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Chan Hyuk Kim, PhD

Department of Biological Sciences, KAIST

면역치료연구실 (Laboratory of Immunothrerapy)

Towards next-generation CAR-T

cell therapy for cancer

| 2

대한혈액학회 Korean Society of Hematology

COI disclosureName of author ： 김찬혁

I am a co-founder of Curocell Inc.

I have a research funding supported by Curocell Inc

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Chimeric Antigen Receptor (CAR)-T cells

Antigen presentation

by MHC

scFv

: recognition of surface antigens

Hinge (CD8, CD28 ECM, IgG4, etc)

: effector–target inter-membrane distance

Transmembrane (CD8, CD28)

: surface expression, multimerization

Intracellular domain (CD28, 4-1BB, OX-40)

(CD3z + costim cytoplasmic domain)

:signal transduction

Surface AntigenA single-chain variable fragment (scFv) is

a fusion protein of the variable regions of

the heavy (VH) and light chains (VL)

of immunoglobulins, connected with a short

linker

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Proliferation and Persistence of 2nd Generation CAR-T

• 41BB-based 2nd generation CD19-targeting CAR-T (CART-19, University of Pennsylvania, Carl June Lab)

• CD19: Pan-B cell marker, commonly expressed in B cell cancers

• 1,000 ~ 10,000 fold expansion in patients

Grupp SA et al. NEJM 2013

Proliferation

• CAR-Ts can persist many years in patients

Maude SL et al. NEJM 2014

Persistence

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Anti-Tumor Activity of CD19-Targeting CAR-T

Pre-infusion Day 27

Maude SL et al. NEJM 2014

10% Blasts Complete Remission

• ~ 90% complete remission rate in relapsed/refractory B-cell ALL patients (average 3-4 months life expectancy with traditional therapies)

B cell chronic lymphoblastic leukemia patient (treated with CART-19, Upenn)

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Approved CAR-T Therapy

https://www.signalsblog.ca/explaining-the-hype-car-t-cells/

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CD19-Targeting CAR-T Therapy: Adverse Events

Persistence

2. Long term normal B cell depletion

1. Severe cytokine release syndrome

CRS

Proliferation

JCAR015 (SJ25C1-28Z, retrovirus)

3. Neurotoxicity

“Live Drug” is difficult to control

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Kill switches to terminate CAR-T

http://www.bellicum.com/technology/caspacide/

VL VH

Dead cellCAR-T cell

VL VH

Kill Switch

Caspase-based kill switches

HSV-TK suicide gene

Transient mRNA transfection of CAR

Clearance of CAR-T cells by mAb specific to the engineered markers

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CAR-T cell

Malignant

cell

CD137

CD3

CD8

scFv

antigen

Conventional CAR-T

cytolysis

VL VH

CD137

CD3

CD8

Switchable

CAR-T cell

Malignant

cell

scFv

Switchable CAR-T

VL VH

CH1CL1

VL VH

Switch

FITC or GCN4 Peptide

TAG

Switchable CAR-T

The activity and specificity of the

Switchable CAR-T cells are dependent upon the switch molecule

(CAR-T activity is inert in the absence of the switch)

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Anti-FITC CAR Design

Pert

er

DL, June C

H –

NE

JM

2011

Anti-FITC scFvCD8

Hinge & TM41BB CD3 zeta

Isotype IgG-FITCAnti-hu-IgG-APC

APC FITC

Primary human T cells transduced with anti-FITC CAR

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FITC-conjugated Anti-CD19 Switches

B

(S74)

C

(T109)

D

(A121)

E

(S202)F

(K136)

A

(G68)

Anti-CD19 Distance/Orientation

Stoichiometry

Site specific conjugation through unnatural amino acid (UAA) methodology

Spatially distinct positions were selected to mediate different geometries between CAR and

target antigen

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Evaluation of FITC-conjugated Anti-CD19 switches

In vitro cytotoxicity of anti-FITC CAR-T cells against NALM6 (CD19pos) at E :T = 5:1 for 24 hrs

EF-FITC

CD19 ~30 kDa

AB-FITC

CD19vs

Target Cell

CAR-T Cell

10 -4 10 -2 100 102 104 1060

20

40

60

80

100

A-FITCB-FITC

C-FITC

D-FITC

E-FITC

F-FITC

Concentration (pM)

