Patent Application
20230203532
The present disclosure relates generally delivery of molecules to cells. More particularly, the present disclosure relates to targeted delivery of molecules to cells.
The targeted delivery of therapeutic molecules within biological systems is an important component of disease treatment. However, targeted delivery of molecules to specific cell types remains a challenge due to instability, off-target effects prior to reaching the target cell, toxicity of molecules in other contexts, as well as other issues.
It is, therefore, desirable to provide a stable targeted therapeutic molecule that only treats the target cell.
It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous approaches.
In a first aspect, the present disclosure provides a recombinant tumor-selective viral particle comprising a nucleic acid encoding a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell, said recombinant polypeptide comprising: at least one targeting moiety for directing said EV to said at least one target molecule expressed by said at least one target cell, at least one EV-anchoring polypeptide, and at least one intravesicular polypeptide.
In another aspect, there is provided a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell comprising: at least one targeting moiety for directing said EV to said at least one target molecule expressed by said at least one target cell, at least one EV-anchoring polypeptide, and at least one intravesicular polypeptide.
In one aspect, there is provided a nucleic acid molecule encoding the recombinant polypeptide as herein.
In one aspect, there is provided a vector comprising the nucleic acid as defined herein.
In one aspect, there is provided a recombinant viral genome comprising the nucleic acid as defined herein.
In one aspect, there is provided a viral particle comprising the nucleic acid as defined herein.
In one aspect, there is provided a host cell comprising the nucleic acid as defined, the vector, the recombinant viral genome, or the viral particle as defined herein.
In one aspect, there are provided targeted extracellular vesicles (EVs) comprising the recombinant polypeptide as defined herein.
In one aspect, there is provided a composition comprising the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein; together with a pharmaceutically acceptable excipient, diluent, or carrier.
In one aspect, there is provided a method of binding a targeting moiety to a target molecule of a target cell comprising contacting said target cell with the targeted EVs as defined herein.
In one aspect, there is provided a method of delivering a payload molecule to a target cell comprising contacting said target cell with the targeted EVs as defined herein.
In one aspect, there is provided a method of delivering a cargo molecule to a target cell comprising contacting said target cell with the targeted EVs as defined herein.
In one aspect, there is provided a method of stimulating an immune response to an antigen comprising administering to a subject the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said target cell comprises an immune cell, and wherein said at least one EV therapeutic payload polypeptide comprises an antigen.
In one aspect, there is provided a method of killing target cells comprising administering to a subject the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.
In one aspect, there is provided a method of reprogramming immune cells comprising contacting the immune cells with the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein the at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.
In one aspect, there is provided a method of directing an immune response to a target disease cell comprising administering to a subject the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said recombinant polypeptide comprises at least two targeting moieties which specifically bind, respectively, to at least two different target molecules, wherein said at least two different target molecules are expressed, respectively, by an immune cell and a disease cell.
In one aspect, there is provided a method of preparing therapeutic targeted EVs for a subject comprising: contacting cells obtained from a subject with the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, or the viral particle as defined herein, and collecting the targeted EVs.
In one embodiment, there is provided a method of producing targeted EVs, wherein said method comprises expressing the nucleic acid as defined herein in cells, or culturing the host cell as defined herein to produce EVs; and collecting the EVs.
Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
Generally, the present disclosure provides a recombinant tumor-selective viral particle comprising a nucleic acid encoding a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell, said recombinant polypeptide comprising: at least one targeting moiety for directing said EV to said at least one target molecule expressed by said at least one target cell; at least one EV-anchoring polypeptide; and at least one intravesicular polypeptide. The viral particle may be from an oncolytic virus. Also provided is a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell comprising: at least one targeting moiety for directing said EV to said at least one target molecule expressed by said at least one target cell, at least one EV-anchoring polypeptide, and at least one intravesicular polypeptide.
In one aspect, there is provided a recombinant tumor-selective viral particle comprising a nucleic acid encoding a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell, said recombinant polypeptide comprising:
In one embodiment, the recombinant tumor-selective viral particle is of an oncolytic virus.
In one aspect, there is provided a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell comprising:
In one embodiment, said at least one EV-anchoring polypeptide comprises an EV-directed transmembrane polypeptide linked to said at least one targeting moiety.
In one embodiment, said EV-directed transmembrane polypeptide comprises a transmembrane domain from LAMP2b, VSVG, CD81, CD82,or LAMP1,.
In one embodiment, said EV-directed transmembrane polypeptide comprises a transmembrane domain from Junin virus glycoprotein, Lassa fever virus glycoprotein, LCMV (lymphocytic choriomeningitis virus) glycoprotein, SARS-CoV-2 glycoprotein, Tamiami virus glycoprotein, Guanarito virus glycoprotein, Paraná virus glycoprotein, Machupo virus glycoprotein, Sabia virus glycoprotein, or CdaA.
In one embodiment, the EV-directed transmembrane polypeptide comprises a transmembrane domain from a Rhabdovirus glycoprotein.
In one embodiment, the EV-directed transmembrane polypeptide comprises a transmembrane domain from a Arenavirus glycoprotein.
In one embodiment, said at least one target cell comprises a mammalian cell.
In one embodiment, said mammalian cell is a human cell.
In one embodiment, said at least one target cell is a tumor cell, a tumor stromal cell, or an immune cell.
In one embodiment, said tumor stromal cell comprises a cancer-associated fibroblast.
In one embodiment, said immune cell is a T-cell, a B-cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil.
In one embodiment, said immune cell is a macrophage. In one embodiment, said at least one target molecule is macrophage receptor (MARCO).
In one embodiment, said T-cell is a regulatory T-cell or a cytotoxic T cell.
In one embodiment, said at least one target molecule is a cell surface marker or a cell surface receptor.
In one embodiment, said at least one target molecule is a TNF-α family receptor, an integrin, a C-type lectin receptor, a leptin, a carcinoembryonic antigen, a CD antigen, a carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, a glycosaminoglycan, a polysaccharide, or a lipid.
In one embodiment, said at least one target molecule comprises a disease-specific cell surface molecule, which is:
In one embodiment, said disease-specific cell surface molecule comprises a tumor-associated antigen.
In one embodiment, said at least one target molecule comprises DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, fibroblast activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, chondroitin sulfate, αv-Integrin, or folate receptor.
In one embodiment, said at least one targeting moiety comprises a receptor ligand, an antibody or a functional fragment thereof, an scFv, a single domain antibody or a DARPin.
In one embodiment, said antibody is a single domain antibody.
In one embodiment, said antibody is a humanized antibody.
In one embodiment, said functional fragment is a Fab′ or a F(ab′)2.
In one embodiment, said at least one targeting moiety comprises anti-DEC205, anti-Clec9A, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CD206, anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPα ectodomain, GE11 peptide, CTX, VAR2Δ,CD40 ligand, CD40-targeting peptide, iRGD, PD1.
In one embodiment, said intravesicular polypeptide may comprise a short amino acid tail for projecting into the intravesicular space. Said intravesicular polypeptide may comprise at least 9 amino acids. Said intravesicular polypeptide may comprise at least 10 amino acids. Said intravesicular polypeptide may comprise at least 11 amino acids. Said intravesicular polypeptide may comprise at least 12 amino acids. Said intravesicular polypeptide may comprise at least 13 amino acids. Said intravesicular polypeptide may comprise at least 14 amino acids. Said intravesicular polypeptide may comprise at least 15 amino acids. Said intravesicular polypeptide may comprise at least 9 amino acids. Said intravesicular polypeptide may comprise 9 to 15 amino acids.
In one embodiment, said intravesicular polypeptide comprises least one EV payload polypeptide linked to said at least one targeting moiety via said EV-anchoring polypeptide. The EV payload polypeptide may comprise, for example, a therapeutic polypeptide, a polypeptide for imaging, a polypeptide for diagnostics, a suicide protein, or a receptor for a biomarker.
In one embodiment, the at least one EV payload polypeptide comprises at least one EV therapeutic payload polypeptide.
In one embodiment, said EV-anchoring polypeptide and said intravesicular polypeptide together comprise an EV-directed recombinant tetraspanin comprising said at least one targeting moiety inserted between two transmembrane domains thereof.
In one embodiment, said recombinant tetraspanin comprises or is derived from human CD63 or CD9.
In one embodiment, said at least one target cell comprises a mammalian cell.
In one embodiment, said mammalian cell is a human cell.
In one embodiment, said at least one target cell is a tumor cell, a tumor stromal cell, or an immune cell.
In one embodiment, said tumor stromal cell comprises a cancer-associated fibroblast.
In one embodiment, said immune cell is a T-cell, a B-cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil.
In one embodiment, said immune cell is a macrophage. In one embodiment, said at least one target molecule is macrophage receptor (MARCO).
In one embodiment, said T-cell is a regulatory T-cell or a cytotoxic T cell.
In one embodiment, said at least one target molecule is a cell surface marker or a cell surface receptor.
In one embodiment, said at least one target molecule is a TNF-α family receptor, an integrin, a C-type lectin receptor, a leptin, a carcinoembryonic antigen, a CD antigen, a carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, a glycosaminoglycan, a polysaccharide, or a lipid.
In one embodiment, said at least one target molecule comprises a disease-specific cell surface molecule, which is:
In one embodiment, said disease-specific cell surface molecule comprises a tumor-associated antigen.
In one embodiment, said at least one target molecule comprises DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, fibroblast activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, chondroitin sulfate, αv-Integrin, or folate receptor.
In one embodiment, said at least one targeting moiety comprises a receptor ligand, an antibody or a functional fragment thereof, an scFv, a single domain antibody. or a DARPin.
In one embodiment, said antibody is a single domain antibody.
In one embodiment, said antibody is a humanized antibody.
In one embodiment, said functional fragment is a Fab′ or a F(ab′)2.
In one embodiment, said at least one targeting moiety comprises anti-DEC205, anti-Clec9A, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CD206, anti- anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPα ectodomain, GE11 peptide, CTX, VAR2Δ,CD40ligand, CD40-targeting peptide, iRGD, PD1.
In one embodiment, the recombinant polypeptide further comprises at least one EV payload polypeptide linked to an N- and/or C-terminus of said recombinant tetraspanin. The EV payload polypeptide may comprise, for example, a therapeutic polypeptide, a polypeptide for imaging, a polypeptide for diagnostics, a suicide protein, or a receptor for a biomarker.
In one embodiment, the at least one EV payload polypeptide comprises at least one EV therapeutic polypeptide.
In one embodiment, said at least one targeting moiety comprises at least two targeting moieties, wherein said EV-anchoring polypeptide and said intravesicular polypeptide together comprise an EV-directed recombinant tetraspanin comprising four transmembrane domains numbered 1, 2, 3, and 4 from N- to C-terminus, wherein a first of said two targeting moieties is inserted between transmembrane domains 1 and 2, and a second of said two targeting moieties is inserted between transmembrane domains 3 and 4.
In one embodiment, said EV-directed recombinant tetraspanin is derived from human CD63 or CD9.
