FIELD OF THE INVENTION

The present invention relates to novel methods for immunization, in particular methods for immunization comprising intradermal administration of immunogenic peptides in combination with an adjuvant.

BACKGROUND OF THE INVENTION

Immunization involves the administration of a substance (an antigen) to a patient in order to induce an immune response against said antigen. The purpose of an immunization can be to prevent a disease (prophylactic immunization) or to treat an existing disease (therapeutic immunization). Antigens can be derived from pathogens or can be disease- related, for example an antigen found in tumour cells but not in normal cells used for a therapeutic immunization against cancer. Specific immune responses against antigens can often be further stimulated by the co-administration of adjuvants. Adjuvants are known in the art to accelerate, prolong, or enhance the quality of the specific immune response to antigens. Many different types of adjuvants have been described in the art.

One set of adjuvants act through toll-like receptors (TLRs). TLRs recognize specific patterns of microbial components, especially those from pathogens, and regulate the activation of both innate and adaptive immunity. Thirteen members of the TLR-family have been identified in man. TLRs are expressed by phagocytic cells such as monocytes, macrophages and dendritic cells. A known lipopeptide adjuvant which interacts with tolllike receptor 2 (TLR2) is the Pam3Cys-lipopeptide. Improved variants of Pam3Cys have been described in Willems et al. (2014) J Med Chem 57:6873 and WO2013051936.

While significant progress has been made in the field of prophylactic and therapeutic immunization, there is still a need for improved methods for immunization that result in more rapid, stronger and/or prolonged immune responses.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an immunogenic peptide for use in combination with a Toll-like receptor 2 agonist in the immunization of a human subject, wherein said Toll-like receptor 2 agonist is a lipopeptide which is not covalently linked to said immunogenic peptide and wherein said immunogenic peptide and said Toll-like receptor 2 agonist are administered intradermally (i.d.) at the same site.

In a further aspect, the invention relates to an immunogenic composition comprising one or more immunogenic peptides of the invention as described herein and a pharmaceutically-acceptable carrier for use in combination with a Toll-like receptor 2 agonist in the immunization of a human subject, wherein said Toll-like receptor 2 agonist is a lipopeptide which is not covalently linked to said immunogenic peptide(s) and wherein said immunogenic peptide(s) and said Toll-like receptor 2 agonist are administered intradermally at the same site.

In a further aspect, the invention relates to a method for immunizing a human subject comprising administration of an immunogenic peptide in combination with a Tolllike receptor 2 agonist to said human subject, wherein said Toll-like receptor 2 agonist is a lipopeptide that is not covalently linked to said immunogenic peptide and wherein said immunogenic peptide and said Toll-like receptor 2 agonist are administered intradermally at the same site.

In an even further aspect, the invention relates to a pharmaceutical composition comprising an immunogenic peptide and a Toll-like receptor 2 agonist, wherein said Tolllike receptor 2 agonist is a lipopeptide which is not covalently linked to said immunogenic peptide.

DESCRIPTION OF THE FIGURES

Figure 1: SLP + Amplivant Administered Intradermally Induces Higher Antigen- Specific CD8 ⁺ T-cell Responses Compared to Subcutaneous Administration. Indicated the AAY-specific responses (to SLP-9) and VNF-specific responses (to SLP-8) over time. C57BL/6 mice were vaccinated i.d. or s.c. at day 0, day 14 and day 28 with a composition of 11 SLPs (50pg/SLP) with or without Amplivant (50pg). One week after each vaccination the antigen-specific CD8 ⁺ T cell response was assessed by flow cytometry. Abbreviations: i.d. intradermal, s.c. subcutaneous, Av Amplivant.

Figure 2: SLP + Amplivant Administered Intradermally Induces Higher IFNy CD8+ and CD4+ T-cell Response Compared to Subcutaneous Administration. In vitro restimulation of splenocytes of mice vaccinated i.d. or s.c., 3x in a prime-boost-boost setting with muSARS-CoV-2 composition (50pg/SLP) with or without Amplivant (50pg). Splenocytes were incubated with individual SLP (SLP-9, SLP-8 or pool M) or SLP pools (pool S, pool N). As negative controls, splenocytes were incubated with medium only (depicted as negative) or irrelevant peptide that was not used for vaccination of the mice (depicted as Ctrl peptide). Shown is the percentage of IFNy ⁺ cells of total CD8 ⁺ T-cells (left) or of total CD4 ⁺ T-cells (right) upon restimulation with muSARS-CoV-2 composition SLP.

