Steroid Acid-Based Immunogenic Enhancers

Abstract

Described herein is an immunogen enhancer for mixing with an antigen of interest.The enhancer generally comprises a sufficient amount of steroid acid and / or steroid acid-peptide conjugate to improve or modify the adaptive immune response to the mixed antigen.In an embodiment, the steroid acid may be a bile acid, and the peptide may comprise one or more functional domains, such as a nuclear localization signal, that can promote antigen presentation and / or antigen cross-presentation, thereby inducing improved cellular immunity or improved cellular and humoral immunity to the antigen.

Description

[Technical field]

[0001] The present specification relates to methods for enhancing the immunogenicity of an antigen, more particularly, the present specification relates to steroid acids and steroid acid-peptide conjugates for improving antigen immunogenicity. [Background technology]

[0002] Although subunit vaccines are generally considered to be the safest vaccines, such antigens may not elicit a sufficiently strong immune response to provide protective and long-lasting immunity. Thus, methods to improve the immunogenicity and efficacy of subunit vaccines are highly desirable. Summary of the Invention

[0003] In a first aspect, described herein is an immunogenic composition comprising an antigen admixed with an enhancer of antigen presentation, the enhancer comprising a steroid acid and / or a steroid acid-peptide conjugate in an amount sufficient to improve presentation of the antigen upon administration of the composition to an antigen-presenting cell compared to administration of a corresponding composition lacking the enhancer. In embodiments, the steroid acid may be a bile acid or a bile acid analog, and the peptide may comprise a functional domain, such as a nuclear localization signal, an endosomal escape signal, and / or a protein transduction domain.

[0004] In further aspects, described herein are cell populations and cell cultures comprising the immunogenic compositions described herein. In further aspects, described herein are vaccines comprising the immunogenic compositions described herein or comprising cells produced using the cell cultures described herein. In further aspects, described herein are methods for eliciting an enhanced adaptive immune response to an antigen of interest in a subject, the method comprising administering to the subject a composition described herein or cells produced using the cell cultures described herein.

[0005] In a further aspect, the present specification describes steroid acid-peptide conjugates for use in admixture with an antigen to enhance immunogenicity or for use in the manufacture of a medicament for generating an immune response in a subject.

[0006] general definition Headings and other identifiers, e.g., (a), (b), (i), (ii), etc., are provided merely to facilitate the readability of the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0007] The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0008] As used in this specification and the claims, the terms "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.

[0009] The term "about" is used to indicate that a value includes the standard deviation of error of the device or method used to determine the value. In general, the term "about" is meant to indicate a possible variation of up to 10%. Thus, variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value are included within the term "about". Unless otherwise indicated, the use of the term "about" before a range applies to both ends of the range.

[0010] Other objects, advantages and features of the present description will become more apparent from a reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. [Brief description of the drawings]

[0011] The accompanying diagram is as follows: [Figure 1] The results of an antigen cross-presentation assay using bone marrow-derived dendritic cells (BMDCs) to evaluate OVA-responsive OT-I (CD8) cells are shown. Mouse BMDCs were pulsed for 3 hours with antigen (ovalbumin (OVA)) alone, cholic acid (CA) alone, cholic acid-NLS peptide conjugate (CA-SV40NLS) alone, various ratios of CA to antigen (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1), or various ratios of cholic acid-NLS peptide conjugate to antigen (CA-SV40NLS:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1). Pulsed BMDCs were then co-cultured with CD8 T cells from OT-I mice, and IFN-γ levels were quantified as a measure of cross-presentation activity. [Diagram 2]Figure 1 shows the effect of various bile acids on the antigen-presenting activity of bile acid-SV40NLS conjugates. For this experiment, BMDCs were used as antigen-presenting cells (n=6), and the molar ratio (bile acid / peptide / conjugate):antigen was 4:1. Controls tested included no antigen ("PBS"), antigen alone ("OVA alone"), unconjugated NLS peptide ("SV40NLS"), unconjugated cholic acid mixed with OVA ("CA"), and the positive control peptide SIINFEKL (SEQ ID NO: 9) mixed with OVA ("SIINFEKL"). The dashed line represents the signal obtained with OVA alone. Bile acids: cholic acid (CA); glycodeoxycholic acid (GDCA); glycochenodeoxycholic acid (GCDCA); ursodeoxycholic acid (UDCA); and lithocholic acid (LCA). [Diagram 3] Figure 1 shows the effect of various NLS peptides on the antigen-presenting activity of cholic acid-NLS peptide conjugates. The dashed line represents the signal obtained with OVA alone. Readout was performed after 24 hours of incubation, and error bars represent SD (n=6). For this experiment, BMDCs were used as antigen-presenting cells. [Figure 4] Figure 1 shows the effect of various NLS peptides on the antigen-presenting activity of cholic acid-NLS peptide conjugates. The dashed line represents the signal obtained with OVA alone. Readout was performed after 24 hours of incubation from a single experiment. The molar ratio of CA-peptide conjugate:OVA was 22:1. For this experiment, BMDCs were used as antigen-presenting cells. [Diagram 5]Figure 1 shows the effect of various NLS peptides on the antigen-presenting activity of cholic acid-NLS peptide conjugates. For this experiment, BMDCs were used as antigen-presenting cells. The dashed line represents the signal obtained with OVA alone. Readouts were taken after 24 hours of incubation from a single experiment. The molar ratios of CA-peptide conjugates:OVA were as follows: CA-GWG-SV40NLS (12:1), CA-hnRNP M NLS (12:1), CA-NLS2-RPS17 NLS (22:1), CA-HuR NLS (22:1), CA-cMyc NLS (2:1), CA-NLS3-RPS17 NLS (22:1), CA-NLS2-RG-RPS17 NLS (2:1), CA-PQBP1 NLS (8:1), CA-hnRNPA1 M9 NLS (22:1), and CA-SV40 NLS (2:1). [Figure 6] Figure 1 shows the effect of different NLS peptides on the antigen-presenting activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as antigen-presenting cells. The dashed line represents the signal obtained with OVA alone. Readout was performed after 24 hours of incubation. The molar ratios of CA-peptide conjugates:OVA were as follows: CA-GWG-SV40NLS (2:1), CA-hnRNP M NLS (8:1), CA-hnRNP D NLS (12:1), CA-NLS2-RG-RPS17 (4:1), CA-cMyc NLS (12:1), CA-HuR NLS (12:1), CA-Tus NLS (2:1), CA-NLS2-RPS17 NLS (4:1), CA-PQBP1 NLS (12:1), CA-hnRNPA1 M9 NLS (2:1), and CA-SV40 NLS (2:1). [Figure 7A]Figure 7 shows the effect of various NLS peptides on the antigen internalization activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with OVA labeled with Alexa Fluor 647 (i.e., OVA647)™. Fluorescence of OVA647 was measured by flow cytometry. Various ratios of CA (NLS1 RPS17 [Figure 7A], NLS3 RPS17 [Figure 7B], PQBP-1 [Figure 7C], and hnRNPA1 M9 NLS [Figure 7D]) to antigen (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) were tested (hnRNPA1 M9 NLS at 2:1). [Figure 7B] Figure 7 shows the effect of various NLS peptides on the antigen internalization activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with OVA labeled with Alexa Fluor 647 (i.e., OVA647)™. Fluorescence of OVA647 was measured by flow cytometry. Various ratios of CA (NLS1 RPS17 [Figure 7A], NLS3 RPS17 [Figure 7B], PQBP-1 [Figure 7C], and hnRNPA1 M9 NLS [Figure 7D]) to antigen (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) were tested (hnRNPA1 M9 NLS at 2:1). [Figure 7C] Figure 7 shows the effect of various NLS peptides on the antigen internalization activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with OVA labeled with Alexa Fluor 647 (i.e., OVA647)™. Fluorescence of OVA647 was measured by flow cytometry. Various ratios of CA (NLS1 RPS17 [Figure 7A], NLS3 RPS17 [Figure 7B], PQBP-1 [Figure 7C], and hnRNPA1 M9 NLS [Figure 7D]) to antigen (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) were tested (hnRNPA1 M9 NLS at 2:1). [Figure 7D]Figure 7 shows the effect of various NLS peptides on the antigen internalization activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with OVA labeled with Alexa Fluor 647 (i.e., OVA647)™. Fluorescence of OVA647 was measured by flow cytometry. Various ratios of CA (NLS1 RPS17 [Figure 7A], NLS3 RPS17 [Figure 7B], PQBP-1 [Figure 7C], and hnRNPA1 M9 NLS [Figure 7D]) to antigen (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) were tested (hnRNPA1 M9 NLS at 2:1). [Figure 8A] Figure 8 shows the effect of various NLS peptides on the antigen processing activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with DQ™ ovalbumin (i.e., OVADQ). The fluorescence of OVADQ was measured by flow cytometry. Various ratios (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) of CA (NLS1 RSP17 [Figure 8A], NLS3 RPS17 [Figure 8B], PQBP-1 [Figure 8C], and hnRNPA1 M9 NLS [Figure 8D]) to antigen were tested (hnRNPA1 M9 NLS at 2:1). [Figure 8B] Figure 8 shows the effect of various NLS peptides on the antigen processing activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with DQ™ ovalbumin (i.e., OVADQ). The fluorescence of OVADQ was measured by flow cytometry. Various ratios (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) of CA (NLS1 RSP17 [Figure 8A], NLS3 RPS17 [Figure 8B], PQBP-1 [Figure 8C], and hnRNPA1 M9 NLS [Figure 8D]) to antigen were tested (hnRNPA1 M9 NLS at 2:1). [Figure 8C]Figure 8 shows the effect of various NLS peptides on the antigen processing activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with DQ™ ovalbumin (i.e., OVADQ). The fluorescence of OVADQ was measured by flow cytometry. Various ratios (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) of CA (NLS1 RSP17 [Figure 8A], NLS3 RPS17 [Figure 8B], PQBP-1 [Figure 8C], and hnRNPA1 M9 NLS [Figure 8D]) to antigen were tested (hnRNPA1 M9 NLS at 2:1). [Figure 8D] Figure 8 shows the effect of various NLS peptides on the antigen processing activity of cholic acid-NLS peptide conjugates. For this experiment, a cross-presenting mesenchymal stromal cell (MSC) line was used as an antigen-presenting cell, which was pulsed with DQ™ ovalbumin (i.e., OVADQ). The fluorescence of OVADQ was measured by flow cytometry. Various ratios (CA:OVA = 22:1, 12:1, 8:1, 4:1, and 2:1) of CA (NLS1 RSP17 [Figure 8A], NLS3 RPS17 [Figure 8B], PQBP-1 [Figure 8C], and hnRNPA1 M9 NLS [Figure 8D]) to antigen were tested (hnRNPA1 M9 NLS at 2:1).

