Patent Application
20250049914
The present description relates to intracellular delivery of biologically active cargoes. More specifically, the present description relates to compositions comprising a steroid acid-peptide conjugate covalently linked to, and/or admixed with, a cargo for improved intracellular, cytosolic and/or nuclear delivery, as well as to the use of such compositions for example in genome editing and the manufacture of cell-based vaccines. The present description further relates to methods of increasing the stability of cargoes via their covalent conjugation to one or more steroid acid-peptide moieties. The present description refers to a number of documents, the contents of which is herein incorporated by reference in their entirety.
Intracellular delivery of biological cargoes such as peptides, proteins, and polynucleotides generally rely on the endocytic pathway as the major uptake mechanism, resulting in a large fraction of the cargoes being trapped inside endosomes/lysosomes. Such trapped cargoes often remain sequestered from their intended targets and may be degraded. Thus, improved strategies for increasing intracellular delivery and avoiding endosomal entrapment would be highly desirable.
In a first aspect, described herein is a composition comprising a steroid acid-peptide conjugate covalently linked to, and/or admixed with, a cargo for intracellular delivery. In some embodiments, covalently linking the cargo to the steroid acid-peptide conjugate increases intracellular delivery and/or cytosolic/nuclear delivery of the cargo, as compared to a corresponding composition lacking the steroid acid-peptide conjugate. In some embodiments, the cargo is admixed with a sufficient concentration of the steroid acid-peptide conjugate to increase intracellular delivery and/or cytosolic/nuclear delivery of the cargo, as compared to a corresponding composition lacking admixture with the steroid acid-peptide conjugate. In particular embodiments, the steroid acid may be a bile acid and the peptide may comprise a functional nuclear localization signal (NLS) or other subcellular targeting domain.
In a further aspect, described herein is a method for delivering a cargo intracellularly, the method comprising providing a composition as defined herein, and administering the composition to target cells in vitro or in vivo.
In a further aspect, described herein is a method for preparing a cargo for intracellular delivery having increased stability, the method comprising covalently linking the cargo to a sufficient number of steroid acid-peptide moieties to produce a covalently-modified cargo that exhibits greater stability (e.g., thermal stability) than the corresponding unmodified cargo.
In a further aspect, described herein is a composition comprising an antigen covalently linked to and/or admixed with a steroid acid-peptide conjugate in an amount sufficient to improve presentation of the antigen upon administration of the composition to non-professional antigen presenting cells (e.g., mesenchymal stromal cells [MSCs]), as compared to administration of a corresponding composition lacking the steroid acid-peptide conjugate.
In a further aspect, described herein is a cell culture comprising non-antigen presenting cells pulsed with an antigen covalently linked to and/or admixed with a steroid acid-peptide conjugate.
In a further aspect, described herein is a vaccine comprising a composition as described herein, or comprising cells produced using a cell culture as described herein.
In a further aspect, described herein is a method for enhancing presentation of an antigen of interest in a subject or cells, the method comprising administering to the subject or in non-antigen presenting cells a composition as described herein, or cells produced using a cell culture as described herein.
In a further aspect, described herein is a method for vaccinating a subject against an infectious disease, the method comprising administering to the subject a composition as described herein or cells produced using a cell culture as described herein, wherein the antigen comprises an antigenic fragment of a pathogen (e.g., virus, bacteria, fungus) causing the infectious disease.
In a further aspect, described herein is a method for treating cancer in a subject, the method comprising administering to the subject a composition as described herein, or cells produced using a cell culture as described herein, wherein the antigen is an overexpressed or aberrantly expressed in cells causing the cancer.
Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.
Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
This application contains a Sequence Listing in computer readable form Dec. 8, 2022. The computer readable form is incorporated herein by reference.
Described herein are compositions and methods relating to cargoes for improved intracellular delivery. In some aspects, the present invention stems from the demonstration herein that total intracellular delivery, cytosolic delivery, and/or nuclear delivery of cargoes may be enhanced by admixture with, or covalent linkage to, a variety of steroid acid-peptide conjugates. In further aspects, the present invention stems from the demonstration herein that covalent conjugation with steroid acid-peptide moieties may improve cargo stability. In further aspects, the present invention stems from the demonstration herein that steroid acid-peptide conjugates described herein may be used for the generation of cell-based vaccines, including in some non-professional antigen-presenting cells that have been previously shown to be immunosuppressive. In a first aspect, described herein is a composition comprising a steroid acid-peptide conjugate covalently linked to and/or admixed with a cargo for intracellular delivery. In some embodiments, covalently linking the cargo to the steroid acid-peptide conjugate increases intracellular delivery and/or cytosolic/nuclear delivery of the cargo, as compared to a corresponding composition lacking the steroid acid-peptide conjugate. In some embodiments, the cargo is admixed with a sufficient concentration of the steroid acid-peptide conjugate to increase intracellular delivery and/or cytosolic/nuclear delivery of the cargo, as compared to a corresponding composition lacking admixture with the steroid acid-peptide conjugate. In some embodiments, steroid acid-peptide conjugate conjugates described herein may increase presentation of an antigenic polypeptide cargo by target cells. In some embodiments, steroid acid-peptide conjugate conjugates described herein may increase intracellular reactive oxygen species production in target cells. In some embodiments, the target cells comprise or consist of professional anti-presenting cells (e.g., dendritic cells, macrophages, B cells, or non-immune cells engineered for overexpression of an immunoproteasome). In some embodiments, the target cells comprise or consist of non-professional antigen-presenting cells (e.g., wild-type, engineered, primary, and/or cultured non-immune cells, such as mesenchymal stromal cells [MCSs; also known as mesenchymal stem cells]. In some embodiments, steroid acid-peptide conjugate conjugates described herein may transform immunosuppressive cells (e.g., immunosuppressive MSCs) into immunostimulatory and/or proinflammatory MSCs, which may then be used, for example, in cell-based immunostimulatory compositions and/or cell-based vaccines.
In some embodiments, the cargo may be or may comprise a protein, peptide, polynucleotide (e.g., DNA, RNA, shRNA, siRNA, antisense oligonucleotides), polynucleotide analog (having cationic, anionic, or charge-neutral backbones), polysaccharide, drug, or any combination thereof. In some embodiments, the cargo for intracellular delivery is a cargo that does not bind specifically to a cell surface receptor or ligand such that increased intracellular delivery is not predominantly the result of receptor- or ligand-mediated internalization/endocytosis (e.g., as is the case with antibody-drug conjugates). In some embodiments, the cargo is not an antibody (e.g., an antibody that binds to a cell surface epitope). In some embodiments, the cargo may comprise an antibody or fragment thereof that specifically binds to an intracellular target or epitope. In some embodiments, the cargo is not an antigen against which an immune response is to be mounted. In some embodiments, compositions described herein do not comprise an adjuvant and/or are not formulated as immunogenic or vaccine compositions.
In some embodiments, the compositions described herein may be for use in genome editing, base editing, transcription control/regulation, o diagnostic compositions. In some embodiments, cargoes described herein may include RNA- or DNA guided nucleases having or lacking DNA/RNA cleavage activity. In some embodiments, cargoes described herein may comprise a CRISPR-Cas nuclease, such as a class 2 CRISPR-Cas nuclease. In some embodiments, the cargoes described herein may comprise Cas9 or Cas12a.
In some embodiments, the cargo described herein may be covalently linked to a sufficient number of steroid acid-peptide moieties such that the cargo exhibits greater stability (e.g., thermal stability) than the unmodified cargo.
In some embodiments, the steroid acid in the steroid acid-peptide conjugates or moieties described herein may enhance endocytosis and/or endosomal escape when internalized. Without being bound by theory, steroid acids (e.g., bile acids and bile acid analogs) have been shown to be utilized/exploited by viruses to facilitate their infection of host cells, such as by increasing their endocytic uptake and/or endosomal escape to gain access to the cytosol (Shivanna et al., 2014; Shivanna et al., 2015; Murakami et al., 2020). For example, bile acids have been shown to trigger the enzyme acid sphingomyelinase (ASM) to cleave sphingomyelin to ceramide on the inner leaflet of endosomes. Increased amounts of ceramide destabilize membranes and facilitate endosomal escape. In some embodiments, steroid acids described herein may comprise those that trigger ceramide accumulation on the inner leaflet of endosomes, thereby destabilizing endosomal membranes and facilitating endosomal escape of the steroid acid upon intracellular delivery. In some embodiments, steroid acids described herein comprise those that trigger increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.