Figure 3: SLP + Amplivant Administered Intradermally Induces Higher IgG Response Compared to Subcutaneous Administration. Final serum from each group was pooled and SLP-specific antibody in the serum was assessed by ELISA. Serum was diluted 1 :500, 1:5000 and 1 :50.000. Shown are OD450nm values of duplicate samples. Figure 4: Intradermal immunisation with 11 SLPs and Amplivant protects against SARS-CoV-2 challenge in K18-hACE2 mice. K18-hACE2 mice were vaccinated 3 times in 2-week intervals. At day 49 after the first vaccination, mice received the SARS-CoV-2 Leiden-08 isolate intranasally. Upon infection, weight was monitored daily. A) Survival of mice. B) Weight per animal.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

When used herein, the term "immunogenic peptide" refers to a peptide capable of inducing (including triggering or boosting) an immune response (such as a local and/or systemic CD4+ and/or CD8+ T cell response and/or an antibody response) in a host organism, typically a mammalian host organism, such as a human host. Likewise, the term "immunogenic composition" means a composition capable of inducing an immune response. An immunogenic peptide may for example have a length of from 8 to 100 amino acids, e.g. 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, 50, 55, 60, 70, 80, 90 amino acids or more. Preferably, a peptide is a synthetic long peptide (SLP), i.e. prepared synthetically (e.g. using solid phase synthesis) having a length of 18 to 100 amino acids, more preferably a peptide having a length of 20-45, e.g. 20-35, amino acid residues. An immunogenic peptide may comprise or consist of a fragment of a protein, typically a protein which is a suitable target for prophylactic or therapeutic immunisation. "Fragment" refers to a sequence of consecutive amino acids that corresponds to, i.e. is identical to, a part of a protein sequence. This does not exclude, however, that the immunogenic peptide or the fragment, while preserving immunogenic properties, may be further modified. For example, the immunogenic peptide of the invention may or may not comprise modified amino acids and/or non-naturally occurring amino acids and/or covalently linked groups.

In the context of describing peptides, the term "in length", for example a peptide of 20 to 45 amino acids or amino acid residues "in length" refers to the number of amino acid residues in the linear peptide chain.

The term "MHC class I ligand" refers to a peptide sequence that can bind to and be presented by an MHC class I molecule. MHC (major histocompatibility complex) class I molecules (in humans including HLA-A, HLA-B and HLA-C) are one of two classes of (MHC) molecules found on the cell surface of nucleated cells. Their function is to present peptide fragments of proteins to cytotoxic T cells, thus trigger an immune response. MHC class I ligands typically have a length of 8-11 amino acids. Proteins or long peptides, such as long peptides of the invention, that comprise MHC class I ligands typically require intracellular processing for the MHC class I ligand to be generated and made available to bind to the MHC Class I molecule and be presented on the cell surface. The intracellular processing typically occurs via proteasomal cleavage in the cytosol.

The term "MHC class II ligand" refers to a peptide sequence that can bind to and be presented by an MHC class II molecule. MHC (major histocompatibility complex) class II molecules (in humans including HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA- DR) are one of two classes of (MHC) molecules found on the cell surface of nucleated cells. Their function is to present peptide fragments of proteins to T helper cells, thus trigger an immune response. MHC class II ligands typically have a length of 11-16 amino acids. Proteins or long peptides, such as long peptides of the invention, that comprise MHC class II ligands typically require intracellular processing for the MHC class II ligand to be generated and made available to bind to the MHC Class II molecule and be presented on the cell surface. The intracellular processing of MHC class II ligands typically occurs via endocytosis and endosomal digestion.

Human Toll-like receptor 2 (TLR2) (UniProtKB-060603) is a member of the Toll-like receptor family. TLR2 is required for recognition of diverse microbial molecules from broad groups of species such as Gram-positive and Gram-negative bacteria, as well as mycoplasma and yeast. Specifically, TLR2 is involved in the recognition of cell-wall components, lipoteichoic acid and lipoprotein, from gram-positive bacteria; lipoarabinomannan, from mycobacteria; and zymosan, from yeast. TLR2 is involved in the specific recognition of a wide range of ligands, either as a homodimer or as a heterodimer with TLR1 or TLR6.

The term "Toll-like receptor 2 agonist" or "TLR2 agonist" when used herein refers to a compound that binds to TLR2 and activates it to produce a biological response. The term includes compounds that bind to TLR2 homodimers as well as compounds that bind to TLR1/2 or TLR2/6 heterodimers.

The term "lipopeptide" refers to a compound comprising, or consisting of, a lipid covalently linked to a peptide.

When used herein, the term "same site", in the context of intradermal administration of two or more compositions, refers to the two or more compositions being administered, for example by injection, to essentially same location on the dermis, i.e. within a distance of 1 cm, such as within 0.5 cm, for example within 0.25 cm of each other.

Further aspects and embodiment of the invention

As described above, in a first aspect, the invention relates to an immunogenic peptide for use in combination with a Toll-like receptor 2 agonist in the immunization of a human subject, wherein said Toll-like receptor 2 agonist is a lipopeptide which is not covalently linked to said immunogenic peptide and wherein said immunogenic peptide and said Tolllike receptor 2 agonist are administered intradermally at the same site.