In one embodiment, said at least two targeting moieties specifically bind to at least two different target molecules.
In one embodiment, said at least two different target molecules are expressed by the same target cell.
In one embodiment, said target cell comprises a mammalian cell.
In one embodiment, said mammalian cell comprises a human cell.
In one embodiment, said target cell comprises a tumor cell, a tumor stromal cell, or an immune cell.
In one embodiment, said tumor stromal cell comprises a cancer-associated fibroblast.
In one embodiment, said immune cell is a T-cell, a B-cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil.
In one embodiment, said immune cell is a macrophage. In one embodiment, said at least one target molecule is macrophage receptor (MARCO).
In one embodiment, said T-cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, each of said at least two target molecules is a cell surface molecule.
In one embodiment, each of said at least two target molecules is a TNF-α family receptor, an integrin, a C-type lectin receptor, a leptin, a carcinoembryonic antigen, a CD antigen, a carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, a glycosaminoglycan, a polysaccharide, or a lipid.
In one embodiment, each of said at least two target molecules comprise a disease-specific cell surface molecule, which is:
In one embodiment, said disease-specific cell surface molecule comprises a tumor-associated antigen.
In one embodiment, each of said at least two target molecules independently comprises DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, fibroblast activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, chondroitin sulfate, αv-Integrin, or folate receptor.
In one embodiment, each of said at least two targeting moieties independently comprises a receptor ligand, an antibody or a functional fragment thereof, an scFv, a single domain antibody or a DARPin.
In one embodiment, said antibody is a single domain antibody.
In one embodiment, said antibody is a humanized antibody.
In one embodiment, said functional fragment is a Fab’ or a F(ab’)2.
In one embodiment, said at least one targeting moiety comprises anti-DEC205, anti-Clec9A, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CD206, anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPα ectodomain, GE11 peptide, CTX, VAR2Δ,CD40 ligand, CD40-targeting peptide, iRGD, PD1.
In one embodiment, said at least two different target molecules are expressed by different target cells.
In one embodiment, said different target cells comprise a disease cell and an immune cell, and wherein said at least two targeting moieties are directed, respectively, to a disease cell surface molecule and an immune cell surface molecule.
In one embodiment, said different target cells comprise a tumor cell and an immune cell, and wherein said at least two targeting moieties are directed, respectively, to a tumor cell surface molecule and an immune cell surface molecule.
In one embodiment, said immune cell is a T cell, and said immune cell surface marker is a T cell surface molecule.
In one embodiment, said T cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, said immune cell is a natural killer (NK) cell, and said immune cell surface marker is an NK cell surface molecule.
In one embodiment, said immune cell is a B cell, and said immune cell surface marker is a B cell surface molecule.
In one embodiment, said immune cell is a macrophage, and said immune cell surface marker is a macrophage cell surface molecule. In one embodiment, said at least one target molecule is macrophage receptor (MARCO).
In one embodiment, said immune cell is a dendritic cell, and said immune cell an surface marker is a dendritic cell surface molecule.
In one embodiment, said immune cell is a neutrophil, and said immune cell surface marker is a neutrophil cell surface molecule.
In one embodiment, said tumor cell surface molecule comprises a tumor-associated antigen.
In one embodiment, said tumor cell surface molecule comprises one of CEACAM5, CD19, CD20, CD22, EGFR, fibroblast activating protein (FAP), CA9, MMP-2, PD-L1, SIRPα, chondroitin sulfate, αv-Integrin, or folate receptor.
In one embodiment, said at least one targeting moiety comprises an antibody or a functional fragment thereof, an scFv, a single domain antibody or a DARPin.
In one embodiment, said antibody is a single domain antibody.
In one embodiment, said antibody is a humanized antibody.
In one embodiment, said functional fragment is a Fab’ or a F(ab’)2.
In one embodiment, the recombinant polypeptide further comprising at least one EV payload polypeptide. The EV payload polypeptide may comprise, for example, a therapeutic polypeptide, a polypeptide for imaging, a polypeptide for diagnostics, a suicide protein, or a receptor for a biomarker.
In one embodiment, the at least one EV payload polypeptide comprises at least one EV therapeutic payload polypeptide.
In one embodiment, said EV therapeutic payload polypeptide is linked to said N-and/or C-terminus of said recombinant tetraspanin.
These payloads are intended to embodiment described herein, as appropriate.
In one embodiment, said at least one EV payload polypeptide is linked via a cleavage site for releasing said at least one EV payload polypeptide.
In one embodiment, said at least one EV therapeutic payload polypeptide is linked via a cleavage site for releasing said at least one EV therapeutic payload polypeptide.
In one embodiment, said cleavage site comprises a self-cleavage peptide, a pH-dependent cleavage site, or a site for enzymatic cleavage.
In one embodiment, said EV therapeutic payload polypeptide comprises an active pharmaceutical ingredient (API).
In one embodiment, said EV therapeutic payload polypeptide comprises a cytotoxic molecule.
In one embodiment, said cytotoxic molecule comprises human GZMB R201K, murine GZMB, diphtheria toxin, a PE38 domain from Pseudomonas exotoxin A, or human TRAIL.
In one embodiment, said payload polypeptide comprises an immunomodulatory molecule.
In one embodiment, the immunomodulatory molecule comprises an enzyme that generates an immunogenic molecule.
In one embodiment, said immunomodulatory molecular comprises a STING or ERAdP pathway activator.
In one embodiment, said STING or ERAdP pathway activator comprises a bacterial dinucleotide cyclase.
In one embodiment, said bacterial dinucleotide cyclase comprises CdaA.
In one embodiment, said payload polypeptide comprises an enzyme.
In one embodiment, said payload polypeptide comprises a nucleic acid-binding domain.
In one embodiment, said nucleic acid binding domain comprises an RNA-binding motif.
In one embodiment, the nucleic acid binding domain comprises an RNA binding motif from a Cas13 family member protein. In one embodiment, the RNA binding motif comprises an RNA binding motif from Cas13a. In one embodiment, the RNA binding motif comprises an RNA binding motif from Cas13b. In one embodiment, the RNA binding motif comprises an RNA binding motif from Cas13d.
In one embodiment, the RNA binding motif comprises an RNA binding motif from Pum (Pumilio-homology domain-1).
In one embodiment, the RNA binding motif comprises an RNA binding motif from Stu1 (Staufen-1).
In one embodiment, the RNA binding motif comprises an RNA binding motif from alphavirus capsid protein L72AE.
In one embodiment, the nucleic acid binding comprises an RNA binding motif from the MS2 coat protein (herein “MS2”).
In one embodiment, the RNA binding motif comprises an RNA binding motif from VEEV capsid protein.
In one embodiment, said RNA binding motif comprises a nucleic acid ligand system.
In one embodiment, said payload polypeptide comprises an antigen.
In one embodiment, said antigen is a tumor-associated antigen.
In one embodiment, said antigen is from a pathogen.
In one embodiment, said EV therapeutic payload polypeptide further comprises an adjuvant.
In one embodiment, said adjuvant comprises a STING or ERAdP pathway activator.
In one embodiment, said STING or ERAdP pathway activator comprises a bacterial dinucleotide cyclase.
In one embodiment, said bacterial dinucleotide cyclase comprises CdaA.
In one embodiment, said at least one EV therapeutic payload polypeptide is linked to at least one further EV payload polypeptide.
In one embodiment, said at least one EV therapeutic payload polypeptide is linked to said at least one further EV payload polypeptide by a cleavage site.
In one embodiment, said at least one EV therapeutic payload polypeptide is separated from said at least one further EV payload polypeptide by at least two EV transmembrane domains.
In one aspect, there is provided a nucleic acid molecule encoding the recombinant polypeptide as herein.
In one embodiment, said nucleic acid further encodes a separate EV cargo molecule.
In one embodiment, said EV cargo molecule comprises a nucleic acid.
In one embodiment, said nucleic acid comprises an RNA.
In one embodiment, the RNA comprises a target sequence from a sequence in Table 12. In one embodiment, the recombinant polypeptide comprises an RNA binding motif from a sequence in Table 12 corresponding to the target sequence of the cargo.
In one embodiment, said RNA comprises an mRNA, an miRNA, or an shRNA.
In one embodiment, said EV cargo molecule comprises a polypeptide.
In one aspect, there is provided a nucleic acid molecule encoding the recombinant polypeptide as defined herein and comprising the EV payload as defined herein.
In one aspect, there is provided a nucleic acid molecule encoding the recombinant polypeptide as defined herein and comprising the EV therapeutic payload as defined herein.
In one embodiment, said nucleic acid further encodes a separate EV cargo molecule.
In one embodiment, said EV cargo molecule comprises a nucleic acid.
In one embodiment, said nucleic acid comprises an RNA.
In one embodiment, said RNA comprises an mRNA, an miRNA, or an shRNA.
In one embodiment, said EV cargo molecule comprises a polypeptide.
In one aspect, there is provided a vector comprising the nucleic acid as defined herein.
In one aspect, there is provided a vector comprising the nucleic acid as defined herein, wherein the recombinant polypeptide comprises an EV therapeutic payload.
In one aspect, there is provided a recombinant viral genome comprising the nucleic acid as defined herein.
In one embodiment, the viral genome is from a Lentivirus, the Tian Tan strain of Vaccinia virus, or Adeno-associated Virus (AAV).
In one embodiment, the viral genome is from a virus that is tumor-selective.
In one embodiment, said viral genome is from an oncolytic virus.
In one embodiment, said oncolytic virus is vesicular stomatitis virus (VSV), Vaccinia virus, Herpes virus simplex 1 (HSV-1), Herpes virus 2 (HSV-2), adenovirus.
In one aspect, there is provided a recombinant viral genome comprising the nucleic acid as defined herein, wherein the recombinant polypeptide comprises an EV therapeutic payload.
In one embodiment, the viral genome is from a virus that is tumor-selective.
In one embodiment, said viral genome is from an oncolytic virus.
In one embodiment, said oncolytic virus is vesicular stomatitis virus (VSV), Vaccinia virus, Herpes virus simplex 1 (HSV-1), Herpes virus 2 (HSV-2), adenovirus.
In one aspect, there is provided a viral particle comprising the nucleic acid as defined herein.
In one embodiment, said viral particle is of a Lentivirus, the Tian Tan strain of Vaccinia virus, or Adeno-associated Virus (AAV).
In one embodiment, said viral particle is of a virus that is tumor-selective.
In one embodiment, said viral particle is of an oncolytic virus.
In one embodiment, said oncolytic virus is vesicular stomatitis virus (VSV), Vaccinia virus, Herpes virus simplex 1 (HSV-1), Herpes virus 2 (HSV-2), adenovirus.
In one aspect, there is provided a viral particle comprising the nucleic acid as defined herein, wherein the recombinant polypeptide comprises an EV therapeutic payload.
In one embodiment, said viral particle is of a virus that is tumor-selective.
In one embodiment, said viral particle is of an oncolytic virus.
