REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 16, 2021, is named 4000_0190001_Seq_Listing.txt and is 1,878 bytes in size.

FIELD OF THE INVENTION

This invention relates to compositions and methods comprising extracellular vesicles harboring nucleic acid that target genes, leading to macrophage polarization of tumor associated macrophages.

BACKGROUND

Immunotherapy is the treatment of disease by inducing, enhancing, or suppressing the immune response. Cancer immunotherapy usually has fewer side effects than traditional cancer therapies, such as chemotherapy and radiation therapy. In one approach, cancer immunotherapy has been used to stimulate the patient's own macrophages to attack cancer cells. Macrophages display different phenotypes, e.g., they can be cancer-promoting or they can possess anti-cancer activity. The M2 phenotype, or “alternatively activated macrophages,” typically exhibit cancer-promoting activities, such as the suppression of the immune system and the production of extracellular matrix- and tissue-remodeling activities. The M1 phenotype, or “classically activated macrophages,” typically exhibit anti-cancer activities such as the phagocytosis of tumor cells and the stimulation of adaptive immunity so that tumor cells can be recognized and attacked. Improvements in immunomodulatory methods and compositions that promote macrophage polarization (i.e., conversion to the M1 phenotype) are needed.

SUMMARY

Aspects of the disclosure encompass an extracellular vesicle comprising one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype. In some aspects the disclosure encompasses an extracellular vesicle that is an exosome comprising one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype

In some aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and the immunomodulating component(s) is a nucleic acid, e.g., an inhibitory RNA, e.g., an antisense RNA, an siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or, e.g., an antisense oligonucleotide (ASO) or, e.g., the immunomodulating component is an antisense oligonucleotide comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs:1-6.

In some aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and the immunomodulating (components), e.g., a nucleic acid, e.g., an inhibitory RNA, e.g., an antisense RNA, an siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or, e.g., an antisense oligonucleotide (ASO) or, e.g., the immunomodulating component is an antisense oligonucleotide comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs:1-6, inhibit(s) at least one macrophage target gene, e.g., at least one gene is selected from the group consisting of: KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, Glutaminase, CEBP/β, Pi3Kγ, and PKM2, e.g., STAT3, STAT6, CEBP/β, Pi3Kγ, KRAS, and HIF1-alpha, e.g., STAT3, e.g., KRAS.

In some aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and the immunomodulating component is an antisense oligonucleotide that targets STAT3.

In some aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and the immunomodulating component is an antisense oligonucleotide that targets KRAS.

In some aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and the immunomodulating component is an inhibitory RNA, e.g., an antisense RNA, an siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA that targets KRAS, e.g., wild-type human KRAS, or wild-type human KRAS and also mouse KRASG12D.

In any of the above-described aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s), e.g., a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and the macrophage is a tumor- (e.g., pancreatic tumor) resident macrophage.

In any of the above-described aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s), e.g., a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and further comprise an additional immunomodulating component, e.g., a small molecule drug, an antibody or active fragment thereof, e.g., an immune checkpoint inhibitor that binds to CTLA-4, PD-1, or PD-L1, or an inhibitor that binds to CSF1-R, or a therapeutic protein or active fragment thereof.

In any of the above-described aspects, the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s), e.g., a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype, and further comprise an additional immunomodulating component, e.g., an antibody or active fragment thereof, e.g., an immune checkpoint inhibitor that binds to CTLA-4, PD-1, or PD-L1, or an inhibitor that binds to CSF1-R, wherein the antibody or active fragment thereof comprises CDRs that are at least 95% identical to the CDRs of Ipilimumab, or at least 95% identical to the CDRs of Nivolumab, or at least 95% identical to the CDRs of Cemiplimab, or at least 95% identical to the CDRs of Pembrolizumab, or at least 95% identical to the CDRs of Atezolizumab, or at least 95% identical to the CDRs of Avelumab, or at least 95% identical to the CDRs of Durvalumab, or at least 95% identical to the CDRs of Pexidartinib, or at least 95% identical to the CDRs of PLX7486, or at least 95% identical to the CDRs of ARRY-382, or at least 95% identical to the CDRs of JNJ-40346527, or at least 95% identical to the CDRs of BLZ945, or at least 95% identical to the CDRs of Emactuzumab, or at least 95% identical to the CDRs of AMG820, or at least 95% identical to the CDRs of IMC-CS4, or at least 95% identical to the CDRs of Cabiralizumab, or wherein the antibody or active fragment thereof is at least one antibody selected from the group consisting of Ipilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab, or wherein the antibody or active fragment thereof is at least one antibody that competes for binding with antibody selected from the group consisting of Ipilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab.

