Nanoparticle-based delivery systems

Abstract

A compound A-L-B, wherein A is a nanoparticle (NP)-forming unit, preferably a NP-forming polypeptide or a NP-forming protein, preferably a NP-forming polypeptide or a NP-forming protein having its C-terminus covalently bound to the N-terminus of L via a peptide bond; L is a polypeptide having the following amino acid sequence written in the single letter code z1–G X1 X2 G X3 G X4 X5 G X6 G X7 G–z2, wherein X1 = any amino acid except H, C, and W, X2 = any amino acid except H, C, and W, X3 = any amino acid except H, C, and W, X4 = any amino acid except H, C, and W, X5 = any amino acid except H, C, and W, X6 = any amino acid except H, C, and W, X7 = any amino acid except H, C, and W, z1 represents the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids, and z2 represents the C-terminus of the polypeptide, or a modification of the C- terminal carboxyl group of the polypeptide, which modification (i) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1 or -C(O)-NR2R3, wherein R1 is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C≡CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2 and R3 is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C≡CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or (ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or (iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond; wherein the side chain of each amino acid of the polypeptide L independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; and B is a compound having biological activity, preferably a protein, which preferably has its N-terminus covalently bound to the C-terminus of L via a peptide bond; wherein the N-terminus of L is covalently bound to A, preferably via a peptide bond, and wherein the C-terminus of L is covalently bound to B, preferably via a peptide bond.

Description

[0001] 251172 WO - 10 September 2025

[0002] Nanoparticle-based delivery systems

[0003] The present invention relates to a compound A-L-B, wherein A is a nanoparticle (NP)-forming unit, wherein L is a polypeptide suitable as linker for nanoparticle- based delivery systems, and wherein B is a compound having biological activity. The invention further relates to nanoparticles formed from a plurality of compounds A-L-B, as well as pharmaceutically active compositions comprising compound A-L-B, or nanoparticles formed from a plurality of compounds A-L-B.

[0004] Nanoparticle-based delivery systems are engineered platforms that use NPs for the targeted delivery and controlled release of compounds, such as, but not limited to, vaccines, therapeutic agents or other compounds having biological activity. Recently, nanoparticles have aroused attention due to their potential application for controlled drug delivery. The modern form of a drug delivery system should minimise side-effects and reduce both dosage and dosage frequency. In nanoparticle drug delivery systems, NPs may be used for the targeted delivery and / or controlled release of therapeutic agents.

[0005] Delivering therapeutic agents to the target site is a major problem in treatment of many diseases. A conventional application of drugs is characterised by limited effectiveness, poor biodistribution, and lack of selectivity. These limitations and drawbacks can be overcome by controlling drug delivery.

[0006] In controlled drug delivery systems (DDS) the drug is transported to the place of action, thus, its influence on vital tissues and undesirable side effects can be minimised. In addition, DDS protects the drug from rapid degradation or clearance and enhances drug concentration in target tissues, therefore, lower doses of drug are required. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects. Cell-specific targeting can be accomplished by attaching drugs to specially designed carriers. Various nanoparticles, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles, have been tested as (nano)carriers in such drug delivery systems (Wilczewska AZ, et al. (2012) Nanoparticles as drug delivery systems, Pharmacological Reports, 64 (5), 10120-1037).

[0007] A nanoparticle is defined as a particle with at least one dimension of <100 nm. It is possible to create NPs featuring distinctive physicochemical features. Size, solubility, shape, hydrophilicity, and surface chemistry, for example, may be regulated, enabling the creation of NPs with specific biological features.

[0008] By way of example, NPs can be constructed to facilitate the inclusion of a wide array of molecules, such as antigens, making them, e.g., useful tools in vaccine development. Antigens can be incorporated into NPs by conjugation (covalent functionalisation) or encapsulation (physical trapping). The conjugation of antigens onto NPs allows the immunogen to be exposed to the immune systems in a manner like how it would be delivered by a virus, prompting a comparable reaction. If the antigenic material is encapsulated in NPs, administration of antigens that would otherwise disintegrate quickly or trigger a localised immune response becomes possible. Moreover, NPs manufactured from certain composites offer not only site-directed antigen presentation but also antigen release over time to enhance immune system exposure (Bezbaruah R, et al. 251172 WO - 10 September 2025

[0009] (2022) Nanoparticle-Based Delivery Systems for Vaccines, Vaccines (Basel), 10 (11), 1946.)

[0010] Presently, a wide range of NPs, particularly liposomes, polymeric and inorganic NPs, self-assembled protein NPs and virus-like particles (VLPs), are being explored as antigen carriers.

[0011] Virus-like particles (VLPs) are self-assembling protein nanoparticles, typically derived from the capsid protein of nonenveloped viruses that infect bacterial, insect, plant, and mammalian cells. VLPs are empty viruses without any infection, maturation, or replication components, making them safe and biocompatible delivery vehicles (Ikwuagwu B, et al. (2022) Virus-like particles for drug delivery: a review of methods and applications, Current Opinion in Biotechnology, 78, 102785).