In one embodiment, said oncolytic virus is vesicular stomatitis virus (VSV), Vaccinia virus, Herpes virus simplex 1 (HSV-1), Herpes virus 2 (HSV-2), adenovirus.
In one aspect, there is provided a host cell comprising the nucleic acid as defined, the vector, the recombinant viral genome, or the viral particle as defined herein.
In one embodiment, the host cell is a prokaryotic cell.
In one embodiment, the host cell is a eukaryotic cell.
In one embodiment, the host cell is a yeast cell or an insect cell.
In one embodiment, the host cell is a mammalian cell.
In one embodiment, the host cell is a human cell.
In one embodiment, the host cell is an immune cell.
In one embodiment, the host cell is a B cell, a T cell, a dendritic cell, a macrophage or a neutrophil.
In one embodiment, the host cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, said host cell further encodes a separate EV cargo molecule.
In one embodiment, said EV cargo molecule comprises a nucleic acid.
In one embodiment, said nucleic acid comprises an RNA.
In one embodiment, the nucleic acid binding domain comprises an RNA binding motif from a Cas13 family member protein. In one embodiment, the RNA binding motif comprises an RNA binding motif from Cas13a. In one embodiment, the RNA binding motif comprises an RNA binding motif from Cas13b. In one embodiment, the RNA binding motif comprises an RNA binding motif from Cas13d.
In one embodiment, the RNA binding motif comprises an RNA binding motif from Pum (Pumilio-homology domain-1).
In one embodiment, the RNA binding motif comprises an RNA binding motif from Stu1 (Staufen-1).
In one embodiment, the RNA binding motif comprises an RNA binding motif from alphavirus capsid protein L72AE.
In one embodiment, the nucleic acid binding comprises an RNA binding motif from the MS2 coat protein (herein “MS2”).
In one embodiment, the RNA binding motif comprises an RNA binding motif from VEEV capsid protein.
In one embodiment, the recombinant polypeptide comprises one of the above-described RNA binding motifs and the EV cargo comprise a cognate RNA target sequence for the RNA binding motif. Examples of RNA binding motifs and cognate target sequences are provided in the sequences of Table 12.
In one embodiment, said RNA comprises an mRNA, an miRNA, or an shRNA.
In one embodiment, said EV cargo molecule comprises a polypeptide.
In one aspect, there is provided a host cell comprising the nucleic acid as defined, the vector, the recombinant viral genome, or the viral particle as defined herein, wherein the recombinant polypeptide comprises an EV therapeutic payload.
In one embodiment, the host cell is a prokaryotic cell.
In one embodiment, the host cell is a eukaryotic cell.
In one embodiment, the host cell is a yeast cell or an insect cell.
In one embodiment, the host cell is a mammalian cell.
In one embodiment, the host cell is a human cell.
In one embodiment, the host cell is an immune cell.
In one embodiment, the host cell is a B cell, a T cell, a dendritic cell, a macrophage or a neutrophil.
In one embodiment, the host cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, said host cell further encodes a separate EV cargo molecule.
In one embodiment, said EV cargo molecule comprises a nucleic acid.
In one embodiment, said nucleic acid comprises an RNA.
In one embodiment, said RNA comprises an mRNA, an miRNA, or an shRNA.
In one embodiment, said EV cargo molecule comprises a polypeptide.
In one aspect, there are provided targeted extracellular vesicles (EVs) comprising the recombinant polypeptide as defined herein.
In one embodiment, the targeted EVs further comprise a separate EV cargo molecule.
In one embodiment, said EV cargo molecule comprises a nucleic acid.
In one embodiment, said nucleic acid comprises an RNA.
In one embodiment, the RNA comprises a target sequence from a sequence in Table 12. In one embodiment, the recombinant polypeptide comprises an RNA binding motif from a sequence in Table 12 corresponding to the target sequence of the cargo.
In one embodiment, said RNA comprises an mRNA, an miRNA, or an shRNA.
In one embodiment, said EV cargo molecule comprises a polypeptide.
In one embodiment, said EV cargo molecule comprises an API.
In one embodiment, the targeted EVS are exosomes.
In one embodiment, the targeted EVS are microvesicles.
In one embodiment, the targeted EVS are ectosomes.
In one embodiment, the targeted EVS are apoptotic bodies.
In one embodiment, the targeted EVS are virus-like particles.
In one embodiment, the targeted EVS are macrovesicles.
In one embodiment, the targeted EVS are oncosomes.
In one embodiment, the targeted EVS are gesicles.
In one aspect, there are provided targeted extracellular vesicles (EVs) comprising the recombinant polypeptide as defined herein, wherein the recombinant polypeptide comprises an EV therapeutic payload.
In one embodiment, the targeted EVs further comprise a separate EV cargo molecule.
In one embodiment, said EV cargo molecule comprises a nucleic acid.
In one embodiment, said nucleic acid comprises an RNA.
In one embodiment, said RNA comprises an mRNA, an miRNA, or an shRNA.
In one embodiment, said EV cargo molecule comprises a polypeptide.
In one embodiment, said EV cargo molecule comprises an API.
In one embodiment, the targeted EVS are exosomes.
In one embodiment, the targeted EVS are microvesicles.
In one embodiment, the targeted EVS are ectosomes.
In one embodiment, the targeted EVS are apoptotic bodies.
In one aspect, there is provided a composition comprising the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein; together with a pharmaceutically acceptable excipient, diluent, or carrier.
In one aspect, there is provided a composition comprising the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein; together with a pharmaceutically acceptable excipient, diluent, or carrier, wherein the recombinant polypeptide comprises an EV therapeutic payload.
In one aspect, there is provided a method of binding a targeting moiety to a target molecule of a target cell comprising contacting said target cell with the targeted EVs as defined herein.
In one aspect, there is provided a use of the targeted EVs as defined herein for binding a targeting moiety to a target molecule of a target cell.
In one aspect, there are provided the targeted EVs as defined herein for use in binding a targeting moiety to a target molecule of a target cell.
In one aspect, there is provided a method of delivering a payload molecule to a target cell comprising contacting said target cell with the targeted EVs as defined herein.
In one aspect, there is provided a use of the targeted EVs as defined herein for delivering a payload molecule to a target cell.
In one aspect, there are provided the EVs as defined herein for use in delivering a payload molecule to a target cell.
In one aspect, there is provided a method of delivering a cargo molecule to a target cell comprising contacting said target cell with the targeted EVs as defined herein.
In one aspect, there is provided a use of the targeted EVs as defined herein for delivering a cargo molecule to a target cell.
In one aspect, there are provided the targeted EVs as defined herein for use in delivering a cargo molecule to a target cell.
In one aspect, there is provided a method of stimulating an immune response to an antigen comprising administering to a subject the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said target cell comprises an immune cell, and wherein said at least one EV therapeutic payload polypeptide comprises an antigen.
In one embodiment, said antigen comprises a disease cell-specific antigen.
In one embodiment, said antigen comprises a tumor-specific antigen
In one embodiment, said antigen is from a pathogen.
In one embodiment, said at least one EV therapeutic payload polypeptide further comprises an adjuvant.
In one aspect, there is provided a use, for stimulating an immune response to an antigen, of the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said target cell comprises an immune cell, and wherein said at least one EV therapeutic payload polypeptide comprises an antigen.
In one embodiment, said antigen comprises a disease cell-specific antigen.
In one embodiment, said antigen comprises a tumor-specific antigen
In one embodiment, said antigen is from a pathogen.
In one embodiment, said at least one EV therapeutic payload polypeptide further comprises an adjuvant.
In one aspect, there is provided the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, for use in stimulating an immune response to an antigen, wherein said target cell comprises an immune cell, and wherein said at least one EV therapeutic payload polypeptide comprises an antigen.
In one embodiment, said antigen comprises a disease cell-specific antigen.
In one embodiment, said antigen comprises a tumor-specific antigen
In one embodiment, said antigen is from a pathogen.
In one embodiment, said at least one EV therapeutic payload polypeptide further comprises an adjuvant.
In one aspect, there is provided a method of killing target cells comprising administering to a subject the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.
In one embodiment, said cytotoxic molecule comprises human GZMB R201K, murine GZMB, diphtheria toxin, a PE38 domain from Pseudomonas exotoxin A, or human TRAIL.
In one embodiment, said target cell comprises a disease cell.
In one embodiment, said disease cell is a tumor cell.
In one aspect, there is provided a use, for killing target cells, the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.
In one embodiment, said cytotoxic molecule comprises human GZMB R201K, murine GZMB, diphtheria toxin, a PE38 domain from Pseudomonas exotoxin A, or human TRAIL.
In one embodiment, said target cell comprises a disease cell.
In one embodiment, said disease cell is a tumor cell.
In one aspect, there is the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, for use in killing target cells, wherein said at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.
In one embodiment, said cytotoxic molecule comprises human GZMB R201K, murine GZMB, diphtheria toxin, a PE38 domain from Pseudomonas exotoxin A, or human TRAIL.
In one embodiment, said target cell comprises a disease cell.
In one embodiment, said disease cell is a tumor cell.
In one aspect, there is provided a method of reprogramming immune cells comprising contacting the immune cells with the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein the at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.
In one embodiment, said immunomodulatory molecular comprises a STING or ERAdP pathway activator.
In one embodiment, said STING or ERAdP pathway activator comprises a bacterial dinucleotide cyclase.
In one embodiment, said bacterial dinucleotide cyclase comprises CdaA.
In one embodiment, said immune cells comprise B cells, a T cells, NK cells, dendritic cells, macrophages, or neutrophils. In one embodiment, said immune cells comprise macrophages.
In one embodiment, said immunomodulatory molecule comprises a STING pathway activator, said immune cells comprise macrophages, and said at least one target molecule is macrophage receptor (MARCO).
In one aspect, there is provided a use, for reprogramming immune cells, of the immune cells with the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein the at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.
In one embodiment, said immunomodulatory molecular comprises a STING or ERAdP pathway activator.
In one embodiment, said STING or ERAdP pathway activator comprises a bacterial dinucleotide cyclase.
In one embodiment, said bacterial dinucleotide cyclase comprises CdaA.
In one embodiment, said immune cells comprise B cells, a T cells, NK cells, dendritic cells, macrophages, or neutrophils. In one embodiment, said immune cells comprise macrophages.
In one embodiment, said immunomodulatory molecule comprises a STING pathway activator, said immune cells comprise macrophages, and said at least one target molecule is macrophage receptor (MARCO).
In one aspect, there is provided the immune cells with the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, for use in reprogramming immune cells, wherein the at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.
In one embodiment, said immunomodulatory molecular comprises a STING or ERAdP pathway activator.
In one embodiment, said STING or ERAdP pathway activator comprises a bacterial dinucleotide cyclase.
In one embodiment, said bacterial dinucleotide cyclase comprises CdaA.
In one embodiment, said immune cells comprise B cells, a T cells, NK cells, dendritic cells, macrophages, or neutrophils. In one embodiment, said immune cells comprise macrophages.
