacy & safety

Ideal target uniquely express on tumor

cells or on cells which are not essential

for survival

Determinants of successful ACT: CAR-T cells

Patient conditioning before ACT

Reduced-intensity or non-

myeloablative

Increased intensity myelo ablative

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

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

`

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

Zou W & Restifo NP Nature Reviews Immunology 2010

IL23R

Pro-tumor:

- enhances vascularization/angiogenesis

- promotes metastasis

- promotes growth

Role of Th17 cells in tumor immunity is

controversial

Anti-tumor:

- enhances tumor immunity by promoting CD8+T cell and DC

functionMartin-Orcazzo et al Immunity 2010

Langowski et al, Nature 2006; Murugayen 2011 J.Immunol; Wang et al. J.Exp.Med 2009

2000

2500

3000

3500

RORγ+/+chimeric mice

RORγ-/-chimeric mice

Tu

mo

r vo

lum

e (

mm

3)

Tumor growth suppression in ROR-/- mice (Th17 cell

deficient)

75

100

RORγ+/+ chimeric miceRORγ-/- chimeric mice

su

rviv

al)

Melanoma tumor growth Survival of tumor bearing host

0 10 20 30 400

500

1000

1500

2000

Tu

mo

r vo

lum

e (

mm

*****

Days after tumor induction

Abrogation of Th17 pathways promotes anti-tumor immune responses

5 10 15 20 25 30 350

25

50

% o

f m

ice (

sDays after tumor induction

**

1

2

3

4

5

6

Nu

mb

ers

of

IL9

+v

e c

ell

s (

%)

Increased IL-9

expression in

RORγ-/- CD4+T

cellsPenk

Psrc1Ptger3

PenkGpr81

Id4Mal2Ly6dIl1f9

Ms4a4bSlc2a4

PgrPhlda3Galntl4

CckbrApobec2

Lgr4Ckmt1Rhod

S100a4Zfp423

Il28raAk1

Foxn4GzmbAbp1

Il9Gzmc

Robo3Robo3

Slc15a2

IL-9

Gene-expression analysis: ROR-/- CD4+T cells

WT RORγ-/-0N

um

be

rs o

f IL

9+

ve

ce

lls

(%

)

cells

-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

Il23rSelenbp1Selenbp2

Cysltr1Serpinb2

Col4a2Il17a

Serpinb2Itga7

Ramp1Col16a1

Ccl24Pkib

Serpinb1aMalat1

Ccr2Il17f

Il17reTbx21 (Tbet)

EomesIl4

IfngIl10

Serpinb6bSerpinb9

Mmp13Spib

GzmdEbf4Il12b

Ms4a4bPenk

CD4+T cells differentiated under Th17 conditions (RORγγγγ-/- / RORγγγγ +/+Fold change)

IL-17f

IL-17a

IL-23r

Th17 genes

Treatment with exogenous rIL-9 suppresses tumor growth

0

500

1000

1500

2000rIL-9 treated

Control

Tu

mo

r vo

lum

e (m

m3)

*

Melanoma (B16F10)

Day 0

Subq

B16F10 (1x105/100 µl)

µ

Day 0 onwards

rIL-9 100

µg/mouse

Follow tumor

development

(3x/week)

Efficacy study plan

IL-9 mediated anti-tumor effects are not

limited to melanoma tumor model

5 10 15 200

200

400

600

800

1000

1200

control

rIL-9 treated

Tu

mo

r vo

lum

e (m

m3)

*

5 10 150T

Lung carcinoma (LLC)

Days after tumor induction

B16F10 (1x10 /100 l)

LLC (1x105/100 µl)µg/mouse

(i.p.),

Every alternate

dayResults

Delayed tumor growth in IL-9 treated group

1,000

1,500

Tum

or

volu

me (

mm

3)

**

Control

rIL-9 treated

Effects of rIL-9 on melanoma tumor growth in Rag1-/- mice

(T cell and B cell deficient host)