[0012] An example for viruses that can be used to produce VLPs are bacteriophages (also known as phages). Bacteriophages are naturally occurring viruses, which are specialised to infect bacteria. As such, bacteriophages are ubiquitous and play a vital role in the ecosystem. They are an integral part of the human commensal microbiome and are well tolerated by the human immune system (Champagne-Jorgensen K, et al. (2023) Immunogenicity of bacteriophages, Trends in Microbiology, 31(10), 1058-1071). This safety property gives bacteriophages and their components a great potential for use in biotechnology and therapy. For example, phage capsid proteins have already been used as vaccine scaffolds for antigen display (Smit JM et al. (2023) First-in-human use of a modular capsid virus-like vaccine platform : an open-label, non-randomised, phase 1 clinical trial of the SARS-CoV-2 vaccine ABNCoV2, The Lancet Microbe, 4(3), el40-el48).

[0013] Fougeroux, C. et al. (2021) Capsid-like particles decorated with the SARS-CoV- 2 receptor-binding domain elicit strong virus neutralization activity, Nature Communications, 12(1), 1-11 discloses two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

[0014] WO 2022 / 229817 Al discloses vaccines comprising virus-like particles displaying at least one SARS-CoV-2 antigen.

[0015] Dixit, A.B. (2019) A viral small terminase subunit (TerS) twin ring pac synapsis DNA packaging model is supported by fluorescent fusion proteins, Virology, 536, 39-48 discloses TerS-eGFP and TerS-mCherry fluorescent fusion proteins.

[0016] Black, L.W. (2015) Old, new, and widely true: The bacteriophage T4 DNA packaging mechanism, Virology, 479-480, 650-656 discloses TerS-eGFP and TerS-mCherry fluorescent fusion proteins.

[0017] However, there is still the need to provide alternative nanoparticle-based delivery systems which can be used, e.g., to present antigens in vaccines against infection diseases, for enzyme immobilisation or as experimental tools for peptide oligomerisation and diagnostics.

[0018] The object of the present invention is therefore to provide a compound which can be used as a linker in nanoparticle-based delivery systems, as well as compounds comprising the said linker useful for nanoparticle-based delivery 251172 WO - 10 September 2025 systems. Further, the object of the invention is to provide nanoparticles, as well as a pharmaceutically active composition comprising the said nanoparticles.

[0019] The object of the present invention is solved by a compound A-L-B, wherein

[0020] A is a nanoparticle (NP)-forming unit, preferably a NP-forming polypeptide or a NP-forming protein, preferably a NP-forming polypeptide or a NP-forming protein having its C-terminus covalently bound to the N-terminus of L via a peptide bond;

[0021] L is a polypeptide having the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, wherein

[0022] X1= any amino acid except H, C, and W,

[0023] X2= any amino acid except H, C, and W,

[0024] X3= any amino acid except H, C, and W,

[0025] X4= any amino acid except H, C, and W,

[0026] X5= any amino acid except H, C, and W,

[0027] X6= any amino acid except H, C, and W,

[0028] X7= any amino acid except H, C, and W, z1represents the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids, and z2represents the C-terminus of the polypeptide, or a modification of the C- terminal carboxyl group of the polypeptide, which modification

[0029] (i) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1or -C(O)-NR2R3, wherein R1is a functional group selected from the group consisting of -(CH2)n-N3, - (C H2)n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2and R3is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, - (C H2)n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or

[0030] (ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or

[0031] (iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond; wherein the side chain of each amino acid of the polypeptide L independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; and

[0032] B is a compound having biological activity, preferably a protein, which preferably has its N-terminus covalently bound to the C-terminus of L via a peptide bond; 251172 WO - 10 September 2025 wherein the N-terminus of L is covalently bound to A, preferably via a peptide bond, and wherein the C-terminus of L is covalently bound to B, preferably via a peptide bond.

[0033] In the compound A-L-B of the present invention, L is a polypeptide having the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, wherein

[0034] X1= any amino acid except H, C, and W,

[0035] X2= any amino acid except H, C, and W,

[0036] X3= any amino acid except H, C, and W,

[0037] X4= any amino acid except H, C, and W,

[0038] X5= any amino acid except H, C, and W,

[0039] X6= any amino acid except H, C, and W,

[0040] X7= any amino acid except H, C, and W, z1represents the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids, and z2represents the C-terminus of the polypeptide, or a modification of the C- terminal carboxyl group of the polypeptide, which modification

[0041] (i) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1or -C(O)-NR2R3, wherein R1is a functional group selected from the group consisting of -(CH2)n-N3, - (C H2)n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2and R3is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, - (C H2)n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or

[0042] (ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or

[0043] (iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond; wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof.