[0012] Sequence Listing This application contains a sequence listing in computer readable form, created on May 5, 2022. The computer readable form is incorporated herein by reference.

[0013] [Table 1] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Described herein are compositions, cells, and methods related to improving or modifying adaptive immune responses to antigens. In some aspects, the invention arises from the demonstration herein that admixing an antigen with a steroid acid or a steroid acid-peptide conjugate improves antigen presentation and / or induces improved cellular immunity or improved cellular and humoral immunity to the antigen. In some embodiments, described herein is the use of a steroid acid or a steroid acid-peptide conjugate as an enhancer of antigen presentation and / or adaptive immunity. Advantageously, the enhancers described herein are not covalently conjugated to the antigen, thereby providing a versatile platform that can be rapidly adapted to formulations with different antigens with enhancer:antigen molar ratios that can be tailored to the antigen of interest.

[0015] In a first aspect, the present specification describes a composition comprising an antigen mixed with an enhancer of antigen presentation. As used herein, the term "mixing" or "mixing" refers to the combination of two separate components into a single composition, which are not covalently conjugated or otherwise reacted together. In some embodiments, the enhancer may comprise a sufficient amount of steroid acid to improve the presentation of the antigen upon administration of the composition to an antigen-presenting cell (e.g., in vitro, ex vivo, or in vivo) compared to administration of a corresponding composition lacking the enhancer. In some embodiments, the enhancer may comprise a sufficient amount of steroid acid-peptide conjugate to improve the presentation of the antigen upon administration of the composition to an antigen-presenting cell (e.g., in vitro, ex vivo, or in vivo) compared to administration of a corresponding composition lacking the enhancer.

[0016] Polypeptide antigens are usually captured by antigen-presenting cells (e.g., dendritic cells), but initially in endosomes. Maturation of endosomes into lysosomes reduces the pH, activating proteolytic enzymes that mediate non-specific antigen degradation. As a result, some of the generated antigenic fragments can then pass through endosomal pores to reach the cytosol, where further antigen degradation occurs by the proteasomal machinery before MHC class I presentation. Although this process occurs naturally, the generated antigenic fragments that are eventually released from the endosomes may be small and / or damaged, making them inappropriate for proteasomal degradation, thereby preventing their MHC class I presentation and therefore the cellular immunity that is based thereon. Without being bound by theory, incorporation of antigens with immunogenic enhancers as described herein may promote antigen internalization / endosomal escape, allowing them (or larger antigenic fragments) to reach the cytosol in a more native conformation and / or in greater amounts. As a result, proteasomal degradation of these more native antigens can result in a greater number and / or diversity of immunogenic and / or stable peptides that are presented via MHC class I on the surface of antigen-presenting cells, thereby eliciting potent T cell activation.

[0017] In some embodiments, the steroid acids described herein (e.g., in enhancers and / or steroid acid-peptide conjugates) may be steroid acids that enhance endocytosis and / or endosomal escape of internalized cargo. 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 viral infection of host cells, for example, by increasing endocytic uptake and / or endosomal escape of the virus 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. Increasing the amount of ceramide destabilizes the membrane and promotes endosomal escape. In some embodiments, steroid acids described herein may include those that induce ceramide accumulation on the inner leaflet of endosomes, thereby destabilizing the endosomal membrane and facilitating endosomal escape of modified polypeptide antigens upon intracellular delivery. In some embodiments, steroid acids described herein may include those that induce increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.

[0018] In some embodiments, the steroid acids described herein may be bile acids, such as primary bile acids or secondary bile acids. In some embodiments, the steroid acids described herein may be bile acid oligomers that contain one or more bile acid moieties (Al-Hilal et al., 2014). In some embodiments, the steroid acid described herein is selected from the group consisting of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TCDCA), ursodeoxycholic acid (UC ... In some embodiments, the steroid acid described herein may be or include a bile acid that is glycodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glycoursodeoxycholic acid (GUDCA). In some embodiments, the steroid acid described herein may be or include glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA), or an analog thereof that enhances antigen presentation and / or adaptive immunity to an antigen when employed in an enhancer described herein.In some embodiments, the steroid acids described herein may be or include analogs of the bile acids described herein that induce endocytosis, induce ceramide accumulation on the inner leaflet of endosomes, induce increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide, and / or have a hydrophobicity greater than that of cholic acid.

[0019] Hydrophobic bile acids such as GCDCA, TCA, GCA, and CA (but not hydrophilic bile acids such as UDCA) have been shown to increase infection and replication of GII.3 human norovirus 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 acids described herein can comprise or consist of bile acids or bile acid analogs that are more hydrophobic than cholic acid. In some embodiments, the steroid acids suitable for conjugation to the polypeptide antigens described herein comprise or consist of bile acids or bile acid analogs that are more hydrophobic than cholic acid (e.g., CDCA, DCA, LCA, TCA, TDCA, TCDCA, GCA, GDCA, or GCDCA; Hanafi et al., 2018).

[0020] In some embodiments, the peptides included in the steroid acid-peptide conjugates described herein may contain one or more domains that confer desired functionality to the conjugates (e.g., intracellular targeting, nuclear localization, nucleolar localization, endosomal escape, and / or protein transduction) that may further enhance immunogenicity. As used herein, a "domain" generally refers to a portion of a protein that has a specific functionality. Some domains preserve their function when separated from the rest of the protein and can therefore be used in a modular manner. The modular nature of many protein domains can provide flexibility with regard to their placement within the peptides described herein. However, some domains can function better when engineered at specific positions of the peptide (e.g., N- or C-terminal regions, or in between). The location of a domain within its endogenous protein can be an indication of where the domain should be engineered into the peptide.