In any of the above-described aspects, wherein the extracellular vesicle, e.g., the exosome, comprises one or more immunomodulating component(s), e.g., a nucleic acid, inhibitory RNA, antisense RNA, siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA, or an ASO that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype and further comprise an additional immunomodulating component, e.g., an antibody or active fragment thereof, e.g., an immune checkpoint inhibitor that binds to CTLA-4, PD-1, or PD-L1, or an inhibitor that binds to CSF1-R, wherein the antibody or active fragment thereof comprises CDRs that are at least 95% identical to the CDRs of Ipilimumab, or at least 95% identical to the CDRs of Nivolumab, or at least 95% identical to the CDRs of Cemiplimab, or at least 95% identical to the CDRs of Pembrolizumab, or at least 95% identical to the CDRs of Atezolizumab, or at least 95% identical to the CDRs of Avelumab, or at least 95% identical to the CDRs of Durvalumab, or at least 95% identical to the CDRs of Pexidartinib, or at least 95% identical to the CDRs of PLX7486, or at least 95% identical to the CDRs of ARRY-382, or at least 95% identical to the CDRs of JNJ-40346527, or at least 95% identical to the CDRs of BLZ945, or at least 95% identical to the CDRs of Emactuzumab, or at least 95% identical to the CDRs of AMG820, or at least 95% identical to the CDRs of IMC-CS4, or at least 95% identical to the CDRs of Cabiralizumab, or wherein the antibody or active fragment thereof is at least one antibody selected from the group consisting of Ipilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab, or wherein the antibody or active fragment thereof is at least one antibody that competes for binding with antibody selected from the group consisting of Ipilimumab, Nivolumab, Cemiplimab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Pexidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820, IMC-CS4 and Cabiralizumab, it further comprises PTGFRN or a fragment thereof, and the antibody or fragment thereof may optionally be fused to the PTGFRN or the fragment thereof.

In any of the above-described aspects, the comparison is determined using an assay selected from the group consisting of an extracellular vesicle uptake assay, a target gene expression assay, a downstream gene expression assay, a cytokine release assay and a macrophage cell surface protein assay.

In any of the above-described aspects, the M2 macrophage is a tumor associated macrophage is a tumor associated macrophage selected from the group consisting of a M2a, M2b, and M2c macrophage.

In any of the above-described aspects, the M1 macrophage exhibits increased secretion of inflammatory cytokines and chemokines selected from the group consisting of INFγ, IL-12, IL-23, TNFα, IL-6, IL-1, CSCL9, CXCL10 and CXCL11 compared to the M2 macrophage prior to polarization, and/or exhibits decreased secretion of immunosuppressive cytokines and chemokines selected from the group consisting IL-10, TGFβ, PGE2, CCL2, CCL17, CCL18, CCL22 and CCL24 compared to the M2 macrophage prior to polarization, and or expresses increased tumor associated antigen compared to the M2 macrophage prior to polarization, and/or increases stimulation of CD8+T-Cells and/or Natural Killer cells compared to the M2 macrophage prior to polarization.

In aspects, the disclosure encompasses a pharmaceutical composition comprising the extracellular vesicle, e.g., exosome, that comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype.

