AXA-042 - A Novel Systemic TLR2/6 Agonist for Anti-Tumor Therapy.
2754

Anna Galkin1, Francesca Mercuri2, Nicholas West3, Ping Zhang3, Weiguang Zeng4, Grant McLachlan1, Michael Bettess5, Ian Holmes5, David Jackson4,Chris Smith5, Christophe Demaison2, Tobias Bald6,7, Phil Kearney1.
1 Axelia Oncology, Melbourne, Victoria, Australia; 2 ENA Respiratory, Melbourne, Victoria, Australia; 3 Systems Biology and Data Science, Mucosal Immunology Research Group, Menzies Health Institute Queensland Griffith University, Gold Coast campus, QLD, Australia; 4 Department of Microbiology and Immunology, the Universityof Melbourne, the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia; 5 Brandon Capital Partners, Melbourne, Victoria, Australia; 6 QIMR Berghofer Medical Research Institute, Australia; 7 Institute for Experimental Oncology, University Medical Center Bonn, Bonn, Germany

INTRODUCTIONRESULTS

1IL-1β

1922

Mean Tumour volume (mm3) ± SEM

153110IL-6

16

TNFα

1466

233

IL-10

25

15

130

20IFNα

2100

22

1

1IL-1RA

2132

465TNFβ13611GROα101021613104

31213

5IP-1016

MIP-1α2328MIP-1β1712220317830FC

3171513

EMT6 (Balb/C)

3500

12500

1000

87.3%

AXA-042CXCL10 (IP10)
6000

150000*<0.0001**** ****

*****

4000

10000050000

0Control0.0074

342/6AXA-042

CXCL9 (MIG)

***

60%

710141721

2000

240Days of Treatment

AXA0TL
R2TL
R9/80****
D34%TGI %CR+PR*
49.2% 22%4020

Control0Human PBMCs were incubated for 6hrs with TLR agonists at 5x
reported EC50: TLR4 CRX-527 (0.25 ng/mL); TLR7/8 R848(0.3µg/mL); TLR9 ODN2006 (5 µg/mL); TLR2/6 Pam2CSK4 (0.15ng/mL), TLR2/6 AXA-042 (0.25 ng/mL). Supernatants wereanalyzed using the multiplex Luminex platform. Data is shown as
median fold change (FC) relative to PBS (4 donors).

2000

0500

1060

1000

2000
1500

0.0373

3000

Saline
AXA-042 1μg IP 1x/wk
AXA-042 3μg IP 1x/wk

3000-6TL
R7

0.00350Control

AXA-042

Control

AXA-042

A. AXA-042 was delivered intraperitoneally (IP) 1x a week (QW) (day 0, 7, 14) for 3 weeks at 3 or 1 μg/mouse. Data is shownas Mean Tumor Volume (mm3) ± SEM (n=9-10 per group). %TGI = % tumor growth inhibition. CR = complete response. PR= ongoing regression. Statistical significance relative to control was determined using Two-Way Anova with Sidak’s Multiple
Comparisons Test, * p<0.05, *** p<0.001, **** p<0.0001. B. Plasma cytokines were assessed on day 14, 6 hrs post AXA-042
3 μg treatment. Statistical significance relative to control was determined using Student’s T-Test

Transporter Functions
TNF Superfamily
TLR
T-Cell Functions
Senescence
Pathogen Response
NK Cell Functions
Microglial Functions
MHCMacrophage Functions
Leukocyte Functions
Interleukins
Interferon
InnateInflammation
HumoralDendritic Cell Functions
Cytokines & Receptors
Complement Pathway
Chemokines & Receptors
Cell Cycle
CD molecules
Cancer Progression
Basic Cell Functions
B-Cell Functions
Apoptosis
Antigen Processing
Adhesion
Adaptive-10Figure 4. AXA-042 Enhances anti-PD-1 Ab Response in the CT26 Tumor ModelA.
Mean Tumour volume (mm3) ± SEM