[0021] In some embodiments, the peptide may include an intracellular targeting signal that facilitates targeting of the modified polypeptide antigen to a specific intracellular compartment. In some embodiments, the peptides described herein may include a nuclear localization signal (NLS). In some aspects, the NLS described herein may include a classical NLS (e.g., including a KK / RXK / R motif), a PY-NLS (e.g., including one or more PY motifs, such as toward the C-terminus of the NLS), a PL-NLS (e.g., including one or more PL motifs, such as toward the C-terminus of the NLS), a ribosomal NLS, an NLS that further includes a nucleolar targeting signal, or any combination thereof.

[0022] In some embodiments, an NLS described herein may comprise the following general consensus sequences: (i) K(K / R)X(K / R), (ii) (K / R)(K / R)X 10~12 (K / R) 3 / 5 , where (K / R) 3 / 5represents three lysine or arginine residues out of five consecutive amino acids; (iii) KRX 10~12 KRRK, (iv) KRX 10~12 K(K / R)(K / R), or (v) KRX 10~12 K(K / R)X(K / R), where X is any amino acid (Sun et al., 2016).

[0023] In some embodiments, the NLS described herein may be a hydrophobic and / or basic NLS. In some aspects, the NLS described herein may comprise at least 3, 4, or 5 acidic residues (e.g., R / K) and / or at least 3, 4, or 5 basic residues (e.g., E / D).

[0024] In some embodiments, the NLS described herein is or can be derived from an NLS from the SV-40 large T antigen (e.g., SV40 NLS), a c-Myc NLS, an acidic M9 domain in the hnRNP A1 protein (e.g., hnRNPA1 M9 NLS), an hnRNP D NLS, an hnRNP M NLS, a PQBP-1 NLS, a HuR NLS, a Tus NLS, a nucleoplasmin NLS, NLS1 RPS17, NLS2 RPS17, NLS3 RPS17, or the NLS2-RG domain RSP17.

[0025] In some embodiments, the nuclear localization signals described herein may comprise or be derived from an NLS from the SV-40 large T antigen (e.g., PKKKRKV; SEQ ID NO: 1 or 2) or other classical NLS. In some embodiments, the nuclear localization signals described herein may comprise or be derived from a non-classical NLS (e.g., the acidic M9 domain in the hnRNP A1 protein, the sequence KIPIK in the yeast transcriptional repressor Matα2; a PY-NLS; a ribosomal NLS; or the composite signal of U snRNP). In some embodiments, the nuclear localization signals described herein comprise or consist essentially of the amino acid sequence of any one of SEQ ID NOs: 1-8 or 10-16 or any portion thereof. In some embodiments, a nuclear localization signal described herein comprises or consists essentially of a nuclear localization signal that is an SV40 NLS (e.g., included in SEQ ID NO: 1 or 2), a GWG-SV40 NLS (e.g., included in SEQ ID NO: 3), an hnRNPA1 M9 NLS (e.g., included in SEQ ID NO: 4), an hnRNP D NLS (e.g., included in SEQ ID NO: 5), an hnRNP M NLS (e.g., included in SEQ ID NO: 10), a PQBP-1 NLS (e.g., included in SEQ ID NO: 6), an NLS2-RG domain RPS17 (e.g., included in SEQ ID NO: 11), NLS1 RPS17 (e.g., included in SEQ ID NO: 15), NLS2 RPS17 (e.g., included in SEQ ID NO: 7), NLS3 RPS17 (e.g., included in SEQ ID NO: 8), a cMyc NLS (e.g., included in SEQ ID NO: 12), a HuR NLS (e.g., included in SEQ ID NO: 13), a Tus NLS (e.g., included in SEQ ID NO: 14), or a nucleoplasmin NLS (e.g., included in SEQ ID NO: 16). In some cases, the SEQ ID NOs referred to above include an N-terminal cysteine ​​residue (e.g., a thiol group of the N-terminal cysteine ​​residue) used to facilitate conjugation to a polypeptide antigen. Thus, in some embodiments, the NLS sequences referred to herein may exclude the N-terminal cysteine ​​residue contained in any one of SEQ ID NOs: 1-8 or 10-16.In some embodiments, other functional groups are also envisioned (e.g., carboxyl groups, synthetic amino acids, etc.) that are added or inserted (e.g., from the N-terminal portion toward the C-terminal portion of the peptides described herein) to facilitate steroid acid-peptide conjugation with a given polypeptide antigen. For example, the peptides may include a C-terminal amide and / or an N-terminal cysteine. In some embodiments, the peptides do not include an endosomal escape motif, or a protein transduction or cell membrane permeability motif.

[0026] In some embodiments, the NLS described herein may be the PQBP-1 NLS (e.g., comprising at least residues 3-21 of SEQ ID NO:6) or another NLS that binds to the nuclear transport receptor Kapβ2. In some embodiments, the NLS described herein comprises the motif RX, which was found to be required for PQBP-1 binding to Kapβ2. 2~5 -PY may also be included (Liu et al., 2020).

[0027] In some embodiments, the NLS described herein may comprise an endosomal degradation motif that facilitates endosomal escape of the antigen upon internalization into an antigen-presenting cell. In some embodiments, the NLS described herein may comprise a protein transduction domain that stimulates endocytosis and / or endosome formation, thereby facilitating internalization into an antigen-presenting cell. In some embodiments, the NLS described herein may lack a protein transduction domain or a cell membrane penetrating peptide, which may be advantageous to avoid inducing a more rapid internalization of the steroid acid conjugate compared to the antigen.

[0028] In some embodiments, the peptides described herein may contain a protein transduction domain (PTD) that stimulates endocytosis, endosome formation, or intracellular delivery in a cell-nonspecific manner.

[0029] In some embodiments, the peptide is preferably a non-immunogenic peptide, thereby supporting the immune response generated against the antigen of interest, rather than against the peptide in the steroid acid-peptide conjugate enhancer. For example, Azuar et al., 2019 conjugated cholic acid to an antigenic peptide from group A streptococcus, which reportedly self-assembled into rod-like nanoparticles and elicited a stronger humoral immune response against the antigenic peptide. Such a humoral immune response against the enhancers described herein is undesirable.

[0030] In some embodiments, the peptides described herein have a length of at least 7, 8, 9, 10, 11, or 12 amino acids, and may have a length of 50-100 or less amino acids, and / or a length of 10-100 amino acids. In some embodiments, the peptides described herein may comprise or consist of a peptide of any one of SEQ ID NOs: 1-8 or 10-16, or a variant thereof, that (a) when conjugated to a steroidal acid confers improved antigen presenting activity to the steroidal acid compared to the antigen presenting activity of the corresponding unconjugated steroidal acid, (b) has nuclear localization and / or endosomolytic activity, (c) differs from a peptide of any one of SEQ ID NOs: 1-8 or 10-16 by the substitution or deletion of no more than 1, 2, 3, 4, or 5 amino acids, or (d) is any combination of (a)-(c).

[0031] In some embodiments, the enhancers described herein include steroid acid-peptide conjugates, where the steroid acid is conjugated to the peptide at a molar ratio of steroid acid:peptide of (a) 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 and 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-terminus of the peptide (e.g., at the free amino group of the N-terminal residue and / or at the thiol group of the N-terminal cysteine ​​residue), or (d) any combination of (a)-(c). In some embodiments, the steroid acid described herein may be conjugated to the peptide at any suitable functional group within the peptide.

[0032] In some embodiments, the molar ratio of enhancer to antigen in the compositions described herein 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, and may be less than or equal to 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 between 1:1 and 1000:1, between 1:1 and 500:1, between 1:1 and 250:1, between 1:1 and 200:1.

[0033] In some embodiments, the antigens described herein may be or include tumor-associated antigens (TAA), tumor-specific antigens (TSA), neoantigens, viral antigens, bacterial antigens, fungal antigens, antigens associated with a disease or disorder suitable for treatment by vaccination and / or immunotherapy, or any antigenic fragment thereof. In some embodiments, the antigens described herein may be or include spike proteins from SARS-CoV-2 or SARS-CoV, or antigenic variants or antigenic fragments thereof. In some embodiments, the TAA, TSA, and / or neoantigen may be a single base variant antigen, a mutant frameshift antigen, a 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 (Smith et al., 2019). In some embodiments, the TSA may be a viral cancer antigen, such as human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV). In some embodiments, the TAA may be or include a cancer-testis antigen, HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), or EGFR (Patel et al., 2017; Tagliamonte et al., 2014). In some embodiments, the antigens described herein may be or include other tumor-derived material, such as cell lysates or tumor-derived exosomes.