[0044] Surprisingly, the polypeptide L of the compound of the present invention may be used as linker for nanoparticle-based delivery systems. It was found that the polypeptide L can be used as a flexible linker, which is able to connect nanoparticle-forming units, such as self-assembling nanoparticle-forming units, and compounds having biological activity, such as proteins or polypeptides, like virus antigens, enzymes, antibodies, growth factors, tumour suppressors, DNA- binding domains, RNA-binding domains, pilins, adhesins, antigenic peptides, 251172 WO - 10 September 2025 cancer neoantigens, diagnostic peptides, and the like. Advantageously, when used as a linker, the polypeptide L provides stability, solubility and flexibility between the NP-forming unit and the compound having biological activity. The terms "polypeptide L", "linker L" and "unit L" are thus used interchangeably herein. Surprisingly, a vaccine composition comprising the nanoparticle-based delivery system of the NP-forming unit, the linker L, and a virus antigen as compound having biological activity provides much stronger immunogenicity already after the first immunisation in comparison to virus antigen alone (Fig. 15) and virus-like particles (Fig. 17).

[0045] According to the invention, L is a polypeptide. A polypeptide is a continuous, unbranched molecular chain of amino acids linked by peptide bonds. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. The polypeptide L of the present invention is characterised by having the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, wherein

[0046] X1, X2, X3, X4, X5, X6, and X7, independently, can be any amino acid except H (histidine), C (cysteine), and W (tryptophan). Accordingly, X1, X2, X3, X4, X5, X6, and X7, independently, can be any amino acid, but must not be H, C, or W. Within the meaning of the present application, an "amino acid" is an organic compound that contains both amino and carboxylic acid functional groups. The amino acid may, e.g., be an alpha- (a-), beta- (p-), or gamma- (y-) amino acid. Within the meaning of the present application, the term "amino acid" encompasses the twenty-two proteinogenic amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine (including / V-formylmethionine), phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine, and pyrrolysine) as well as non-proteinogenic amino acids and modified amino acids which may arise from post-translational modification (e.g., phosphorylation, amidation, acylation (like acetylation or formylation), alkylation, glycosylation, lipidation, isoprenylation, etc.) or another chemical modification. Preferably, the "amino acids" of the present application encompass the twenty-two proteinogenic amino acids as well as their derivatives arising from post-translational modification. The amino acids X1, X2, X3, X4, X5, X6, and X7may be, independently from each other, selected from any of the aforementioned amino acids (as long as they are not H, C, or W).

[0047] Within the above amino acid sequence, z1represents either the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids. The term "N- terminus of the polypeptide" (also known as "amino-terminus of the polypeptide") refers to the N-terminal -NH2 group of the first amino acid of the polypeptide chain, which in case of compound L is the first glycine (G). The term "group consisting of 1 to 10 amino acids" refers to a group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, which may be the same or different amino acids. The amino acids in the group consisting of 1 to 10 amino acids may be 251172 WO - 10 September 2025 linked via peptide bonds to a peptide chain, which may be branched or unbranched, preferably unbranched.

[0048] In a preferred embodiment, z1represents the N-terminus of the polypeptide.

[0049] Within the above amino acid sequence, z2represents either the C-terminus of the polypeptide, or a modification of the C-terminal carboxyl group of the polypeptide. The said modification either (i) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1or -C(O)-NR2R3, wherein R1is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C=CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2and R3is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, - (C H 2) n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or (ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or (iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond.

[0050] The term "C-terminus of the polypeptide" (also known as "carboxy-terminus of the polypeptide") refers to the C-terminal -COOH group of the last amino acid of the polypeptide chain, which in case of compound L is the last glycine (G).

[0051] As an alternative to being the C-terminus of the polypeptide, z2may represent a modification of the C-terminal carboxyl group of the polypeptide.

[0052] The said modification may include a functional group which may be used as a functional group in a so-called click-chemistry reaction. In chemical synthesis, "click-chemistry" is a class of simple, atom-economy reactions commonly used for joining two molecular entities of choice. Click-chemistry is not a single specific reaction but describes a way of generating products that follow examples in nature, which also generates substances by joining small modular units. Click reactions occur in one pot, are not disturbed by water, generate minimal and inoffensive byproducts, and are "spring-loaded" (i.e., characterised by a high thermodynamic driving force that drives it quickly and irreversibly to high yield of a single reaction product, with high reaction specificity (in some cases, with both regio- and stereo-specificity)). These qualities make click reactions particularly suitable to the problem of isolating and targeting molecules in complex biological environments. Functional groups which may be used in click-chemistry reactions are azide group (-N3), alkyne group (-C=CH) (for Copper(I)-catalysed azide-alkyne cycloaddition), strained difluorooctyne (DIFO), or dibenzylcyclooctyne (DIBO) (for strain-promoted azide-alkyne cycloaddition).

[0053] Alternatively, the said modification may be a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction. Suitable catcher / tag pairs are known to the person skilled in the art (e.g., Fan R and Aranko AS (2024) Catcher / Tag Toolbox: Biomolecular Click- Reactions For Protein Engineering Beyond Genetics, ChemBioChem, 25(1), e202300600). 251172 WO - 10 September 2025

[0054] Alternatively, the said modification may be a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the five amino acids LPXTG (Popp MWL, et al. (2009) Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation, Current Protocols in Protein Science, 15.3.1-15.3.9). Sortase A may catalyse the cleavage of the recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine Gly4 peptide and the compound L.