Immunogenic peptides

Immunogenic peptides used in the invention are typically derived from a protein (also termed target or target protein herein) such as a human protein, for example a human protein present on tumour cells, or a protein from a pathogen, such as a virus.

Proteins that are specifically expressed by infected, pre-cancerous and/or cancerous cells are suitable targets for therapeutic immunization. Examples of viral proteins that are targets for prophylactic and therapeutic vaccines are proteins derived from hepatitis B virus (HBV), human papilloma virus (HPV) and coronavirus (SARS-CoV-2). Thus, in one embodiment, the immunogenic peptide used in the invention comprises or consists of a fragment of a HBV, HPV or SARS-CoV-2 protein.

Suitable target proteins from HBV e.g. genotypes A, B, C or D, including Core protein (HBcAg) (e.g. UniProtKB Q21360), Polymerase (Pol protein) (e.g. UniProtKB 011885), protein HBsAg (e.g. UniProtKB Q773S4), X-protein (e.g. UniProtKB Q8V1H6), Large envelope protein (e.g. UniProtKB P03138) and capsid protein (e.g. UniProtKB P03147). Suitable target protein antigens from HPV e.g. genotypes 6 and 11 which induce genital warts and oncogenic genotypes 16, 18, 31, 33, 45, 52 and 58 include L2 protein (e.g. UniProtKB P03107; P06793), E2 protein (e.g. UniProtKB P03120, P06790), E6 oncoprotein (e.g. UniProtKB P03126; P06463) and E7 oncoprotein (e.g. UniProtKB P03129; P06788).

In a preferred embodiment, the target protein is an HBV protein, in particular X- protein, Polymerase (Pol protein) or Core protein (HBcAg) or splice variants thereof. Suitable HBV-derived immunogenic peptides have been described in W02015187009 and W02021110919 (both ISA Pharmaceuticals) (herein incorporated by reference).

In another preferred embodiment, the target protein is an HPV protein, in particular E6 or E7. Suitable HPV-derived immunogenic peptides have been described in WO2017220463 and WO2021156404 (both ISA Pharmaceuticals).

In another preferred embodiment, the target protein is a SARS-CoV-2 protein, in particular S protein, M protein or N protein. Suitable SARS-Cov-2-derived immunogenic peptides have been described in WO2021214297 (ISA Pharmaceuticals).

Non-viral proteins that are suitable targets for prophylactic and therapeutic immunization may be tumour specific proteins and/or tumour associated proteins, often termed tumour specific antigens and tumour associate antigens. In one embodiment, the target protein is PRAME or Melanoma antigen preferentially expressed in tumours (e.g. UniProtKB P78395). Suitable PRAME-derived immunogenic peptides have been described in W008118017 (LUMC).

In one embodiment, the immunogenic peptide used in the invention comprises or consists of a fragment of a protein selected from the group consisting of: HBV protein X, HBV Polymerase, HBV Core, HBV Surface antigen, HPV E6, HPV E7 and human PRAME, SARS-CoV-2 S protein, SARS-CoV-2 M protein and SARS-CoV-2 N protein.

In one embodiment, the immunogenic peptide used in the invention is from 20 to 45 amino acid residues in length, such as 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 or 45 amino acids in length.

In one embodiment, the immunogenic peptide used in the invention comprises at least one MHC class I ligand and/or least one MHC class II ligand.

In one embodiment, the immunogenic peptide used in the invention has the ability to induce a CD8+ response and/or a CD4+ response and/or an antibody response (such as an IgG response) in a human subject.

In one embodiment, the immunogenic peptide used in the invention comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 1-157.

In one embodiment, the immunogenic peptide used in the invention comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 1-146.

Table 1: Sequence listing

Toll-like receptor 2 agonists

As described above, the method of the invention comprises administration of a Toll-like receptor 2 (TLR.2) agonist, wherein said Toll-like receptor 2 agonist is a lipopeptide.

Suitable TLR2agonists for use in the invention have inter alia been described in Willems et al. (2014) J Med Chem 57:6873, WO2013051936, W02019122050, van den Ende et al. ChemBioChem 10.1002/cbic.202000687, Lu et al. Eur. J. Org. Chem. 2021, 5415-5423, Lu et al. J. Med. Chem. 2020, 63, 2282-2291 and Zom et al. (2016) Oncotarget 7 (41) : 67087.

In one embodiment, said Toll-like receptor 2 agonist is a di- or triacylated lipopeptide, preferably wherein one, two or all of the acyl groups is a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms.

In one embodiment, said Toll-like receptor 2 agonist comprises a N- tetradecylcarbamyl chain.

In one embodiment, said Toll-like receptor 2 agonist further comprises one or two palmitoyl chains.

Suitable Toll-like receptor 2 agonists for use in the method of the invention have e.g. been described in WO2013051936 (herein incorporated by reference).