[0034] In some embodiments, the enhancers described herein may allow for increased cytosolic delivery of an antigen compared to a corresponding composition lacking the enhancer. In some embodiments, the enhancers described herein may allow for increased total cellular delivery of an antigen compared to a corresponding composition lacking the enhancer. In some embodiments, the enhancers described herein may allow for enhanced cellular immunity to an antigen compared to a corresponding composition lacking the enhancer. In some embodiments, the enhancers described herein may allow for increased IFN-γ production by CD8+ T cells upon exposure to an antigen compared to a corresponding composition lacking the enhancer. In some embodiments, the enhancers described herein may allow for enhanced humoral immunity to an antigen compared to a corresponding composition lacking the enhancer. In some embodiments, the enhancers described herein may allow for increased antibody species diversity (or biological diversity) to an antigen compared to a corresponding composition lacking the enhancer (e.g., comprising antibodies to poorly immunogenic epitopes).

[0035] In some aspects, described herein are cell cultures comprising cell populations and compositions described herein (e.g., comprising an antigen and an enhancer of antigen presentation). In some embodiments, the cells may comprise immune cells (e.g., T cells), antigen-presenting cells (e.g., dendritic cells, macrophages, engineered antigen-presenting cells), MHC class I expressing cells, MHC class II expressing cells, or any combination thereof.

[0036] In some embodiments, the compositions described herein may further comprise a pharma- ceutically acceptable excipient and / or an adjuvant (eg, a vaccine adjuvant suitable for human or veterinary use).

[0037] In some aspects, described herein are vaccines comprising the compositions described herein or comprising cells produced using the cell cultures or cell populations described herein. In some embodiments, the vaccine can be a therapeutic or prophylactic vaccine (e.g., an anti-cancer vaccine, an anti-viral vaccine, or an anti-bacterial vaccine). In some embodiments, the immunogenic enhancers described herein can reduce the amount of antigen and / or antigen-presenting cells that are incorporated into an immunogenic composition (e.g., a vaccine) required to generate an immune response compared to the absence of the immunogenic enhancer.

[0038] In some aspects, described herein are methods of eliciting an enhanced adaptive immune response to an antigen of interest in a subject, the methods comprising administering to the subject a composition described herein or cells produced using the cell culture described herein.

[0039] In some aspects, described herein are methods for vaccinating a subject against an infectious disease, the methods comprising administering to the subject a composition described herein or a cell produced using the cell culture described herein, wherein the antigen comprises an antigenic fragment of a pathogen (e.g., a virus, a bacterium, a fungus) that causes the infectious disease.

[0040] In some aspects, described herein are methods for treating cancer in a subject, the methods comprising administering to the subject cells produced using the compositions described herein or the cell cultures described herein, wherein an antigen is overexpressed or aberrantly expressed in the cancer causing cells.

[0041] In a further aspect, the present specification describes a method for treating or preventing a disease or disorder suitable for treatment by vaccination and / or immunotherapy, the method comprising administering to a subject an immunogenic composition as described herein.

[0042] In some aspects, described herein are steroid acid-peptide conjugates for use in admixture with an antigen. In some embodiments, admixture of the steroid acid-peptide conjugate with the antigen allows for (i) increased cytosolic delivery of the antigen compared to a corresponding composition lacking the enhancer, (ii) increased total cellular delivery of the antigen compared to a corresponding composition lacking the enhancer, (iii) enhanced cellular immunity to the antigen compared to a corresponding composition lacking the enhancer, (iv) increased IFN-γ production by CD8+ T cells upon exposure to the antigen compared to a corresponding composition lacking the enhancer, (v) enhanced humoral immunity to the antigen compared to a corresponding composition lacking the enhancer, (vi) increased antibody species diversity to the antigen compared to a corresponding composition lacking the enhancer, or (vii) any combination of (i)-(vi).

[0043] In some aspects, described herein are cell populations produced using the compositions described herein, or the cultures described herein, for use in therapy.

[0044] In some aspects, described herein is the use of a composition described herein or a cell population produced using a culture described herein for generating an immune response in a subject, or for the manufacture of a medicament (e.g., a vaccine) for generating an immune response in a subject. In some embodiments, the immune response may include enhanced cellular immunity to an antigen, increased IFN-γ production by CD8+ T cells upon exposure to said antigen, enhanced humoral immunity to said antigen, or any combination thereof, compared to that generated from a corresponding composition or cell culture lacking the enhancer.