In some embodiments, the steroid acid described herein may comprise or consist of a bile acid (e.g., a primary bile acid or a secondary bile acid). In some embodiments, the steroid acid may be or comprise: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA), or any analog thereof that: induces endocytosis; triggers ceramide accumulation on the inner leaflet of endosomes; triggers increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide; and/or has a hydrophobicity greater than that of cholic acid.
Hydrophobic bile acids such as GCDCA, TCA, GCA, and CA (but not hydrophilic bile acids such as UDCA) were shown to increase GII.3 human norovirus infection and replication in host intestinal cells by enhancing endosomal uptake and endosomal escape via ASM-mediated ceramide accumulation on the apical membrane (Murakami et al., 2020). In some embodiments, the steroid acid described herein comprises or consists of a bile acid or bile acid analog that is more hydrophobic than cholic acid. In some embodiments, the steroid acid described herein comprises or consists of a bile acid or bile acid analog that is more hydrophobic than cholic acid (e.g., CDCA, DCA, LCA, TCA, TDCA, TCDCA, GCA, GDCA, or GCDCA; Hanafi et al., 2018).
In some embodiments, the cargoes described herein are covalently linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 steroid acid-peptide moieties. Covalent modification of proteinaceous antigens with different steroid acid-NLS peptides has been shown in previous studies to enhance intracellular delivery and antigen presentation in a variety of antigen-presenting cells (U.S. Pat. No. 11,291,717).
In some embodiments, the steroid acid-peptide conjugate is covalently linked to the cargo via a linker (e.g., bifunctional, trifunctional linker, or multi-functional linker). In some embodiments, the linker may be a cleavable or non-cleavable linker.
In some embodiments, the steroid acid may be conjugated to the peptide, for example at or towards a free N-terminal or C-terminal amino group of the peptide or at some other functional group within the peptide.
In some embodiments, the peptide may be a non-immunogenic peptide. In some embodiments, the peptide may be a water-soluble peptide, wherein conjugation of the peptide to the steroid acid increases the water solubility of the steroid acid-peptide moiety as compared to the steroid acid moiety alone. In some embodiments, the peptide may be a cationic peptide. In some embodiments, the peptide may comprise one or more domains that impart an additional functionality to the modified polypeptide antigen. As used herein, a “domain” generally refers to a part of a protein having a particular functionality. Some domains conserve their function when separated from the rest of the protein, and thus can be used in a modular fashion. The modular characteristic of many protein domains can provide flexibility in terms oftheir placement within the peptides described herein. However, some domains may perform better when engineered at certain positions of the peptide (e.g., at the N- or C-terminal region, or therebetween). The position of the domain within its endogenous protein may be an indicator of where the domain should be engineered within the peptide.
In some embodiments where non-specific delivery may be desired, the peptide may comprise a protein transduction domain (PTD) that stimulates endocytosis, endosomal formation, or intracellular delivery in a non-cell-specific manner. In some embodiments, the peptide may comprise a subcellular targeting signal promoting targeting of the modified polypeptide antigen to a specific subcellular compartment. In some embodiments, the peptide may comprise a nuclear localization signal (NLS) that targets the modified polypeptide antigen to the nucleus.
In some embodiments, the nuclear localization signals described herein may comprise or be derived from the NLS from SV-40 large T-antigen (e.g., PKKKRKV; SEQ ID NO: 1 or 2) or from other classical NLSs. In some embodiments, the nuclear localization signals described herein may comprise or be derived from non-classical NLS (e.g., acidic M9 domain in the hnRNP A1 protein; the sequence KIPIK in yeast transcription repressor Matα2; PY-NLS; ribosomal NLS; or the complex signals of U snRNPs). In some embodiments, the nuclear localization signal described herein comprises or consists essentially of the amino acid sequence of any one of SEQ ID NOs: 1 to 15, or any portion thereof. In some embodiments, the nuclear localization signal described herein comprises or consists essentially of a nuclear localisation signal which is SV40 NLS (e.g., comprised in SEQ ID NO: 1 or 2), GWG-SV40 NLS (e.g., comprised in SEQ ID NO: 3), hnRNPA1 M9 NLS (e.g., comprised in SEQ ID NO: 4), hnRNP D NLS (e.g., comprised in SEQ ID NO: 5), hnRNP M NLS (e.g., comprised in SEQ ID NO: 6), PQBP-1 NLS (e.g., comprised in SEQ ID NO: 7), NLS2-RG Domain RPS17 (e.g., comprised in SEQ ID NO: 8), NLS1 RPS17 (e.g., comprised in SEQ ID NO: 9), NLS2 RPS17 (e.g., comprised in SEQ ID NO: 10), NLS3 RPS17 (e.g., comprised in SEQ ID NO: 11), cMyc NLS (e.g., comprised in SEQ ID NO: 12), HuR NLS (e.g., comprised in SEQ ID NO: 13), Tus NLS (e.g., comprised in SEQ ID NO: 14), or Nucleoplasmin NLS (e.g., comprised in SEQ ID NO: 15). In some instances, the SEQ ID NOs referred to above comprise an N-terminal cysteine residue that was or that may be used to facilitate conjugation to the polypeptide antigen (e.g., the thiol group of the N-terminal cysteine residue). Thus, in some embodiments, the NLS sequences referred to herein may exclude the N-terminal cysteine residue comprised in any one of SEQ ID NOs: 1 to 15. In some embodiments, other functional groups added or inserted (e.g., towards the N or C terminal portions of the peptides described herein) to facilitate steroid acid-peptide conjugation to a given polypeptide antigen are also envisaged (e.g., carboxyl groups, synthetic amino acids, etc.). For example, the peptide may include a C-term amide and/or an N-term cysteine.
In some embodiments, the peptide describe herein may comprise one or more cysteine residues (e.g., at or towards the peptide's N- and/or C terminus) having a free thiol group (—SH) or a thiol group that is protected in a cleavable manner (e.g., by a pharmaceutically acceptable protecting group). Such modifications may be introduced, for example, during chemical synthesis of the peptides or via chemical modification with one or more functional or protecting groups following peptide synthesis. In some embodiments, free thiol groups facilitate further conjugations and/or reactivities in reducing environments, such as the peri-cellular and/or intracellular environments (e.g., of cancer cells). In some embodiments, the free thiol group may be conjugated or protected by conjugation to a different peptide or to the same peptide (e.g., via a disulphide bridge between two cysteine-comprising peptides). In some embodiments, the steroid acid-peptide conjugates described herein may be comprised in oligomer form (e.g., dimer, trimer, tetramer, pentamer, etc.). In some embodiments, the steroid acid-peptide conjugates described herein may be comprised in oligomer form via cleavable linkages (e.g., disulphide or other linkages cleavable in peri-cellular and/or intracellular environments). In some embodiments, the cleavable linkages maybe motifs recognizable by intracellular proteases.
In some embodiments, peptides described herein do not comprise an endosomal escape motif, or protein transduction, or cell penetrating motif.
In some embodiments, the nuclear localization signals described herein may comprise the general consensus sequence: (i) K(K/R)X(K/R); (ii) (K/R)(K/R)X10-12(K/R)3/5, wherein (K/R)3/5 represents three lysine or arginine residues out of five consecutive amino acids; (iii) KRX10-12KRRK; (iv) KRX10-12K(K/R)(K/R); or (v) KRX10-12K(K/R)X(K/R), wherein X is any amino acid (Sun et al., 2016).