In one embodiment, the method or use of the invention comprises administration of a TLR.2 agonist of the formula (I) :

wherein R ¹ and R ² are each, independently, a branched or straight group having up to 17 atoms selected from C (carbon), N (nitrogen), O (oxygen) and S (sulphur); q is from 0 to and including 18; X ¹ is S or O; Y ¹ is S or Se (selenium); and R ³ comprises an amino acid or a peptide of two or more amino acids; or a pharmaceutically acceptable salt thereof.

In one embodiment of the molecule of formula (I), X ¹ is O.

In one embodiment of the molecule of formula (I), Y ¹ is S.

In one embodiment of the molecule of formula (I), X ¹ is O and/or Y ¹ is S.

In one embodiment of the molecule of formula (I), R ¹ and R ² are preferably each independently branched or straight aliphatic groups having up to 17 atoms (i.e. each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 atoms) in length containing one or more selected from C, N, O and S. The person skilled in the art knows that the term "up to 17 atoms in length" means in the context of the invention that the backbone of the branched or straight group comprises up to 17 atoms; thus, the hydrogen atoms of an aliphatic group are not included in calculating the number of up to 17 atoms.

In one embodiment of the molecule of formula (I), R ¹ and R ² are each independently a straight alkyl group having 10 to 17 carbon atoms, preferably 15 carbon atoms.

In another embodiment of the molecule of formula (I), q is from 11 to and including 18, e.g. 11 to and including 15, such as 11, 12, 13 or 14, e.g. 12 or 14.

In one embodiment of the molecule of formula (I), R ³ comprises a peptide of two or more amino acids. Such peptides have been described to promote agonistic activity (Bessler et al. (1985) J Immunol 135: 1900).

In one embodiment, the R ³ group comprises a Ser(Lys) _r peptide wherein r is 1, 2, 3, 4 or 5. In one embodiment of the molecule of formula (I), group R ³ is represented by formula (VI): wherein R ¹⁰ comprises or consists of a (Lys)r peptide part, wherein r is 0, 1, 2, 3, 4 or 5 and wherein R ⁹ is hydrogen or a group comprising one to six atoms chosen from C, N and O.

Examples of possible R ⁹ groups are hydrogen, C1-C6 alkyl, preferably a C1-C4 alkyl, C2-C6 alkenyl, preferably a C2-C3 alkenyl, C2-C6 alkynyl, preferably a C2-C3 alkynyl, Cl- C5 hydroxyalkyl, C1-C5 mercaptoalkyl, C1-C5 aminoalkyl, Cl-C4-cyanoalkyl, C1-C3- azidooalkyl, for example a -CH2N3 group, Cl-C6-haloalkyl, for example -CH2X group (X = F, Cl, Br), aromatic 5 or 6-membered rings containing one or more selected from C, N, O and S, and 3- to 6-membered (hetero)cyclic rings containing one or more selected from C, N, O and S. Preferably, R ⁹ is a -CH2-OH group, a -CH2-CH3 group, a -(CH2)3-CH3 group, a- CH2C=CH group, a -CH2CH=CH2 group or a -(CH2)2NH2 group. In one embodiment, R ⁹ is not hydrogen and the asymmetric carbon to which R ⁹ is attached has the L configuration.

In one embodiment, the method or use of the invention comprises administration of a TLR2 agonist of the formula (II): wherein R ¹ and R ² are each, independently, a branched or straight group having up to 17 atoms selected from C, N, O and S; Y ¹ is S or Se; R ³ comprises an amino acid or a peptide of two or more amino acids; R ⁴ is H, acetyl or a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms, for example a palmitoyl group (CH3(CH2)i4CO); and n is 0, 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof.

In one embodiment of the molecule of formula (II), X ¹ is O.

In one embodiment of the molecule of formula (II), Y ¹ is S.

In one embodiment of the molecule of formula (II), X ¹ is O and/or Y ¹ is S.

In one embodiment of the molecule of formula (II), R ¹ and R ² are preferably each independently branched or straight aliphatic groups having up to 17 atoms (i.e. each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 atoms) in length containing one or more selected from C, N, O and S.

In one embodiment of the molecule of formula (II), R ¹ and R ² are each independently a straight alkyl group having 10 to 17 carbon atoms, preferably 15 carbon atoms.

In one embodiment of the molecule of formula (II), R ³ comprises a peptide of two or more amino acids.

In one embodiment, the R ³ group comprises a Ser(Lys) _r peptide wherein r is 1, 2, 3, 4 or 5.

In one embodiment of the molecule of formula (II), group R ³ is represented by formula (VI): wherein R ¹⁰ comprises or consists of a (Lys)r peptide part, wherein r is 0, 1, 2, 3, 4 or 5 and wherein R ⁹ is hydrogen or a group comprising one to six atoms chosen from C, N and O.