In some aspects, the disclosure encompasses a method of treating a disease (e.g., cancer such as, e.g., pancreatic cancer) in a patient (e.g., a human) in need thereof, comprising administering (e.g., via a route selected from the group consisting of intravenous, intraperitoneal and intratumoral administration) the extracellular vesicle of any of the above-described aspects, e.g., the exosome that comprises one or more immunomodulating component(s) (e.g., an inhibitory RNA targeting a proto-oncogene (e.g., KRAS)) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype of any of the above-described aspects, or comprising administering a pharmaceutical composition comprising the extracellular vesicle of any of the above-described aspects, e.g., the exosome that comprises one or more immunomodulating component(s) that, upon contact with a macrophage, selectively repolarizes the macrophage from an M2 to an M1 phenotype of any of the above-described aspects.

In some aspects, the disclosure encompasses the methods of treatment described above and further comprises a second therapy, e.g., a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

In some aspects, the disclosure encompasses methods of modulating gene expression in a macrophage comprising contacting the macrophage with an extracellular vesicle comprising one or more immunomodulating components that inhibit at least one gene and thereby increase macrophage polarization from an M2 to an M1 phenotype, as compared to contacting the macrophage with equimolar amount(s) of the immunomodulating components alone.

In some aspects, the methods of modulating gene expression in a macrophage comprising contacting the macrophage ex vivo or in vitro with an extracellular vesicle comprising one or more immunomodulating components that inhibit at least one gene and thereby increase macrophage polarization from an M2 to an M1 phenotype, as compared to contacting the macrophage with equimolar amount(s) of the immunomodulating components alone.

In some aspects, the methods of modulating gene expression in a macrophage comprising contacting the macrophage in vivo, with an extracellular vesicle (e.g., by administering the extracellular vesicle to a subject, e.g., a human subject, e.g., by a route selected from the group consisting of intravenous, intraperitoneal and intratumoral administration) comprising one or more immunomodulating components that inhibit at least one gene and thereby increase macrophage polarization from an M2 to an M1 phenotype, as compared to contacting the macrophage with equimolar amount(s) of the immunomodulating components alone.

In some aspects, the subject of the above-disclosed methods of modulating gene expression in a macrophage, is suffering from a condition selected from cancer (e.g., pancreatic cancer) and fibrosis

In some aspects, the extracellular vesicle used in any of the above-disclosed methods of modulating gene expression in a macrophage is an exosome, and the immunomodulating component is a nucleic acid, e.g., an inhibitory RNA, such as, e.g., an antisense RNA, an siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA.

In some aspects, the extracellular vesicle used in any of the above-disclosed methods of modulating gene expression in a macrophage is an exosome, and the immunomodulating component is a nucleic acid, e.g., an ASO.

In some aspects, the extracellular vesicle used in any of the above-disclosed methods of modulating gene expression in a macrophage is an exosome, and the immunomodulating component is a nucleic acid, e.g., an antisense oligonucleotide comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs: 1-6 or an antisense oligonucleotide comprising a sequence selected from SEQ ID NOs: 1-6, and the at least one gene is selected from the group consisting of KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STAT6, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, Glutaminase, CEBP/β, Pi3Kγ, and PKM2, or from the group consisting of: STAT3, STAT6, CEBP/β, Pi3Kγ, KRAS, and HIF1-alpha, or is STAT3, or is KRAS, and if KRAS, the immunomodulatory component can optionally be an inhibitory RNA that targets wild-type human KRAS.

In some aspects, the disclosure provides a method of treating pancreatic cancer in a subject comprising: administering to the subject an extracellular vesicle comprising an inhibitory RNA targeting human wild-type KRAS; wherein the treatment increases the percentage of polarization of tumor-resident macrophages from an M2 to an M1 phenotype to a greater level than that observed in a patient treated with an inhibitory RNA targeting human KRASG12D.

In some aspects the disclosure provides the above-described method of treating pancreatic cancer in a subject, wherein the percentage of polarization of tumor-resident macrophages is determined using an ex-vivo assay of tumor-resident macrophages obtained from a tumor sample.

