Extracellular Vesicles from Cardiosphere-Derived Cell and from Mesenchymal

Stem Cells Show Different Immunomodulatory Capabilities and Distinct RNA Cargo

Introduction

Summary and Conclusions

Methods

Results• Cardiosphere-derived cells (CDCs) possess cardioprotective, regenerative, and

immunomodulatory characteristics when delivered to the heart post-myocardial

infarction (MI) which appear to be unique traits to CDCs

• Human CDC-derived extracellular vesicles (CDC-EVs) recapitulate the effects of

CDCs in acute and chronic in vivo models of MI suggesting most of the

therapeutic effects of CDCs are mediated by CDC-EVs

• Here we tested the hypothesis that a distinct cargo profile will define the

functional efficacy between CDC-EVs and mesenchymal stem cell-derived EVs

(MSC-EVs)

• CDCs derived from different donors possess variable regenerative potency in an

in vivo MI animal model thus we compared EVs derived from potent and non-

potent CDCs (Cheng et al, JACC Heart Failure, 2014)

• To assess the immunomodulatory role of CDCs and CDC-EVs, we investigated

the immunomodulatory effect of EVs in a macrophages and a T-cell based assay

(de Couto et al, JCI, 2015; de Couto et al, Circ, 2017)

• The size and particle and protein concentration of CDC-EVs are significantly greater and higher than MSC-EVs irrespective from potency

• CDC-EVs and MSC-EVs show a differential cargo composition with a higher Y-RNA and miRNA content in CDC-EVs compared to 15 days and 48 hour

MSC-EVs

• miRNA clustering analysis showed that CDC-EVs and MSC-EVs cluster separately with a sub-cluster present in MSC-EVs (MSC-EVs 48h)

• CDC-EVs demonstrated differential clustering between in vivo potent and non-potent CDCs

• Potent CDC-EVs showed a significant upregulation of miR#1 and miR#2 and downregulation of miR#3 compared to non-potent CDC-EVs

• EVs from potent CDCs showed a stronger dose-dependent upregulation of anti-inflammatory genes in activated rat and mouse macrophages compared

to EVs obtained from non-potent CDCs or from MSCs

• EVs from potent CDC cell lines reduce the increased accumulation of activated macrophages in an in vivo peritonitis mouse model

• CDC-EVs have strong immunomodulatory capabilities on human activated T-lymphocytes Acknowledgements: DoD Award # (PR150618)

Isolation of MSCs and CDCs

• MSCs were obtained from Lonza

• CDCs:

Isolation and characterization of EVs

In vivo macrophage assay (mouse)

Group 1 Plasmalyte (P) Plasmalyte (P)

Group 2 CDC-EV (E) Plasmalyte (P)

Group 3 CDC-EV (E) CDC-EV (E)

• EV characterization:

- Particle size and concentration (Nanoparticle tracking technology)

- Protein concentration (DC protein assay)

• CDC-EV samples: n=15 (15 days); MSC-EV samples: n=4 (15 days), n=2 (48h)

EV characterization

Exosomal miRNA of CDC-EVs and MSC-EVs

Donor heart from organ

procurement organization

Explant formation

Atria and septum

Cardiosphere formation Cardiosphere-Derived

Cells

Zymosan injection (i.p.)Sacrifice

D1 D2 D3

10kDa MWCO0.45 µm

PES

Confluent P5

CDCs/MSCs4x washes + culture

with serum-free medium

Concentrated

CM with EVs

Storage at -80°C

48 hours or

15 days

• EV isolation:

Immunomodulation: In vivo mouse macrophage assay

A.S. Walravens1, S. Smolgovsky1, L. Kelly1, H. Rachid2, R. Al-Daccak2, K. Peck1, G. de Couto1, L. Marbán1, L. R.-Borlado1

1Capricor Therapeutics, 8840 Wilshire Blvd, Beverly Hills, CA 902112HLA et Medicine Jean Dausset Laboratory, Hopital Saint Louis, Paris, France

In vitro macrophage assay (mouse and rat)

3% Brewer’s Thioglycollate (i.p.)