[0055] In a preferred embodiment, z2represents the C-terminus of the polypeptide.

[0056] Within the above amino acid sequence, the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof. The chemical modification may be introduced by posttranslational modification, e.g., if a eukaryotic host cell is used to produce the polypeptide, or by (subsequent) chemical modification, e.g., if the polypeptide is synthetised by peptide synthesis (such as solid-phase synthesis), or if a host cell unable to introduce posttranslational modifications is used to produce the polypeptide.

[0057] In a preferred embodiment of the present invention, the polypeptide L has the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, wherein

[0058] X1= L, I, M, or V,

[0059] X2= E or D,

[0060] X3= S, T, N, or Q,

[0061] X4= F or Y,

[0062] X5= Q, N, S, or T,

[0063] X6= P,

[0064] X7= S, T, N, or Q, wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; and wherein z1and z2have the same meaning as described previously herein. Preferably, z1represents the N-terminus of the polypeptide and / or z2represents the C-terminus of the polypeptide.

[0065] In a further preferred embodiment of the present invention, the polypeptide L has the following amino acid sequence written in the single letter code

[0066] GLEGSGFQGPGSG-z2, wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; 251172 WO - 10 September 2025 wherein z2has the same meaning as described previously herein. Preferably, z2represents the C-terminus of the polypeptide.

[0067] In a further preferred embodiment of the present invention, the polypeptide L has the following amino acid sequence

[0068] GLEGSGFQGPGSG (SEQ ID NO: 3).

[0069] In the present invention, the N-terminus of L is covalently bound to A, preferably covalently bound via a peptide bond.

[0070] In the present invention, A is a nanoparticle (NP)-forming unit, preferably a NP- forming polypeptide or a NP-forming protein, preferably a NP-forming polypeptide or a NP-forming protein having its C-terminus covalently bound to the N-terminus of L via a peptide bond.

[0071] The part A-L of the compound A-L-B of the present invention is a fusion molecule of the units L and A. Preferably, the part A-L of A-L-B is a fusion protein. In this case, the NP-forming unit A needs to be or needs to comprise a NP-forming polypeptide or a NP-forming protein. Within the meaning of the present application, a "nanoparticle (NP)-forming unit" is a compound which is able to form nanoparticles, e.g. by spontaneous oligomerisation, which may also be called spontaneous organisation or association. The spontaneous oligomerisation or organisation is also known as "self-assembly". Spontaneous oligomerisation (self-assembly) is defined as a process in which individual units of material associate with themselves spontaneously into a defined and organised structure or larger units with minimal external direction. It is known that many proteins or polypeptides have the potential to spontaneously oligomerise (or "self-assemble") and thereby generating nanoparticles, e.g. ferritin, bacteriophage Qp coat protein, lumazine synthase, hepatitis B virus surface antigen small-envelope protein (HbsAg) and influenza Matrix 1 (Ml) protein (Nguyen B and Tolia NH (2021) Protein-based antigen presentation platforms for nanoparticle vaccines, npj Vaccines, 6(1), 1-11). Accordingly, the unit A may be any of said known spontaneously oligomerising proteins or polypeptides. However, the unit A may also be any other compound capable of forming nanoparticles, e.g., by spontaneous oligomerisation, and being suitable to being covalently bound to the N-terminus of L, e.g. DNA origami, gold nanoparticles (AuNPs), quantum dots (QDs), carbon nanotubes (CNTs), and silicon or iron oxide nanoparticles (Hess KL, Medintz IL and Jewell CM (2019) Designing inorganic nanomaterials for vaccines and immunotherapies, Nano today, 27, 73-98). Accordingly, the NP-forming unit A may be a compound selected from the group consisting of spontaneously oligomerising polypeptides, spontaneously oligomerising proteins, DNA origami, gold nanoparticles (AuNPs), quantum dots (QDs), carbon nanotubes (CNTs), silicon oxide nanoparticles, and iron oxide nanoparticles.

[0072] In a preferred embodiment, A is a bacteriophage-derived small terminase protein subunit (TerS) capable of spontaneous oligomerisation or a derivative thereof being capable of spontaneous oligomerisation. Preferably, A is a bacteriophage-derived TerS capable of spontaneous oligomerisation. The genome packaging motor of bacteriophages is built by two terminase subunits, 251172 WO - 10 September 2025 known as large subunit (TerL) and small subunit (TerS), which are both essential for viral genome packaging. Small terminase subunits are involved in bacteriophage genome delivery into empty capsids during infection of host bacteria. Phage DNA is replicated in a way that a large DNA concatemer is produced and cut during virus assembly. There, TerS recognises phage's own DNA and delivers it to the TerL subunit for genome cutting and packing into individual viral capsids (Wangchuk J, et al. (2021) The coevolution of large and small terminases of bacteriophages is a result of purifying selection leading to phenotypic stabilization, Virology, 564, 13-25). Crystal structures of isolated TerS reveal ring-like nanoparticle structures, composed of TerS homooligomers, indicating that TerS is capable of spontaneous oligomerisation (selfassembly of nanoparticles). Therefore, small terminase protein subunits (TerSs) are regarded as "NP-forming units" within the meaning of the present application.