Examples of possible R ⁹ groups are hydrogen, C1-C6 alkyl, preferably a C1-C4 alkyl, C2-C6 alkenyl, preferably a C2-C3 alkenyl, C2-C6 alkynyl, preferably a C2-C3 alkynyl, Cl- C5 hydroxyalkyl, C1-C5 mercaptoalkyl, C1-C5 aminoalkyl, Cl-C4-cyanoalkyl, C1-C3- azidooalkyl, for example a -CH2N3 group, Cl-C6-haloalkyl, for example -CH2X group (X = F, Cl, Br), aromatic 5 or 6-membered rings containing one or more selected from C, N, O and S, and 3- to 6-membered (hetero)cyclic rings containing one or more selected from C, N, O and S. Preferably, R ⁹ is a -CH2-OH group, a -CH2-CH3 group, a -(CH2)3-CH3 group, a- CH2C=CH group, a -CH2CH=CH2 group or a -(CH2)2NH2 group. In one embodiment, R ⁹ is not hydrogen and the asymmetric carbon to which R ⁹ is attached has the L configuration. In one embodiment, the method or use of the invention comprises administration of a TLR.2 agonist of the formula (III): wherein R ² is a branched or straight group having up to 17 atoms selected from C, N, O and S; X ¹ is S or O; Y ¹ is S or Se; R ⁵ is NH2 or CH3; and R ⁶ comprises an amino acid, a peptide of two or more amino acids, OH, OCH3 or NHCH2CH2N(CH3)2; or a pharmaceutically acceptable salt thereof.

In one embodiment of the molecule of formula (III), X ¹ is O.

In one embodiment of the molecule of formula (III), Y ¹ is S.

In one embodiment of the molecule of formula (III), X ¹ is O and/or Y ¹ is S.

In one embodiment of the molecule of formula (III), R ² is a branched or straight aliphatic group having up to 17 atoms (i.e. each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 atoms) in length containing one or more selected from C, N, O and S.

In one embodiment of the molecule of formula (III), R ² is a straight alkyl group having 10 to 17 carbon atoms, preferably 15 carbon atoms.

In one embodiment of the molecule of formula (III), R ⁶ comprises a peptide of two or more amino acids.

In one embodiment, the R ⁶ group comprises a (Lys) _r peptide wherein r is 1, 2, 3, 4 or 5.

In one embodiment, the method or use of the invention comprises administration of a TLR2 agonist of the formula (IV): wherein R ² is a branched or straight group having up to 17 atoms selected from C, N, O and S; Y ¹ is S or Se; R ⁴ is H, acetyl or a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms, for example a palmitoyl group (CH3(CH ₂ )i4CO); and R ⁶ comprises an amino acid, a peptide of two or more amino acids, OH, OCH3 or NHCH ₂ CH ₂ N(CH ₃ ) ₂ ; or a pharmaceutically acceptable salt thereof.

In one embodiment of the molecule of formula (IV), Y ¹ is S.

In one embodiment of the molecule of formula (IV), R ² is a branched or straight aliphatic group having up to 17 atoms (i.e. each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 atoms) in length containing one or more selected from C, N, O and S.

In one embodiment of the molecule of formula (IV), R ² is a straight alkyl group having 10 to 17 carbon atoms, preferably 15 carbon atoms.

In one embodiment of the molecule of formula (IV), R ⁶ comprises a peptide of two or more amino acids.

In one embodiment, the R ⁶ group comprises a (Lys) _r peptide wherein r is 1, 2, 3, 4 or 5.

In one embodiment, the method or use of the invention comprises administration of a TLR2 agonist of the formula (V):

wherein R ⁷ is H or CO(CH) _m CH3 with m is 10, 11, 12, 13, 14, 15, 16 or 17; R ⁸ is H or

CO(CH) _n CH ₃ with n is 10, 11, 12, 13, 14, 15, 16 or 17; Y ¹ is S or Se; R ⁴ is H, acetyl or a fatty-acyl group having 8 or more carbon atoms, e.g. from 12 to 20 carbon atoms, for example a palmitoyl group (CH3(CH2)i4CO); and R ⁶ comprises an amino acid, a peptide of two or more amino acids, OH, OCH ₃ or NHCH2CH2N(CH3)2 or a group of the formula (VII): wherein X ² is O or NH; or a pharmaceutically acceptable salt thereof.

In one embodiment of the molecule of formula (V), Y ¹ is S.

In one embodiment, the R ⁶ group comprises a (Lys) _r peptide wherein r is 1, 2, 3, 4 or 5.

In one embodiment, the method or use of the invention comprises administration of a TLR2 agonist of the formula (VIII), also termed Amplivant:

wherein SKKKK is a SerLysLysLysLys (Ser(Lys)4) peptide sequence.

In one embodiment, the method or use of the invention comprises administration of a TLR.2 agonist of the formula (VIII), wherein the compound of formula (VIII) is a mixture of compounds comprising a Cys((R)-2,3-di(palmitoyloxy)-propyl) moiety and compounds comprising a Cys((S)-2,3-di(palmitoyloxy)-propyl) moiety.