101
10100Concentration pg/ml

1000

2000%TGI D34 % CR

1500

56.4%

1000500****

0%

70.5%

10%

86.6%

40%

****

****
10036********
13********

2000

100017Saline0.0181

1000
800
600
4002000Saline

IFITM180000.0093
6000
4000
200001Saline

AXA-042 10μg
IL12B

8000.0139

600
4002000Saline

0.0015

2000Saline

010202000
1500
1000
5000Saline

1AXA-042 10μg

CD69

GZMB

Saline
AXA-042 10μg

AXA-042 10μg

40010.0352

IFITM1TIGIT
600

AXA-042 10μg

2500

1AXA-042 10μg

1AXA-042 10μg

6000.0094

4002000Saline

1AXA-042 10μg

%TGI42.5%
52.1%
72.1%

**

Figure 8. AXA-042 Target Engagement Signature Identified in Whole Blood

D34 %CR0%0%10%

Non-Responders Display a Distinct Whole Blood DEG Signature
*****
****

1317A.

-603610B.
Day 8Day 15

Days of Treatment

AXA-042 10 μg/mouse IP (A) or IV (B) in combination with anti-PD-1 antibody (Ab) (10 mg/kg IP 2x/wk). Data is shown as Mean Tumor Volume (mm3) ± SEM (n=10 per group).
%TGI = % tumor growth inhibition. CR = complete response Statistical significance relative to control was determined using Two-Way Anova with Sidak’s Multiple Comparisons Test,* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 5. AXA-042 Induces DC and TAM Activation% M2

M2 PD-L1

M1 CD800.00810.00150.0056

AXA-042 + aTNFα
10000

6000

AXA-042 + aLy6G+aRatκ Ab

4000

2000

00AXA-042 + aCD8A Ab

5000

2000

AXA-042 + aCSF1R Ab

0Control AXA-042

0Control AXA-042

Control AXA-042

AXA-042 + Isotype Ab

Both NR and CR

CR only

NR only

Not significant in CR or NR

020104000

AXA-042 + Saline
0.005550MFI6000

40150008000

06010000

CT26 (Balb/C) Day 10

DC CD80

MFI800080

Figure 6. AXA-042 CT26 Efficacy isMacrophage and TNFα Dependent

Mean % TGI ± SEM

10.1

110100 1000 10000
Concentration pg/ml

TLR

EC50 (pg/ml)

TLR

EC50 (pg/ml)

hTLR2

10.4

TLR2

26mTLR2

17.4

TLR2/665

TLR2 NullNot active

TLR1/2

Not active

www.PosterPresentations.com

0AXA-042 10μg

TMEM173 (STING)

CT26 tumor tissues were collected 24 hrs after the 3rd dose of AXA-042 (10 μg/mouse, day 7) forFACS analysis. M2: CD45+CD11b+F4/80+ CD206+ MHCII low; M1: CD45+CD11b+F4/80+
CD206- MHCII +; DC: CD45+CD11c+ MHCII+. Data is presented as either mean % of events
relative to the indicated gate or mean fluorescence intensity ± SEM (n=4-5 per group). Statisticalsignificance relative to control was determined using Student’s T-Test.

AXA-042 (10μg IP 1x/wk) efficacy was performed in presence of CSF1R (400μg 3x/wk),
Ly6G (25μg QD +50 μg anti-Rat κ Ab EOD), CD8A (250 μg every 3 days) or TNFα (200μg every 3 days) depletion Abs. Mean %TGI ± SEM is shown. % TGI for individual
animals was calculated according to the following formula: ((Mean CD10-CD0) – (TD10TD0)) / (Mean CD10-CD0)*100, where T are AXA-042 treated tumors, and C are respectivecontrols (saline + depletion Ab).

AXA-042 10 μg/mouse was delivered IP 1x/wk to CT26 tumor bearing mice for three weeks. Whole blood was collected on days 8 and 15 (24hrs post 2ndand 3rd dose) and immune gene expression analysis was performed using the Nanostring nCounter Mouse PanCancer Immune Profiling panel. A
comparison of the differentially expressed genes (DEGs) between the complete responders vs control and the non-responders vs control on day 8 (A) orday 15 (B) is shown. The color coding indicates whether the DEG was significant (corrected p value < 0.05) in either or both cohorts relative to the control.