72h Isolation

peritoneal

macrophages

Plating 2x106 cells / well

on a 6-well plate

dosing CDC/MSC-

EVs per cell

1h Gene expression

of Arg1 and

Nos2

6h

EV dosing: 500 or 2500 particles per cell

EV treatment: 1.55x1010 particles per mouse

EV or plasmalyte administration: tail vein

Exosomal small RNA sequencing

• miRNeasy Serum/Plasma kit to isolate total RNA

• NextGen 500 Sequencing (Illumina); 30 ng/sample RNA input

• CDC-EV samples: n=10 (15 days); MSC-EV samples: n=4 (15 days), n=2 (48h)

Mode size Particle concentrationProtein

concentration

Cargo of CDC-EVs vs MSC-EVs

K-means (K=3)MSC1 15d

MSC2 15d

MSC3 15d

MSC4 15d

MSC3 48h

MSC4 48h

CDC1

CDC2

CDC8

CDC3

CDC7

CDC9

CDC4.1

CDC11

CDC4.2

CDC5

CDC10.1

CDC10.2

CDC12

CDC6

MS

C1 1

5d

MS

C2 1

5d

MS

C3 1

5d

MS

C4 1

5d

MS

C3 4

8h

MS

C4 4

8h

CD

C1

CD

C2

CD

C8

CD

C3

CD

C7

CD

C9

CD

C4.1

CD

C11

CD

C4.2

CD

C5

CD

C10

.1

CD

C10

.2

CD

C12

CD

C6

#, p<0.0001

* vs Potent 2500p, p<0.01

Exosomal miRNA cargo of potent vs non-potent CDC-EVs

miR#1

CD

C8

CD

C1

CD

C2

CD

C6

CD

C9

CD

C7

CD

C5

CD

C11

CD

C4

.1

CD

C4

.2

CD

C3

CD

C1

0.1

CD

C1

0.2

Co

un

ts

PotentNon-potent

miR#2

CD

C8

CD

C1

CD

C2

CD

C6

CD

C9

CD

C7

CD

C5

CD

C11

CD

C4

.1

CD

C4

.2

CD

C3

CD

C1

0.1

CD

C1

0.2

PotentNon-potent

miR#3

CD

C8

CD

C1

CD

C2

CD

C6

CD

C9

CD

C7

CD

C5

CD

C11

CD

C4

.1

CD

C4

.2

CD

C3

CD

C1

0.1

CD

C1

0.2

PotentNon-potent

Immunomodulation: In vitro rat macrophage assay Immunomodulation: In vitro mouse macrophage assay

T

T

MoMo

NK B

B

B

T

T

T

T

Human PBMC T cells

Negative

Selection

PHA

CDC-EVs

T

T T

T T

T

Proliferation

quantification

NK

In vitro T-cell assay

CFSE labelling of T cells

Analysis with flow cytometry

Mediu

mPHA

CDC

5x109 E

V/ml

10x109 E

V/ml

20x109 E

V/ml

0

20

40

60

80

100

% p

rolif

erat

ing

cells

CD4+

CD8+

*

****

***

****

*

Mediu

mPHA

CDC

5x109 E

V/ml

10x109 E

V/ml

20x109 E

V/ml

0

20

40

60

80

100

% p

rolif

erat

ing

cells

CD4+

CD8+

*

****

***

****

*

Mediu

mPHA

CDC

5x109 E

V/ml

10x109 E

V/ml

20x109 E

V/ml

0

20

40

60

80

100

% p

rolif

erat

ing

cells

CD4+

CD8+

*

****

***

****

*

PHA

% p

rolife

rati

ng

cells

Immunomodulation: In vitro T-cell assay

*, p<0.05

***, p<0.005

****, p<0.001

Potent

Non-potent

K-means (K=3)CDC10.1

CDC10.2

CDC3

CDC4.1

CDC4.2

CDC11

CDC5

CDC7

CDC9

CDC1

CDC2

CDC8

CDC6

CDC12

CD

C10

.1

CD

C10

.2

CD

C3

CD

C4.1

CD

C4.2

CD

C11

CD

C5

CD

C7

CD

C9

CD

C1

CD

C2

CD

C8

CD

C6

CD

C12

Potent

*, p<0.05

Jennifer Moseley, Chris Sakoda, Saravana Kanagavelu, Sharon Vaturi, Liang Li, Linda Marban, Luis Rodriguez-Borlado

Capricor Therapeutics, 8700 Beverly Boulevard, Davis Building, Los Angeles, CA 90048

Efficacy and in vitro Uptake of EVs from Cardiosphere-Derived Cells

CONCLUSIONS

REFERENCES

1. Ibrahim, A. G. E., Cheng, K., and Marban, E. (2014) Exosomes as Critical Agents of Cardiac Regeneration Triggeredby Cell Therapy. Stem Cell Reports. 2, 606-619

• CDC-EVs are effective in a variety of pre-clinical mouse models,including models of radiation dermatitis, GvHD and Duchennemuscular dystrophy.

