D35 Project

Ihsan Gursel,

Dennis Klinman,
Cevayir Coban,
Ken J. Ishii,
Fumihiko Takeshita,
Daniela Verthelyi,
Mayda Gursel

Ajinomoto Bio-Pharma
Services, GeneDesignWhat is CpG DNA?
CpG DNA is a general term of immunostimulatory DNA
containing unmethylated CG dinucleotides with certain
flanking sequences (=CpG motifs).
Bacterial DNA contains these CpG motifs ~20 fold morethan vertebrate (mammalian) DNA due to CpG methylation
and CpG suppression in mammalian DNA.
Oligonucleotides containing the CpG motifs (CpG ODN)
mimic the activity of bacterial DNA.

What are CpG Motifs?
CpG motifs (mouse) Pu-Pu-C-G-Py-Py Immunostimulatory

ActivityCpG ODN

T-C-A-A-C-G-T-T-G-A++++Non CpG ODN

T-C-A-A-G-C-T-T-G-A-

CpG ODN

T-C-G-A-C-G-T-C-G-A++++

Methyl CpG ODN T-C-GA-mC-G-T-C-G-A-

Non CpG ODN-T-C-T-C-C-G-G-A-G-A

Molecular Basis of TLR-Ligand Interaction

4Ishii KJ and Akira S Cell Host Microbe 2008

Therapeutic applications of CpG ODNCpG ODN

Innate immune activation(­ pDC, B, Mf Þ ­ NK cell)
PathogenDC maturation
B cell activation

Tumor
Th1,IFNs, IL12, NO, Chemokines
NK killing, phagocytosis

Th2

VaccineAllergen

IgG2a, IFNg, CTL
IgE, Eosinophilia

Anti-infection
Anti-Cancer

Anti-Allergen

Th1 Adjuvant

Ishii KJ et al Curr. Op. Mol Ther. 2004

CpG ODN Improves Host Resistanceto a Variety of Pathogens
CpG ODN(50ug)

Challenge

Treatment
SalineCpG ODN

Organism
L. monocytogenes
L. monocytogenes

Challenged Dose103LD50103

SalineCpG ODN

Malaria sporozoites
Malaria sporozoites

102102088

SalineCpG ODN

Ebola virus
Ebola virus

103103050

SalineCpG ODN

P. aeruginosa
P. aeruginosa

102102003 days

Time% protected0100

Klinman DM, Verthelyi D, Takeshita F and Ishii KJ Immunity 1999 11(2):123-9

24h

CpG ODN

2-3daysB

Polyreactive

TLR9/MyD88

Phago
cytosisC-G-Py-Py-T-A

M!

G-A-Pu-Pu-

1-3wks

IgM

, NO?

NK

IL-12

NK killing, IFNg

Pathogensorcancer

cDC
IL-12
IL-15
CD40L

pDC

Thy1.2 DC
IL-12, IFNg

IFNg, NO?

(IFNa =X)
IKDC

Activation, IL-12, IFNa(?), IFNg

CpG ODN acts as an immunoprophylaxis against variety of inf. Org.
AnimalOrganism% of protection

Mouse

L. monocytogenes

Mouse

F. tularensis

Mouse

M. tuberculosis

60-100ChickenE. Coli

90-100Mouse

P. yoelii

80

Mouse

L. major

100

Rhesus macaqueL. major

0 (K) or 100 (D)Mouse

Friend virus

74

Mouse

HSV-250

Mouse

Ebola virus

50MouseRML prion

100

0 or 100100Ishii KJ et al., Curr Opin Mol Ther. 2004 6(2):166-74.

Obstacles• Optimal CpG motifs for mouse are not active
in human immune system.
• Low efficacy of DNA vaccines are observed
currently in human clinical trials (than that ofmouse).
• TLR9 expression in immune cells is different
between human and mouse .

®Needs humanized CpG motifs

Humanization of CpG DNA (ODN): Distinct types of CpG
Mouse CpG
NN-Pu-Pu-CpG-Py-PyNN

B cellmDC

pDC

K-Type CpG ODN (PS)
NNNTCGT/ANNN

D-type CpG ODN (mix)GGnnnatcgatnnngggGG

B cellpDC

Proliferation
Polyclonal activationIL-6, IgM, chemokines

MHC, CD40, CD86
IFNa, TNFa, chemokinesmDC
APC function
CD8 T cell activation­ IFNaNK
NK activity
IFNg

Klinman DM et al Patented 1999,Verthelyi D, Ishii KJ et al J. Immunol. 2001, Ishii KJ et al Curr Opin Mol Ther. 2004 6(2):166-74.