In some embodiments, the peptide does not include an endosomal escape motif (e.g. -GFFG, -GWG, -GFWG, -GFWFG, -GWWG, -GWGGWG, and -GWWWG), or protein transduction, or cell penetrating motif (such as a cell penetrating peptide).
In some embodiments, the steroid acid described herein is not or does not comprise cholic acid; the NLS peptide is not or does not comprise an SV40 NLS; and/or the steroid acid-peptide conjugate is not or does not comprise CA-SV40.
In some embodiments, the composition may further comprise any pharmaceutically or physiologically acceptable carrier and/or excipient. In some embodiments, the compositions described herein may be formulated within a hydrogel, liposome, lipid-based transfection agent, or nanoparticle (e.g., lipid nanoparticle).
In some embodiments, the compositions, methods and uses described herein may be formulated or adapted for any route of administration, such as but not limited to oral, intravenous, intranasal, intramuscular, subcutaneous, intradermal, intratumoral, intracranial, topical, and intrarectal administration.
In some embodiments, the compositions described herein may be for use in increasing the intracellular, cytosolic, and/or nuclear delivery of a biologically active cargo (e.g., therapeutic cargo or diagnostic cargo) in vitro or in vivo.
In a further aspect, described herein is a method for delivering a cargo intracellularly, the method comprising providing a composition as defined herein, and administering the composition to target cells in vitro or in vivo.
In a further aspect, described herein is a method for preparing a cargo for intracellular delivery having increased the stability, the method comprising covalently linking the cargo to a sufficient number of steroid acid-peptide moieties to produce a covalently-modified cargo that exhibits greater stability (e.g., thermal stability) than the corresponding unmodified cargo. In some embodiments, cargo may be reacted with between a 2-fold and 100-fold molar excess of the steroid acid-peptide conjugate; between a 2-fold and 50-fold molar excess of the steroid acid-peptide conjugate; or between a 5-fold and 25-fold molar excess of the steroid acid-peptide conjugate. In some embodiments, the mean number of steroid acid-peptide moieties conjugated per cargo is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; or is between about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and n, wherein n is the total number of accessible sites on the cargo available for conjugation.
In another aspect, described herein is a composition comprising non-antigen presenting cells or non-professional antigen presenting cells and an antigen covalently linked to or admixed with an enhancer of antigen-presentation (e.g., the steroid acid-peptide conjugate defined herein). As used herein, the term “admixture” or “admixing” refers to the combination of two separate components into a single composition, wherein the components are not covalently conjugated or otherwise reacted together. In some embodiments, the enhancer may comprise a steroid acid or steroid acid-peptide conjugate in an amount sufficient to improve presentation (e.g., cross presentation or classical antigen presentation) of the antigen upon administration of the composition to non-antigen-presenting cells (e.g., in vitro, ex vivo, or in vivo), as compared to administration of a corresponding composition lacking the enhancer. In some embodiments, the enhancer may comprise a steroid acid-peptide conjugate in an amount sufficient to improve presentation (e.g., cross presentation or classical antigen presentation) of the antigen upon administration of the composition to antigen-presenting cells (e.g., in vitro, ex vivo, or in vivo), as compared to administration of a corresponding composition lacking the enhancer.
As used herein, the term “non-antigen presenting cells (APCs)” or “non-professional antigen presenting cells” refer to cells that do not efficiently present antigen or possess the ability/machinery to efficiently present antigen when unstimulated, untreated, or unmodified (e.g., genetically). For example, untreated wild type mesenchymal stem cells do not efficiently present antigen to T cells, whereas they can be genetically engineered to possess specific machinery (e.g., proteasomes or immunoproteosomes) to enhance their antigen presentation capabilities. Professional APCs generally refer to dendritic cells (DCs), macrophages, and B cells, which express high levels of MHC-II and express sufficient levels of the proteins/machinery involved in efficient antigen presentation.
As used herein, the term “antigen presentation” may refer to the classical antigen presentation pathways of extracellular (via MHC class II) and/or intracellular antigens (via MHC class I), as well as the cross presentation pathway (presentation of extracellular antigen via MHC class I).
Polypeptide antigens are normally captured by antigen-presenting cells (e.g., dendritic cells) but are initially entrapped in endosomes. Endosomal maturation towards lysosomes results in a decrease in pH and an activation of proteolytic enzymes that mediate non-specific antigen degradation. As a result, some of the antigen fragments generated may then pass through endosomal pores to reach the cytosol where further antigen degradation takes place by the proteasomal machinery prior to MHC class I presentation. Although this process occurs naturally, the generated antigen fragments that ultimately leave the endosomes may be small and/or damaged, rendering them unsuitable for proteasomal degradation, thereby precluding their MHC class I presentation and thus cellular immunity based thereon. Without being bound by theory, admixture of antigens with immunogen enhancers described herein may facilitate internalization/endosomal escape of the antigens, allowing them (or larger antigen fragments) to reach the cytosol in a more native conformation and/or in greater quantities. As a result, proteasomal degradation of these more native antigens may result in a higher amount and/or variety of immunogenic and/or stable peptides presented via MHC class I at the surface of antigen-presenting cells, thereby eliciting potent T-cell activation.
As used herein, “polypeptide antigen” refers to an immunogenic peptide-linked chain of amino acids of any length, but generally at least 8, 9, 10, 11, or 12 amino acids long. For greater clarity, polypeptide antigens referred to herein exclude antigen-binding antibodies or fragments thereof. As used herein, a “protein antigen” refers to a polypeptide antigen having a length of at least 50 amino acid residues, while a “peptide antigen” refers to a polypeptide antigen having a length of less than 50 amino acid residues. For greater clarity, polypeptides, proteins, and peptides described herein may or may not comprise any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc.) or incorporate one or more synthetic or non-natural amino acids, to the extent that the modification or synthetic or non-natural amino acids does not destroy the antigenicity of the polypeptide antigen or the desired functionality of the peptide (or domain comprised therein).
1. A composition comprising a steroid acid-peptide conjugate covalently linked to and/or admixed with a cargo for intracellular delivery.
2. The composition of item 1, wherein the cargo is or comprises a protein, peptide, polynucleotide, polynucleotide analog, polysaccharide, drug, or any combination thereof.
3. The composition of item 1 or 2, wherein: (a) the cargo does not bind specifically to a cell surface receptor or ligand; (b) the cargo is not an antibody (e.g., an antibody that binds to a cell surface epitope); (c) the cargo is not or does not comprise an antigen; or (d) any combination of (a) to (c).
4. The composition of any one of items 1 to 3, wherein the cargo is or comprises a nuclease, such as a CRISPR-Cas nuclease (e.g., a class 2 CRISPR-Cas nuclease, such as Cas9 or Cas12a).
5. The composition of any one of items 1 to 4, wherein: (a) covalently linking the cargo to the steroid acid-peptide conjugate increases intracellular delivery and/or cytosolic/nuclear delivery of the cargo, as compared to a corresponding composition lacking the steroid acid-peptide conjugate; or (b) the cargo is admixed with a sufficient concentration of the steroid acid-peptide conjugate to increase intracellular delivery and/or cytosolic/nuclear delivery of the cargo, as compared to a corresponding composition lacking admixture with the steroid acid-peptide conjugate.
6. The composition of any one of items 1 to 5, wherein the cargo is covalently linked to a sufficient number of steroid acid-peptide moieties such that the cargo exhibits greater stability (e.g., thermal stability) than the unmodified cargo.
7. The composition of any one of items 1 to 6, wherein the steroid acid is or comprises a bile acid (e.g., a primary bile acid or a secondary bile acid).
8. The composition of any one of items 1 to 7, wherein the steroid acid is or comprises: (a) a bile acid which is: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glycoursodeoxycholic acid (GUDCA); (b) an analog of the bile acid of (a) that: induces endocytosis; triggers ceramide accumulation on the inner leaflet of endosomes; triggers increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide; and/or has a hydrophobicity greater than that of cholic acid; (c) a bile acid or bile acid analog that is more hydrophobic than cholic acid (e.g. CDCA, DCA, LCA, TCA, TDCA, TCDCA, GCA, GDCA, or GCDCA); or (d) any combination of (a) to (c).