[0045] item In some embodiments, one or more of the following items are described herein. 1. A composition comprising an antigen admixed with an enhancer of antigen presentation, the enhancer comprising a steroid acid and / or a steroid acid-peptide conjugate in an amount sufficient to improve presentation of the antigen upon administration of the composition to an antigen-presenting cell compared to administration of a corresponding composition lacking the enhancer. 2. The composition described in item 1, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a steroid acid that induces ceramide accumulation on the inner leaflet of the endosome, thereby destabilizing the endosomal membrane in the antigen-presenting cell and promoting endosomal escape of the antigen into the cytosol. 3. The composition according to item 1 or 2, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a steroid acid that induces an increase in acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide. 4. The composition according to any one of items 1 to 3, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a bile acid. 5. The composition according to any one of items 1 to 4, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a primary bile acid or a secondary bile acid. 6. The steroid acid in the enhancer or in the steroid acid-peptide conjugate is (a) cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), 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 glucocholic acid (GLCA). 6. The composition of any one of items 1 to 5, which is or comprises a bile acid that is 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)-(c). 7. The composition according to any one of items 1 to 6, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is or comprises glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UDCA), or lithocholic acid (LCA). 8. The composition according to any one of items 1 to 7, wherein the peptide comprises a nuclear localization signal (NLS). 9. The NLS is a classical NLS (e.g., containing a KK / RXK / R motif), a PY-NLS (e.g., containing one or more PY motifs, such as those toward the C-terminus of the NLS, or containing the motif RX 2~59. The composition of claim 8, wherein the NLS is a PL-NLS (e.g., a ribosomal NLS, an NLS further comprising a nucleolar targeting signal, or any combination thereof), a PL-NLS (e.g., a PL-PY and / or having Kapβ2 binding activity ... 10. The composition according to item 8 or 9, wherein the NLS is or is derived from SV40 NLS, cMyc NLS, hnRNPA1 M9 NLS, hnRNP D NLS, hnRNP M NLS, PQBP-1 NLS, HuR NLS, Tus NLS, nucleoplasmin NLS, NLS1 RPS17, NLS2 RPS17, NLS3 RPS17, or NLS2-RG domain RSP17. 11. The composition according to any one of items 8 to 10, wherein the NLS comprises at least three acidic residues (e.g., R / K) and / or at least three basic residues (e.g., E / D). 12. The composition according to any one of items 1 to 11, wherein the peptide comprises an endosomolytic motif that promotes endosomal escape. 13. The composition according to any one of items 1 to 12, wherein the peptide comprises a protein transduction domain that stimulates endocytosis and / or endosome formation. 14. The composition according to any one of items 1 to 12, wherein the peptide lacks a protein transduction domain or a cell membrane penetrating peptide. 15. The composition according to any one of items 1 to 14, wherein the peptide is a non-immunogenic peptide. 16. The composition according to any one of items 1 to 15, wherein the peptide has a length of at least 7, 8, 9, 10, 11, or 12 amino acids and has a length of 50 to 100 or less amino acids and / or has a length of 10 to 100 amino acids. 17. The composition according to any one of items 1 to 16, comprising or consisting of a peptide or variant thereof, which (a) when conjugated to a steroid acid, confers improved antigen-presenting activity to the steroid acid compared to the antigen-presenting activity of the corresponding unconjugated steroid acid, (b) has nuclear localization and / or endosomolytic activity, (c) differs from a peptide of any one of SEQ ID NOs: 1 to 8 or 10 to 16 by substitution or deletion of not more than 1, 2, 3, 4, or 5 amino acids, or (d) is any combination of (a) to (c). 18. The composition according to any one of items 1 to 17, wherein the enhancer comprises a steroid acid-peptide conjugate, the steroid acid being conjugated to a peptide (a) in 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 from 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-terminus of the peptide (e.g., at the free amino group of the N-terminal residue and / or at the thiol group of the N-terminal cysteine ​​residue), or (d) any combination of (a) to (c). 19. The composition according to any one of items 1 to 18, wherein the molar ratio of enhancer 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 and is not 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 and 1000:1, between 1:1 and 500:1, between 1:1 and 250:1, between 1:1 and 200:1. 20. The composition according to any one of items 1 to 19, wherein the antigen is a polypeptide antigen comprising one or more MHC class I epitopes and / or MHC class II epitopes. 21. The polypeptide antigen is (a) a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or disorder suitable for treatment by vaccination and / or immunotherapy, or any antigenic fragment thereof, or (b) a coronavirus 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 base variant antigen, a mutated frameshift antigen, a splice variant antigen, a gene fusion antigen, an endogenous retroelement antigen, or a human 21. The composition according to item 20, which is or comprises another class of antigen, such as human leukocyte antigen (HLA)-somatic mutation-derived antigens or post-translational TSA, cancer antigens derived from viruses (e.g. human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV)), cancer-testis antigens, HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), EGFR, cell lysates or other substances derived from tumors (e.g. exosomes derived from tumors). 22. The composition according to any one of items 1 to 21, wherein the enhancer allows (i) increased cytosolic delivery of the antigen compared to a corresponding composition lacking the enhancer, (ii) increased total cellular delivery of the antigen compared to a corresponding composition lacking the enhancer, (iii) enhanced cellular immunity to the antigen compared to a corresponding composition lacking the enhancer, (iv) increased IFN-γ production by CD8+ T cells upon exposure to the antigen compared to a corresponding composition lacking the enhancer, (v) enhanced humoral immunity to the antigen compared to a corresponding composition lacking the enhancer, (vi) increased antibody species diversity to the antigen compared to a corresponding composition lacking the enhancer, or (vii) any combination of (i) to (vi). 23. The composition according to any one of items 1 to 22, further comprising a pharma- ceutically acceptable excipient and / or adjuvant. 24. A cell culture comprising a cell population and a composition according to any one of items 1 to 23. 25. The cell culture of item 24, wherein the cells comprise immune cells (e.g., T cells), antigen-presenting cells (e.g., dendritic cells, macrophages, engineered antigen-presenting cells), MHC class I-expressing cells, MHC class II-expressing cells, or any combination thereof. 26. A vaccine comprising a composition according to any one of items 1 to 22 or comprising cells produced using the cell culture according to item 24 or 25. 27. The vaccine according to item 26, which is a therapeutic or prophylactic vaccine (e.g. an anti-cancer vaccine, an anti-viral vaccine, or an anti-bacterial vaccine). 28. A method for inducing an enhanced adaptive immune response to an antigen of interest in a subject, comprising administering to the subject a composition according to any one of items 1 to 23, or a cell produced using the cell culture according to item 24 or 25. 29. A method for vaccinating a subject against an infectious disease, comprising administering to the subject a cell produced using the composition according to any one of items 1 to 23 or the cell culture according to item 24 or 25, wherein the antigen comprises an antigenic fragment of a pathogen (e.g. virus, bacterium, fungus) causing the infectious disease. 30. A method for treating cancer in a subject, comprising administering to the subject cells produced using the composition according to any one of items 1 to 23 or the cell culture according to item 24 or 25, wherein the antigen is overexpressed or aberrantly expressed in the cancer-causing cells. 31. A steroid acid-peptide conjugate for use in admixture with an antigen. 32. A steroid acid-peptide conjugate for use according to item 31, wherein the steroid acid-conjugate and / or the antigen is as described in any one of items 1 to 22. 33. Use of the composition according to any one of items 1 to 23 or the cell culture according to item 24 or 25 for generating an immune response in a subject or for the manufacture of a medicament (e.g. a vaccine) for generating an immune response in a subject. 34. The use according to item 33, wherein the immune response comprises enhanced cellular immunity to the antigen, increased IFN-γ production by CD8+ T cells upon exposure to the antigen, enhanced humoral immunity to the above, or any combination thereof, compared to that generated from a corresponding composition or cell culture lacking the enhancer. 35. A method for improving the immunogenicity of a polypeptide antigen, the method comprising admixing the polypeptide antigen with an enhancer of antigen presentation, the enhancer comprising a bile acid-peptide conjugate in an amount sufficient to improve presentation of the polypeptide antigen upon administration to an antigen-presenting cell or a subject compared to administration of a corresponding composition lacking the enhancer, and the peptide comprised in the bile acid-peptide conjugate comprises a nuclear localization signal (NLS). 36. The method according to item 35, wherein the bile acid is cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), 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). 37. The method of claim 35 or 36, wherein the bile acid is an analog of CA, CDCA, DCA, LCA, GCA, TCA, GDCA, GCDCA, TDCA, GLCA, TLCA, THDCA, TCDCA, UCA, TUDCA, UDCA, or GUDCA, and the analog induces endocytosis, induces ceramide accumulation on the inner leaflet of endosomes, or causes an increase in acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide. 38. The method according to item 35 or 36, wherein the steroid acid is or comprises glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UDCA), or lithocholic acid (LCA). 39. The method according to any one of items 35 to 38, wherein the NLS is SV40 NLS (SEQ ID NO: 1 or 2), GWG-SV40NLS (SEQ ID NO: 3), hnRNPA1 M9 NLS (SEQ ID NO: 4), hnRNP D NLS (SEQ ID NO: 5), hnRNP M NLS (SEQ ID NO: 10), PQBP-1 NLS (SEQ ID NO: 6), NLS2-RG domain RPS17 (SEQ ID NO: 11), NLS1 RPS17 (SEQ ID NO: 15), NLS2 RPS17 (SEQ ID NO: 7), NLS3 RPS17 (SEQ ID NO: 8), cMyc NLS (SEQ ID NO: 12), HuR NLS (SEQ ID NO: 13), Tus NLS (SEQ ID NO: 14) or nucleoplasmin NLS (SEQ ID NO: 16). 40. The method according to any one of items 35 to 38, wherein the NLS is a mutant of an NLS having nuclear localization activity, and the NLS comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 8 or 10 to 16. 41. The method according to any one of items 35 to 40, wherein the peptide is a non-immunogenic peptide. 42. The method according to any one of items 35 to 41, wherein the peptide has a length of 10 to 100 amino acids. 43. The method according to any one of items 35 to 41, wherein the molar ratio of enhancer to antigen in the composition is from 1:1 to 1000:1. 44. The method according to any one of items 35 to 41, wherein the molar ratio of enhancer to antigen in the composition is from 1:1 to 500:1. 45. The method according to any one of items 35 to 41, wherein the molar ratio of enhancer to antigen in the composition is from 1:1 to 250:1. 46. ​​The method according to any one of items 35 to 45, wherein the antigen is a polypeptide antigen comprising one or more MHC class I epitopes and / or MHC class II epitopes. 47. The method according to any one of items 35 to 46, wherein the polypeptide antigen is or comprises a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a tumor-derived cell lysate, a tumor-derived exosome, a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, or other antigen associated with a disease or disorder suitable for treatment by vaccination and / or immunotherapy. 48. The method according to any one of items 35 to 47, wherein the polypeptide antigen is or comprises a SARS-CoV spike protein or an antigenic fragment thereof. 49. The method of any one of items 35-48, wherein the enhancer allows for (i) increased cytosolic delivery of the antigen compared to a corresponding composition lacking the enhancer, (ii) increased total cellular delivery of the antigen compared to a corresponding composition lacking the enhancer, (iii) enhanced cellular immunity to the antigen compared to a corresponding composition lacking the enhancer, (iv) increased IFN-γ production by CD8+ T cells upon exposure to the antigen compared to a corresponding composition lacking the enhancer, (v) enhanced humoral immunity to the antigen compared to a corresponding composition lacking the enhancer, (vi) increased antibody species diversity to the antigen compared to a corresponding composition lacking the enhancer, or (vii) any combination of (i)-(vi). 50. An immunogenic composition comprising a polypeptide antigen and an antigen presentation enhancer according to item 35, or a cell population comprising the polypeptide antigen and the antigen presentation enhancer according to item 1, and a pharma- ceutically acceptable excipient and / or adjuvant. 51. The immunogenic composition of item 50, wherein the cell population comprises dendritic cells, B cells, T cells, macrophages, genetically engineered antigen-presenting cells, MHC class I expressing cells, MHC class II expressing cells, or any combination thereof. 52. A method for inducing an enhanced adaptive immune response to an antigen of interest in a subject, comprising administering to the subject an immunogenic composition according to item 50 or 51. EXAMPLES

[0046] Example 1: General Materials and Methods Generation of bile acid-NLS moiety The bile acid-NLS moiety was synthesized similarly to the synthesis of cholic acid-NLS (ChAcNLS) previously described by Beaudoin et al., 2016. For example, in CA-SV40NLS, 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) containing the nuclear localization signal from the SV40 large T antigen (SEQ ID NO: 2) adjacent to linker amino acids.