GC

A

T

T

A

T

D

GC

GC

CGGGTC

C

GG

GGGGC
A

A GTK

TT

G

Required parameters for activity:-Minimum length 12 bp-PS backbone- multiple CpG motifs

Required parameters for activity:- Minimum length 18 bp- PO Backbone- Unmethylated CpG- Central hexameric motif- Selfcomplementary regions flanking the hexamer- 3’ Poly Gs

Individual innate immune response modifiers induce distinctresponses in human PBMC.
MPL

K ODN

(TLR4) (TLR9)

D35 ODN(TLR9)

Proliferation++++++-(B cells)

IgM++++++-( B cells)

IL-10+++++(B cells)

TNFα+++++++/-(Monocytes)

IL-6++++++++(Monocytes)

IP-10++++++(Monocytes)

IFNa+-++++(pDC)

IFNg++++++(NK cells)

Humanization of CpG DNA (ODN): Distinct types of CpG
K(B) -Type CpG ODN (PS) D(A)-type CpG ODN (mix)Mouse CpG
GGnnnatcgatnnngggGG
NN-Pu-Pu-CpG-Py-PyNN NNNTCGT/ANNN

B cell mDC

pDC

B cellpDC

Proliferation
Polyclonal activation
IL-6, IgM, chemokines

MHC, CD40, CD86
IFNa, TNFa, chemokines

mDC
APC function
CD8 T cell activation­ IFNaNK
NK activity
IFNg

Ishii KJ, Gursel I, Gursel M, Klinman DM. Curr Opin Mol Ther. 2004 6(2):166-74. Ishii KJ and Akira S Trends in Immunology 200613

K3 is on the way to human
1) Malaria vaccine BK-SE36 (Prof. T Horii) with
Humanized CpG ODN (K3) / Aluminum hydroxide）Ph1a(Osaka, 2013), Ph-2 (Uganda (Ph1-b) now in Ph-2
in Burkina Faso (AMED/GHIT/EVI)2) used in human clinical trial for cancer
immunotherapy and soon for vaccine adjuvant against
AMR, Pandemic Flu,

Humanization of CpG DNA (ODN): Distinct types of CpG
K(B) -Type CpG ODN (PS) D(A)-type CpG ODN (mix)Mouse CpG
GGnnnatcgatnnngggGG
NN-Pu-Pu-CpG-Py-PyNN NNNTCGT/ANNN

B cell mDC

pDC

B cellpDC

Proliferation
Polyclonal activation
IL-6, IgM, chemokines

MHC, CD40, CD86
IFNa, TNFa, chemokines

mDC
APC function
CD8 T cell activation­ IFNaNK
NK activity
IFNg

Ishii KJ, Gursel I, Gursel M, Klinman DM. Curr Opin Mol Ther. 2004 6(2):166-74. Ishii KJ and Akira S Trends in Immunology 200615

!"#$%&'"#()'&*)"+)*,-.+/&,"*00(#$0$,('+1$2"345"
6$2"12&+10&#1"$6"7&*/80+#*+/*/

Experience in murine models of leishmaniasis:Lesions size

§ CpG ODN can cure mice of lethal L. major infection., even whentreatment is delayed until day 20 (Zimmermann et al., J. Immunol.,1998)§ Systemic or local CpG therapy prevents death in mice with footpad L.
major infection. (Walker et al., PNAS, 1999)

UntreatedCpG ODN d1
CpG ODN d20weeks• Clinical study BCG improved outcome as treatment
adjuvant in CL and PKDL≠

How about Monkey?

In vivo D35 induces increase levels ofIFNa and IFNg in serumIFNa2Saline
D35
Control ODN6020Saline
D35
Control ODNTimes Above Saline Average

Times Above Saline Average

70IFNg

25

*50

1540

1030
20

51000-3 days

3hrs

4-8 days

12 days

17 days-3 daysTime Point

Macaque treated in vivo with D35(SC 0.5mg/kg on days 1, 3 & 7).
5 macaques per group.