9. The composition of any one of items 1 to 8, wherein each cargo molecule is covalently linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 steroid acid-peptide moieties.
10. The composition of any one of items 1 to 9, wherein the steroid acid-peptide conjugate is covalently linked to the cargo via a cleavable or non-cleavable linker (e.g., bifunctional, trifunctional linker, or multi-functional linker).
11. The composition of any one of items 1 to 10, wherein the steroid acid-peptide conjugate is covalently linked to the cargo via said peptide (e.g., the steroid acid is conjugated at or towards the N- or C-terminus of the peptide).
12. The composition of any one of items 1 to 11, wherein the peptide: (i) comprises a protein transduction domain that stimulates endocytosis and/or endosomal formation; (ii) comprises a subcellular targeting signal; (iii) is a cationic peptide (e.g., a non-cell-penetrating cationic peptide); (iv) is a non-immunogenic peptide; (v) comprises at least one cysteine residue (e.g., at or towards the peptide's N- and/or C terminus) having a free thiol group or a thiol group that is protected in a cleavable manner (e.g., by a pharmaceutically acceptable protecting group); or (vi) any combination of (i) to (v).
13. The composition of any one of items 1 to 12, wherein: the steroid acid is not or does not comprise cholic acid; the NLS peptide is not or does not comprise an SV40 NLS; and/or the steroid acid-peptide conjugate is not or does not comprise CA-SV40.
14. The composition of any one of items 1 to 13, wherein the peptide is or comprises a nuclear localization signal which is a classical NLS (e.g., NLS from SV-40 large T-antigen (e.g., PKKKRKV; SEQ ID NO: 1 or 2) or from other classical NLSs) or a non-classical NLS (e.g., acidic M9 domain in the hnRNP A1 protein; the sequence KIPIK in yeast transcription repressor Matα2; PY-NLS; ribosomal NLS; and the complex signals of U snRNPs).
15. The composition of any one of item 1 to 14, wherein the peptide is or comprises a nuclear localization signal which is a/an: SV40 NLS (e.g., comprised in SEQ ID NO: 1 or 2), GWG-SV40NLS (e.g., comprised in SEQ ID NO: 3), hnRNPA1 M9 NLS (e.g., comprised in SEQ ID NO: 4), hnRNP D NLS (e.g., comprised in SEQ ID NO: 5), hnRNP M NLS (e.g., comprised in SEQ ID NO: 6), PQBP-1 NLS (e.g., comprised in SEQ ID NO: 7), NLS2-RG Domain RPS17 (e.g., comprised in SEQ ID NO: 8), NLS1 RPS17 (e.g., comprised in SEQ ID NO: 9), NLS2 RPS17 (e.g., comprised in SEQ ID NO: 10), NLS3 RPS17 (e.g., comprised in SEQ ID NO: 11), cMyc NLS (e.g., comprised in SEQ ID NO: 12), HuR NLS (e.g., comprised in SEQ ID NO: 13), Tus NLS (e.g., comprised in SEQ ID NO: 14), or Nucleoplasmin NLS (e.g., comprised in SEQ ID NO: 15), or is a variant of an NLS having nuclear localization activity, the NLS comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 15.
16. The composition of any one of items 1 to 15, wherein the steroid acid comprises CA or DCA, and the peptide comprises an hnRNPA1 M9 NLS or a variant thereof having nuclear localization activity.
17. The composition of any one of item 1 to 16, wherein the peptide does not comprise an endosomal escape motif, or protein transduction motif, or cell penetrating motif.
18. The composition of any one of items 1 to 17, wherein the composition or conjugate is formulated within a hydrogel, liposome, lipid-based transfection agent, or nanoparticle (e.g., lipid nanoparticle).
19. The composition of any one of items 1 to 18, further comprising pharmaceutically or physiologically acceptable carrier and/or excipient.
20. The composition of any one of items 1 to 19, for use in: (a) increasing the intracellular, cytosolic, and/or nuclear delivery of a biologically active cargo (e.g., therapeutic cargo or diagnostic cargo) in vitro or in vivo, as compared to a corresponding composition lacking the steroid acid-peptide conjugate; (b) increasing presentation of an antigenic polypeptide cargo by target cells, such as by professional anti-presenting cells (e.g., dendritic cells, macrophages, B cells, or non-immune cells engineered for overexpression of an immunoproteasome), or by non-professional antigen-presenting cells (e.g., wild-type, engineered, primary, and/or cultured non-immune cells, such as mesenchymal stromal cells (MCSs)); (c) increasing intracellular reactive oxygen species production in target cells, such as by professional anti-presenting cells (e.g., dendritic cells, macrophages, B cells, or non-immune cells engineered for overexpression of an immunoproteasome), or by non-professional antigen-presenting cells (e.g., wild-type, engineered, primary, and/or cultured non-immune cells, such as MCSs); (d) transforming immunosuppressive cells (e.g., immunosuppressive MSCs) into immunostimulatory and/or proinflammatory MSCs; or (e) any combination of (a) to (d).
21. The composition for use of item 20, wherein the composition is adapted or formulated for oral, intravenous, intranasal, intramuscular, subcutaneous, intradermal, intratumoral, intracranial, topical, intrarectal administration, or any other route of administration.
22. A method for delivering a cargo intracellularly, the method comprising providing a composition as defined in any one of items 1 to 21, and administering the composition to target cells in vitro or in vivo.
23. A method for preparing a cargo for intracellular delivery having increased stability, the method comprising covalently linking the cargo to a sufficient number of steroid acid-peptide moieties to produce a covalently-modified cargo that exhibits greater stability (e.g., thermal stability) than the corresponding unmodified cargo.
24. The method of item 23, wherein the cargo and/or the steroid acid-peptide is as defined in any one of items 1 to 17.
25. The method of item 23 or 24, wherein the cargo is reacted or admixed with between a 2-fold and 1000-fold, 2-fold and 500-fold, 2-fold and 200-fold, 2-fold and 100-fold molar excess of the steroid acid-peptide conjugate; between a 2-fold and 50-fold molar excess of the steroid acid-peptide conjugate; or between a 5-fold and 25-fold molar excess of the steroid acid-peptide conjugate.
26. The method of any one of items 23 to 25, wherein the mean number of steroid acid-peptide moieties conjugated per cargo, or the molar ratio of cargo: steroid acid-peptide conjugate admixed, is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; or wherein the mean number of steroid acid-peptide moieties conjugated per cargo is between about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and n, wherein n is the total number of accessible sites on the cargo available for conjugation.
27. A composition comprising an antigen covalently linked to and/or admixed with a steroid acid-peptide conjugate in an amount sufficient to improve presentation of the antigen upon administration of the composition to non-antigen presenting cells (e.g., mesenchymal stromal cells [MSCs]), as compared to administration of a corresponding composition lacking the steroid acid-peptide conjugate.
28. The composition of item 27, wherein the steroid acid or peptide is as defined in any one of items 7, 8, or 10 to 17.
29. The composition of item 27 or 28, wherein the molar ratio of steroid acid-peptide conjugate to antigen in the composition is at least 0.01:1, 0.05:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1; is no more than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 50:1, 100:1, 250:1, 500:1, 1000:1, and/or is between 1:1 to 1000:1; 1:1 to 500:1, 1:1 to 250:1, 1:1 to 200:1.
30. The composition of any one of items 27 to 29, wherein the steroid acid is conjugated to the peptide: (a) at a molar ratio of steroid acid: peptide of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or between 1:1 to 10:1; (b) at a free amino group and/or a free thiol group (e.g., of a lysine or cysteine) of the peptide; (c) at or towards the N-terminal end of the peptide (e.g., at the free amino group of N terminal residue and/or at the thiol group of an N-terminal cysteine residue); or (d) any combination of (a) to (c).
31. The composition of any one of items 27 to 30, wherein the antigen is a polypeptide antigen comprising one or more MHC class I epitopes and/or MHC class II epitopes.