[0047] Generation of bone marrow-derived dendritic cells Mouse bone marrow-derived dendritic cells (BMDCs) were generated by flushing whole bone 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. After lysis of red blood cells, cells were cultured in medium supplemented with 50 ng / mL mouse recombinant GM-CSF. Medium was changed on days 2, 4, 6, and 8. On day 9, medium was replaced to contain recombinant mouse GM-CSF and LPS from E. coli O111 (1 ng / mL) to stimulate dendritic cell (DC) maturation. Mature DCs were assessed by flow cytometry for their surface expression of CD3, CD19, NK1.1, CD11c, CD80, CD86, and I-Ab.

[0048] Antigen cross-presentation assay To assess antigen cross-presentation, cells were plated in 24-well plates (Corning; Massachusetts, United States) at 25 × 10 cells per well. 3 The mice were seeded with 10 cells and then pulsed with various concentrations of antigen or antigen-containing admixtures for 3 h. At the end of the pulse period, the cells were washed to remove excess antigen and incubated with 10 T cells purified from the spleens of OT-I mice using a T cell isolation kit according to the manufacturer's protocol. 6The cells were co-cultured with CD8 T cells at 100 / mL. After 72 hours, the supernatants were collected and used to quantify cytokine production by commercially available enzyme-linked immunosorbent assays (ELISAs).

[0049] Antigen presentation assay using the B3Z reporter system Various bile acid-NLS conjugates were screened using the B3Z reporter system. The B3Z cell line expresses H2-K b -SIINFEKL complex specific T cell hybridoma. Upon activation via its TCR, a LacZ reporter gene (under the control of the NFAT promoter) is expressed. Briefly, 1.5 × 10 5 BMDCs or 2.5 x 10 5 5 × 10 MSCs were treated with a mixture of ovalbumin (OVA) and bile acid-NLS conjugate. 4 B3Z cells and incubated at 37°C, 5% CO 2 The next day, all cells were washed twice with PBS (pH 7.4) and the cell pellet was resuspended in 0.15 mM chlorophenol red-β-D-galactopyranoside (CPRG) substrate (Calbiochem, La Jolla, CA), 0.125% NP40 (EMD Sciences, La Jolla, CA), 9 mM MgCl 2 The OVA was dissolved by adding 100 μL of dissolution buffer containing 10 mM 2-mercaptoethanol (Aldrich, USA) and 100 mM 2-mercaptoethanol in PBS. After 5 or 24 h of incubation at 37°C, the absorbance was measured at 570 nm with a reference wavelength of 636 nm. For these experiments, OVA was resuspended in PBS (pH 7.3) at 5–10 mg / mL. The various bile acid-NLS conjugates were dissolved in H2SO4 at 10 mg / mL. 2 The bile acid-NLS conjugate:antigen mixtures were prepared at various molar ratios according to Table 2.

[0050] [Table 2]

[0051] Example 2: Cholic acid and cholic acid-SV40NLS enhance OVA antigen cross-presentation / immunogenicity Cross-presentation assays were performed to screen for agents that may improve the cross-presentation and / or immunogenicity of antigens in non-covalent admixture with antigen. Mouse BMDCs were pre-pulsed with either antigen alone or antigen mixed with candidate immunogenic enhancers at various ratios. Pulsed BMDCs were then co-cultured with CD8 T cells from the spleens of OT-I mice, and the amount of IFN-γ produced by CD8 T cells was quantified as a measure of cross-presentation activity. Preliminary screening identified cholic acid and cholic acid-NLS peptide conjugates as potential immunogenic enhancers. Figure 1 shows the results of a cross-presentation assay in which BMDCs were pre-pulsed with the antigen ovalbumin alone ("OVA alone"), cholic acid alone ("CA alone"), cholic acid-NLS peptide conjugate alone ("CA-SV40NLS alone"), or various ratios of cholic acid:antigen admixture ("CA:OVA") or cholic acid-NLS peptide conjugate:antigen admixture ("CA-SV40NLS:OVA"). Interestingly, BMDCs prepulsed with the highest ratio of cholic acid:antigen admixture (CA:OVA) tested resulted in up to a 3-fold increase in the amount of IFN-γ produced compared to BMDCs prepulsed with OVA antigen alone. Strikingly, BMDCs prepulsed with the highest ratio of cholic acid-NLS peptide conjugate:antigen admixture (CA-SV40NLS:OVA) tested resulted in a 6-7-fold increase in the amount of IFN-γ produced compared to BMDCs prepulsed with OVA antigen alone.

[0052] Example 3: Enhancement of antigen presentation in the presence of SV40NLS conjugated to various bile acids Mutants of CA-SV40NLS were synthesized to investigate the structure-activity relationship for the antigen cross-presentation enhancement activity observed for this conjugate. More specifically, conjugates with various bile acids conjugated to the SV40NLS peptide (SEQ ID NO: 1) were synthesized and their effects on antigen presentation were evaluated by using the B3Z reporter system with OVA antigen as described in Example 1. The results in Figure 2 show that when OVA was mixed with the CA-SV40NLS conjugate, an increase in antigen cross-presentation was observed compared to OVA antigen alone ("OVA alone"; dashed line). These results were consistent with those observed using the OT-I CD8 T cell-based assay (Figure 1). Interestingly, when cholic acid was replaced with the bile acids: glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UDCA), or lithocholic acid (LCA), antigen cross-presentation comparable to or higher than CA-SV40NLS was observed. In Figure 2, when OVA was mixed with either unconjugated cholic acid ("CA") or SV40NLS peptide ("SV40NLS"), no increase in antigen cross-presentation was observed over antigen alone ("OVA"), which may have been due to the lower sensitivity of the B3Z reporter system compared to the OT-I CD8 T cell-based assay used in Figure 1. Interestingly, a subsequent assay using the same B3Z reporter system showed that when OVA was mixed with unconjugated glycoursodeoxycholic acid (GUDCA; 22:1), up to approximately 30% of the B3Z response (OD 20:1) was greater than OVA alone (data not shown). 570 Furthermore, the immunogenicity enhancer activity of GUDCA was observed at all GUDCA:OVA molar ratios tested (i.e., 2:1, 4:1, 8:1, 12:1, and 22:1).

[0053] Example 4: Enhancement of antigen presentation in the presence of SV40NLS conjugated to various bile acids Further mutants of CA-SV40NLS were synthesized in which the SV40NLS peptide was replaced with peptides containing other NLS peptides (Table 3), and the antigen presentation activity of the CA-NLS peptide conjugates was 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 Figures 3-6 show that the antigen presentation activity of the various conjugates was correlated with the highest B3Z response (OD ) for that conjugate. 570 ) are compared in terms of the conjugate:antigen ratio that resulted in

[0054] [Table 3]

[0055] The results in Figures 3-6 generally show that increased antigen presentation can be achieved by exposing antigen-presenting cells to antigen in the presence of cholic acid conjugated to peptides containing various types of nuclear localization signals and having various amino acid sequences.