Provided herein are compositions and methods comprising extracellular vesicles selected, enriched, or engineered with one or more immunomodulating components that can modify the activity of macrophages, promoting switching of macrophages from the M2 to the M1 phenotype (macrophage polarization) and boosting the patient's immune system to fight cancer.

Accordingly, in a first aspect, provided herein is an extracellular vesicle comprising one or more immunomodulating components, e.g., nucleic acid molecules that inhibits at least one gene in a target cell. In certain embodiments the target cell is a macrophage and the gene inhibition increases macrophage polarization from the M2 to M1 phenotype as compared to an equimolar amount of the immunomodulating component(s) alone. In certain embodiments, the extracellular vesicle is an exosome. In certain embodiments, the nucleic acid is an inhibitory RNA. In certain embodiments, the inhibitory RNA is an antisense RNA, an siRNA, an shRNA, a miRNA, a lncRNA, a pri-miRNA or a pre-miRNA. In certain embodiments, the at least one gene is selected from the group consisting of: KRAS, HRAS, NRAS, HIF1-alpha, HIF1-beta, Sp1, P300, LKB1, AMPK, STAT3, STATE, n-MYC, c-MYC, HCAR1, A2AB, IDO, TDO, Arginase, Glutaminase, CEBP/β, Pi3Kγ, and PKM2. In certain embodiments, the gene is KRAS. In certain embodiments, the nucleic acid is an inhibitory RNA that targets wild-type human KRAS. In certain embodiments, the inhibitory RNA also targets mouse KrasG12D. In certain embodiments, the macrophage is a tumor resident macrophage. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the extracellular vesicle further comprises an additional immunomodulating component. In certain embodiments, the additional immunomodulating component is a small molecule drug, an antibody or a therapeutic protein. In certain embodiments, the antibody is an immune checkpoint inhibitor. In certain aspects, provided herein is a pharmaceutical composition comprising any of the above mentioned extracellular vesicles.

In certain aspects, described herein is a method of treating a disease in a patient in need thereof comprising administering the extracellular vesicles or the pharmaceutical compositions described herein to the patient, thereby treating the disease in the patient. In certain embodiments, the disease is a cancer. In certain embodiments, the cancer is pancreatic cancer. In some embodiments, the disease is a fibrotic condition. In some embodiments, the fibrotic condition is lung fibrosis, liver fibrosis, or pancreatic fibrosis. In certain embodiments, the liver fibrosis is non-alcoholic steatohepatitis, or NASH. In certain embodiments, the patient is human. In certain embodiments, the nucleic acid is an inhibitory RNA targeting a proto-oncogene. In certain embodiments, the proto-oncogene is human KRAS. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the methods further comprise performing at least a second therapy. In certain embodiments, the second therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

In certain embodiments, the target cell M2 macrophage is a tumor associated macrophage selected from the group consisting of a M2a, M2b, and M2c macrophage. In certain embodiments, the M1 macrophage exhibits increased secretion of inflammatory cytokines and chemokines selected from the group consisting of INFγ, IL-12, IL-23, TNFα, IL-6, IL-1, CSCL9, CXCL10 and CXCL11 compared to the M2 macrophage prior to polarization. In certain embodiments, the M1 macrophage exhibits decreased secretion of immunosuppressive cytokines and chemokines selected from the group consisting IL-10, TGFβ, PGE2, CCL2, CCL17, CCL18, CCL22 and CCL24 compared to the M2 macrophage prior to polarization. In certain embodiments, the M1 macrophage expresses increased tumor associated antigen compared to the M2 macrophage prior to polarization. In certain embodiments, the M1 macrophage increases stimulation of CD8+T-Cells and/or Natural Killer cells compared to the M2 macrophage prior to polarization.

In certain aspects, described herein is a method of treating pancreatic cancer in a subject comprising administering to the subject an extracellular vesicle comprising an inhibitory RNA targeting human wild-type KRAS; wherein the treatment increases the percentage of polarization of tumor-resident macrophages from the M2 to M1 phenotype to a greater level than that observed in a patient treated with an inhibitory RNA targeting human KRASG12D.