32. The composition of any one of items 27 to 31, wherein the antigen is or comprises: (a) a tumor-associated antigen (TAA), tumor-specific antigen (TSA), tumor lysate, a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or disorder amenable to treatment by vaccination and/or immunotherapy; or any antigenic fragment thereof; or (b) a corona viral antigen (e.g., SARS-CoV-2 Spike protein, SARS-CoV Spike protein, or an antigenic fragment thereof; or a cancer antigen, such as a single-nucleotide variant antigen, a mutational frameshift antigen, splice variant antigen, a gene fusion antigen, an endogenous retroelement antigen, or another class of antigen, such as a human leukocyte antigen (HLA)-somatic mutation-derived antigen or a post-translational TSA, a viral-derived cancer antigen (e.g., from human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV)), a cancer-testis antigen, HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), EGFR, cell lysates or other material derived from a tumor (e.g., tumor-derived exosomes).
33. The composition of any one of items 27 to 32, further comprising a pharmaceutically acceptable excipient and/or adjuvant.
34. A cell culture comprising non-antigen presenting cells (e.g., mesenchymal stromal cells [MSCs]) and the composition as defined in any one of items 27 to 33.
35. A cell culture comprising non-antigen presenting cells (e.g., mesenchymal stromal cells [MSCs]) pulsed with an antigen covalently linked to and/or admixed with a steroid acid-peptide conjugate.
36. A vaccine comprising the composition as defined in any one of items 27 to 32, or comprising cells produced using the cell culture as defined in item 34 or 35.
37. The vaccine of item 36, which is a therapeutic or prophylactic vaccine (e.g., anti-cancer vaccine, anti-viral vaccine, or anti-bacterial vaccine).
38. A method for enhancing presentation of an antigen of interest in a subject or cells, the method comprising administering to the subject or in non-antigen presenting cells (e.g., mesenchymal stromal cells [MSCs]) the composition as defined in any one of items 27 to 33, or cells produced using the cell culture as defined in item 34 or 35.
39. A method for vaccinating a subject against an infectious disease, the method comprising administering to the subject the composition as defined in any one of items 27 to 33 or cells produced using the cell culture as defined in item 34 or 35, wherein the antigen comprises an antigenic fragment of a pathogen (e.g., virus, bacteria, fungus) causing the infectious disease.
40. A method for treating cancer in a subject, the method comprising administering to the subject the composition as defined in any one of items 27 to 33 or cells produced using the cell culture as defined in item 34 or 35, wherein the antigen is an overexpressed or aberrantly expressed in cells causing the cancer.
41. The composition as defined in any one of items 27 to 33, or the cell culture as defined in item 34 or 35, for use in: (i) generating enhancing presentation of an antigen of interest in a subject or in non-professional antigen presenting cells (e.g., mesenchymal stromal cells [MSCs]); (ii) the manufacture of a medicament (e.g., vaccine) for generating an immune response in a subject; (iii) increasing presentation of an antigenic polypeptide cargo by non-professional antigen-presenting cells (e.g., wild-type, engineered, primary, and/or cultured non-immune cells, such as mesenchymal stromal cells [MCSs]); (iv) increasing intracellular reactive oxygen species production in by non-professional antigen-presenting cells (e.g., wild-type, engineered, primary, and/or cultured non-immune cells, such as MCSs); (v) transforming immunosuppressive cells (e.g., immunosuppressive MSCs) into immunostimulatory and/or proinflammatory MSCs; or (vi) any combination of (i) to (v).
All cell culture media and reagents were purchased from Wisent Bioproducts (St-Bruno, QC, Canada) unless otherwise indicated. All flow cytometry antibodies were purchased from BD Biosciences (San Jose, CA, USA) unless otherwise indicated. The albumin from chicken egg white (ovalbumin; OVA) and LPS was purchased from Sigma-Aldrich (St-Louis, MI, USA). DQ™ OVA was purchased from ThermoFisher (Waltham, MA, USA). Recombinant GM-CSF was purchased from Peprotech (Rocky Hill, NJ, USA).
Sterilized microscopy coverslips were placed in 24-well cell culture plates and seeded overnight with 25,000 cells per well. The following day, cells were treated with a cargo solution (e.g., containing unconjugated cargo; cargo conjugated to one or more steroid acid-peptide moieties; or unconjugated cargo mixed with steroid acid-peptide moieties) in a final volume of 250 μL per well for a specified incubation time. Following incubation, cells were washed three times with PBS and then fixed for 30 minutes in a 1% paraformaldehyde/sucrose solution on ice. The fixed cells were permeabilized with 0.05% Triton X-100/PBS, washed three times with PBS, and then blocked with a 10% normal goat serum/PBS solution for 1 h in a humidified chamber. For Cas9 cargoes, cells were then treated with an anti-Cas9-AF488 antibody and incubated at room temperature in the dark for 1 h, washed three times in PBS, and then incubated with Hoescht nuclear stain diluted in PBS for 15 minutes. After a final washing step, the cells were mounted on microscopy slides with a drop of SlowFade™ reagent and sealed.
Mouse bone marrow derived DCs (BMDCs) were generated by flushing the whole marrow from mouse femurs using RPMI™ 1640 supplemented with 10% fetal bovine serum (FBS), 50 U/mL Penicillin-Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1% MEM Non-essential Amino Acids, 1 mM Sodium Pyruvate, 0.5 mM beta-mercaptoethanol. Following red blood cell lysis, cells were then cultured in media supplemented with 50 ng/mL murine recombinant GM-CSF. The media was changed on days 2, 4, 6 and 8. On day 9, the media was replaced to include recombinant murine GM-CSF and LPS from Escherichia coli 0111 (1 ng/mL) to stimulate DC maturation. Mature DCs were assessed by flow cytometry for their surface expression of CD3, CD19, NK1.1, CD11c, CD80, CD86, and I-Ab.
To assess the expression of cell surface markers, BMDCs were incubated with various antibodies diluted according to manufacturer's instructions using the staining buffer (PBS containing 2% FBS) for 30 min at 4° C. in the dark. After extensive washing using the staining buffer, the cells were re-suspended in 400 μL of staining buffer. The samples were acquired by BD FACSDiva™ on CANTOII™, then analyzed using FlowJo™ v10.
Steroid acid-peptide conjugates (e.g., CA-SV40 NLS) were synthesized as previously described in Beaudoin et al., 2016, in U.S. Pat. No. 11,291,717, and in PCT application publication number WO/2022/232945, unless otherwise indicated. For example, for CA-SV40 NLS, cholic acid was conjugated to the free amino group of the N-terminal cysteine residue of a 13-mer peptide (CGYGPKKKRKVGG; SEQ ID NO: 1) that comprises a nuclear localization signal from SV40 large T-antigen (SEQ ID NO: 2) flanked by linker amino acids. For cargo conjugations, cargoes were solubilized at 1-10 mg/mL in sterile PBS with or without other formulation components, but free of amine (NH3) or sulfhydryl (SH) groups. The SM(PEG)4 cross-linker was added to the reaction for 1 h using different molar excess ratios (10× for Cas9-NLS or Cas9-GFP cargoes; 50× for OVA cargoes). The free SM(PEG)4 cross-linker was discarded by Centricon™ filtration and Sephadex™ column. Steroid acid-peptide conjugates were added in the same molar excess ratios and incubated for 1 h to obtain different amounts of steroid acid-peptide moieties per cargo. Free unconjugated steroid acid-peptide conjugates were removed by Centricon™ filtration and Sephadex™ column. Steroid acid-peptide-cargo conjugates were concentrated in sterile PBS to obtain final concentration 5-10 mg/mL as determined by UV absorbance.