[0073] Within the meaning of the present application, the term "derivative of the bacteriophage-derived small terminase protein subunit (TerS)" refers to all lengths fragments including truncations at the N- and / or C-terminus, bacteriophage-derived TerS containing amino acid residue substitutions including D-amino acid residues and modified amino acid residues as well as bacteriophage-derived TerS containing disulfide bonds and extensions at the N- and / or C-terminus. It is to be understood that all these derivatives should still be capable of spontaneous oligomerisation.

[0074] The TerS structure consists of terminal DNA-binding and regulatory domains with a central oligomerisation domain that mediates the nanoparticle formation or self-assembly of nanoparticles (see Fig. 1). Surprisingly, it was found that recombinantly expressed minimised deletion mutants of TerS which lack the DNA-binding domain and the regulatory domain, i.e., mutants (derivatives) of TerS only comprising the central domain (oligomerisation domain) of TerS, show increased solubility in comparison to full-length TerS (see, e.g., Fig. 7 A-D). Thus, the said central domains of TerS are especially suitable for nanoparticle formation, as more TerS central domain monomers are present in solution. Moreover, the central domains of TerS do not show any unwanted TerS-DNA interaction which could contribute to host cell DNA contamination in the final composition comprising the self-assembled nanoparticles. Therefore, in another preferred embodiment of the present invention, A is a central domain of a bacteriophage-derived TerS capable of spontaneous oligomerisation or a derivative thereof being capable of spontaneous oligomerisation. The term "derivative of the central domain of the bacteriophage-derived TerS" refers to all lengths fragments including truncations at the N- and / or C-terminus, central domains of bacteriophage-derived TerS containing amino acid residue substitutions including D-amino acid residues and modified amino acid residues as well as central domains of bacteriophage-derived TerS containing disulfide bonds. It is to be understood that all these derivatives should still be capable of spontaneous oligomerisation. Preferably, A is a central domain of a bacteriophage-derived TerS capable of spontaneous oligomerisation.

[0075] The bacteriophage-derived TerS may be selected from the group consisting of Aeromonas phage 44R.R.2 TerS, Shigella phage Sf6 TerS, Staphylococcus phage 80B TerS, Staphylococcus phage A411 TerS, and Campylobacter phage PC5 251172 WO - 10 September 2025

[0076] TerS, preferably selected from the group consisting of Aeromonas phage 44RR2 TerS, Shigella phage Sf6 TerS, and Campylobacter phage PC5 TerS. It is to be understood that the bacteriophage-derived TerS capable of spontaneous oligomerisation or their central domains being capable of spontaneous oligomerisation recited previously herein are "nanoparticle (NP)-forming units" within the meaning of the present invention. Preferably, A is Campylobacter phage PC5 TerS (CaPC5 TerS), more preferably the central domain of Campylobacter phage PC5 TerS (cdCaPC5 TerS; also designated as NCACaPC5 TerS herein).

[0077] In a preferred embodiment of the present invention, A is a polypeptide comprising one of the following amino acid sequences of SEQ ID NOs: 4, 5, or 6: i) LKXXFXXMRXXLVXXIXXGQXXLXXISXXIXXXXXXXXXXXVXXYXXLVXXI XXSTXXLXXIYXXIXXI (SEQ ID NO: 4), wherein each X, independently of each other, denotes any amino acid; ii) YXXVRXXMXXQSXXLLXXAXXALXXAXXXXXXXXXXXFXXLMXXMXXTNX XMXXMHXXM (SEQ ID NO: 5), wherein each X, independently of each other, denotes any amino acid; iii) PDAXEVAKARLRVDTXKWXLARMNPRKYGDXVTNELXXXXXXAIQIETS (SEQ ID NO: 6), wherein each X, independently of each other, denotes any amino acid.

[0078] The amino acid sequence of SEQ ID NO: 4 includes the conserved amino acids of the central domain of Campylobacter phage PC5 TerS (cdCaPC5 TerS) which are necessary for spontaneous oligomerisation (self-assembly). Amino acids which are not conserved within the sequence of the central domain of CaPC5 TerS (cdCaPC5 TerS) are designated as "X" and may be, independently of each other, any amino acid.

[0079] The amino acid sequence of SEQ ID NO: 5 includes the conserved amino acids of the central domain of Aeromonas phage 44RR2 TerS (cdAe44RR2 TerS) which are necessary for spontaneous oligomerisation (self-assembly). Amino acids which are not conserved within the sequence of the central domain of Ae44RR2 TerS (cdAe44RR2 TerS) are designated as "X" and may be, dependently of each other, any amino acid.