In another embodiment, the method or use of the invention comprises administration of a TLR.2 agonist of the formula (VIII), wherein the compound of formula (VIII) is chirally pure and only consists of compounds comprising a Cys((R)-2,3- di(palmitoyloxy)-propyl) moiety (R-diastereoisomer).

In another embodiment, the method or use of the invention comprises administration of a TLR2 agonist selected from the group consisting of: PamsCysSer, PamsCysSerLys, Pam3CysSer(Lys)4 (also termed Pam3CSK4), Pam2CysSer(Lys)4 (also termed Pam2CSK4), PamiCysSer(Lys)4 (also termed Pam lCSK4).

Immunogenic compositions

In a further aspect, the invention relates to an immunogenic composition comprising one or more immunogenic peptides of the invention as described herein above and a pharmaceutically-acceptable carrier for use in combination with a Toll-like receptor 2 agonist in the immunization of a human subject, wherein said Toll-like receptor 2 agonist is a lipopeptide which is not covalently linked to said immunogenic peptide(s) and wherein said immunogenic peptide(s) and said Toll-like receptor 2 agonist are administered intradermally at the same site.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides according to the invention, for example two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 1-157, preferably derived from the same organism, such as the same virus, optionally from same protein. In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides according to the invention, for example two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 1-146, preferably derived from the same organism, such as the same virus, optionally from same protein.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides according to the invention, for example two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 1-69.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO:70-113 and 152-157.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO:70-113.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 114-133 and 147-151.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 114-133.

In one embodiment, the immunogenic composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 134-146.

In one embodiment, the immunogenic composition is used in combination with a lipopeptide Toll-like receptor 2 agonist having any one of the additional features described herein above.

In a further aspect, the invention relates to a composition comprising one or more immunogenic peptides according to the invention and a Toll-like receptor 2 agonist according to the invention. Such a composition allows combined administration to the human subject.

Accordingly in a further aspect, the invention relates to a pharmaceutical composition comprising an immunogenic peptide and a Toll-like receptor 2 agonist, wherein said Toll-like receptor 2 agonist is a lipopeptide which is not covalently linked to said immunogenic peptide. Preferably, the composition is suitable for intradermal administration and/or formulated for intradermal administration.

Uses of the immunogenic peptides and immunogenic compositions and methods of treatment

As described above, the immunogenic peptide(s) and the Toll-like receptor 2 agonist are used, or for use, in the immunization of a human subject, wherein said immunogenic peptide(s) and said Toll-like receptor 2 agonist are administered intradermally at the same site. Administration may be performed simultaneously or sequentially. Optionally all components are mixed before simultaneous administration.

In some embodiments, all immunogenic peptides of the plurality to be administered are comprised within one immunogenic composition. In other embodiments, the immunogenic peptides of the invention are distributed over two or more compositions, e.g. distributed over two or more vials. In such embodiments, the compositions may be mixed before administration to the patient or the compositions may be administered separately. Thus, in a further main aspect, the invention relates to a vaccine (i.e. a vaccine product) comprising two or more compositions which together comprise the plurality of immunogenic peptides as defined herein.

In one embodiment, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides according to the invention are administered, for example two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 1-69.

In one embodiment, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 70- 113 and 152-157 are administered.

In one embodiment, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 70- 113 are administered.

In one embodiment, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 114-133 and 147-151 are administered.

In one embodiment, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 114-133 are administered.

In one embodiment, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more immunogenic peptides selected from the group consisting of SEQ ID NO: 134-146 are administered. In one embodiment, the invention relates to an immunogenic peptide for use according to the invention or an immunogenic composition for use according to the invention in combination with a said Toll-like receptor 2 agonist as defined herein, wherein the administration of said Toll-like receptor 2 agonist and said immunogenic peptide or said immunogenic composition is simultaneous or sequential within a time interval of 24 hrs or less, such as 2 hrs or less, e.g. 1 hr or less, such as 30 min or less.

In one embodiment, said Toll-like receptor 2 agonist and said immunogenic peptide or said immunogenic composition are mixed prior to administration.

In a further aspect, the invention relates to a method for immunizing a human subject comprising administration of an immunogenic peptide in combination with a Tolllike receptor 2 agonist to said human subject, wherein said Toll-like receptor 2 agonist is a lipopeptide that is not covalently linked to said immunogenic peptide and wherein said immunogenic peptide and said Toll-like receptor 2 agonist are administered intradermally at the same site.

The immunization according to the invention may be for prophylactic or therapeutic purposes. For example, the immunization may be performed for the prevention or treatment of cancer. Alternatively, the immunization may be performed for the prevention or treatment of infectious diseases, such as virus-induced diseases.

The immunization according to the invention may induce a CD8+ response and/or a CD4+ response and/or an antibody response in the human subject.

The immunization according to the invention may induce a protective response, such as a response that protects against infection, against disease, against severe disease or death.