To evaluate steroid acid-peptide-cargo loading, 10 μg of unconjugated or conjugated cargoes were separated under reducing conditions on a 12% polyacrylamide gel and stained with Coomassie brilliant blue R-250™ (Bio-Rad, Mississauga, ON, Canada). The migration distance in the gel relative to the blue dye front (Rf) was measured and the numbers of steroid acid-peptide moieties conjugated per cargo were estimated by reference to a logarithm plot of molecular weight versus 1/Rf for Kaleidoscope pre-stained standards (Bio-Rad) electrophoresed under identical conditions. In addition, Western blot analysis against the cargoes were performed to confirm the Coomassie results.
For this assay, 15×103 DC2.4 cells were seeded on a sterile cover slide in a 24-well plate. Two days following transfection of DC2.4 cells with the eGFP-hGal3 mammalian expression vector, 0.1 mg/mL of cargo was added to cells then incubated for 3 h at 37° C. The cells were then washed twice to remove excess protein prior to being mounted on a slide. The slides were viewed by fluorescent microscopy (Nikon, Eclipse™ Ti2-U) and the results analyzed using the ImageJ™ software.
To evaluate OVA degradation/processing, cells were incubated with 10 g/mL DQ™ OVA (with or without steroid acid-peptide modification) at 37° C. 30 minutes later, cells were washed, and regular media was added. At the end of the indicated incubation time, cells were collected and washed with cold PBS containing 2% FBS. Fluorescence was monitored by analyzing the cells by flow cytometry.
An Applied Photophysics (Leatherhead, Surrey, UK) Chirascan™ Q100 circular dichroism (CD) spectrometer was used for intrinsic tryptophan fluorescence (ITF) analysis and a VWR digital heatblock (Radnor, PA) was used for dry block temperature incubations. The Chirascan™ Q100 autosampler rack cooling system was used for all 4° C. incubations. Data was analyzed using MATLAB™ software (Natick, MA). Briefly, samples were removed from storage at −20° C. and allowed to equilibrate to room temperature. Samples were then diluted to 0.8 mg/mL in PBS from stock concentrations in the range of 4 to 5 mg/mL. Diluted samples were then analyzed for ITF without exposure to thermal stress (Native) or after ten minutes of thermal stress by dry block incubation. An aliquot of each diluted sample was incubated at 4° C., a second aliquot was incubated at 37° C., while a third aliquot was incubated at 80° C. BSA, diluted to 0.8 mg/mL, was included with the samples under each of the thermal conditions described above. All samples were re-equilibrated to room temperature after incubation. ITF Analysis was performed in 8 triplicates by excitation at 280 nm with an emission scan range of 200-600 nm with a bandwidth of 1.0 nm, a Time-per point of 1 s, and a Step of 0.5. The triplicate spectra were blank subtracted, averaged, and converted from units of mdeg to relative fluorescence intensity using MATLAB software. Diluted BSA solutions were assayed as controls preceding and following the sample sequence.
To evaluate antigen cross-presentation, cells were seeded at 25×103 cells per well in 24-well plates (Coming; Massachusetts, United States), then pulsed with antigens or antigen-containing mixtures at different concentrations for 3 h. At the end of the pulsing period, the cells were washed to remove excess antigen and co-cultured with 106/mL CD8 T-cells purified from the spleens of OT-I mice using T-cell isolation kits according to the manufacturer's protocol. After 72 hours, supernatants were collected and used to quantify cytokine production by commercial enzyme-linked immunosorbent assays (ELISAs).
Various bile acid-NLS conjugates were screened using the B3Z reporter system. The B3Z cell line is a T-cell hybridoma specific for the H2-Kb-SIINFEKL complex. Once activated via its TCR, the LacZ reporter gene (under the NFAT promoter control) is expressed. Briefly, 1.5×105 BMDCs or 2.5×105 MSCs were co-cultured with 5×104 B3Z cells treated with the mixing conditions of ovalbumin (OVA) and bile acid-NLS conjugates for overnight at 37° C. with 5% CO2. The following day, all cells were washed twice with PBS (pH 7.4), and the cell pellets were lysed by adding 100 μL of a lysis buffer containing 0.15 mM chlorophenol red-beta-D-galactopyranoside (CPRG) substrate (Calbiochem, La Jolla, CA), 0.125% NP40 (EMD Sciences, La Jolla, CA), 9 mM MgCl2 (Aldrich, USA) and 100 mM 2-mercaptoethanol in PBS. After a 5- or 24-h incubation at 37° C., absorbance was taken at 570 nm with 636 nm as the reference wavelength. For these experiments, OVA was re-suspended in PBS (pH 7.3) at 5-10 mg/mL. The different bile acid-NLS conjugates were re-suspended in H2O at 10 mg/mL. Bile acid-NLS conjugate: antigen mixtures were prepared at different molar ratios according to Table 1.
All female Balb/c and C57BL/6 (6-8 weeks old) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in a pathogen-free environment at the animal facility located at the Institute for Research in Immunology and Cancer (IRIC). All experimental procedures and protocols were approved by the Animal Ethics Committee (CDEA) of Universite de Montreal.
The flow-cytometry antibodies (CD44, CD45, CD73, CD90, H2-Kb, and I-Ab) were purchased from BD Biosciences (San Jose, CA, USA). OVA-AF647 and OVA-DQ® were purchased from Life Technologies (Waltham, MA USA) and used according to manufacturer's instructions. The annexin-V staining kit was purchased from Cedarlane (Burlington, ON, CANADA). Recombinant Cytochorme C was purchased from Sigma Aldrich (Oakville, ON, CANADA).
The EG.7 cell line used in this study was obtained from ATCC. The B3Z cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum (FBS). EG.7 cells were cultured RPMI 16400 supplemented with 2 g/L Glucose, 10% FBS, 50 U/mL Penicillin-Streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM Sodium Pyruvate, and 0.5 mM 0-Mercaptoethanol, and kept under selection using 80 mg/mL of G418. All cells were maintained at 37° C. in a 5% CO2 incubator. All cell culture media and reagents were purchased from Wisent Bioproducts (St-Bruno, QC, Canada).
In order to generate Bone marrow (BM)-derived mouse MSCs, the femurs of 6-8-week-old female Balb/c or C57BL/6 mice were isolated and flushed with Alpha Modification of Eagle's Medium (AMEM) supplemented with 10% FBS and 50 U/mL Penicillin-Streptomycin in a 10 cm cell culture dish, then incubated at 37° C. Two days later, non-adherent cells were removed and the media replaced every 3 to 4 days until plastic-adherent cells reached 80% confluency. The generated cells were detached using 0.05% trypsin and expanded until a uniform MSC population was obtained. The generated MSCs were validated for their innate phenotype by flow-cytometry for the expression of CD44, CD45, CD73, and CD90. The cells were frozen in liquid nitrogen until use.
To assess cross-presentation assay, 25×103 cells MSCs were seeded per well in 24-well plate then pulsed with steroid acid peptide-conjugated for 6 h admixed with OVA. At the end of the pulsing period, the cells were washed to remove excess antigen then 5×104 B3Z cells. The cells were incubated for 17-19 hours prior to their lysis and incubation for another 4-6 hours at 37° C. with a CPRG solution. The optical density signal was detected at wavelength 570 using a SynergyH1™ microplate reader (Biotek, Winooski, VT, United States).
To evaluate OVA uptake, MSCs were first treated with 1 g/mL of OVA-AF647 admixed with CA-hnRNPA1 for 1 hour at 37° C. then assessed for their fluorescence intensity by flow-cytometry. To evaluate antigen processing, MSCs were incubated with 10 g/mL OVA-DQ® admixed with A1 at 37° C. Half an hour later, cells were washed, and regular media added. At the end of the indicated incubation time, cells were collected and washed with cold PBS containing 2% FBS. Fluorescence was monitored by flow cytometry.
Endosomal leakage was assessed using an apoptosis assay. Briefly, 10′ MSCs were first supplemented with 10 mg/mL of exogenous rCyt-C for 6 h at 37° C. in the presence or absence of CA-hnRNPA1 (50 μM). Once the incubation period completed, the cells were collected using Accutase®, washed with ice cold PBS, then stained for Annexin-V according to manufacturer's instructions prior to analysis using BD FACS Diva™ on CANTOII™.