[0080] The amino acid sequence of SEQ ID NO: 6 includes the conserved amino acids of the central domain of Shigella phage Sf6 TerS (cdShSf6 TerS) which are necessary for spontaneous oligomerisation (self-assembly). Amino acids which are not conserved within the sequence of the central domain of ShSf6 TerS (cdShSf6 TerS) are designated as "X" and may be, independently of each other, any amino acid.

[0081] In another preferred embodiment, A is a polypeptide comprising or consisting of one of the following amino acid sequences:

[0082] 0 z3-SNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIA; 251172 WO - 10 September 2025 ii) z3-NDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD;

[0083] Hi) z3-ATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWI_AKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGK; wherein z3represents the N-terminus of the polypeptide, or an additional N- terminal group consisting of 1 to 10 amino acids, preferably wherein z3= Gly- Pro-. The additional N-terminal group consisting of 1 to 10 amino acids may be a (single) methionine, an N-terminal purification tag (such as His-tag or Strep- tag), or the (C-terminal) amino acids of a protease cleavage site remaining after protease cleavage. Preferably, the additional N-terminal group is Gly-Pro- (i.e., a group consisting of the two amino acids Gly and Pro connected via a peptide bond). The ability of unit A to spontaneously oligomerise or self-assemble into nanoparticles is not affected by the said additional N-terminal group.

[0084] It is to be understood that if the respective polypeptide is synthesised by solidphase synthesis or liquid-phase synthesis by methods known to the person skilled in the art, the group z3may be the N-terminus of the respective polypeptide. Accordingly, by such synthetic methods, polypeptides of the following SEQ ID NOs: 7 to 9 may be obtained :

[0085] D

[0086] SNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLVDT INNSTKLLTDIYKNIVDIQIKIA (SEQ ID NO: 7);

[0087] H)

[0088] NDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNMHY QSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGAAT VAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD (SEQ ID NO: 8); iii)

[0089] ATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFRDK YAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVTNEL VGKDGGAIQIETSPMSTLFGK (SEQ ID NO: 9).

[0090] SEQ ID NO: 7 is the sequence of the central domain of CaPC5 TerS, SEQ ID NO: 8 is the sequence of the central domain of Ae44RR2 TerS, and SEQ ID NO: 9 is the sequence of the central domain of ShSf6 TerS.

[0091] However, if the above polypeptides of this embodiment are to be produced by heterologous expression, it is to be understood that z3should be the additional N-terminal group consisting of 1 to 10 amino acids. In one embodiment, z3may be a (single) methionine (M) or / V-formylmethionine. In another embodiment, z3may be an N-terminal purification tag (such as His-tag or Strep-tag) or a part or fragment thereof. In another embodiment, z3may be the (C-terminal) amino acid(s) of a protease cleavage site remaining after protease cleavage. Heterologously or recombinantly expressed polypeptides or proteins usually contain purification tags, which may be located at the N-terminal portion of the 251172 WO - 10 September 2025 said polypeptides or proteins, often between the initial methionine and the amino acids of the polypeptide or protein responsible for its biological function. When studying or utilising the heterologously expressed polypeptides or proteins, however, it is often preferred that the purification tags are removed. To achieve this, protease cleavage sites can be introduced into the sequence of a polypeptide or protein to be expressed - between the purification tag and the actual polypeptide or protein of interest. Suitable protease cleavage sites (and their corresponding proteases) are known to the person skilled in the art. After heterologous expression and subsequent purification of the polypeptide, a protease can be used to cleave off the purification tag. Proteases recognise the amino acid sequence of their respective cleavage site and cleave the peptide bond between the amino acids of the cleavage site by hydrolysis (this process is also referred to as proteolysis). The amino acids downstream of the cleaved peptide bond remain in the polypeptide or protein of interest. It is to be understood that the remaining (C-terminal) amino acids of the cleavage site do not interfere with the biological activity of the polypeptide or protein of interest. One example of a protease cleavage site is the amino acid sequence LEVLFQGP, which is the cleavage site of recombinant type 14 3C protease from human rhinovirus (HRV 3C). HRV 3C protease cleaves the cleavage site between Q and G, so that the two amino acids GP (Glycine and Proline) will remain at the N- terminus of the actual polypeptide or protein of interest.

[0092] Accordingly, in another preferred embodiment, A is a polypeptide comprising or consisting of one of the following amino acid sequences of SEQ ID NOs: 12 to 14:

[0093] D

[0094] GPSNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIA (SEQ ID NO : 12);

[0095] H)

[0096] GPNDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD (SEQ ID NO: 13);

[0097] Hi)

[0098] GPATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGK (SEQ ID NO: 14).

[0099] The sequences of SEQ ID NOs: 12 to 14 include the sequences of SEQ ID NOs: 7 to 9, respectively, as well as the two remaining amino acids GP of the protease cleavage site after proteolysis.

[0100] Preferably, A is a polypeptide consisting of one of the amino acid sequences of SEQ ID NOs: 12 to 14.