In one embodiment, said immunomodulatory molecule comprises a STING pathway activator, said immune cells comprise macrophages, and said at least one target molecule is macrophage receptor (MARCO).
In one aspect, there is provided a method of directing an immune response to a target disease cell comprising administering to a subject the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said recombinant polypeptide comprises at least two targeting moieties which specifically bind, respectively, to at least two different target molecules, wherein said at least two different target molecules are expressed, respectively, by an immune cell and a disease cell.
In one embodiment, said disease cell comprises a tumor cell.
In one embodiment, one of said at least two different target molecules comprises a tumor-associated antigen.
In one embodiment, said immune cell comprises a T-cell, a B-cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil.
In one embodiment, said immune cell is a T cell.
In one embodiment, said immune cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, said immune cell is an NK cell.
In one aspect, there is provided a use, for directing an immune response to target disease cells, of the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, wherein said recombinant polypeptide comprises at least two targeting moieties which specifically bind, respectively, to at least two different target molecules, wherein said at least two different target molecules are expressed, respectively, by an immune cell and a disease cell.
In one embodiment, said disease cell comprises a tumor cell.
In one embodiment, one of said at least two different target molecules comprises a tumor-associated antigen.
In one embodiment, said immune cell comprises a T-cell, a B-cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil.
In one embodiment, said immune cell is a T cell.
In one embodiment, said immune cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, said immune cell is an NK cell.
In one aspect, there is provided the vector as defined herein, the recombinant viral genome as defined herein, the viral particle as defined herein, or the targeted EVs as defined herein, for use in directing an immune response to target disease cells, wherein said recombinant polypeptide comprises at least two targeting moieties which specifically bind, respectively, to at least two different target molecules, wherein said at least two different target molecules are expressed, respectively, by an immune cell and a disease cell.
In one embodiment, said disease cell comprises a tumor cell.
In one embodiment, one of said at least two different target molecules comprises a tumor-associated antigen.
In one embodiment, said immune cell comprises a T-cell, a B-cell, a natural killer (NK) cell, a dendritic cell, a macrophage, or a neutrophil.
In one embodiment, said immune cell is a T cell.
In one embodiment, said immune cell is a regulatory T cell or a cytotoxic T cell.
In one embodiment, said immune cell is an NK cell.
Preparation of Therapeutic Targeted EVs
In one aspect, there is provided a method of preparing therapeutic targeted EVs for a subject comprising:
In one embodiment, said cells are tumor cells.
In one embodiment, said cells are immune cells.
In one embodiment, said immune cells comprises T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.
In one aspect, there is provided a use, for preparing therapeutic targeted EVs for a subject comprising, of the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, or the viral particle as defined herein.
In one embodiment, said cells are tumor cells.
In one embodiment, said cells are immune cells.
In one embodiment, said immune cells comprises T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.
In one aspect, there is provided the nucleic acid as defined herein, the vector as defined herein, the recombinant viral genome as defined herein, or the viral particle as defined herein for use in preparing therapeutic targeted EVs for a subject.
In one embodiment, said cells are tumor cells.
In one embodiment, said cells are immune cells.
In one embodiment, said immune cells comprises T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils
In one embodiment, there is provided a method of producing targeted EVs, wherein said method comprises expressing the nucleic acid as defined herein in cells, or culturing the host cell as defined herein to produce EVs; and collecting the EVs.
The following definitions are provided to facilitate understanding of the terms used herein.
By a “tumor-selective” virus is meant a virus that preferentially grows or replicates in tumor cells.
By “oncolytic virus” is meant any one of a number of viruses that have been shown, when active, to replicate and kill tumor cells in vitro or in vivo. These viruses may naturally oncolytic viruses, or virus that have been modified to produce or improve oncolytic activity. Oncolytic viruses include Rhabdoviruses. Rhabdoviruses include: Carajas virus, Chandipura virus, Cocal virus, Isfahan virus, Piry virus, Vesicular stomatitis Alagoas virus, BeAn 157575 virus, Boteke virus, Calchaqui virus, Eel virus American, Gray Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joya virus, Malpais Spring virus, Mount Elgon bat virus, Perinet virus, Tupaia virus, Farmington, Bahia Grande virus, Muir Springs virus, Reed Ranch virus, Hart Park virus, Flanders virus, Kamese virus, Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus, Kern Canyon virus, Nkolbisson virus, Le Dantec virus, Keuraliba virus, Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus, Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus, Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus, Charleville virus, Coastal Plains virus, DakArK 7292 virus, Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus, Joinjakaka virus, Kannamangalam virus, Kolongo virus, Koolpinyah virus, Kotonkon virus, Landjia virus, Manitoba virus, Marco virus, Nasoule virus, Navarro virus, Ngaingan virus, Oak- Vale virus, Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, Rio Grande cichlid virus, Sandjimba virus, Sigma virus, Sripur virus, Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata virus, Rhode Island, Adelaide River virus, Berrimah virus, Kimberley virus, Maraba virus, Bovine ephemeral fever virus, or engineered variants thereof.
“Extracellular vesicles” (EVs) are cell-derived membranous structures, including exosomes, microvesicles, virus-like particles, macrovesicles, oncosomes, gesicles, and apoptotic bodies. These extracellular vesicles generally are categorized based on their size, specific markers, cellular origin and biogenesis processes. Exosomes are 30-160 nm vesicles of endosomal-origin released from the cell upon fusion of a multivesicular body (MVB) membrane with the plasma membrane. Exosomes are produced by every cell type and their release can be induced by a variety of stimuli, including stress, hypoxia, cell death, and viral infection. Classical microvesicles (also known as microparticles) are 100 nm-1 µm vesicles released from the cell by shedding of the plasma membrane. Cancer cells can also secrete larger microvesicles (>1 µm) called oncosomes, which only differ from classical microvesicles in regard to their size. Like exosomes, microvesicle release can be induced by stress and viral infection, and their contents are heterogeneous. Apoptotic bodies are large EVs that are released from apoptotic cells by blebbing and range in size from 200 nm to 5 µm. These phosphatidylserine and Annexin V-coated EVs contain cytoplasmic contents from the dying cell. Traditionally, EVs that pelleted at 100,000 g were referred to as exosomes, but in fact this pellet contains a combination of microvesicles and exosomes. Though their biogenesis pathways are distinct, exosomes and microvesicles have many similarities and are difficult to distinguish from one another once released from the cell. Recently, the International Society for Extracellular Vesicles suggested the term Small EVs (sEVs) should be used for particles less than 200 nm in size, while the term Large EVs (IEVs) should be used for particles greater than 200 nm.
The terms “programmed EVs” (PEVs) and “targeted EVs” are used synonymously herein to refer to EVs comprising the recombinant polypeptide, as defined herein, and therefore having an engineered acquired affinity (provided by the targeting moiety) for a target molecule.
By “recombinant” is meant a nucleic acid or polypeptide molecule that contains segments of different origins, such as (but not limited to) the products of genetic engineering through recombinant DNA technology.
By “EV-anchoring polypeptide” is meant a polypeptide that tethers the recombinant polypeptide to an EV membrane.
By “EV-directed transmembrane polypeptide” is meant the portion of a transmembrane protein that spans the entirety of a phospholipid bilayer membrane of an EV, and which innately targets (is trafficked to) EV membranes.
Proteins containing such EV-directed transmembrane domains can originate from viruses (e.g. VSVG), or originate in cells (e.g. CD63 and lamp2b). Membrane spanning domains may be single pass or may pass through the membrane multiple times, such as four times (quadruple pass, or tetraspanin).
Single pass and tetraspanin domains can be engineered via linker sequences to carry a single or multiple payloads, and single pass domains can be similarly engineered to carry a single or multiple targeting moieties in tandem.
Likewise, by “EV-directed tetraspanin” is meant a subset of tetraspanins that are trafficked to EV membranes. Tetraspanins are a family of membrane proteins found in all multicellular eukaryotes, and also referred to as the transmembrane 4 superfamily (TM4SF) proteins. They have four transmembrane alpha-helices and two extracellular domains, one short extracellular domain or loop, and one longer extracellular domain/loop. Although several protein families have four transmembrane alpha-helices, tetraspanins are defined by conserved amino acid sequences including four or more cysteine residues in the EC2 domain, with two in a highly conserved ‘CCG’ motif.
Tetraspanins can be engineered to carry up to 2 targeting moieties, and up to 2 payloads directly, or more if linked together.
Table 1 provides some examples of proteins that specifically direct to, and are enriched in, EV membranes. These examples include single pass and tetraspanin domains.
By “derived from”, in the context of a recombinant tetraspanin, it would be understood that the native tetraspanin is modified to include exogenous sequences, such as a targeting moiety inserted into one or both extracellular loop(s) and/or a payload linked to the tetraspanin.
By “targeting moiety” is meant a molecule capable of binding to a target molecule with sufficient affinity and specificity so as to be able to target EVs to a target cell expressing the target molecule. Non-limiting examples of targeting moieties include antibodies, functional fragments thereof, engineered fragments thereof, ligands (which target receptors), designed ankyrin repeat proteins (DARPins) (which bind target proteins), and domains that mediate specific protein-protein interactions. It would be understood that the targeting moiety of the recombinant polypeptide is, in the context of an EV, intended to be externally-orientated.
Table 2 sets for some example targeting moieties.
A “single domain antibody” (sdAbs), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies composed of two heavy chains and two light chains. sdABs are produced by immunization of dromedaries, camels, llamas, alpacas or sharks, or can be engineered from common IgGs with four chains.
By “functional fragment” is meant a portion of an antibody that maintains the paratope (comprising the complementary determining regions or CDRs) and is capable of binding to the same target molecule as the parent antibody from which is it derived. Examples include Fab and F(ab′)2 fragments.
By “engineered fragment” is meant a recombinant polypeptide derived from a parent antibody and retaining the paratope, thus being able to bind to the same target molecule as the parent antibody. An example is a single-chain variable fragment (scFv), which is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide, of typically 10 to about 25 amino acids.
“DARPins” are repeat proteins comprising several repeating structural domains (generally 4 to 6 repeats) of usually 33 amino acids. DARPins can be selected and used as alternative scaffolds for specific targeting because they can bind to their target antigens with high affinity and specificity. A key advantage of using DARPins compared to monoclonal antibodies is that DARPins generally possess low molecular weights, containing between 40 to 100 amino acid residues. For example, HER2 is frequently overexpressed in breast cancer cells. DARPins binding to the extracellular domains of HER2 can be selected and used to direct therapeutic EVs towards malignant cells expressing HER2.
By “target molecule” is meant a molecule to which the targeting moiety binds. Such molecules may be cell surface molecules, such as, e.g., polypeptides, lipids, or polysaccharides that can be specifically bound by the targeting moiety.
By “target cell” is meant a cell that expresses the target molecule that is bound by the targeting moiety, and to which the payload (if applicable) and/or cargo (if applicable) is/are directed.