• CDC-EVs are taken up by immune cells with increased uptake indendritic cells and macrophages.

• These results demonstrate the therapeutic potential of CDC-EVsfor several indications and allow for a better understanding of theirfate in vivo.

Gordon EV August 2018

INTRODUCTION

CDC-EVs are made from Cardiosphere-derived cells

Cardiosphere-derived cells (CDCs), a cell product currently inclinical trials (Regress-HFpEF, HOPE 2, ALPHA PAH) secreteextracellular vesicles. CDC-EVs contain known EV markers (Figure1A) and are <200 nm in size (Figure 1B). CDC-EVs show a uniquemiRNA expression profile when compared with fibroblast (NHDF)EVs (Figure 1C).

Figure 2. CDC mechanism of action. CDCs secrete EVs containing bioactive moleculesable to regulate different responses involved in tissue regeneration.

Capricor Therapeutics has an ongoing clinical trial for the treatment of Duchennemuscular dystrophy (DMD) using an allogeneic cell therapy product, cardiosphere-derived cells (CDCs). It is widely accepted that most of the therapeutic effectsobserved in cell therapies using non-engrafting cells are caused by paracrine factorssecreted by the delivered cells early after administration. In early studies in a mousemodel of myocardial infarction, the therapeutic benefits of CDCs were recapitulatedusing the extracellular vesicles (EVs) they secrete. In more recent studies, we haveshown that CDC-EVs have a strong immunomodulatory capacity on macrophagesand T cells, making them very attractive for the treatment of inflammatorydisorders.

Here, we show that CDC-EVs administered systemically also recapitulate thebeneficial therapeutic effects of CDCs in a mouse preclinical model of DMD. mdxmice treated with CDC-EVs have a significant increase in exercise capacity comparedwith mice treated with placebo. CDC-EVs also elicit therapeutic effects in models ofradiation dermatitis and graft-versus-host disease.

As we explore the efficacy of CDC-EVs in different preclinical disease models,understanding the uptake and biodistribution of CDC-EVs becomes criticallyimportant. Here we show the in vitro uptake of CFSE-labeled EVs in Jurkat (T cells),THP-1 (monocytes), dermal fibroblasts, epithelial cells, and endothelial cells. Wehave also tested uptake in cell mixtures in vitro and in splenocytes ex vivo. In vitro,we see a dose response in CFSE-labeled EV uptake in Jurkat cells, and little uptake infibroblasts and epithelial cells. In splenocytes ex vivo, uptake of labeled CDC-EVsoccurs rapidly and all cell types tested appear to take up EVs, though phagocyticcells take up CDC-EVs to a larger extent compared with other cells tested.

Taken together, these data aid in the understanding of the target cells and tissuesof CDC-EVs. A better understanding of the fate of EVs will be invaluable in choosingappropriate indications and delivery methods for our EV therapeutic.

ABSTRACT

CDC-EVs recapitulate the regenerative properties of CDCs2.

Figure 1. A) Mass spectrometry of CDC-EVs showing expression of known EVmarkers. B) CDC-EV particle size was measured using images obtained from cryo-transmission electron microscopy. C) Differential expression of miRNAs in CDC-EVscompared to fibroblast-EVs.

CDC-EVs improve exercise capacity in mouse model of DMD

Figure 6. A) CDC-EVs were labeled with CFSE then added to jurkat T cells overnight.CDC-EVs are taken-up by jurkat T cells in a dose dependent manner. B) CDC-EVswere labeled with CFSE then added to T-cells (Jurkat), or monocytes (THP-1). After 1hour, 4 hours, or overnight, cells were analyzed by flow cytometry to detect uptakeof labeled EVs.

CDC-EVs are internalized by immune cells

IN VITRO UPTAKE

D

Figure 7. Mouse splenocytes were incubated with Cell Trace Far Red-labeled CDC-EVs for 10 minutes at 37 °C. Immune cell markers were added and samples wereanalyzed by flow cytometry to assess CDC-EV uptake in the different cellpopulations.