CONCLUSIONSRESEARCH POSTER PRESENTATION DESIGN © 20191200

CT26 tumors were collected 6 hrs after the 3rd dose of AXA-042 (10 μg/mouse, day 7) and immune gene expression analysis was performed usingthe Nanostring nCounter Mouse PanCancer Immune Profiling panel. A. Pathway scores were calculated using the Nanostring Pathway Module.
B. Data is shown as mean normalized counts ± SEM (n=4/group). Statistical significance relative to control was determined using Student’s T-Test.

0Days of Treatment

Control AXA-042

00.1

2500

0SalinePathway Scores

SalineaPD-1 Ab
AXA-042 10μg IV 1x/wk
aPD1-Ab + AXA-042 10μg IV 1/wk02

3000

500400CT26 (Balb/C)

3000-52hTLR2
hTLR2/6hTLR1/2

3500-6% of CD11b+F4/80+3hTLR2mTLR2
TLR2 null

OD 650 nM +/- SD

OD 650 nM +/- SD3Saline
aPD-1 AbAXA-042 10 μg IP 1x/wk
aPD-1 Ab + AXA-042 10 μg IP 1x/wk

0

RESULTS
Figure 1. AXA-042 is a potent and selective TLR2/6 Agonist
A.
B.

4000

MFI

• In vitro potency and selectivity of AXA-042 was assessed
across a panel of human and mouse HEK-blue TLR
reporter assays.
• AXA-042 activity was profiled relative to other TLR
agonists in human PBMC cytokine release assays.
• AXA-042 in vivo efficacy was evaluated in the EMT6 andCT26 syngeneic tumor models.
• The Nanostring nCounter™ Mouse PanCancer Immune
Profiling Panel was used to identify AXA-042 tumorlocalized and peripheral response signatures.

B.

CT26 (Balb/C)

4500

Mean Tumour volume (mm3) ± SEM

MATERIALS AND METHODS

1000

Mean Normalized Counts ± SEM

11500

Mean Normalized Counts ± SEM

1320000.0408600

Mean Normalized Counts ± SEM

280

4000

0.03632500

Mean Normalized Counts ± SEM

IL-12p70

B.

Mean Normalized Counts ± SEM

20A.

Mean Normalized Counts ± SEM

1pg/ml1pg/ml2pg/ml2IL6

MRC1 (CD206)

IL12IL6pg/ml

2Figure 7. AXA-042 Engages the Innate and Adaptive Immune Response Pathways

Mean Normalized Counts ± SEM

A.

25IFNγFigure 3. Systemic AXA-042 Efficacy in the EMT6 Tumor
Model is Associated with Cytokine Release
B.

Mean Normalized Counts ± SEM

Figure 2. AXA-042 Displays a Distinct
PBMC Cytokine Release Profile

TL
R4• Treatment approaches that engage both the innate andadaptive immune response have the potential totransform anti-cancer therapy, especially in settings ofcheckpoint inhibitor insensitivity or acquired resistance.
• Toll-like receptors (TLRs) mediate the initial cellularresponse to external pathogens or endogenous
alarmins, activating downstream pro-inflammatorycascades and leading to the activation and recruitmentof key innate subsets.
• TLR2 is a cell surface receptor, expressed predominantly
on macrophages, dendritic cells (DC), neutrophils andsubsets of NK and T cells. TLR2 signaling plays animportant role in NK and T cell cytolytic cell functions,as well as DC and macrophage activation.
• AXA-042 is a novel synthetic TLR2/6 agonist designed
for systemic delivery to re-engage the innate immune
response to help overcome tumor immune escape.

• Systemic delivery of AXA-042 was well tolerated in vivo, demonstrated innate response engagement andanti-tumor efficacy as monotherapy and in combination with anti-PD-1 Ab immunotherapy.
• AXA-042 has completed GLP toxicology studies and initiation of Phase 1/1b trial is planned for early 2022.