By “intravesicular polypeptide” is meant the polypeptide portion of the recombinant polypeptide that extends internally to the EV. It will be understood that the intravascular polypeptide may comprise a short polypeptide (e.g. of at least 9 amino acids) that projects into the intravesicular space. However, in other configurations described herein, the it will be understood that the intravascular polypeptide may comprise an EV payload polypeptide. In yet other configurations the EV-directed transmembrane domain and the intravascular polypeptide may together comprise an EV-directed recombinant tetraspanin, which may or may not comprise at least one EV payload polypeptide, which may be linked to the N- and/or C-terminus.
“Monotargeted” indicates that a population of EVs is targeted to a target molecule. However, where “at least one” target is specified, it will be understood that this is also intended to encompass EVs directed to more than one target molecule, so that the EVs are minimally monotargeted.
Likewise, “bispecific” means that an EV targets two target molecules. Where “at least two” is specified, it will be understood that this is also intended to encompass EVs directed to more than two target molecules, such that the EVs are minimally bispecific.
By “cell surface molecule” is meant any molecule that is anchored or otherwise associated with a cell surface to permit targeting of the cell by the recombinant polypeptide via the targeting moiety. Such molecules may include, for example, polypeptides, polysaccharides, or lipids (including polysaccharide and lipid modifications to polypeptides). Examples include integral membrane proteins, peripheral membrane proteins, and modifications thereof.
A “cell surface marker” is a cell surface molecule particular to (or enriched in) a particular cell type. A cell surface marker or a combination of cell surface markers may be unique to a given cell type, or cell state (such as a disease state).
By “tumor stroma” is meant cells in the tumor environment other than cancer cells per se, such as, e.g., cancer associated fibroblasts.
By “tumor-associated antigen” (TAA) is meant any immunogen that is associated with tumor cells, and that is either absent from or less abundant in healthy cells or corresponding healthy cells (depending on the application and requirements). For instance, the tumor associated antigen may be unique, in the context of the organism, to the tumor cells. A TAA may be, for example, a tumor-specific mutation, an aberrantly spliced protein, an oncofetal antigen, or an endogenous retroviral protein. A TAA may be a neoantigen comprising neoepitope. Neoantigens are newly formed (non-autologous) antigens that have not been previously recognized by the immune system, and can arise, e.g., from tumor mutations.
The terms “payload” and “cargo” are used differentially here. The former are part of the recombinant polypeptide, while the latter are intended to be separate molecules to be carried in the EVs.
By “EV payload polypeptide” is meant any polypeptide that is part of the recombinant polypeptide itself, and that would therefore be co-encoded by the same nucleic acid molecule. EV payload polypeptides include any polypeptides for which EV loading or EV-mediated targeting or delivery would be desirable.
By “EV therapeutic payload polypeptide” is meant a therapeutic polypeptide that is part of the recombinant polypeptide itself, and that would therefore be co-encoded by the same nucleic acid molecule. Categories of payloads include (but are not limited to) cytotoxic molecules (e.g. GZMB variants, Diphtheria Toxin, pe38 (domain from pseudomonas exotoxin A), or TRAIL), immune reprogramming molecules (e.g. STING or ERAdP pathway activators, such as bacterial cyclases), enzymes, nucleotide binding domains, and antigens (such as tumor antigens or antigens from infectious pathogens, such as Dengue virus, Malaria, or Rotavirus). Non-limiting examples are presented in Table 3.
By “EV cargo”, in contrast, is meant a molecule to be carried in the EV, but that is otherwise separate from the recombinant polypeptide that directs the EV to a target. Accordingly, the cargo may be encoded by the same or a different nucleic acid that encodes the recombinant polypeptide (in the case of the latter they would be understood to be expressed as separately polypeptides). It is envisaged, for example, that host cells manipulated to express the recombinant polypeptide could separately encode (or be modified to express) the cargo (or vice versa). Being a separate molecule to the recombinant polypeptide, cargo molecule need not be polypeptides. For example, the cargo molecule could be a small molecule, e.g. a small molecule drug or imaging agent. The cargo could be a nucleic acid, such as an mRNA, miRNA, shRNA, or siRNA. Nucleic acids could be preferentially loaded into vesicles, for example, in embodiments in which the payload comprises a nucleic acid binding domain. Such binding domains may be sequence-specific, binding to a sequence motif within the nucleic acid molecule. Cargo molecules could also comprise polypeptides, such as cytotoxic molecules, immune reprogramming molecules, enzymes, or antigens.
The term “linked” indicates that two moieties are covalently linked, though such linkage need not be direct. For example, if “A” and “B” are “linked”, it would be understood that the linkage could comprise additional amino acids residues or polypeptides. Likewise, linked “via” a feature, such as a linker polypeptide or payload, indicates that the feature lies between (and separates) “A” and “B” in the context of the recombinant polypeptide. However, neither “A” nor “B” need be directly attached to the intervening feature.
By “adjuvant” will be understood a molecule that potentiates the immune response to an antigen and/or modulates it towards the desired immune response.
Where nucleic acid and amino acid molecules are referred to herein, it will be appreciated the embodiments encompassing sequence variants thereof are also expressly contemplated. For example, such sequence variants may be for polypeptides (or nucleic acid molecule encoding polypeptides) that retain substantially the same function as the parent molecule from which they are derived, or the same function. Such sequence variants may be at least 70% identical to the parent molecule. They may be at least 80% identical to the parent molecule. They may be at least 90% identical to the parent molecule. They may be at least 95% identical to the parent molecule. They may be at least 96% identical to the parent molecule. They may be at least 97% identical to the parent molecule. They may be at least 98% identical to the parent molecule. They may be at least 99% identical to the parent molecule. Sequence variants contemplated herein may comprise conservative amino acid substitutions (or nucleic acid sequence changes encoding them). Sequence variants contemplated herein may comprise silent mutations.
The following Examples outline embodiments of the invention and/or studies conducted pertaining to the invention. While the Examples are illustrative, the invention is in no way limited the following exemplified embodiments.
Attempts have been made to program EVs to carry or deliver therapeutics using multiple molecules, mechanisms and means, which can be expensive, required to be ex vivo, and time-consuming (such as via electroporation), and which are not shelf-stable and lack validation.
There is therefore a need for a means to deliver molecules to cells, including, but not limited to, therapeutics and toxins, to targeted cells in vivo, or prepared simply and continually in vivo, in vitro, or ex vivo, in a simple, inexpensive, stable way.
Recombinant peptides have been designed for targeted delivery of molecules to cells.
Application: Blocking or activating function of cell surface receptors.
How these platforms work: They can act as competitive binding or blocking drugs.
Advantages: Stability and lack of immunogenicity. In cases that the PEV is produced from a virus platform, this has the potential for in-situ delivery. Incidentally, the approach is also more cost-effective due to these solutions.
Deliverv/manufacturina modalities: Viral-based platforms (e.g. Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, HSV-1, etc.). Plasmids (e.g. pcDNA 3.1) and free PEVs.
Example 1(a): Programmed cell death protein 1 (PD1) is a surface protein preferentially expressed in immune cells such as T, B, NK cells, and myeloid-derived dendritic cells. Upon engagement with its ligand, PD-L1, PD1 transmits immune inhibitory signals. Molecules (monoclonal antibodies) that antagonize the PD1:PD-L1 interaction by binding to PD1 and PD-L1 have been shown to facilitate T cell-mediated killing of tumor cells. While these strategies are showing positive results in some clinical indications, these approaches tend to yield a large amount of wasted antigen that do not make it to their final destination/use (e.g. exosomes expressing PD-L1). These factors reduce bioavailability and increase the risk of toxicities of anti-PD1 and anti-PD-L1 monoclonal antibodies.
As such, presently contemplated is a PEV targeting PD1 with PD-L1 as the targeting moiety, thereby blocking the function. Targeting moiety: PD-L1 (blocking application/adjuvant for ICIs).
Payloads: None or optional.
Transmembrane domain (TD domain): All the examples listed in Table 1 could be used.
Example 1(b): Similarly, T cells could be activated via the CD3 surface protein using a CD3 targeting moiety, such as an anti-CD3 antibody (T cell activation/engager application).
Payloads: None or optional.
Transmembrane domain (TD domain): All the examples listed in Table 1 could be used.
Results: Data for PD1 targeting can be seen in
It is noted that that only the EVs derived from cells infected with a VV expressing the mPD-1-LAMP2B-HA tag construct were pulled down with anti-PD1 antibodies. This data shows that the construct is not only expressed and incorporated into EVs, but also the “topology or orientation” of the PEV constructs in the EVs is as expected.
Background: In nature, the Major Histocompatibility Complex (MHC) is required for T cells to recognize and kill tumor cells. However, most tumors downregulate the expression of the (MHC) to escape immune attack. One existing strategy in the art to circumvent the tumor’s escape mechanism is by way of engineered bi-specific antibodies which draw T-cells and Tumor cells to close proximity. These bi-specific antibodies are also referred to as Bi-specific T cell Engagers or BiTEs.
These BiTEs are able to mediate the T cell’s capacity to recognize and kill tumor cells in an MHC independent fashion. BiTEs consist of linked variable chain antibody fragments directed against the T cell antigen CD3 and a specific tumor-associated antigen (TAA). Similarly, Bi-specific NK cell engagers or BiKEs can mediate simultaneous binding to an activating receptor on NK cells and a surface tumor antigen to thus promote NK cell-dependent killing of tumor cells. Although existing BiKE and BiTE technologies are promising, many that are currently in clinical development have issues with associated toxicity during systemic administration, drug stability issues (short half-life), and challenges to reaching high enough local concentrations to be effective in most solid cancers
Application: PEV constructs with two targeting moieties: one that recognizes T (or NK) cell targets, and the other targeting tumor cells (cancer cell or CAFs).
How these platforms work: These PEVs will promote the synapsis between T cells and tumor cells or between NK cells and tumor cells, thus promoting the directed killing of tumor cells by these immune cell types.
Advantages: Displaying BiTEs and BiKEs in a PEV format is more stable than the bi-specific antibody constructs.
Special Features: Generally, payload-less - the PEV construct itself is a stable bi-specific cell engager bringing T or NK cells closer to cancer cells. These PEVs can be produced in vivo or ex vivo.
Delivery modalities: Using OVs as delivery vehicles in patients to secrete BiTEs and BiKEs in the infected cancer cell. As such, the PEV is delivered to the exact site where needed, and therefore likely to be effective at picomolar concentrations. i.e., lower dose treatment than the current bi-specific antibody approaches. Viral-based platforms such as: Vaccinia virus (abbreviated as VacV or VV), lentivirus, adeno-associated virus [AAV], VSV, HSV-1, etc. could be used.
Plasmids (e.g. pcDNA 3.1) for preparing the virus and infecting cells, as well as for manufacturing isolated PEVs are also contemplated.
Targeting moieties: Single chain variable fragments or nanobodies as described above. These bind to:
Payloads: None
Transmembrane Domain: All the examples listed in Table 1 could be used. Examples included here are with tetraspanin proteins, however single pass TM proteins may be used “multimerization technology” (see special features for details).
Results:
Backaround/Context: Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes such as inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. The platforms described herein may represent effective means to augment and prolong the cellular and tumoral immune responses evoked by infectious disease and cancer vaccines, respectively.
