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Systemic administration of AXA-042, a novel TLR2/6 agonist, reshapes the tumor
microenvironment, as revealed by single cell sequencing analysis.
Bedrich Eckhardt1*, Kellie Mouchemore1,2,*, Yang Liao1,2, Wei Shi1,2, Francesca Mercuri3, Phil Kearney4, Robin L Anderson1,2 Anna Galkin4.
1 Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; 2School of Cancer Medicine, La Trobe University, Bundoora, Victoria, Australia; 3 ENA Respiratory, Melbourne, Victoria, Australia; 4Axelia Oncology, Melbourne, Victoria, Australia.

* B Eckhardt and K Mouchemore contributed equally as first author contributors

RESULTSINTRODUCTION

MATERIALS AND METHODS• Female Balb/c mice bearing orthotopic EMT6.5
mammary tumors received a single treatment of salineor AXA-042 (10 μg/mouse IV)• Equal numbers of viable cells were recovered fromcollagenase-digested tumors (n=3/group) 24 hours aftertreatment and pooled prior to loading onto a Chromium
Single Cell Chip

Figure 1. AXA-042 Treatment Alters the Composition of the EMT6.5 Tumor Environment
A.

C.

Figure 3. AXA-042 Alters the Composition of Myeloid Population Subclusters
A.

C.

B.

TLR2

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A. Single-cell transcriptomes for all cells dissociated from ETM6.5 tumors, visualized on a Uniform Manifold Approximation and Projection (UMAP)
plot. Numbers displayed next to each cell type in the legend reflect actual cell counts included in each cluster. B. AXA-042 treatment redistributes
the cell composition of the EMT6.5 tumor. Data is shown as % of each cell type C UMAP plot demonstrating enrichment of TLR2 expression inmacrophages, neutrophils and dendritic cell subsets. D. Impact of AXA-042 treatment on TLR2 expression in indicated subsets. Data is shown as %of each cell type.

B.
Saline

AXA-042

Saline

CD4+
CD4T
Treg
CD8T Naive
CD8T Memory Like
CD8T Effector Memory
CD8T Exhausted
All Other Cell TypesA.

AXA-042

AXA-042

Cluster 01
Cluster 02
Cluster 03
Cluster 04
Cluster 05
Cluster 06
Cluster 07
Cluster 08
Cluster 09

A. UMAP of myeloid cell subsets. B. UMAP of granulocytes (03, 04, 06,07), macrophage (01, 02, 08, 09) and monocyte (05) subclusters indissociated EMT6.5 tumors. Numbers displayed next to each cell subset orcluster in the UMAP legends reflect actual cell counts included in eachcluster. C. Heatmap showing expression of marker genes in clusters. Topmarker genes in each cluster were selected based on number ofsignificant comparisons and average FDR value calculated across allcomparisons. No unique marker genes were identified for Macrophagecluster 2. D. AXA-042 redistributes the composition of the myeloid cell
subclusters 24hr post treatment: decreasing macrophage sub-clusters 01,02, 08 and 09 and increasing the neutrophil subsets 03, 04, 06 and 07.

Figure 4. GO Pathway Enrichment Analysis for Differentially Expressed Genes
in TLR2+ Myeloid Subsets Post AXA-042 Treatment

Figure 2. AXA-042 Increases Proportion of Activated T Cell Subsets in EMT6.5 Tumors
A.

SalineTLR2-Gr50D.
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Epithelial Cells
MacrophagesMonocytes
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%

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• 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 onmacrophages, 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.
• Once-a-week treatment with AXA-042 led to 87%
growth inhibition of syngeneic EMT6 tumors (AACR
Annual Meeting 2022 Poster 3502)

C.

CD69+
CD69-B.
CD69+
CD69-

CD8+

• 10x Genomics single-cell transcriptome libraries wereprepared from each sample and sequenced withIllumina NextSeq 550 sequencing platform.
• Cell type annotation based on the ImmGen database
was determined using the SingleR package. T cellsubsets were defined using TILPRED.
• Differential gene expression analyses were completed toidentify AXA-042 responsive gene signatures in cellsubsets that included at least 10 cells. The identified
genes were required to be expressed in at least 3 cells.
RESEARCH POSTER PRESENTATION DESIGN © 2019www.PosterPresentations.com

A. Impact of AXA-042 treatment on proportion of T cell subsets in EMT6.5 dissociated tumors. Data is shown as % of each cell type relative to alltumor cells. B. AXA-042 increases proportion of activated CD69+ CD4+ and CD8+ T cells 24hr post treatment. Data is shown as % of each cell type.

A-C. Plots of the top enriched GO terms for differentially expressed genes in TLR2+ Monocytes (A), Granulocytes (B) and Macrophages (C). Significanceset at p<0.001, data is shown as –log10(p value) or gene ratio (cut-off set at 10% of the GO term signature genes)

CONCLUSIONS• Systemic AXA-042 treatment led to a reorganization of the tumor microenvironment within 24 hours of treatment.
• AXA-042 altered the myeloid cell subset profiles, promoted the influx of activated T cells and reduced tumor cell viability.
• AXA-042 has completed GLP toxicology studies and is undergoing evaluation in a Phase 1 clinical trial (ACTRN12622000993796) in advanced solid tumors.