[0056] Using BMDCs as antigen-presenting cells, the glutamate-rich peptide PQBP-1 NLS was associated with a significantly higher antigen-presenting activity (Figures 3 and 5). Furthermore, the NLS2-RG domain RPS17, NLS3-RPS17, cMyc NLS, and HuR NLS peptides were also associated with high antigen-presenting activity. Interestingly, the peptide GWG-SV40NLS was associated with a higher antigen-presenting activity than SV40NLS, suggesting that the addition of adjacent aromatic amino acids (WW or GWWG) is beneficial for activity (see Figures 3-5). Similar results were observed using a DC cell line (DC2.4) as antigen-presenting cells.

[0057] Using a cross-presenting cell line of MSCs as antigen-presenting cells, various cholic acid peptide conjugates enhanced antigen presentation of OVA (Figure 6). Similar to BMDCs, PQBP-1 NLS, HuR NLS, and GWG-SV40NLS were associated with significantly higher antigen-presenting activity compared to OVA alone or OVA mixed with CA-SV40NLS.

[0058] To further investigate the effect of bile acid peptide conjugates on antigen presentation, antigen internalization and processing were evaluated. MSC cell lines were pulsed with OVA labeling with AF647 in the presence of various bile acid peptide conjugates (NLS1-RPS17 [Fig. 7A], NLS3 RPS17 [Fig. 7B], PQBP-1 [Fig. 7C], and hnRNPA1 M9 NLS [Fig. 7D]) at various molar ratios, and fluorescence was evaluated by flow cytometry. Bile acid conjugates were shown to enhance OVA internalization, generally with increasing ratios. OVA processing was evaluated by pulsing MSC cell lines with DQ™-ovalbumin (OVA-DQ) in the presence of the same bile acid peptide conjugates as in Fig. 7A-7D. Bile acid conjugates NLS1-RPS17 [Figure 8A], NLS3 RPS17 [Figure 8B], PQBP-1 [Figure 8C], and hnRNPA1 M9 NLS [Figure 8D] were shown to enhance OVA processing, generally with increasing ratios.

[0059] Altogether, these data demonstrate the versatility and potency of bile acid peptide conjugates in enhancing antigen presentation.

[0060] References Al-Hilal et al.,(2014).Functional transformations of bile acid transporters induced by high-affinity macromolecules.Scientific Reports,4:4163.doi:10.1038 / srep04163. Azuar et al.,(2019).Cholic Acid-based Delivery System for Vaccine Candidates against Group A Streptococcus.ACS Medicinal Chemistry Letters,10:1253-1529。 Beaudoin et al.,(2016).ChAcNLS,a novel modification to antibody-conjugates permitting target cell-specific endosomal escape,localization to the nucleus and enhanced total intracellular accumulation.Molecular Pharmaceutics,13(6):1915-26。 Hanafi et al.,(2018).Overview of Bile Acids Signaling and Perspective on the Signal of Ursodeoxycholic Acid,the Most Hydrophilic Bile Acid,in the Heart.Biomolecules,8(4):159。 Liu et al.,(2020),The Renpenning syndrome-associated protein PQBP1 facilitates the nuclear import of splicing factor TXNL4A through the karyopherin 2 receptor.Journal of Biological Chemistry,295(13):4093-4100。 Murakami et al.,(2020).Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.Proceedings of the National Academy of Sciences USA.117(3):1700-1710。 Patel et al.,(2017).Next generation approaches for tumor vaccination.Chinese Clinical Oncology.6(2):19。 Shivanna et al.,(2014)The crucial role of bile acids in the entry of porcine enteric calicivirus.Virology 456~457,268~278。 Shivanna et al.,(2015).Ceramide formation mediated by acid sphingomyelinase facilitates endosomal escape of caliciviruses.Virology,4483,218~228。 Smith et al.,(2019).Alternative tumor-specific antigens.Nature Review Cancer.19(8):465-478。 Sun et al.,(2016).Factors influencing the nuclear targeting ability of nuclear localization signals.Journal of Drug Targeting,24(10):927-933。 Tagliamonte et al.,(2014).Antigen-specifc vaccines for cancer treatment.Human Vaccines & Immunotherapeutics,10(11):3332-3346。

Claims

1. A composition comprising a polypeptide antigen admixed with an enhancer of antigen presentation, said enhancer comprising a steroid acid-peptide conjugate in an amount sufficient to improve presentation of said antigen upon administration of said composition to an antigen presenting cell compared to administration of a corresponding composition lacking said enhancer; A composition, wherein the peptide contained in the steroid acid-peptide conjugate comprises a nuclear localization signal (NLS), and the peptide contained in the steroid acid-peptide conjugate is a non-immunogenic peptide.

2. The steroid acid in the enhancer or in the steroid acid-peptide conjugate is (i) induces ceramide accumulation on the inner leaflet of endosomes, thereby destabilizing endosomal membranes in said antigen-presenting cells and promoting endosomal escape of said antigen into the cytosol; and / or (ii) a steroid acid that induces an increase in acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.

3. The composition of claim 1 , wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a bile acid.

4. The composition of claim 1, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a primary or secondary bile acid.

5. The steroid acid in the enhancer or in the steroid acid-peptide conjugate is (a) a bile acid which is cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), 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) above, which induces endocytosis, induces ceramide accumulation on the inner leaflet of the endosome, induces an increase in 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 analogue that is more hydrophobic than cholic acid; or (d) The composition of claim 1 which is or comprises any combination of (a)-(c).

6. 2. The composition of claim 1, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is or comprises glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UDCA), or lithocholic acid (LCA).

7. The NLS is (i) a classical NLS comprising a K-K / R-X-K / R motif, a PY-NLS comprising one or more PY motifs, such as towards the C-terminus of said NLS, or comprising the motif R-X 2-5 -PY and / or having Kapβ2 binding activity, a PL-NLS comprising one or more PL motifs, such as towards the C-terminus of said NLS, a ribosomal NLS, an NLS further comprising a nucleolar targeting signal, or any combination thereof; (ii) is or is derived from SV40 NLS, cMyc NLS, hnRNPA1 M9 NLS, hnRNP D NLS, hnRNP M NLS, PQBP-1 NLS, HuR NLS, Tus NLS, nucleoplasmin NLS, NLS1 RPS17, NLS2 RPS17, NLS3 RPS17, or NLS2-RG domain RSP17; or (iii) the composition of claim 1 comprising at least three acidic residues and / or at least three basic residues.

8. The peptide is (i) contains an endosomolytic motif that facilitates endosomal escape; (ii) comprises a protein transduction domain that stimulates endocytosis and / or endosome formation; (iii) lacking a protein transduction domain or a cell membrane penetrating peptide; (iv) at least 7, 8, 9, 10, 11, or 12 amino acids in length and no more than 50-100 amino acids in length, and / or 10-100 amino acids in length; or (v) The composition of claim 1 which is any combination of (i) to (iv).

9. The peptide is any one of the peptides of SEQ ID NOs: 1 to 8 or 10 to 16, or a variant thereof; (a) when conjugated to a steroidal acid, it confers improved antigen-presenting activity to the steroidal acid compared to the antigen-presenting activity of the corresponding unconjugated steroidal acid; (b) has nuclear localization and / or endosomolytic activity; (c) differs from any one of the peptides of SEQ ID NOs: 1-8 or 10-16 by not more than 1, 2, 3, 4, or 5 amino acid substitutions or deletions; or (d) The composition of claim 1, comprising or consisting of a peptide or variant thereof, which is any combination of (a) to (c).

10. The enhancer comprises a steroid acid-peptide conjugate, the steroid acid being (a) 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 and 10:1; (b) at free amino and / or free thiol groups of the peptide, (c) at or towards the N-terminus of the peptide, or The composition of claim 1, wherein any combination of (d) (a) to (c) is conjugated to a peptide.