Application: Immunologic adjuvants (e.g. STING or ERAdP activators, which generate immunogenic molecules that stimulate the immune system) payloads can be specifically delivered to antigen presenting cells (APCs) such as dendritic cells (DCs) by targeting specific DC surface molecules.
How these platforms work: Antigen presenting cells (APC), such as DCs exhibit a largely immature or immunologically tolerizing phenotype (not yet functionally ready to accept presented-antigens, or serving to suppress immune responsiveness). Delivery of immunologic adjuvants (i.e. STING or ERAdP pathway activators, e.g. bacterial dinucleotide cyclases such as CdaA and MtbDisa which are c-di-AMP cyclases, and VCA0848, which is a c-di-GMP cyclase, or mouse/human cGAS) to DCs via PEVs may result in activation of STING and/or ERAdP, which enhances DC antigen presentation capacity, and increases expression of T cell co-stimulatory molecules, thereby boosting the APC activity. In some instances, these platforms can be used in combination with vaccine approaches.
Advantages: Stability, less off-target toxicity, tailored delivery.
Targeting moieties: The targets are antigen presenting cell-surface molecules, including but not limited to CD40, a TNF-α family receptor, DEC-205, a C-type lectin receptor and CD11c, an integrin receptor, by way of targeting moieties including specific monoclonal antibodies, scFvs, single domain antibodies, nanobodies (i.e. anti-DEC205, anti-Clec9A, anti-CD11 c, anti-lectin receptor). Peptides and ligands represent a suitable alternative to antibodies as active targeting agents (e.g. CD40 ligand or CD40-targeted peptide).
Payloads: Bacteria dinucleotide cyclases (i.e. CdaA, etc.) (Note: these payloads are enzymes, thus these examples indicate that functionally active enzymes could also be delivered by PEVs).
Transmembrane domain: All the examples listed in Table 1 could be used. Thus far, all our examples are built with VSV-G and CD63.
Delivery/manufacturing modalities: Viral-based platforms such as, Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, HSV-1, etc. could be used. Plasmids (e.g. pcDNA 3.1) for preparing recombinant virus and transfecting cells, as well as manufactured and isolated PEVs are also contemplated.
Results:
Isolated PEVs containing anti-DEC205-VSVG-CdaA chimeric constructs lead to STING activation in a dose dependent fashion. The STING (stimulator of interferon genes) pathway contributes to the activation of antigen presenting cells, including DCs. STING activation is mediated by its phosphorylation. In DCs, activation of STING is important for IFN-β expression and IL-12 production as well as for the surface expression of the activation markers CD40 and CD86. The role of the cGAS-STING pathway is important in pathogen detection and in cancer immunity. STING activation, as well as ERAdP activation, appear to be an essential component in the recruitment of immune cells to the tumor microenvironment, which is paramount to immune clearance of the tumor. STING activation provides an adjuvant function during vaccination as well.
The data shown here demonstrates that only EVs decorated with anti-DEC205-VSVG-CdaA constructs can activate the STING-TBK1-IRF3 signaling axis in primary murine dendritic cells (
Background/Context: Tumor-associated antigens and/or immune reprograming moieties (e.g. STING or ERAdP pathway activators) can be specifically delivered to surface molecules on APCs, such as dendritic cells via PEVs. This construct would express a targeting moiety to target PEVs to DCs (dendritic cells) and it could concomitantly carry one or multiple payloads.
Application: These platforms will represent effective means to elicit robust tumor antigen-specific immunity.
How these platforms work: DCs exhibit a largely immature or tolerizing phenotype. Tumor antigen delivery via PEVs (as payloads or cargo), in conjunction with co-administration of an adjuvant (DC maturation stimuli such as agonistic anti-CD40 mAbs, poly(I:C), cytosine-phosphate-guanine (CpG), lipo-polysaccharide (LPS), or toll-like receptor ⅞ (TLR⅞) agonists) or targeted co-delivery of PEVs containing STING or ERAdP pathway activators as described above (e.g. Bacterial dinucleotide cyclases such as CdaA and MtbDisa which are c-di-AMP cyclases, and VCA0848, which is a c-di-GMP cyclase) results in enhanced tumor-associated antigen presentation capacity and increased expression of T cell costimulatory molecules
Tumor-associated antigens alone or in combination with adjuvants or in combination with immune reprograming moieties (e.g. STING or ERAdP pathway activators) can be specifically delivered to surface molecules on dendritic cells via PEVs
Targeting moieties: Targets: Antigen presenting cell-surface molecules, including CD40, a TNF-α family receptor, DEC205, a C-type lectin receptor (CLEC9) and CD11c, an integrin receptor, are targeted by targeting moieties including specific monoclonal antibodies, scFvs, single domain antibodies, nanobodies (i.e. anti-DEC205, anti-Clec9A, anti-CD11c), ligands or targeted peptides (e.g. CD40 ligand or CD40-targeted peptide).
Payloads: Specific tumor-associated antigens (For proof-of-concept in mouse tumor models: DCT and OVA are being explored). Human tumor-associated antigens relevant for clinical testing can be used (e.g. HPV-E6 and E7, NY-ESO-1, etc.). Cancer-specific neoantigens can also be used.
Concomitant expression of specific disease cell antigens is contemplated, such as tumor-associated/specific antigens (e.g. OVA, DCT, mERKm9 etc.). Also, adjuvant molecules such as a STING or ERAdP activator could be concomitantly delivered with disease-specific antigens or tumor-associated/specific antigens
Transmembrane domain: All the examples listed in Table 1 could be used. Thus far, all our examples are built with VSV-G.
Delivery/manufacturing modalities: Viral-based platforms such as, Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, HSV-1 etc. could be used. Plasmids (e.g. pcDNA 3.1) for transfecting cells, as well as manufactured isolated PEVs are also contemplated.
Background/Context: Pathogen-specific antigens and/or immune reprograming moieties (e.g. STING or ERAdP pathway activators) can be specifically delivered to surface molecules on dendritic cells via PEVs. This construct would express a targeting moiety to tailor PEVs to DCs and it could concomitantly carry multiple payloads.
Application: These platforms will represent effective means to elicit robust pathogen-specific antigen-driven immunity.
How these platforms work: Similar to above, DCs exhibit a largely immature phenotype. Pathogen-specific antigen delivery via PEVs, in conjunction with co-administration of adjuvant (DC maturation stimuli such as agonistic anti-CD40 mAbs, poly(I:C), cytosine-phosphate-guanine (CpG), lipo-polysaccharide (LPS), or toll-like receptor ⅞ (TLR⅞) agonists) or targeted co-delivery of PEVs containing STING or ERAdP pathway activators (e.g. Bacterial dinucleotide cyclase, such as CdaA and MtbDisa which are c-di-AMP cyclases, and VCA0848, which is a c-di-GMP cyclase) results in enhanced pathogen-specific antigen presentation capacity and increased expression of T cell costimulatory molecules
Targeting moieties: Targets include Antigen presenting cell-surface molecules, including CD40, a TNF-α family receptor, DEC-205, a C-type lectin receptor and CD11c, an integrin receptor, are targeted by means of targeting moieties such as specific monoclonal antibodies, scFvs, single domain antibodies, nanobodies (i.e. anti-DEC205, anti-Clec9A, anti-CD11c), ligands or targeted peptides (e.g. CD40 ligand or CD40-targeted peptide).
Payloads: Pathogen-specific antigens [e.g. Dengue PM & E antigens, Malaria CS30, Rotavirus VP6, etc.). Concomitant expression of specific infectious disease-associated antigens with adjuvant molecules such as STING or ERAdP activator could be pursued to boost vaccination activity.
Transmembrane domain: All the examples listed in Table 1 could be used. Thus far, all our examples are built with VSV-G.
Delivery/manufacturing modalities: Viral-based platforms (e.g. Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.). Plasmids (e.g. pcDNA 3.1) and free PEVs.
Background/Context: Immune reprograming molecules (e.g. cytokines, miRNAs) can be specifically delivered as payloads or cargoes to surface molecule targets on specific immune cell populations via PEVs. This construct would express a targeting moiety to tailor PEVs to specific-immune cell populations and it could concomitantly carry multiple payloads.
Application: These platforms represent effective means to reprogram or educate (e.g. activate, phenotype change, etc.) immune cells to play specific functions and thus fight inflammatory diseases and cancer. In addition, these PEVs could be used to augment the visibility (immunogenicity) of cancer cells to immune cells (e.g. promoting immunogenic cell death).
How these platforms work: Immune-suppressive M2 macrophages will be turned into immune-boosting M1 macrophages that are ready to engulf tumor cells. Also, certain subsets of macrophages are important in causing inflammatory diseases such as asthma, atherosclerosis, rheumatoid arthritis, osteoarthritis, endometriosis, diabetes type 1 and 2, and obesity. Macrophage reprograming can be done with PEVs.
Targeting moieties: Single chain variable fragments or a binding peptide for CD206 (Mannose receptor) can be used to specifically target M2 macrophages (also known as tumor-promoting macrophages).
Payloads: either payload-less with cargo, or payload being an RNA-binding motif to specifically capture cargo that modifies macrophage polarization to reduce inflammatory gene expression through RNAi, as multiple genes can be downregulated simultaneously. Cargo targets may include inflammatory mediators such as cytokines (e.g., TNF-α, IL-6, IL-1β), chemokines (e.g., CCL2, CCL3, CCL5), and transduction targets involved in promoting inflammation, such as members of the NF-KB signaling cascade. miRNA cassettes targeting IKBα, siRNA directed toward mitogen-activated protein kinase4 4 (Map4k4) reduced systemic inflammation by reducing Tnf-α mRNA in macrophages.
How these platforms work: Regulatory T cells (Tregs) are known to restrict the function of effector T cells. In the context of cancer, Tregs are powerful inhibitors of anti-tumor immunity and the presence of these cells in the tumor microenvironment leads to tumor growth. Directed targeting of regulatory molecules in Tregs with PEVs will lead to the conversion of these cells into IFNg-secreting effector cells (cancer-fighting cells).
Targeting moieties: Single chain variable fragments directed to CTL4 (cytotoxic T-lymphocyte-associated antigen 4) on the surface of immune suppressive T cells.
Payloads: either payload-less with cargo, or payload being an RNA-binding motif to specifically capture cargo that convert immunosuppressive regulatory T cells (Tregs) into cancer fighting T cell by downregulating CARMA1 and/or MALT1. For example, a miRNA cassettes containing a shRNA against CARMA 1 and/or MALT1. T cell activation. These miRNA cassettes may be EV-directed miRNA cassettes with or without RNA sequences corresponding to the payload RNA-binding motif recognition site. Alternatively, these miRNA cassettes may be regular non-EV directed cassettes which include an RNA sequence corresponding to the payload’s RNA-binding motif recognition site.
Transmembrane Domain: All the examples listed in Table 1 could be used.
Delivery/manufacturing modalities: Viral-based platforms (e.g. Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.). Plasmids (e.g. pcDNA 3.1) and free PEVs.