For the turbidity assay to track protein aggregation, OVA (1 mg/mL) and CA-hnRNPA1 (50 μM) were diluted in serum-free AMEM. 100 μL of each sample were added to a polystyrene flat bottom 96-well plate (Coming). The wavelength for measurement was defined according to examination of the absorbance spectra of the buffer (serum-free AMEM) in which no significant peak was observed. Thus, turbidity was assessed at 420 nm using a Synergy H1 microplate reader (BioTek). Plates were incubated at 37° C. and shaken for 5 s before each reading, that was taken in every 15 minutes. The experiment was conducted 4 times and in each 6 technical replicates were performed.
For cytokine and chemokine profiling, 15 cm cell culture dishes containing 80-90% confluent MSCs were grown in serum-free AMEM for 24 h at 37° C. and 5% CO2. MSCs were then treated with 50 μM of CA-hnRNPA1 in serum-free AMEM for 6 h. The supernatant was collected and fresh serum-free AMEM was replenished, without CA-hnRNPA1. After 24 h of the initial CA-hnRNPA1 treatment, the supernatant was collected and gathered with the previous one collected. Collected supernatants were then concentrated using the Amicon Ultra-4™ centrifugal filters (3000 NMWL) for 1 h at 4° C. Collected concentrates (80×) were then frozen at −80° C. until shipped to EveTechnologies™ (Calgary, AB, Canada) for cytokine/chemokine assessment by Commerc™.
For therapeutic vaccination, female C57BL/6 mice (n=10/group) received a SC injection of 5×105 EG.7 cells at day 0. Five days later (appearance of palpable tumors ˜35-50 mm3), mice were SC-injected with 5×105 CA-hnRNPA1+OVA-pulsed MSCs (two injections 1 week apart). Control animals received 5×105 tumor cells alone. Treated animals were followed thereafter for tumor growth. For therapeutic vaccination in combination with the immune-checkpoint inhibitors (anti-PD-1), mice received SC-injections of the antibody or its isotype at 200 g/per dose every 2 days for a total of 6 doses over two weeks. A similar approach was conducted for allogeneic dosing vaccination in C57BL/6 mice but using Balb/c-derived MSCs.
For RNA-seq, control MSCs or MSCs treated with CA-hnRNPA1 alone or CA-hnRNPA1+OVA for 6 h were used to extract RNA a commercial RNA extraction kit. Quantification of total RNA was made by QuBit™ (ABI) and 500 ng of total RNA was used for library preparation. Quality of total RNA was assessed with the BioAnalyzer™ Nano (Agilent) and all samples had a RIN above 8. Library preparation was done with the KAPA™ mRNAseq stranded kit (KAPA, Cat no. KK8420). Ligation was made with 9 nM final concentration of Illumina index and 10 PCR cycles was required to amplify cDNA libraries. Libraries were quantified by QuBit and BioAnalyzer. All libraries were diluted to 10 nM and normalized by qPCR using the KAPA library quantification kit (KAPA; Cat no. KK4973). Libraries were pooled to equimolar concentration. Sequencing was performed with the Illumina Hiseq2000 using the Hiseq™ Reagent Kit v3 (200 cycles, paired-end) using 1.7 nM of the pooled library. All Fastq files (strand-specific sequencing, N=4 per group) were aligned to GRCm38 (mouse genome Ensembl release 102) with STAR (v2.7). Raw reads mapping to genomic features (summarized per gene) were extracted with featureCounts (strand specific option). Expression matrices were filtered, genes with very low counts were removed and protein coding genes were kept for further analyses. Gene expression in both CA-hnRNPA1—and CA-hnRNPA1+OVA-treated MSCs were compared to BM-Derived MSC controls with DESeq2™ to generate a ranked list of differentially expressed genes based on the log 2 fold change. Gene set enrichment on either ranked lists of genes, or a number of significantly up- or down-unregulated genes perturbed by CA-hnRNPA1 alone or admixed with CA-hnRNPA1 variant compared to MSC controls were performed using the Reactome collection of pathways. The variance stabilizing transformation was applied to gene expression matrices prior to visualization. If not mentioned in the text, significance threshold is set to 5% after p-value adjustment with the Benjamini-Hochberg method to control for false positives among differentially expressed genes (DEGs). All custom scripts including prediction of putative targets were written in R programming and statistical language. Data visualization was made with ggplot2, enrichplot, Upset plots and Pheatmap R functions.
p-values were calculated using one-way analysis of variance (ANOVA). Results are represented as average mean with standard deviation (S.D.) error bars and statistical significance is represented with asterisks: * p<0.05, ** p<0.01, *** p<0.001.
Recombinant Cas9-NLS protein conjugated with a molar excess of CDCA-SV40 NLS steroid acid-peptide moieties ([CDCA-SV40]-Cas9-NLS) was produced as described in Example 1. HEK293 cells were incubated with 2 μM of either unconjugated Cas9-NLS cargo or [CDCA-SV40]-Cas9-NLS cargo for 0, 3 or 6 hours at 37° C., and then intracellular Cas9-NLS delivery was assessed by fluorescence microscopy as described in Example 1. Representative microscopy images for cells incubated with unconjugated Cas9-NLS or conjugated [CDCA-SV40]-Cas9-NLS as cargo are shown in
A GFP-galectin-3 (GFP-Gal3) based detection system was utilized to explore the effect on endosomal membranes following intracellular delivery of steroid acid-peptide-conjugated cargoes. Briefly, Gal3 is a cytosolic protein that exhibits high affinity towards β-galactoside sugars, which are normally present on the cell surface, Golgi apparatus, and in the lumen of endocytic compartments (i.e., compartments sequestered from the cytosol). When expressed under normal conditions, Gal3 is evenly distributed across the cytosol but disruption of endosomal membranes allows Gal3 to access and bind luminal glycoproteins. We thus transiently transfected the murine dendritic cell line DC2.4 with a construct expressing Gal3 fused to enhanced green fluorescent protein (eGFP-Gal3). Two days later, DC2.4 cells were incubated with 0.1 mg/mL of either unconjugated OVA cargo or [CA-SV40]-OVA conjugated cargo for 3 h at 37° C. and the cells were observed by fluorescent microscopy. As shown in
The fluorogenic substrate DQ™ ovalbumin (DQ™ OVA) was employed to study the intracellular fate of steroid acid-peptide-conjugated cargoes. Briefly, while a strong fluorescence quenching effect is observed when the DQ™ OVA substrate remains intact, hydrolysis of DQ™ OVA into single dye-labeled peptides by proteases relieves this quenching, thereby producing brightly fluorescent products. Recombinant DQ™ OVA conjugated with a molar excess of CA-SV40 NLS steroid acid-peptide moieties ([CA-SV40]-DQ™ OVA) was produced as described in Example 1. Primary bone marrow-derived DCs were incubated with either DQ™ OVA unconjugated cargo or [CA-SV40]-DQ™ OVA conjugated cargo for 3 or 6 h at 37° C. Cells were then collected and fluorescence was monitored by flow cytometry. As shown in
Mesenchymal stromal cells (MSCs) were incubated with either unconjugated fluorescently labeled OVA-AF647 alone or mixed with 45 μM of CA-HnRPA1 NLS steroid acid-peptide for nine hours and then intracellular OVA-AF647 delivery was assessed by flow cytometry as described in Example 1. As shown in
Cytochrome C is a protein that is normally entrapped in the mitochondria but its release into the cytosol in known to induce cell death. An experiment was performed in which EL4 cells were incubated with recombinant cytochrome C either alone (
[CA-SV40]-OVA prepared as described in Example 1 using different molar excess ratios of OVA cargo to CA-SV40 NLS conjugate. SDS-PAGE followed by Coomassie staining revealed that [CA-SV40]-OVA prepared using a 25× molar excess of CA-SV40 NLS had an average of about four [CA-SV40] moieties conjugated per OVA, corresponding to a MW increase of about 8.6 kDa compared to unconjugated OVA. [CA-SV40]-OVA prepared using a 50× molar excess of CA-SV40 NLS had an average of about eight [CA-SV40] moieties conjugated per OVA, corresponding to a MW of about 19.2 kDa. Furthermore, to assess the overall stability of [CA-SV40]-OVA, ITF analysis was conducted to measure its unfolding following thermal stress. In this assay, changes in peak shifts or intensities are indicative of unfolding as polypeptide residues may become solvent-exposed and undergo change in orientation (
A Cas9-GFP fusion cargo protein was conjugated to (e.g., [CA-SV40]-Cas9-GFP), or mixed with (e.g., Cas9-GFP+[Bile acid-NLS]), different bile acid-NLS moieties and then evaluated for intracellular delivery as described in Example 1. Delivery experiments were performed in JIMT-1 cells, which are generally considered difficult to transfect, and intracellular cargo delivery was measured via flow cytometry based on GFP fluorescence. Briefly, JIMT-1 cells were co-incubated with 5 μg (0.0257 nmol) Cas9-GFP cargo and 0.275 μmol different bile acid-NLS moieties for 48 hours and then intracellular Cas9-GFP delivery was assessed with a Biotek Spectrometer (final volume 2 mL). The results are shown in Table 2 with GFP fluorescence values being normalized to that of cells incubated with the unconjugated cargo alone (Cas9-GFP alone). The results in Table 2 suggest that bile acid-NLS moieties can increase intracellular delivery of proteinaceous cargoes, even in some cells/cell lines traditionally considered difficult to transfect. The increase in intracellular delivery was observed with the moieties being conjugated to or simply mixed with the cargoes. Fluorescence microscopy experiments in live cells confirmed that the Cas9-GFP cargoes (which were engineered to contain an NLS) were successfully delivered to the nucleus of JIMT-1 cells (data not shown).