The treatment of the human may or may not include administration of further compounds, for example further antigens or adjuvants. In one embodiment, the use or method of treatment of the invention does not comprise administration of iNKT agonist.

Preferably, a dose of immunogenic peptides applied to a subject at a given time point, either in a single or in multiple injections at a certain time point, comprises an amount of peptides in the range from 0.1 pg to 20 mg, such as about 0.1 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 15 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 150 pg, 200 pg, 500 pg, 1 mg, 20 mg or any value in between.

A single injection volume (i.e. volume applied on one location at a certain time point), comprising a peptide dose, may be between 50 pL and 1 ml_. The single injection volume may be 50 pL, 75 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 600 pL, 700 pL, 800 pL, or any value in between. In one embodiment, the volume is between 75 pL and 250 pL or between 50 pL and 75 pL. In one embodiment, the peptide(s) and TLR.2 agonists are administered in a molar ratio from 10: 1 to 1: 10, such as a molar ratio between 5: 1 and 1:5, e.g. a molar ratio between 2: 1 and 1 :2, such as an equimolar ratio.

Immunization of a human subject may comprise administration of more than one dose, such as two or three doses of the adjuvanted immunogenic peptide. The interval between doses may for example be between about two and four weeks, for example about two, three or four weeks. In one embodiment, a further (booster) dose is administered between 6 and 18 months after the initial administration(s), such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 months after the initial administration(s).

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application herein is not, and should not be, taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

EXAMPLES

Example 1

In this study, the impact of administration route (intradermal or subcutaneous) and addition of a lipopeptide TLR.2 agonist, the R-diastereoisomer (see above) of Amplivant (uPaml4), (Willems et al. 2014 J Med Chem 57(15):6873; Zom et al. (2016) Oncotarget 7 (41): 67087), on the immunogenicity of SLPs (Synthetic Long Peptides) was investigated. For this study, a composition of SARS-CoV-2 antigen based SLPs was used (Table 1), which was proven immunogenic in C57BL/6 mice (muSARS-CoV-2 compositions). C57BL/6 mice were vaccinated with SLPs with or without Amplivant 3 times in 2-week intervals. Mice were vaccinated with 50 pg per SLP per vaccination (the 11 SLP were lyophilized as a pool) with or without 50 pg Amplivant. SLPs were dissolved in 20% DMSO in water for injection and mixed with Amplivant before administration. Compositions were administered via intradermal tail base (30pl) or subcutaneous injection in the flank (200pl).

Table 2. SLP sequences >

The impact of the administration route and addition of adjuvant Amplivant was assessed by studying the SLP-specific T cell response and the induction of SLP-specific IgG antibodies. Epitope specific tetramers for 2 MHC-I epitopes enabled real-life monitoring of antigen-specific CD8 ⁺ T cell responses elicited by 2 different SLPs (as described in Pardieck et al, Nature Comm 2022: 13(1) :3966). The following tetramers were used for the identification of antigen-specific CD8 ⁺ T cells: VNFNFNGL H2-Kb APC (VNF tetramer) and AAYYVGYL H2-Kb PE (AAY tetramer), whereby the amino acid sequence depicts the epitope to which the T cells are reactive followed by the MHC molecule in which the epitope is bound, followed by the fluorochrome to which the epitope-MHC complex is conjugated that can be measured by flow cytometry. The AAY tetramer allowed for the identification of SLP-9 specific CD8 ⁺ T cells, whereas the VNF tetramer was used to study SLP-8 specific CD8 ⁺ T cell responses. One week after each vaccination the antigen-specific CD8 ⁺ T cell response towards 2 SLPs was characterized by flow cytometry (Figure 1). Figure 1 shows that the intradermal vaccination route of SLP with Amplivant resulted in higher percentages of antigen-specific CD8 ⁺ T cells, compared to subcutaneous administration of SLP with adjuvant.

In addition to the identification of antigen-specific CD8 ⁺ T cells using tetramers, the presence of SLP-specific CD8 and CD4 T-cells was measured in an in vitro restimulation assay. Around day 50 after the first vaccination, mice were sacrificed and in vitro restimulation of splenocytes was performed by incubating splenocytes with SLP (pools). In this restimulation study, we analysed the T cell response upon stimulation with SLP-9 or SLP-8 (to correlate the data to the tetramer responses measured in blood), or the remaining SLPs of protein S (Pool S: SLP-1, SLP-3, SLP-5, SLP-7, SLP-10, SLP-11), protein N (Pool N : SLP-2, SLP-4) or protein M (Pool M : SLP-6). To compare SLP-specific responses, all SLP were tested at 1 pg per SLP in in vitro restimulation. After lysis of the erythrocytes using lysis buffer, splenocytes were washed and incubated overnight with SLP (pools). The next day, intracellular cytokine staining (for IFNy) was performed to study the induction of SLP-specific T cell responses as measured by flow cytometry together with surface markers like CD3, CD4 and CD8. Like the kinetics of tetramer responses in blood, mice that received intradermal vaccination of SLP in combination with Amplivant showed higher CD8 ⁺ T-cell responses towards SLPs upon in vitro restimulation with single or pooled SLP, compared to subcutaneous administration of the composition (Figure 2). Even though CD4 ⁺ T-cell responses were lower, IFNy production was seen upon restimulation with pool S in the intradermal administration group (SLP + Amplivant), but not in the subcutaneous administration group. Vaccination with SLP only (no Amplivant), resulted in low responses, as detected by tetramer binding and IFNy production upon restimulation (Figure 2).