3hrs

4-8 daysTime Point

12 days

17 daysFold increase in gene expression in skin over time in macaques injected with D35 or L.
major (note response in hours vs. days)
SALINEpDC
APC
Type I IFN
TLR & IRF
TcellsT cellactivation
Tcellchemotaxis
Cytotoxicity<0.05
0.05-0.1Interleukins
0.1-0.3
0.3- 33-5
5-10
10-50
50-100100-500
500-1000
1000-5000>5000

Adhesion

gene
CD209
CD80
CD83
CD86
IFNa2
IFNB1
IRF7
TLR7
TLR9
CD3e
CD4
CD8
CD28
CD40
CD69
CCL5
CXCL10
IFNG
GZMB
PRF1
IL1A
IL1B
IL2
IL2RA
IL6IL8
IL10
IL-12AIL12B
TNF-ALPHA
TGFb
FAS
Fas Ag CD95
FOXP3
NOS9A
SELE
SELP

0624

D-ODN (hours)
4806

Leishmania(days post infection)
C-ODN
6 d1 24 d448 d1072d24

24 gene48 720d0pDC
CD209CD80
CD83
APC
CD86
IFNa2
Type I IFN
IFNB1
IRF7
TLR7
TLR and IRF
TLR9CD3eTcells
CD4CD8CD28
Tcell activation
CD40
CD69
Tcell chemotaxis CCL5CXCL10IFNG
Cytotoxicity
GZMB
PRF1
IL1A
IL1B
IL2
IL2RA
Interleukins
IL6
IL8IL10
IL-12AIL12B
TNF-ALPHA
TGFb
FAS
FasL
FOXP3
NOS9A
SELE
Adhesion
SELP

d66

Mean Lesion Area (mm2)

CpG as an immunoprotective agent: D35 reduces the severity of a
Leishmania amazonensis infection
1000

D ODN
K ODN
SALINE

8006004002000-3 0 37142228354249

63

Day after infectious challenge

Note: Lesion size of D ODN treated animals was significantly
reduced when compared to saline treated controls or macaques treatedwith a different ODN sequence (p <.03, N=6/group).
J.Immunol.2003

D35 made by GeneDesign
reduces lesion size and improves healing in response to L. major infection.

Thacker SG, McWilliams IL, Bonnet B, Halie L, Beaucage S, et al. (2020) CpG ODN D35 improves the response to abbreviated lowdose pentavalent antimonial treatment in non-human primate model of cutaneous leishmaniasis. PLOS Neglected Tropical Diseases
14(2): e0008050. https://doi.org/10.1371/journal.pntd.0008050
https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0008050

New project funded by GHIT

Clinical development of CpG-D35 for combined
treatment of cutaneous leishmaniasis• RFP Year
• Disease• Intervention

2020
NTD (Leishmaniasis)
Drug• Development Stage
Phase 1 Clinical Development• Collaboration Partners
Drugs for Neglected Diseases initiative (DNDi)
The University of Tokyo
Ajinomoto Bio-Pharma Services
GeneDesign (GeneDesign)D35 project will address global health challenges
The expectation based on preclinical data is that combining
CpG-D35 with chemotherapy for the treatment of patientswith CL will;1) speed lesion re-epithelization,2) minimize scarring,3) reduce the rate of relapse,
and hopefully4) reduce the risk of developing drug resistance

1. Flynn B, Wang V, Sacks DL, Seder RA, Verthelyi D. 2005. Prevention and treatment of cutaneous leishmaniasis in primates byusing synthetic type D/A oligodeoxynucleotides expressing CpG motifs. Infect Immun;73(8):4948-54.
2. Verthelyi D, Gursel M, Kenney RT, Lifson JD, Liu S, Mican J, Klinman DM. 2003. CpG oligodeoxynucleotides protect normal
and SIV-infected macaques from Leishmania infection. J Immunol; 170(9):4717-23.
3. Miranda-Verastegui C, Tulliano G, Gyorkos TW, Calderon W, Rahme E, Ward B, Cruz M, Llanos-Cuentas A, Matlashewski G.
2009. First-line therapy for human cutaneous leishmaniasis in Peru using the TLR7 agonist imiquimod in combination withpentavalent antimony. PLoS Negl Trop Dis; 3(7):e491. doi: 10.1371/journal.pntd.0000491

Innovation in D35 project
Our proposed approach is clearly differentiated from the current
treatment recommendations for CL.
Developing a novel D class CpG to promote the immune
response required for the control of Leishmania infection, incombination with chemotherapy, will provide a major step
forward over existing monotherapies or combination therapies
targeting the parasite only.
Conventional chemotherapy -> KILL most Leishmania
parasites
D35 promotes the HOST immune response in the host, theimmune system -> LIMIT and REMOVE any remaining
parasites.

Special thanks toDaniela Verthelyi at FDA and her teamDennis Klinman, Ihsan Gursel and the lab alumni
Special thanks toByron and Steve

THANK YOU!
⼀期⼀会 ICHI GO ICHI E ; Live every day as though it were last.