To assess whether the enhanced intracellular delivery conferred by bile acid-NLS moieties is compatible with other delivery technologies, a delivery experiment in JIMT-1 cells was performed using Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol, except that the cargo was Cas9-GFP alone or conjugated to/mixed with different bile acid-NLS moieties. The results are shown in Table 3 with GFP fluorescence values being normalized to that of cells incubated with the unconjugated cargo/transfection reagent alone (Cas9-GFP alone). The results in Table 3 suggest that bile acid-NLS moieties can increase intracellular delivery of proteinaceous cargoes in the context of lipid-based delivery systems. The increase in intracellular delivery was observed with the moieties being conjugated to or simply mixed with the cargoes before formulation with the transfection reagent. Fluorescence microscopy experiments in live cells confirmed that the Cas9-GFP cargoes were successfully delivered to the nucleus of JIMT-1 cells (data not shown). Strikingly, mixture with the moiety CA-HuR resulted in a 17-fold increase in intracellular Cas9-GFP delivery as compared to Cas9-GFP/transfection reagent alone.
To assess whether Cas9 retains endonuclease activity following conjugation with bile acid-NLS moieties, a delivery experiment was performed by treating JIMT-1 cells with Cas9 complexed with TrueGuide™ CDK4 gRNA (Invitrogen). Endonuclease activity was assessed following delivery using the GeneArt™ Genomic Detection Kit (Cat. No: A24372, Life Technologies), with positive endonuclease activity being observable by the detection of genomic DNA cleavage products, as shown using the manufacturer's positive (+) and negative (−) controls in
Further delivery experiments were performed to assess the impact of bile acid-NLS moieties on cytosolic/nuclear delivery of polynucleotide cargoes. Poly-D-lysine (“poly-K”; MW: 110 kDa, Thermo Fisher Scientific) was conjugated with a 10-fold molar excess of [CA-SV40] moieties as described in Example 1. Plasmid DNA encoding GFP was used as cargo, with cytosolic/nuclear delivery being evaluated by flow cytometry based on GFP fluorescence. Briefly, plasmid DNA complexes were prepared by adding, dropwise and with constant mixing, 7.5 μg poly-K or 5 μg of [CA-SV40]-poly-K in 0.3 mL of serum-free DMEM to 8 μg plasmid DNA in 0.7 mL of serum-free DMEM (NH3:phosphate=2:1). The mixed solutions were kept for 30 min at 20° C. before use. HEK293 cells were seeded (day 0) into wells of culture plates. On day 1, the medium was removed and 1 mL of a solution containing a plasmid/poly-K complexes in serum-free DMEM was added. After 6 h and 24 h incubation at 37° C. in a humidified atmosphere (95% air, 5% CO2), 1 mL of complete medium was added and cells were further incubated at 37° C. in 0.5 mL of the relevant complete culture medium for another 24 h before the cells were collected and subjected to flow cytometry analysis. The results shown in Table 4 demonstrate the ability of bile acid-NLS moieties to delivery polynucleotide cargoes intracellularly to the cytosol/nucleus.
Variants of CA-SV40NLS were synthesized in order to explore structure-activity relationships relating to the antigen cross-presentation enhancing activity observed for this conjugate, and therefore a measure of antigen/cargo delivery. More particularly, conjugates having different bile acids conjugated to the SV40NLS peptide (SEQ ID NO: 1) were synthesized and their effect on antigen presentation was evaluated by using the B3Z reporter system with the OVA antigen as described in Example 1. The results in
Further variants of CA-SV40NLS were synthesized in which the SV40NLS peptide was replaced with peptides comprising other NLS's (Table 5) and the antigen presentation activities of the CA-NLS peptide conjugates were evaluated using the B3Z reporter system as described in Example 1. The following conjugate: antigen molar ratios were tested for each conjugate: 2:1, 4:1, 8:1, 12:1 and 22:1. The results in
The results in
Using BMDCs as antigen presenting cells, the glutamate-rich peptide PQBP-1 NLS was associated with strikingly high antigen-presentation activity (
Using a cross-presenting cell line of MSCs (i.e., immortalized MSCs genetically engineered to possess cross-presenting capabilities, “cpMSCs”) as antigen presenting cells, various cholic acid peptide conjugates enhanced antigen presentation of OVA (
To further dissect the effect of bile acid peptide conjugates on antigen presentation, antigen internalization and processing were evaluated. cpMSCs were pulsed with OVA-labelled with AF647 in the presence of various molar ratios of different bile acid peptide conjugates, NLS1-RPS17 [
In summary, these data demonstrate the versatility and capability of bile acid peptide conjugates in enhancing cargo/antigen delivery, processing, and presentation.
In
Next, the mechanism involved in cross presentation enhancement of steroid acid-peptide conjugates was further dissected.
A variant of CA-hnRNPA1 in which the bile acid CA was replaced with the bile acid DCA (i.e., DCA-hnRNPA1) yielded similar results as CA-hnRNPA1 in terms of antigen cross-presentation in WT MSCs and induction of intracellular ROS.
These data demonstrate the striking enhancement of cross presentation of antigen by steroid acid-peptide conjugates in non-professional cross-presenting cells.
To determine the effectiveness of steroid acid peptide-conjugates in cell-based therapeutic vaccines, mice were first implanted with EG.7 lymphoma cells then immunized with WT MSCs (that were previously pulsed with OVA in the presence or absence of CA-hnRNPA1) and/or treated with the immune checkpoint inhibitor/anti-cancer agent, anti-PD-1 antibody. The immunization scheme is shown in
Mice immunized with syngeneic WT MSCs previously pulsed with OVA and CA-hnRNPA1 had significantly smaller tumors (
Even stronger positive results were observed after immunization of allogeneic WT MSCs in EG.7 implanted mice, whereby mice immunized with WT MSCs previously pulsed with OVA and CA-hnRNPA1 had strikingly lower tumor volumes (
Overall, these findings suggest that “off-the-shelf” allogeneic or syngeneic MSCs previously pulsed with tumor antigens in the presence of steroid acid-peptide conjugates may be effectively exploited as universal vaccines to trigger potent anti-tumoral responses.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051795 | 12/8/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63265125 | Dec 2021 | US | |
| 63362487 | Apr 2022 | US |