5 10 15 20 250

500

Tum

or

volu

me (

mm

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

400

600

800control

rIL-9 treated

um

e (m

m3

)

Lung carcinoma (LLC)

1000

1500 rIL-9 treated

Control

um

e (m

m3)

Melanoma (B16F10)

ns

5 10 150

200

400

Tu

mo

r vo

lu10 15 20

0

500

1000

Tu

mo

r vo

lu ns

Days after tumor induction

IL-9 mediated tumor growth suppression is dependent of mast cells

Engineering Th9 cells: TAA specific Tumor model

OT2 TCR transgenic mice

(CD4 cells recognize ova)

Normal WT or Rag1-/-

CD4+CD25-

Th9TGFβ + IL-4

Naïve

OT2

Engineer tumor specific Th9 cells

B16F10-Ova cells

Follow tumor

development

CD4+CD25-

CD62L+

Th17TGFβ + IL-6

Generation of Ovalbumin expressing B16 tumor cells(Lentiviral method)

1500

2000 no T cells

Th9

Th17

Tu

mo

r vo

lum

e (

mm

3)

Treatment with Th9 cells suppresses tumor growth

Immunocompetent host (Wild type)

No T cells

Th17 cells

0 5 10 15 200

500

1000

Tu

mo

r vo

lum

e (

mm

***

Days after tumor induction

Th17 cells

Th9 cells

4000

5000

Th0 cells

Th9

Th9 plus a-IL-9

no T cells

e (m

m3

)

Rag1-/- (T cells and B cells deficient)

Th9 cell therapy: efficacy studies in immunodeficient host

10 15 200

1000

2000

3000

Tu

mo

r v

olu

me

Days after tumor induction

**

*

Th9 cells suppresses tumor growth independent of T cell and B cell

presence

No Th cells

+Th0 cells

Effects of Th9 cells on CD8+T cells (OT-1 cells) proliferation

Mechanism of anti-tumor effects of Th9 cells

CFSE labeled

OT1-CD8-T cells

irradiated B16-

Ova cells

OT2-Th0 or OT2-

Th9 cells

3 days

+ ±

90%

45%

+ Th9 cells

+Th0 cells

CFSE

cou

nts

Evaluation of OT1-CD8-T cells by flow

cytometry

OT1 cells: ovalbumin specific CD8+T cells

CFSE: Carboxyfluorescein succinimidyl ester

Presence of Th9 cells promotes the OT1-

CD8-T cells proliferation.5%

No Th cells

+ Th0 cells

69 31

71 29

EL-4 B16-Ova5mM CFSE labeled B16F10-ova cells + 0.5 mM CFSE labeled EL-4

cells

±±±± OT2-Th0 or OT2-Th9

Examining Cytotoxic activity of Th9 cells

Mechanism of anti-tumor effects of Th9 cells

36 h.

Th9 cells lyse tumor cells in antigen/

target dependent manner

+ Th0 cells

(25:1)

+ Th9 cells

(25:1)89 10

71 29

CFSE

cou

ntsTumor cell lysis was measured by flow cytometry.

Target cells: B16-Ova

B16 ova specific Th cells: OT2-Th cells

Translational research

What is the relevance of these findings in human?

IL-1

7

Gate on CD4+IFNγγγγ-IL-4-

Skin

4

6

8

+IF

Ng

-IL

-4-I

L-1

7- (%

) **

Th9 cells are present in human ““““skin”””” and ““““blood””””

Th9 cellsIL-9

PU.1

Blood

IL-9

0

2

CD

4+

IL-9

+

Blood Skin T cells from

metastasis

Healthy subjects Melanoma

Th9 cells are not just a murine phenomenon

Summary

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

Limitation of CAR-T cells

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.

and clinical findings � Immunosuppressive agents

Laboratory members at IIT Bombay

Richa Agarwal

Mukti Vats

Farha Memon

Sathya

Atish

Department of Dermatology, Brigham &

Women’s Hospital (BWH)

Thomas S Kupper

Acknowledgement

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!!