[0101] In a preferred embodiment, A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 12. Preferably, A consists of the amino acid sequence of SEQ ID NO: 12.

[0102] In a preferred embodiment of the present invention, the part A-L of the compound A-L-B has the following amino acid sequence of SEQ ID NO : 16: 251172 WO - 10 September 2025

[0103] GPSNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIAGLEGSGFQGPGSG

[0104] In another preferred embodiment of the present invention, the part A-L of the compound A-L-B has the following amino acid sequence of SEQ ID NO : 17:

[0105] GPNDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDDGLEGSGFQGPGSG

[0106] In another preferred embodiment of the present invention, the part A-L of the compound A-L-B has the following amino acid sequence of SEQ ID NO : 18:

[0107] GPATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGKGLEGSGFQGPGSG

[0108] In the compound A-L-B of the present invention, A-L is the part A-L previously described herein and B is a compound having biological activity. The C-terminus of L is covalently bound to B, preferably via a peptide bond. The compound having biological activity B is preferably a protein, which preferably has its N- terminus covalently bound to the C-terminus of L via a peptide bond.

[0109] The compound A-L-B of the present invention is a fusion molecule of the units A, L and B. Preferably, the compound A-L-B is a fusion protein. As described previously herein, in the latter case, the NP-forming unit A needs to be or needs to comprise a NP-forming polypeptide or a NP-forming protein.

[0110] The term "biological activity" refers to any activity of a substance that is demonstrable in living organisms. In pharmacology, biological activity describes the beneficial or adverse effects of a drug on living matter. The biological activity of a substance is usually measured by a bioassay and the biological activity is generally dosage-dependent, which is investigated via dose-response curves.

[0111] In a preferred embodiment of the present invention, B is selected from the group consisting of small molecules, small molecule drugs, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, siRNA, miRNA, shRNA, DNA vaccines, or nucleic acid mimetic molecules. It is to be understood that the compounds recited previously herein (i.e., small molecules, small molecule drugs, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, siRNA, miRNA, shRNA, DNA vaccines, or nucleic acid mimetic molecules) are "compounds having biological activity" within the meaning of the present invention.

[0112] It is to be understood that if B is a peptide, a polypeptide, or a protein, it may be bound to the C-terminus of L via a peptide bond (between its N-terminus and the C-terminus of L). However, in principle it is also possible that the peptide, polypeptide, or protein is attached to the C-terminus of L via a bond (or group) formed in a chemical reaction, for example in a "click-chemistry" reaction or another bioconjugation reaction. In case of a "click-chemistry" reaction, z2of L should represent a modification of the C-terminal carboxyl group of the polypeptide L which is a functional group which may be used in a click-chemistry reaction. Moreover, the peptide, polypeptide, or protein should include at least one functional group which can react with the aforementioned functional group in the click-chemistry reaction. It is possible that this functional group is present 251172 WO - 10 September 2025 at the N-terminus of the peptide, polypeptide, or protein, or at any other position of the peptide, polypeptide, or protein, as long as it is able to react with the functional group of polypeptide L. Also, B can be conjugated to the C- terminus of L via a "catcher / tag" system. In this case, z2of L is selected from known or newly designed "catcher" polypeptides and B includes a "tag" subunit and a subunit with biological activity. It is possible that the "tag" subunit is present at the N-terminus of the peptide, polypeptide, or protein, or at any other position of the peptide, polypeptide, or protein, as long as it is able to react with the "catcher" group of polypeptide L. Further, B can be conjugated to the C- terminus of L via enzymatic transpeptidation rection mediated by the sortase A enzyme from Staphylococcus aureus or its derivatives (e.g., Hirakawa H, et al. (2015) Ca2+-independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli., Biotechnology journal, 10(9), 1487-92). In this case, z2of L should be a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of L via a peptide bond. In this case (B being a peptide, a polypeptide, or a protein), the compound A-L-B of the present invention comprises either the peptide bond as covalent bond between L and B, or the bond (or group) formed in one of the aforementioned conjugation reactions between L and B.

[0113] If B is a small molecule, a small molecule drug, a nucleic acid, DNA, RNA, siRNA, miRNA, shRNA, a DNA vaccine, or a nucleic acid mimetic molecule, it may be bound to the C-terminus of L via a bond (or group) formed in a chemical reaction, for example in a "click-chemistry" reaction. In this case, z2of L should represent a modification of the C-terminal carboxyl group of the polypeptide L which is a functional group which may be used in a click-chemistry reaction. Moreover, the small molecule, small molecule drug, nucleic acid, DNA, RNA, siRNA, miRNA, shRNA, DNA vaccine, or nucleic acid mimetic molecule should include at least one functional group which can react with the aforementioned functional group in the click-chemistry reaction. It is possible that this functional group is present at any position of the small molecule, the small molecule drug, the nucleic acid, DNA, RNA, siRNA, miRNA, shRNA, the DNA vaccine, or the nucleic acid mimetic molecule, as long as it is able to react with the functional group of polypeptide L. It is also possible that the small molecule, the small molecule drug, the nucleic acid, DNA, RNA, siRNA, miRNA, shRNA, the DNA vaccine, or the nucleic acid mimetic molecule may be bound to the C-terminus of L via enzymatic transpeptidation rection mediated by the sortase A enzyme from Staphylococcus aureus or its derivatives. In this case, z2of L should be a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of L via a peptide bond. In this case (B being a small molecule, a small molecule drug, a nucleic acid, DNA, RNA, siRNA, miRNA, shRNA, a DNA vaccine, or a nucleic acid mimetic molecule), the compound A-L-B of the present invention comprises the bond (or group) formed in one of the aforementioned conjugation reactions between L and B.