Cyto

toxic

ity (

%)

EC50

A-FITC

3.529

B-FITC

0.9711

C-FITC

2.823

D-FITC

3.387

E-FITC

3.795

F-FITC

2.429

Site Effect

10 -4 10 -2 100 102 104 1060

20

40

60

80

100

A-FITC

B-FITC

E-FITC

F-FITC

EF-FITC

AB-FITC

Concentration (pM)

Cyto

toxic

ity (

%)

EC50

A-FITC

3.529

B-FITC

0.9711

E-FITC

3.795

F-FITC

2.429

EF-FITC

2.535

AB-FITC

0.07031

Valency Effect

10 -4 10 -2 100 102 104 1060

20

40

60

80

100

B-FITCAB-FITC

Random DAR 0.5

Random DAR 1

Random DAR 2

Concentration (pM)

Cyto

toxic

ity (

%)

EC50

B-FITC

0.9711

AB-FITC

0.07031

DAR 0.5

2.602

DAR 1

2.194

DAR 2

1.463

*

Site-specific vs Random

*Random FITC

conjugation via NHS

method

B

C D

E F

A

antigen

binding

Anti-CD19 Switch

“AB”

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In vivo Anti-Tumor Activity

In vivo xenograft• Luciferized NALM6 cells (0.5x106) were IV injected to NSG mice

Anti-FITC CAR-T

Day 6

Day 12

Day 19

Day 33

Day 47

CART 19

CART-19 CD19 Fab B-FITC AB-FITC E-FITC EF-FITCRandom

DAR 2

Optimized anti-CD19-AB switch with anti-FITC CAR-T cells exhibits equivalent tumor regression as

UPenn CART-19 in NALM-6 xenograft model

Day 1: Tumor

Inoculation, IV

Day 7: 40x106 (~70% CAR+) CAR-T infusion, IV

Fab switch dose: 0.5 mg/kg q.o.d (Day 7 ~ 17)

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Application to other systems:

Optimization of anti-Her2 switches

Anti-Her2 (Herceptin Fab) switches

Her2 ECD

~ 46kDa

Herceptin Fab

A

B

F

E

S K B R 3 (H e r2 3 + )

1 0 -4 1 0 -2 1 0 0 1 0 2 1 0 4 1 0 6

0

2 0

4 0

6 0

8 0

1 0 0 B -F IT C

E -F IT C

A B -F IT C

E F -F IT C

C o n c e n tra t io n (p M )

Cy

toto

xic

ity

(%

)

EC50

B-FITC

9.286

E-FITC

4.298

AB-FITC

10.39

EF-FITC

5.871

M D A M B 2 3 1 (H e r2 1 + )

1 0 -4 1 0 -2 1 0 0 1 0 2 1 0 4 1 0 6

0

2 0

4 0

6 0

8 0

1 0 0 B -F IT C

E -F IT C

A B -F IT C

E F -F IT C

C o n c e n tra t io n (p M )

Cy

toto

xic

ity

(%

)

EC50

B-FITC

125.0

E-FITC

25.48

AB-FITC

33.34

EF-FITC

3.521

B

C D

E F

A

antigen

binding

Anti-Her2 Switch

“EF”

• Larger Extracellular Domain

• Membrane-Proximal Epitope

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Application to other systems:

Optimization of anti-CD22 switches

CD22 ECD

~ 75 kDa

Orentas et al. Blood 2013

Like Herceptin, M971 binds to membrane-proximal epitope of CD22

Anti-CD22 (M971 Fab) switches

*

*

10 -2 100 102 104 106

0

20

40

60

Concentration (pM)

% C

yto

tox

icit

y

A-FITC

E-FITC

B-FITC

F-FITC

AB-FITC

EF-FITC

EC50

A-FITC

1268

E-FITC

1611

B-FITC

7965

F-FITC

1418

AB-FITC

1820

EF-FITC

56.57

NALM6 (CD22 low)

* Conjugation site (B) that kills the activity

B

C D

E F

A

antigen

binding

Anti-CD22 Switch

“EF”

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Application to other systems:

