M Reprogramming of tumor associated M2 macrophages with ... · STAT6 and C/EBP mRNA silencing by...

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Reprogramming of tumor associated M2 macrophages with antisense oligonucleotide-loaded exosomes results in potent single-agent antitumor activity

Sushrut Kamerkar, Dalia Burzyn, Charan Leng, Olga Burenkova, Su Chul Jang, Raymond Yang, Adam Boutin, Katherine Kirwin, Tong Zi, William Dahlberg, Eric Zhang, Kelvin Zhang, Scott

Estes, Kyriakos Economides, Timothy Soos, Sriram Sathyanarayanan.

Codiak BioSciences, 500 Technology Square 9th Floor, Cambridge, MA 02139

Introduction

exoASO, a novel engineered exosome

Selective delivery of ASO to tumor associated myeloid cells

Effective M2 to M1 macrophage reprograming in vitro by exoASO . A: Human primary M2 macrophages were incubated for 48 hr. with equivalent doses of exoASO and free ASO targeting STAT6 (top) or C/EBP (bottom). Gene expression levels were analyzed by qPCR. One representative donor of five is shown . B: Primary human M2 macrophages were treated with 4 mM exoASO or free ASO and cytokine production was analyzed after 24 hr treatment with LPS (10 ng/ml) using a multiplex flow cytometry assay. One representative donor of three is shown. ****, P < 0.0001 and ***, P < 0.001 by one-way ANOVA with Tukey’s multiple comparison test, compared to exoASO-scramble or free ASO. exoASO-C/EBP or exoASO-STAT6 as compared to exoASO-Scramble induced TNF-a by 41-fold and 8-fold respectively. IL-10 levels were decreased by 28-fold and 2.5-fold by exoASO-C/EBP and exoASO-STAT6, respectively, compared to exo-Scramble.

Exosome tropism to myeloid cells promotes selective delivery of ASO. A-B: BALB/c mice bearing CT26 subcutaneous (sc) tumors received one intravenous dose (8 mg) of fluorescently labeled exoASO or free ASO. One hour later, peripheral blood (C) and tumor (D) were collected and analyzed by flow cytometry. C: CT26 sc tumors received one intratumoral (it) dose (4 mg) of fluorescently-labeled exoASO. One hour later, tumors were dissected and enzymatically digested, and tumor cell suspensions analyzed by flow cytometry. MDSC=myeloid-derived suppressor cells. mMDSC= monocytic MDSC. gMDSC= granulocytic MDSC. cDC2= type 2 conventional dendritic cells. cDC1= type 1 conventional dendritic cells

Anti-tumor activity of exoASO-STAT6 and –C/EBP as single agents. CT26 tumor cells were implanted subcutaneously in the flanks of mice (n =

10 per group). ExoASO and free ASO were dosed intratumorally and antibodies intraperitoneally following dosing regimen in A. A: Dosing

scheme. B-C: exoASO-STAT6 or free ASO. D-E: exoASO- C/EBP or free ASO. Geometric means of tumor volumes are depicted in B and D.

Individual tumor growth curves are shown in C and E. CR: Number of complete responses.

• exoASO is a novel, engineered exosome that can selectively deliver antisense oligonucleotides to M2 macrophages.

• exoASO enables selective silencing of key transcription factors, e.g., those that control the immunosuppressive program, STAT6 and C/EBPβ in M2 macrophages.

• Effective silencing of STAT6 and C/EBP by exoASO in M2 macrophages results in reprograming to an M1 phenotype, with up to 15-fold induction of proinflammatory cytokines and up to 19-fold down regulation of anti-inflammatory cytokines in vitro.

Summary

Presented at the AACR Tumor Immunology and Immunotherapy, November 17-20th, 2019 in Boston, MA USA. All inquiries can be directed to presenting authors or by visiting www.codiakbio.com.

A

B

STAT6 and C/EBP mRNA silencing by exoASO repolarizes human M2 macrophages in vitro

Effective reprogramming of M2 macrophages in vivo by knockdown of STAT6 and C/EBP

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In vivo knockdown and reprogramming by exoASO. CT26 tumors were treated intratumoral (IT) with 4 mg of free ASO or exoASO according to schematic in A). After treatment, tumor-associated myeloid cells were isolated using CD11b-positive selection magnetic bead isolation (80% enrichment). A: Dosing scheme. B-C: Target gene KD in pre- and post-enrichment samples were analyzed by qPCR in whole tumor and in tumor CD11b+ cells. D-E: . Gene expression analysis in tumor myeloid cells was performed by Nanostring using a mouse myeloid panel. ***, P < 0.001 by two-way ANOVA with Tukey’s multiple comparison test, compared to exoASO-scramble or free ASO.

STAT6 C/EBPA B C

Monotherapy with exoASO-STAT6 or exoASO-C/EBPβ results in significant anti-tumor activity

• Highly immunosuppressive (M2 phenotype) macrophages are potent divers of tumor growth

• Targeting M2 macrophages with anti-CSF1R therapies show modest single agent activity

• Decreasing the expression of key immunosuppressive transcription factors STAT6 and C/EBPβ could effectively reprogram M2 macrophages to a pro-inflammatory M1 phenotype

• We have developed novel, engineered exosomes that selectively deliver antisense oligos (ASOs) to M2 macrophages to selectively silence STAT6 and C/EBPexpression

• Exosomes are natural intercellular messengers that can deliver bioactive payloads

• exoASO is a novel engineered exosome stably loaded with antisense oligonucleotides (ASOs)

• Cholesterol-tagged ASOs targeting STAT6 or C/EBP are exogenously loaded onto the exosome surface

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Blood-IV

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Free STAT6 ASO IC50 596.1 nM

exoASO-STAT6 IC50 351.6 nM

Free C/EBP ASO IC50 789.3 nM

exoASO-C/EBP IC50 373.6 nM

STAT6 Free ASO

Scramble ExoASO

C/EBPbExoASO

STAT6 ExoASO

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• exoASO-STAT6 and exoASO-C/EBP treatment of mice with CT26 tumors resulted in 50-60% complete response as a monotherapy.

• No significant anti-tumor activity was observed with anti-PD-1 or anti-CSF1R monotherapy in this model.

• Collectively, exoASOs against STAT6 and C/EBPβ represent a first-in-class strategy to target tumor associated myeloid cells in a highly selective manner.

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Exosome

Antisense oligonucleotide (ASO)

D0

CT26, SC

D8 D13D10 D14

Tumor CD11b+ cell

isolation

exoASO, free ASOIT injection

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— Unlabeled exo

— Macrophages_exoASO

— gMDSC_exoASO

— mMDSC_exoASO

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— cDC2_exoASO

— cDC1_exoASO

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— Unlabeled exo

— Macrophages_exoASO

— Tumor cells_exoASO

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exoASO-

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STAT6

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STAT6

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exoASO-

ScrambleFree STAT6

ASOexoASO-

STAT6

exoASO-

C/EBP

0

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1 0 0 0 0 0

1 5 0 0 0 0

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F r e e A S O

T cells Dendritic cells

Monocytes Neutrophils

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Tumorcells

Macrophages T cells Dendritic cells

Myeloid-derived

suppressor cells

15-fold 4-fold

19-fold 6-fold

D0

CT26, SC

exoASO, free ASOIT injection, TIW

D8 D20

60-75 mm3

mAbsIP injection, DIW

D14

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