How these platforms work: Decreased T cell function has been described in chronic viral, bacteria and parasitic infections and in cancer. CD3-targeting PEVs can be used to stimulate the activity of disease-fighting T cells in the immune system.
Targeting moieties: T cell activation: Single chain variable fragments or single-domain antibodies targeting CD3 on T cells.
Payloads: The CD3 targeting construct is payless- Engaging CD3 in T cells may be sufficient to activate them and mobilize them to kill cancer cells. Anti-CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T-cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Cai2+).
Background/Context: These provide an EV that functions like a targeted cytotoxic T cell (akin to CAR-T therapy, but removing the T cell from the equation). This enables the killing of highly immunosuppressive, immunologically “cold” tumors and MHC-I deficient cancers by our PEVs.
Application: PEVs with a cytotoxic function, used as a drug to target specific tumor cell types as described in the examples below. Other cell types could be contemplated.
How these platforms work:
Advantages: not autologous, stable, specifically targeted, can be virally delivered or shelf-stably produced.
Targeting moieties: Single chain variable fragments or single domain antibodies (i.e., anti-CD19, anti-CD20, anti-CD22, anti-EGFR, anti-FAP, anti-CEA, anti-CA9) or through targeting peptides [i.e. MMP2-targeted chlorotoxin (CTX), proteoglycan-targeted VAR2Δ (VAR2Δ also named as VAR2CSA, binds to a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta and also found on a high proportion on cancer cells), GE11 peptide, which targets with high affinity EGFR].
Payloads: Cytotoxic payloads such as murine granzyme B (mGZMB), human granzyme B (hGZMB R201K) - note that the R201K mutation is to confer resistance against the endogenous human granzyme B inhibitor-, diphtheria toxin (DT), TRAIL (a cytokine that causes cell death primarily in tumor cells), and the truncated pseudomonas exotoxin 38 (PE38).
Transmembrane Domain: All the examples listed in Table 1 could be used.
Delivery/manufacturing modalities: Viral-based platforms (e.g. Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.). Plasmids (e.g. pcDNA 3.1) and free PEVs.
Results:
Leftmost blot - 293T cells transfected with pcDNA3.1 plasmids encoding the constructs.
Middle blot - U2OS human osteosarcoma cells infected with vaccinia virus (VACV) encoding these constructs
Rightmost blot - small extracellular vesicles (sEVs) isolated from 786-0 human renal cell adenocarcinoma cells infected with the viruses.
As expected, no signal is picked up in cells infected by the VACV-eGFP which does not express any granzyme B protein.
These blots show that not only are the chimeric granzyme B fusion constructs expressed by the plasmid, but that they are also successfully sorted and packaged in the sEVs through the transmembrane VSVG linker.
Background/Context: Reprograming moieties can be specifically delivered as free cargoes (therapeutic miRNAs, mRNAs) or by binding to RNA binding proteins/domains payloads (e.g. RNA binding proteins/domains MS2, CAS13, or others), linked to surface molecule targets on specific tumor (e.g. immune cell populations, CAFs or cancer cells) via PEVs. This construct would express a targeting moiety to tailor PEVs to the desire cell type and it could concomitantly carry a single or multiple payloads and/or these constructs can be combined with specific cargoes with corresponding sequences.
Application: These platforms represent effective means to reprogram or educate (e.g. activate, phenotype change, etc.) tumor resident cells to play specific functions and thus fight cancer.
How these platforms work: For example, these PEVs could be used to augment the visibility (immunogenicity) of cancer cells to immune cells (e.g. promoting immunogenic cell death) or could be used to re-program T cell as CAR-T cells in situ in the tumor microenvironment.
Special Features: “nucleic acid ligand system” between a “RNA binding payloads (e.g. MS2, CAS13) and a therapeutic RNA molecule cargo (i.e. mRNAs, IncRNAs, microRNAs) containing the “matching” RNA binding motif (RNA ligand domain) bound by the RNA binding payload.
Targeting moieties: All the examples listed above.
Payloads: RNA binding proteins or their RNA-binding motifs (e.g. Cas13, MS2 coat protein, Staufen-1, human Pumilio-homology domain-1).
Transmembrane domain: All the examples listed in Table 1 could be used.
Delivery/manufacturing modalities: Viral-based platforms (e.g. Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.). Plasmids (e.g. pcDNA 3.1) and free PEVs.
Results:
Background/Context: The term “cargo” is defined herein as a molecule that is coexpressed with but it is not part of the chimeric protein construct. As such, cargo can be included/co-expressed whether or not there is a payload in the construct. Cargo can be nucleic acids or proteins that are preferentially directed to EVs, and/or RNAs that may include a special sequence recognized by a specific RNA-recognizing payload included in the PEV construct.
Application: Can be for any application where it might be suitable to target molecules to the target cell, particularly if they cannot maintain activity/function in the PEV construct. In other cases, could promote specific delivery of an EV cargo molecule.
Special Features: RNA can either have an RNA-binding motif recognition site to bind to RNA-binding motif payload or have an EV-directing motif. Proteins may be preferentially directed to EVs by way of specific sequences that are known in the art to target them.
Targeting moieties: Various, depending on situation
Payload: RNA-binding motif, for instances where the cargo has RNA-binding motif recognition sequence.
To produce EVs that are tailored and concomitantly carry specific nucleic acid molecules of interest (cargos; e.g. microRNAs and mRNAs) a PEV can be designed to carry a payload that contains 1 or more RNA-binding domains (RBDs). The RNA cargo can display the binding motif recognized by the RBD (also referred to as the RNA ligand domain) and thus specifically interact and be carried by the PEV containing RBDs (“RNA nucleic acid ligand system” system). For example, well characterized RNA-binding domains found in cellular RNA binding proteins such as the RRM, KH, cold shock domain (CSD), and the zinc finger CCHC domains could be used. Similarly, RNA-binding domains found in Viral coat or capsid proteins (e.g. the MS2 bacteriophage coat protein) or bacterial RNA-binding Cas proteins (e.g. Cas13) can be designed as part payloads in PEV constructs that function as RNA-carrying or nucleic acid ligand systems. The cognate RNA binding ligands will be included in the RNA cargo molecules.
Transmembrane domain: the PEV can contain any transmembrane domain according to the other categories outlined in this document- cargos are not part of the presently described chimeric construct
These constructs are used as controls for various experiments in multiple categories. Some of these are independently proof of concept constructs, e.g. functional payload delivery (mCherry or Nanoluc™) or placement of targeting molecules within tetraspanin transmembrane domains for a single target.
Results:
It has been demonstrated that virus infection (e.g. Vaccinia virus infection) increases small EV secretion.
Results:
As previously described in Example 9.
Background/Context: Cargo can be included/co-expressed whether or not there is a payload in the construct.
Application: Can be for any application where it might be suitable to target molecules to the target cell, particularly if they cannot maintain activity/function in the PEV construct. In other cases, could promote specific delivery of an EV cargo molecule to the target cell.
Special Features: Cargo RNA molecules can either have an RNA-binding motif recognition site to bind to RNA-binding motif payload or have an EV-directing motif. Proteins may be preferentially directed to EVs by way of specific sequences that are known in the art to target them.
Targeting moieties: Various, depending on the application.
Payload: RNA-binding motif, for instances where the cargo has an RNA-binding motif recognition sequence.
To produce EVs that are tailored and concomitantly carry specific nucleic acid molecules of interest (cargos, e.g., microRNAs and mRNAs) a PEV can be designed to carry a payload that contains 1 or more RNA-binding domains (RBDs). The RNA cargo can display the binding motif recognized by the RBD (also referred to as the RNA ligand domain) and thus specifically interact and be carried by the PEV containing RBDs (“RNA nucleic acid ligand system” system). For example, well characterized RNA-binding domains found in cellular RNA binding proteins such as the RRM domain, K homology (KH) domain, cold shock domain (CSD), and the zinc finger CCHC domains could be used. Similarly, RNA-binding domains found in viral coat or capsid proteins (e.g., the MS2 bacteriophage coat protein) or bacterial RNA-binding Cas proteins (e.g., Cas13) can be designed as part payloads in PEV constructs that function as RNA-carrying or nucleic acid ligand systems. The cognate RNA binding ligands will be included in the RNA cargo molecules.
Transmembrane domain: the PEV can contain any transmembrane domain according to the other categories outlined in this document- cargos are not part of the presently described chimeric construct.
Results:
Table 12 provides amino acid motifs (RNA binding motif) that were experimentally shown in
ENENGITGRMRGKTVKNNKGEEKYVSGE VDKIYNENKQNEVKENLKMFYSYDFNMD NKNEIEDFFANIDEAISSIAHGIVHFNL ELEGKDIFAFKNIAPSEISKKMFQNEIN EKKLKLKIFKQLNSANVFNYYEKDVIIK YLKNTKFNFVNKNIPFVPSFTKLYNKIE DLRNTLKFFWSVPKDKEEKDAQIYLLKN IYYGEFLNKFVKNSKVFFKITNEVIKIN KQRNQKTGHYKYQKFENIEKTVPVEYLA IIQSREMINNQDKEEKNTYIDFIQQIFL KGFIDYLNKNNLKYIESNNNNDNNDIFS KIKIKKDNKEKYDKILKNYEKHNRNKEI PHEINEFVREIKLGKILKYTENLNMFYL ILKLLNHKELTNLKGSLEKYQSANKEET FSDELELINLLNLDNNRVTEDFELEANE IGKFLDFNENKIKDRKELKKFDTNKIYF DGENIIKHRAFYNIKKYGMLNLLEKIAD KAKYKISLKELKEYSNKKNEIEKNYTMQ QNLHRKYARPKKDEKFNDEDYKEYEKAI GNIQKYTHLKNKVEFNELNLLQGLLLKI LHRLVGYTSIWERDLRFRLKGEFPENHY IEEIFNFDNSKNVKYKSGQIVEKYINFY KELYKDNVEKRSIYSDKKVKKLKQEKKD LYIANYIAHFNYIPHAEISLLEVLENLR KLLSYDRKLKNAIMKSIVDILKEYGFVA TFKIGADKKIEIQTLESEKIVHLKNLKK KKLMTDRNSEELCELVKVMFEYKALEAA ARV
IYSEDLPVELPRQMFDNEIKSHLKSLPQ MEGIDFNNANVTYLIAEYMKRVLDDDFQ TFYQWNRNYRYMDMLKGEYDRKGSLQHC FTSVEEREGLWKERASRTERYRKQASNK IRSNRQMRNASSEEIETILDKRLSNSRN EYQKSEKVIRRYRVQDALLFLLAKKTLT ELADFDGERFKLKEIMPDAEKGILSEIM PMSFTFEKGGKKYTITSEGMKLKNYGDF FVLASDKRIGNLLELVGSDIVSKEDIME EFNKYDQCRPEISSIVFNLEKWAFDTYP ELSARVDREEKVDFKSILKILLNNKNIN KEQSDILRKIRNAFDANNYPDKGVVEIK ALPEIAMSIKKAFGEYAIMKGSLQLPPL ERLTLGSSYPYDVPDYAYPYDVPDYAYP YDVPDYA
DKNAGYKIGNAKFSHPKGYAWANNPLY TGPVQQDMLGLKETLEKRYFGESADGND NICIQVIHNILDIEKILAEYITNAAYAV NNISGLDKDIIGFGKFSTVYTYDEFKDP EHHRAAFNNNDKLINAIKAQYDEFDNFL DNPRLGYFGQAFFSKEGRNYIINYGNEC YDILALLSGLAHWVVANNEEESRISRTW LYNLDKNLDNEYISTLNYLYDRITNELT NSFSKNSAANVNYIAETLGINPAEFAEQ YFRFSIMKEQKNLGFNITKLREVMLDRK DMSEIRKNHKVFDSIRTKVYTMMDFVIY RYYIEEDAKVAAANKSLPDNEKSLSEKD IFVINLRGSFNDDQKDALYYDEANRIWR KLENIMHNIKEFRGNKTREYKKKDAPRL PRILPAGRDVSAFSKLMYALTMFLDGKE INDLLTTLINKFDNIQSFLKVMPLIGVN AKFVEEYAFFKDSAKIADELRLIKSFAR MGEPIADARRAMYIDAIRILGTNLSYDE LKALADTFSLDENGNKLKKGKHGMRNFI INNVISNKRFHYLIRYGDPAHLHEIAKN EAVVKFVLGRIADIQKKQGQNGKNQIDR YYETCIGKDKGKSVSEKVDALTKIITGM NYDQFDKKRSVIEDTGRENAEREKFKKI ISLYLTVIYHILKNIVNINARYVIGFHC VERDAQLYKEKGYDINLKKLEEKGFSSV TKLCAGIDETAPDKRKDVEKEMAERAKE SIDSLESANPKLYANYIKYSDEKKAEEF TRQINREKAKTALNAYLRNTKWNVIIRE DLLRIDNKTCTLFANKAVALEVARYVHA YINDIAEVNSYFQLYHYIMQRIIMNERY EKSSGKVSEYFDAVNDEKKYNDRLLKLL CVPFGYCIPRFKNLSIEALFDRNEAAKF DKEKKKVSGNSGSGAAARV
As previously described in Example 7.