Optimization of anti-CLL1 switches

Anti-CLL1 (1075.9 Fab) switches

The size of CLL1 antigen is relatively small (~ 24 kDa), similar to CD19 (~30 kDa)

1 0 -4 1 0 -2 1 0 0 1 0 2 1 0 4 1 0 6

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

C o n c e n tra tio n (p M )

cy

toto

xic

ity

(%

)

A -F IT C

B -F IT C

A B -F IT C

EC50

A-FITC

47.05

B-FITC

57.83

AB-FITC

5.446

Valency Effect

1 0 -4 1 0 -2 1 0 0 1 0 2 1 0 4 1 0 6

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

C o n c e n tra tio n (p M )

cy

toto

xic

ity

(%

)

A -F IT C

B -F IT C

C -F IT C

D -F IT C

E -F IT C

F -F IT C

EC50

A-FITC

48.70

B-FITC

33.41

C-FITC

130.3

D-FITC

69.58

E-FITC

152.8

F-FITC

139.4

Site Effect

B

C D

E F

A

antigen

binding

Anti-CLL1 Switch

“AB”

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Impact of the Size of Antigen and the Location of Epitope:

Optimization of CAR vs Switch

CAR optimization

(scFv and spacer)vs

Universal CAR +

Switch optimization

(location of TAG)

B

C D

E F

A

Wang R et al. J Immunol 2009

TCR-MHC

(Fixed)CAR-TAA

(Variable)

Guest RD al. J Immunother 2005

Spacer

CAR

TAA

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Can we control the activity of switchable CAR-T cells to

address safety issues associated with conventional CD19-

targeting CAR-T?

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Control of in vivo CAR-T Activity

Anti-FITC CAR-T

+ Anti-CD19-FITC (AB)

Day 12

Day 19

Day 6

Day 25

PBS CART-19 0.05 0.0050.5 mg/kg

Day 31

1 0 1 8 2 7

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

P B L T c e ll c o u n t

D a y s a fte r N a lm -6 iv

Re

lati

ve

ev

en

ts/u

L

C A R T -1 9

0 .5 m g /k g

0 .0 5 m g /k g

0 .0 0 5 m g /k g

0 .5 m g /k g ( n o t u m o r )

in vivo activity and proliferation of anti-FITC CAR-T cells can be regulated in a switch dose-

dependent manner

Anti-FITC CAR-T

+ Anti-CD19-FITC (AB)

Day 12

Day 19

Day 6

Day 25

PBS CART-19 0.05 0.0050.5 mg/kg

Day 31

Dose titration of switch (treatment day 7 ~ 17)

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1. Severe cytokine release syndrome (CRS) due to the rapid,

uncontrolled activation of CAR-T

2. Long term normal B cell depletion due to the persistent activity

of CAR-T

Adverse Effects of CD19-targeting CAR-T

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Treatment-Related Toxicity Observed in Xenograft Model

Anti-FITC CAR-T

+ Anti-CD19-FITC (AB)

Day 12

Day 19

Day 6

Day 25

PBS CART-19 0.05 0.0050.5 mg/kg

Day 31 CA

RT

-19

0.5

mg

/kg

0.0

5m

g/k

g

0.0

05m

g/k

g

0.5

mg

/kg

(n

o t

um

or )

8 0

9 0

1 0 0

1 1 0

1 2 0

D a y 1 0 : B o d y w e ig h t % c h a n g e

We

igh

t (%

)

**

Rapid tumor clearance by CART19 or high dose switch (0.5 mg/kg) group developed

acute toxicity (BW% change)

Development of toxicity is tumor-dependent (no tumor group)

Suboptimal dose (0.05 mg/kg) slowly, but yet significantly, reduced the tumor burden

with minimal BW loss

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Time

Switch dose

Tumor burden

Switch Dose Escalation: Prevention of the Acute Toxicity

Anti-FITC CAR-T

Day 12

Day 18

Day 6

Day 25

PBS 0.05 (x6)0.05 (3)

+ 0.5 (3)0.5 (x6) mg/kg

Day 33

0.005 (x6)

Anti-FITC CAR-T

Day 12

Day 18

Day 6

Day 25

PBS 0.05 (x6)0.05 (3)

+ 0.5 (3)0.5 (x6) mg/kg

Day 33

0.005 (x6)