11. 2. The composition of claim 1, wherein the molar ratio of enhancer 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, or 20:1 and is no greater 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, or 1000:1 and / or is between 1:1 and 1000:1, between 1:1 and 500:1, between 1:1 and 250:1, or between 1:1 and 200:

1. (i) the polypeptide antigen comprises one or more MHC class I epitopes and / or MHC class II epitopes; or (ii) the polypeptide antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or disorder suitable for treatment by vaccination and / or immunotherapy, or any antigenic fragment thereof; or (iii) The composition of claim 1, wherein the polypeptide antigen is or comprises a coronavirus antigen, such as SARS-CoV-2 spike protein, SARS-CoV spike protein, or an antigenic fragment thereof; a single base mutant cancer antigen; a mutated frameshift cancer antigen; a splice variant cancer antigen; a gene fusion cancer antigen; an endogenous retroelement cancer antigen; a human leukocyte antigen (HLA)-somatic mutation derived cancer antigen; a post-translational TSA; a cancer antigen derived from a virus, such as human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV); a cancer-testis antigen, such as HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), or EGFR; a cell lysate; or a tumor-derived material.

13. The enhancer comprises: (i) increased cytosolic delivery of the antigen compared to a corresponding composition lacking the enhancer; (ii) increased total cellular delivery of the antigen compared to a corresponding composition lacking the enhancer; (iii) enhanced cellular immunity against the antigen, as compared to a corresponding composition lacking the enhancer; (iv) increased IFN-γ production by CD8+ T cells upon exposure to said antigen, compared to a corresponding composition lacking said enhancer; (v) enhanced humoral immunity to the antigen, as compared to a corresponding composition lacking the enhancer; (vi) an increase in the diversity of antibody species against the antigen compared to a corresponding composition lacking the enhancer; or (vii) The composition of claim 1, which enables any combination of (i) to (vi).

14. The composition of claim 1 further comprising a pharma- ceutically acceptable excipient and / or adjuvant.

15. 13. A cell culture comprising a population of cells and the composition of claim 1.

16. 16. The cell culture of claim 15, wherein the cells comprise immune cells, antigen presenting cells, MHC class I expressing cells, MHC class II expressing cells, or any combination thereof.

17. A vaccine comprising the composition of claim 1.

18. 18. The vaccine of claim 17 which is a therapeutic or prophylactic vaccine.

19. eliciting an enhanced adaptive immune response to an antigen of interest in a subject; vaccinating a subject against an infectious disease, wherein the antigen comprises an antigenic fragment of a pathogen causing the infectious disease; Treating cancer in a subject, wherein the antigen is overexpressed or aberrantly expressed in cells causing the cancer; or For generating an immune response in a subject, or for the manufacture of a medicament for generating an immune response in a subject.

18. A composition according to claim 1, a cell produced using the cell culture according to claim 15, or a vaccine according to claim 17 for use in

20. 20. The composition, cell, or vaccine of claim 19, wherein the immune response comprises enhanced cellular immunity to the antigen, increased IFN-γ production by CD8+ T cells upon exposure to the antigen, enhanced humoral immunity to the antigen, or any combination thereof, compared to that generated from a corresponding composition or cell culture lacking the enhancer.

21. A method for improving the immunogenicity of a polypeptide antigen, the method comprising admixing the polypeptide antigen with an enhancer of antigen presentation, the enhancer comprising a steroid acid-peptide conjugate in an amount sufficient to improve presentation of the polypeptide antigen upon administration to an antigen presenting cell or a subject compared to administration of a corresponding composition lacking the enhancer, wherein the peptide comprised in the steroid acid-peptide conjugate comprises a nuclear localization signal (NLS), and wherein the peptide in the steroid acid-peptide conjugate is a non-immunogenic peptide.

22. The steroid acid in said enhancer or in said steroid acid-peptide conjugate, (i) induces ceramide accumulation on the inner leaflet of endosomes, thereby destabilizing endosomal membranes in said antigen-presenting cells and promoting endosomal escape of said antigen into the cytosol; and / or 22. The method of claim 21, wherein (ii) the steroid acid induces an increase in acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.

23. The method of claim 21, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a bile acid.

24. The method of claim 21, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is a primary bile acid or a secondary bile acid.

25. The steroid acid in said enhancer or in said steroid acid-peptide conjugate, (a) a bile acid which is cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), 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) above, which induces endocytosis, induces ceramide accumulation on the inner leaflet of the endosome, induces an increase in 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 analogue that is more hydrophobic than cholic acid; or (d) The method of claim 21, which is or includes any combination of (a)-(c).

26. The method of claim 21, wherein the steroid acid in the enhancer or in the steroid acid-peptide conjugate is or comprises glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UDCA), or lithocholic acid (LCA).

27. ​​The NLS, (i) a classical NLS comprising a K-K / R-X-K / R motif, a PY-NLS comprising one or more PY motifs, such as towards the C-terminus of said NLS, or comprising the motif R-X 2-5 -PY and / or having Kapβ2 binding activity, a PL-NLS comprising one or more PL motifs, such as towards the C-terminus of said NLS, a ribosomal NLS, an NLS further comprising a nucleolar targeting signal, or any combination thereof; (ii) an SV40 NLS, a cMyc NLS, hnRNPA1 M9 NLS, hnRNP D NLS, hnRNP M NLS, PQBP-1 NLS, HuR NLS, Tus NLS, nucleoplasmin NLS, NLS1 RPS17, NLS2 RPS17, NLS3 RPS17, or the NLS2-RG domain RSP17, or derived therefrom, or (iii) at least three acidic residues and / or at least three basic residues.

28. The peptide, (i) contains an endosomolytic motif that facilitates endosomal escape; (ii) comprises a protein transduction domain that stimulates endocytosis and / or endosome formation; (iii) lacking a protein transduction domain or a cell membrane penetrating peptide; (iv) at least 7, 8, 9, 10, 11, or 12 amino acids in length and no more than 50-100 amino acids in length, and / or 10-100 amino acids in length; or (v) any combination of (i) to (iv).

29. The peptide is any one of the peptides set forth in SEQ ID NOs: 1 to 8 or 10 to 16, or a variant thereof, (a) when conjugated to a steroidal acid, it confers improved antigen-presenting activity to the steroidal acid compared to the antigen-presenting activity of the corresponding unconjugated steroidal acid; (b) has nuclear localization and / or endosomolytic activity; (c) differs from any one of the peptides of SEQ ID NOs: 1-8 or 10-16 by not more than 1, 2, 3, 4, or 5 amino acid substitutions or deletions; or (d) The method of claim 21, comprising or consisting of a peptide, or variant thereof, which is any combination of (a) to (c).

30. The enhancer comprising a steroid acid-peptide conjugate, the steroid acid being (a) 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 and 10:1; (b) at free amino and / or free thiol groups of the peptide, (c) at or towards the N-terminus of the peptide, or 22. The method of claim 21 , wherein (d) any combination of (a)-(c) is conjugated to a peptide.

31. The method of claim 21, wherein the molar ratio of enhancer 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, or 20:1 and is not greater 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, or 1000:1, and / or is 1:1 to 1000:1, 1:1 to 500:1, 1:1 to 250:1, or 1:1 to 200:

1.

32. (i) the polypeptide antigen comprises one or more MHC class I epitopes and / or MHC class II epitopes; or (ii) the polypeptide antigen is a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or disorder suitable for treatment by vaccination and / or immunotherapy, or any antigenic fragment thereof; or 22. The method of claim 21, wherein the polypeptide antigen is or comprises a coronavirus antigen, such as SARS-CoV-2 spike protein, SARS-CoV spike protein, or an antigenic fragment thereof; a single base mutant cancer antigen; a mutated frameshift cancer antigen; a splice variant cancer antigen; a gene fusion cancer antigen; an endogenous retroelement cancer antigen; a human leukocyte antigen (HLA)-somatic mutation derived cancer antigen; a post-translational TSA; a cancer antigen derived from a virus, such as human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV); a cancer-testis antigen, such as HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), or EGFR; a cell lysate; or a tumor-derived material.

33. The enhancer, (i) increased cytosolic delivery of the antigen compared to a corresponding composition lacking the enhancer; (ii) increased total cellular delivery of the antigen compared to a corresponding composition lacking the enhancer; (iii) enhanced cellular immunity against the antigen, as compared to a corresponding composition lacking the enhancer; (iv) increased IFN-γ production by CD8+ T cells upon exposure to said antigen, compared to a corresponding composition lacking said enhancer; (v) enhanced humoral immunity to the antigen, as compared to a corresponding composition lacking the enhancer; (vi) an increase in the diversity of antibody species against the antigen compared to a corresponding composition lacking the enhancer; or (vii) The method of claim 21, which enables any combination of (i) to (vi).

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