IN VIVO EFFICACY

CDC-EVs improve radiation dermatitis score in irradiated mice

CDC-EVs improve weight and survival in model of GvHD

A B C

CDC-EV to NHDF-EVNHDF-EV to CDC-EV

miR146amiR-22miR-24

mir-210miR-150miR-140-3pmiR-19amir-27bmiR-19b

miR-27a

let-7fmiR-423-5p

let-7bmiR-155

miR-125a-5p

let-7clet-7a

miR-125blet-7e

miR-26a

-200 -100 0 100 200 300 400 500

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

CD63 CD81 LAMP1 Ezrin Alix

CDC

NHDF

No

rmal

ize

d t

o t

ota

l pro

tein

Fold Change

Extracellular vesiclesCardiosphere-Derived Cells

Immunomodulatory

MacrophagesT cells

Anti-apoptotic

Proliferative

Cardiomyocytes

Angiogenic

Vessels

Antifibrotic

Fibroblasts

Regenerative

Progenitor cells

0Days

g radiation Follow up

Sacrifice

Histology

35 43 50 54

Transplant of:5x106 CD3- BM cells2x106 splenocytes

282147

High dose1x1010 EVs

Low dose1x109 EVs

3x109 EVs

n=15 (3x)

2 1 2 8 3 5 4 2 4 9 5 6

0

2 5

5 0

7 5

1 0 0

D a y

Pe

rc

en

t s

urv

iva

l

V e h ic le

L o w d o s e

H ig h D o s e

21 28 35 42 49 56Days

100

75

25

0

% S

urv

ival

50

Percentage Weight Change

2 1 2 8 3 5 4 2 4 9 5 6

-3 0

-2 5

-2 0

-1 5

-1 0

-5

D a y

Pe

rc

en

t w

eig

ht

ch

an

ge

V e h ic le

L o w d o s e

H igh D o se

*

**

***

***

21 28 35 42 49 56Days

-5

-10

-15

-20

-25

-30

% W

eig

ht

chan

ge

VehicleLow doseHigh dose

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 10 20 30 40

* p<0.05** p<0.01*** p<0.001

******

**** *

Day

Mea

n b

lind d

erm

score

±S

EM

Vehicle

CAP-2003

Figure 3. A) Study timeline. Mice were given radiation on Day 0 then a transplant ofCD3+ BM cells and splenocytes. Mice were injected with either low dose of CDC-EVs(1x109, n=15), high dose of CDC-EVs (3x109 -1x1010, n=15), or vehicle control (n=15)as indicated. Mice were sacrificed on day 54. B) Percent weight change for 3 groupsover the 54-day study. C) Percent survival for 3 groups over the 54-day study.

Figure 5. A) Study timeline. Mice were exercised on a treadmill prior to the start ofthe study to assess baseline exercise capacity. 2x109 CDC-EVs or vehicle control wereadministered weekly for 3 weeks by systemic injection. Exercise capacity wasassessed again 1 week after the final injection. B) Exercise capacity of mice giveneither vehicle control or CDC-EVs before administration (week 0) and one week after3 administrations (week 3). C) Exercise capacity on weeks 0 and 3 represented as apercent change from baseline.

0 1 3Week 2

Treatment

Treadmill exercise

Co

un

ts

CFSE signal

Ove

rnig

ht

4 h

ou

rs1

ho

ur

THP-1Monocytes

JurkatT-cells

0 1 3Days

DamageFollow up

Sacrifice

Histology

Treatment

2 4 5 6 7 22 29 36 40

Cel

l Mar

ker

EV uptake

Control ControlCDC-EV CDC-EV

B c

ells

CD

8+

T ce

llsM

on

ocy

tes

Den

dri

tic

cells

Mac

rop

hag

es

Figure 4. A) Study timeline. Radiation was given to mice on days 0, 1, 2, 5, 6, and 7.Mice were injected with either CDC-EVs or vehicle control on days 7, 22, 29, and 36.B) Mean derm score for each group ± SEM. The score reflects the severity of theradiation dermatitis and the groups were blinded for score assessment.

A

B

A

B C

A

B C

A B

01,0005,00010,00025,00050,000

CDC-EVs/cell

CDC-EV1 CDC-EV2