In conclusion, using the muSARS-CoV-2 composition, we showed increased T-cell responses upon intradermal vaccination of the SLP composition in combination with Amplivant compared to subcutaneous administration of the composition.

Additionally, final serum was obtained to investigate the induction of SLP-specific IgG antibodies upon vaccination. Generation of IgG antibody responses requires the presence of antigen specific T helper cells. Serum from each group was pooled to characterize the SLP-specific IgG antibody production per vaccination group using ELISA. In brief, ELISA plates were coated overnight with 1 pM SLP. The next day, a-specific binding was blocked using 150 pL PBS/5% Bovine Serum Albumin and incubated for 2 hours at room temperature. After blocking, a serial dilution of mouse serum was added to the plate. Subsequently, the plate was incubated for 1 hour with goat-anti-mouse IgG antibody conjugated to biotin. Next, the plate was incubated with streptavidin -HRP and after one hour TMB substrate was added. After 20 minutes the reaction was stopped by adding 100 pL of IM sulfuric acid and the OD450nm value was measured. The SLP-specific antibody responses are depicted in Figure 3 and Table 3. Figure 3 and Table 3 show that intradermal vaccination of SLP in combination with Amplivant generally induced the most potent antibody responses, compared to subcutaneous vaccination of SLP + Amplivant and compared to vaccinations without Amplivant. In addition, antibody responses to Amplivant were present upon both intradermal and subcutaneous administration of muSARS-CoV-2 composition in combination with Amplivant.

Table 3: Antibody responses

Example 2

Since the SLPs in combination with Amplivant administered intradermally showed strong CD8+ T cell responses in C57BL/6 mice, we aimed to investigate the protective capacity of the pool of 11 SLPs in K18-hACE2 transgenic mice. Methods

K18-hACE2 transgenic mice, expressing the human ACE2 receptor (hACE2) under control of the cytokeratin 18 (K18) promoter, were obtained from the Jackson Laboratory (B6.Cg- Tg(K18-ACE2)2Prlmn/J) and bred in-house at LUMC. At the start of the experiments, mice were six to eight weeks old. Animals were housed in individually ventilated cages under specific-pathogen free conditions at the animal facility at Leiden University Medical Center (LUMC). All animal experiments were approved by the Animal Experiments Committee of the LUMC and performed according to the recommendations and guidelines set by LUMC and by the Dutch Experiments on Animals Act.

Mice received a vaccination at day 0, day 14 and day 28 (2-week interval). These vaccines contained 11 SLPs with Amplivant. The SLP dose was 50 pg per SLP per mouse per vaccination. The 11 SLPs were lyophilized as a pool. The dose of Amplivant per mouse per vaccination was 50 pg. The SLPs were dissolved in 20% DMSO/water for injection (WFI) and mixed with Amplivant.

SARS-CoV-2 infection was performed similar to previous study (Pardieck et al. (2022) Nature Communications 2022 13: 1. 2022; 13(l): l-ll). Clinical isolate SARS-CoV- 2/human/NLD/Leiden-0008/2020 (here named SARS-CoV-2) was used for the SARS-CoV- 2 infection of mice. Isolate Leiden-0008 was propagated and titrated in Vero-E6 cells (ATCC CRL-1586). K18-hACE2 transgenic were anaesthetized with iso-flurane gas and intranasally infected. Mice received 10.000 plaque forming units (PFU) of the SARS-CoV-2 Leiden-08 isolate in a total volume of 50p I DMEM.

Mouse weight was monitored daily. Euthanasia criteria were weight loss of >20 percent of body weight compared to the pre-study weight and a moribund state. All experiments with SARS-CoV-2 were performed in the Biosafety Level 3 (BSL3) Laboratories at the LUMC.

Results

K18-hACE2 transgenic mice were prophylactically vaccinated with the 11 SLPs and the protection against SARS-CoV-2 infection was investigated. These mice are susceptible to SARS-CoV-2 infection, because they express the human ACE2 receptor under the murine K18 promoter thereby inducing expression of the receptor in epithelial cells, including the lung epithelia.

Sequential intradermal vaccination with the SLPs in combination with Amplivant resulted in protection against SARS-CoV-2 infection in K18-hACE2 transgenic mice shown by their improved survival and individual weight (Figure 4A and Figure 4B).