Application: Can be for any application where it might be suitable to target cytotoxic molecules [e.g., Granzyme B (GZMB)] to the target cell, in this example the targeted cells are cancer-associated fibroblasts or activated fibroblasts, cancer cells and pancreatic cancer patient tumour samples that express the fibroblast activating protein (FAP).
Results
As previously described in Example 3.
As previously described in Examples 3, 4, and Example 6a.
Application: Can be for any application where it might be suitable to target “immune reprogramming” molecules to the target immune cell. In this example the targeted cells are primary macrophages. In this particular example, macrophages treated with EVs loaded with PEV constructs that targeted these EVs specifically to macrophages (via the anti-Marco targeting moiety) and simultaneously deliver the STING pathway activator bacterial enzyme, CdaA. Of note, macrophages, especially tumour-associated macrophages or TAMs which are characterized by expressing high levels of MARCO on their surface, are known in the literature to be reprogrammed (or polarized) into a pro-inflammatory phenotype upon STING activation.
Results
As previously described in Example 2.
Background: The Major Histocompatibility Complex (MHC) is required for T cells to recognize and kill tumor cells. However, most tumors downregulate the expression of the (MHC) to escape immune attack. One existing strategy in the art to circumvent the tumor’s escape mechanism is by way of engineered bi-specific antibodies which draw T-cells and tumor cells to close proximity. These bi-specific antibodies are also referred to as Bi-specific T cell Engagers or BiTEs.
BiTEs are able to mediate the T cell’s capacity to recognize and kill tumor cells in an MHC independent fashion. BiTEs consist of linked variable chain antibody fragments directed against the T cell antigen CD3 and a specific tumor-associated antigen (TAA). Similarly, Bi-specific NK cell engagers or BiKEs can mediate simultaneous binding to an activating receptor on NK cells and a surface tumor antigen to thus promote NK cell-dependent killing of tumor cells. Although existing BiKE and BiTE technologies are promising, many that are currently in clinical development have issues with associated toxicity during systemic administration, drug stability issues (short half-life), and challenges to reaching high enough local concentrations to be effective in most solid cancers
Application: PEV constructs with two targeting moieties: one that recognizes T cell targets, and the other targeting tumor cells (cancer cell or CAFs).
How these platforms work: These PEVs promote the synapsis between T cells and tumor cells, thus promoting the directed killing of tumor cells by these immune cell types.
Advantages: Displaying BiTEs and BiKEs in a PEV format is more stable than the bi-specific antibody constructs.
Special Features: Generally, payload-less - the PEV construct itself is a stable bi-specific cell engager bringing T or NK cells closer to cancer cells. These PEVs can be produced in vivo or ex vivo.
Delivery modalities: Using tumor-selective viruses as delivery vehicles in patients to secrete BiTEs and BiKEs in the infected cancer cell. As such, the PEV is delivered to the exact site where needed, and therefore likely to be effective at picomolar concentrations. i.e., lower dose treatment than the current bi-specific antibody approaches. Viral-based platforms such as: Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, HSV-1, etc. could be used.
Plasmids (e.g. pcDNA 3.1) for preparing the virus and infecting cells, as well as for manufacturing isolated PEVs are also contemplated.
Targeting moieties: Single chain variable fragments or nanobodies as described above. These bind to: tumor cells through surface tumor antigen targets (e.g. anti-CEA, anti-CA9, anti-FAP, etc.) and T cells through molecules that bind to T cells (e.g. CD3 target, via an anti-CD3 scFV targeting moieties.
Payloads: None
Transmembrane Domain: All the examples listed in Table 1 could be used. Examples included here are with tetraspanin proteins, however single pass TM proteins may be used “multimerization technology” (see special features for details).
Results
As previously described in Examples 3 and 4.
Background/Context: Tumor-associated antigens and/or immune reprograming moieties (e.g. STING or ERAdP pathway activators) can be specifically delivered to surface molecules on APCs, such as dendritic cells via PEVs. This construct would express a targeting moiety to target PEVs to DCs (dendritic cells) and it could concomitantly carry one or multiple payloads.
Application: These platforms will represent effective means to elicit robust tumor antigen-specific immunity.
How these platforms work: DCs exhibit a largely immature or tolerizing phenotype. Tumor antigen delivery via PEVs (as payloads or cargo), in conjunction with coadministration of an adjuvant (DC maturation stimuli such as agonistic anti-CD40 mAbs, poly(l:C), cytosine-phosphate-guanine (CpG), lipo-polysaccharide (LPS), or toll-like receptor ⅞ (TLR⅞) agonists) or targeted co-delivery of PEVs containing STING or ERAdP pathway activators as described above (e.g. Bacterial dinucleotide cyclases such as CdaA and MtbDisa which are c-di-AMP cyclases, and VCA0848, which is a c-di-GMP cyclase) results in enhanced tumor-associated antigen presentation capacity and increased expression of T cell costimulatory molecules
Tumor-associated antigens alone or in combination with adjuvants or in combination with immune reprograming moieties (e.g. STING or ERAdP pathway activators) can be specifically delivered to surface molecules on dendritic cells via PEVs.
Targeting moieties: Targets: Antigen presenting cell-surface molecules, including CD40, a TNF-α family receptor, DEC205, a C-type lectin receptor (CLEC9) and CD11c, an integrin receptor, are targeted by targeting moieties including specific monoclonal antibodies, scFvs, single domain antibodies, nanobodies (i.e. anti-DEC205, anti-Clec9A, anti-CD11c), ligands or targeted peptides (e.g. CD40 ligand or CD40-targeted peptide).
Payloads: Specific tumor-associated antigens (For proof-of-concept in mouse tumor models: DCT and OVA are being explored). Human tumor-associated antigens relevant for clinical testing can be used (e.g. HPV-E6 and E7, NY-ESO-1, etc.). Cancer-specific neoantigens can also be used.
Concomitant expression of specific disease cell antigens is contemplated, such as tumor-associated/specific antigens (e.g. OVA, DCT, mERKm9 etc.). Also, adjuvant molecules such as a STING or ERAdP activator could be concomitantly delivered with disease-specific antigens or tumor-associated/specific antigens
Transmembrane domain: All the examples listed in Table 1 could be used.
Delivery/manufacturing modalities: Viral-based platforms such as, Vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, HSV-1 etc. could be used. Plasmids (e.g. pcDNA 3.1) for transfecting cells, as well as manufactured isolated PEVs are also contemplated.
Results
As previously described in Example 3.
The “response receiver-modulated diguanylate cyclase” of Geobacter sulfurreducens [GsPCA] produces cyclic AMP-GMP (3′,3′-cGAMP) in this common soil bacteria. The REC (signal receiving/dimerizing) regulatory domain was deleted and the diguanylate-cyclase (DGC) or GGDEF domain were expressed yielding a unique, constitutively active form. When this active form was expressed in a PEV construct targeted to dendritic cells by the anti-DEC205 scFV and loaded into EVs by the VSVG transmembrane domain, functional activation of the interferon response was observed.
Where features are named herein, it will be understood that corresponding example sequences for the features (or sequences that comprise) may found in Table 13.
It will be understood that functional variants are encompassed in some embodiments. The functional variant may comprise sequences that are at least 80% identical to the example sequences set forth in Table 13, wherein said variants retain substantially the same functional as the parent molecule from which they are derived. The functional variants may be at least 90% identical to the respective parent molecule. The functional variants may be at least 95% identical to the respective parent molecule. The functional variants may be at least 96% identical to the respective parent molecule. The functional variants may be at least 97% identical to the respective parent molecule. The functional variants may be at least 98% identical to the respective parent molecule. The functional variants may be at least 99% identical to the respective parent molecule. Likewise, certain embodiment encompass functional fragments that retain substantially the same functional as the full-length parent molecule from which they are derived.
In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required.
The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole. All references referred to herein are incorporated by reference in their entireties.
Stickney, Z., Losacco, J., McDevitt, S., Zhang, Z., and Lu, B. (2016) Development of exosome surface display technology in living human cells. Biochem Biophys Res Commun. 472(1): 53-9.
Meyer, C., Losacco, J., Stickney, Z., Li, L., Marriott, G., and Lu, B. (2017) Pseudotyping exosomes for enhanced protein delivery in mammalian cells. Int J Nanomedicine. 12:3153-3170.
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U.S. Pat. Application No. 16/900,383.
This application claims the benefit of priority of U.S. Provisional Pat. Application No. 62/941,768 entitled “RECOMBINANT POLYPEPTIDES FOR PROGRAMMING EXTRACELLULAR VESICLES”, which was filed Nov. 28, 2019, and which is hereby incorporated by reference. All publications, patents, and patent applications mentioned in this specification and exhibits are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/051630 | 11/27/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62941768 | Nov 2019 | US |
