M Reprogramming of tumor associated M2 macrophages with ... · STAT6 and C/EBP mRNA silencing by...
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8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 0 250 500 750 1000 1250 1500 1750 2000 2250 Days Post Tumor Implantation Tumor Volume (mm 3 ) (GeoMean) PBS Free STAT6 ASO exoASO-STAT6 anti-PD1 exoASO-Scramble anti-CSF1R 8 10 12 14 16 18 20 22 24 26 28 30 0 250 500 750 1000 1250 1500 1750 2000 2250 Days Post Tumor Implantation Tumor Volume (mm 3 ) GeoMean PBS Free C/EBP ASO exoASO-C/EBP anti-PD1 exoASO-Scramble anti-CSF1R 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 9 th 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 17-20 th , 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 exoASO-Scramble exoASO-C/EBP exoASO-Scramble exoASO-C/EBP 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Normalized Gene Expression (mCebpB) ** Non enriched CD11b enriched * exoASO-Scramble Free STAT6 ASO exoASO-STAT6 exoASO-Scramble Free STAT6 ASO exoASO-STAT6 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Normalized Gene Expression (mStat6) Non enriched CD11b Enriched *** ** * 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/EBP A 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/EBP expression • 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 B A C D E Tumor- IV Blood-IV A B ASO (nM) Normalized % gene expression (hCEBPB) 100 1000 10000 0 25 50 75 100 125 exoASO-C/EBP Free C/EBP ASO exoASO-Scramble ASO (nM) Normalized % gene expression (hSTAT6) 100 1000 10000 0 25 50 75 100 125 exoASO-STAT6 Free STAT6 ASO exoASO-Scramble 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/EBPb ExoASO STAT6 ExoASO 0 500 1000 1500 2000 2500 CSF1R mRNA normalized linear counts 0 50 100 150 200 250 Nos2 mRNA normalized linear counts 0 2000 4000 6000 8000 Arg1 mRNA normalized linear counts E 0 500 1000 1500 TGF 1 mRNA normalized linear counts exoASO- Scramble Free STAT6 ASO exoASO- STAT6 exoASO- C/EBP • 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 . **** Untreated exoASO-Scramble exoASO-STAT6 Free STAT6 ASO exoASO-C/EBP Free C/EBP ASO 0 2000 4000 6000 8000 TNF- a TNF- a pg/ml **** **** **** **** Untreated exoASO-Scramble exoASO-STAT6 Free STAT6 ASO exoASO-C/EBP Free C/EBP ASO 0 1000 2000 3000 4000 IL-10 IL-10 pg/ml **** **** *** **** *** *** *** Untreated exoASO-Scramble exoASO-STAT6 Free STAT6 ASO exoASO-C/EBP Free C/EBP ASO 0 10 20 30 40 50 TARC/CCL17 TARC/CCL17 (pg/ml) Exosome Antisense oligonucleotide (ASO) D0 CT26, SC D8 D13 D10 D14 Tumor CD11b+ cell isolation exoASO, free ASO IT injection 0 250 500 750 1000 1250 1500 1750 2000 exoASO-Scramble Tumor Volume, mm3 0 250 500 750 1000 1250 1500 1750 2000 PBS Tumor Volume, mm3 Free STAT6 ASO exoASO-STAT6 anti-PD1 anti-CSF1R 0 250 500 750 1000 1250 1500 1750 2000 2250 exoASO-Scramble Tumor Volume, mm3 Free C/EBP ASO exoASO-C/EBP anti-PD1 anti-CSF1R 0 250 500 750 1000 1250 1500 1750 2000 2250 PBS Tumor Volume, mm3 CR 0/10 CR 0/10 CR 0/10 CR 0/10 CR 5/10 CR 0/10 CR 0/10 CR 0/10 CR 0/10 CR 0/10 CR 6/10 CR 0/10 **** **** Untreated exoASO-Scramble exoASO-STAT6 Free STAT6 ASO exoASO-C/EBP Free C/EBP ASO 0 50 100 150 200 250 IL-12 p40 IL12-p40 pg/ml XXXXXXXXXXXXXXXXXXXX — Unlabeled exo — Macrophages_exoASO — gMDSC_exoASO — mMDSC_exoASO ASO-Cy5 — cDC2_exoASO — cDC1_exoASO C Normalized counts — Unlabeled exo — Macrophages_exoASO — Tumor cells_exoASO ASO-Cy5 Normalized counts Tumor- IT D exoASO- Scramble Free STAT6 ASO exoASO- STAT6 exoASO- C/EBP exoASO- Scramble Free STAT6 ASO exoASO- STAT6 exoASO- C/EBP exoASO- Scramble Free STAT6 ASO exoASO- STAT6 exoASO- C/EBP exoASO- Scramble Free STAT6 ASO exoASO- STAT6 exoASO- C/EBP 0 50000 100000 150000 ASO uptake (Cy5 MFI) exoASO Free ASO T cells Dendritic cells Monocytes Neutrophils 0 10000 20000 30000 40000 ASO uptake (Cy5 MFI) exoASO Free ASO Tumor cells Macrophages T cells Dendritic cells Myeloid- derived suppressor cells 15-fold 4-fold 19-fold 6-fold D0 CT26, SC exoASO, free ASO IT injection, TIW D8 D20 60-75 mm3 mAbs IP injection, DIW D14
Transcript of 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.
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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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exoASO-STAT6 IC50 351.6 nM
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exoASO-C/EBP IC50 373.6 nM
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Scramble 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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D0
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D14