Initial low dose (0.05mg/kg) switch reduced a majority of tumor with minimal toxicity and

subsequent dose escalation (0.5 mg/kg) eliminated the remaining tumor burden

Dose

increased

to 0.5mg

/kg

Anti-FITC CAR-T

Day 12

Day 18

Day 6

Day 25

PBS 0.05 (x6)0.05 (3)

+ 0.5 (3)0.5 (x6) mg/kg

Day 33

0.005 (x6)

Dose-dependent elevation of inflammatory cytokines (human and mouse)

0 1 0 2 0 3 0

6 0

8 0

1 0 0

1 2 0

B o d y W e ig h t C h a n g e

D a y s a fte r N a lm -6 iv

% B

od

y w

eig

ht

ch

an

ge

P B S

0 .5 m g /k g (x 6 )

0 .0 5 m g /k g (x 6 )

0 .0 5 m g /k g (x 3 )

+ 0 .5 m g /k g ( x 3 )

0 .0 0 5 m g /k g (x 6 )

PB

S

0.5

mg

/kg

0.0

5 m

g/k

g

0.0

05m

g/k

g

0

1 0 0 0

2 0 0 0

3 0 0 0

h IF N g

pg

/mL

***

***

PB

S

0.5

mg

/kg

0.0

5 m

g/k

g

0.0

05 m

g/k

g

0

1 0 0

2 0 0

3 0 0

h T N F

pg

/mL

**

n .s .

Inflammatory Cytokines

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1. Severe cytokine release syndrome (CRS) due to the rapid,

uncontrolled activation of CAR-T

2. Long term normal B cell depletion due to the persistent activity

of CAR-T

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A proof-of-principle in vivo experiment: Repopulation of

B cells in immunocompetent mice

FITC

CAR mTcells

αCD19-FITC

αCD19

CAR mTcells

normal B cell depletion

B-aplasia

Syngeneic mouse (B6) CART cells

B cell

repopulation

Allow time

For recovery

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Overcoming B cell Aplasia by Termination of Switch Dosing

Day 0: CTX

preconditioning

Anti-mCD19 switch dose: 1 mg/kg qd (Day 2 ~ 11)

Day 1: mCART-19 or mCART-FITC

D a y 5 D a y 1 2 D a y 2 2

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

P B L C D 1 9+ c o u n ts

Re

lati

ve

ev

en

ts/u

L

C o n tro l

a n ti-C D 1 9 (1 D 3 ) C A R -T

a n ti-F IT C C A R -T +

a n ti-C D 1 9 -F IT C

n s n s

*

**

PNAS 2016

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Platform Expansion:

Peptide Neo-Epitope (PNE)-based Switches

14-aa PNE sequence from the yeast transcription factor GCN4

IgG4m sCAR-T cells

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Effect of the CAR Hinge Design (Length and Dimerization)

CD8: 45-aa CD8-based hinge

IgG4: 12-aa sequence derived from the hinge of IgG4 (short cell-to-cell distance)

mIgG4: IgG4 with serine-to-proline mutation (S228P) to enhance CAR dimerization

PNAS 2016

| 28

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Summary

Optimization of the structure of the switch molecules as well as the CAR

hinge sequence is crucial for the robust anti-tumor activity of switchable

CAR-T cells.

The ability to control the temporal activation of the CAR-T cell response

by switch dosage may be helpful clinically to ameliorate adverse events

associated with severe CRS.

the ability to “turn-off” the activity of the CAR-T cell by discontinuation

of switch dosing can potentially prevent adverse effects associated with

the persistent activity of CAR-T cells, such as B-aplasia.

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Young-Ho Lee

Yujean Lee

Sang Jun Lah

Hyun Cheol Jeong

Segi Kim

Jung Hyun Cha

Hyeong Ji Lee

Hyung Ryul Choi

Acknowledgments

TSRI

Pete Schultz

Hwayoung Yun

Sei Hyun Choi

Josh Cao

Brian Lawson

Calibr Kim group

Jennifer Ma

Minsoo Kim

Ji Young Kim

Xinxin Wang

Eduardo Laborda

Holly Pugh

Collaborators

Travis Young

Stephanie Pinkerton

KAIST Kim group