[0114] In a further preferred embodiment of the present invention, B is a polypeptide or protein selected from the group consisting of virus antigens, enzymes, antibodies, growth factors, tumour suppressors, DNA-binding domains, RNA- 251172 WO - 10 September 2025 binding domains, pilins, adhesins, antigenic peptides, cancer neoantigens, and diagnostic peptides. In the resulting compounds A-L-B, all units A, L, and B are preferably completely peptidic compounds (i.e., they are polypeptides or proteins) and connected via peptide bonds, resulting in a polypeptide chain A- L-B.

[0115] In a further preferred embodiment of the present invention, B is a virus antigen, more preferably Nipah virus receptor binding protein extracellular domain having the following amino acid sequence of SEQ ID NO: 10 :

[0116] KPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAY SHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEF YYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFALRNIEKGKYDKV MPYGPSGIKQGDTLYFPAVGFLVRTEFTYNDSNCPIAECQYSKPENCRLSMGIRPNSHYI LRSGLLKYNLSDEENSKIVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGD VQTVNPLVVNWRDNTVISRPGQSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFL DSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDN VI PKLFAVKIPEQCT (SEQ ID NO: 10).

[0117] Nipah virus is a WHO high-priority pathogen with pandemic potential and a very high mortality rate over 60%. Despite early outbreaks in Bangladesh and India, there are no available vaccines against Nipah disease (Eaton BT, et al. (2006) Hendra and Nipah viruses: different and dangerous, Nature Reviews Microbiology, 4(1), 23-35). The Nipah virus receptor binding protein extracellular domain (also called Nipah virus attachment glycoprotein G ectodomain) is exposed on the Nipah virus surface and is the dominant viral antigen for vaccines (Amaya M and Broder CC (2020) Vaccines to Emerging Viruses: Nipah and Hendra, Annual Review of Virology, 7(1), 447-473).

[0118] In the resulting compounds A-L-B comprising Nipah virus receptor binding protein extracellular domain having the following amino acid sequence of SEQ ID NO: 10 as unit B, all units A, L, and B are preferably completely peptidic compounds (i.e., they are polypeptides or proteins) and connected via peptide bonds, resulting in a polypeptide chain A-L-B. In an embodiment of the invention, the compound A-L-B has the following amino acid sequence of SEQ ID NO: 11 :

[0119] GPATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGKGLEGSGFQGPGSGKPQTEGVSNLVGLPNNICLQKT SNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLEKIGSCSRGVSKQRIIGVG EVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEFYYVLCAVSVVGDPILNSTYWS GSLMMTRLAVKPKNNGESYNQHQFALRNIEKGKYDKVMPYGPSGIKQGDTLYFPAVGF LVRTEFTYNDSNCPIAECQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDEENSKIVFIE ISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVQTVNPLVVNWRDNTVISRPG QSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEVLYR AQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT (SEQ ID NO: 11)

[0120] Surprisingly, a compound A-L-B being a fusion protein including a central domain of TerS (as unit A), a linker L and the Nipah virus receptor binding protein extracellular domain of SEQ ID NO: 10 (Nipah Gecd; as unit B), showed 251172 WO - 10 September 2025 superior immunogenicity compared to the soluble form of Nipah Gecd in a mouse model, indicating that the said compound A-L-B may be a suitable vaccine candidate (see Fig. 15 and Fig. 17). The compound A-L-B spontaneously oligomerises to form ring-like nanoparticles. There the Nipah Gecd antigens are exposed to the outside to boost immunogenicity.

[0121] The part A-L described herein and the compound A-L-B of the present invention advantageously self-assemble into nanoparticles. The object of the present invention is therefore further solved by a nanoparticle, formed from a plurality of compounds A-L-B of the present invention. Also disclosed herein are nanoparticles formed from a plurality of parts A-L.

[0122] The nanoparticles of the present invention may be formed from more than one of compounds A-L-B. Alternatively, the nanoparticles of the present invention may be formed from one or more compounds A-L-B and additionally comprise A-L. It is to be understood that the nanoparticles of the present invention are self-assembled nanoparticles. The nanoparticles may be formed from a total number of 8 to 12 compounds A-L-B. In a preferred embodiment, unit A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 and the nanoparticle is formed from 10 or 11 or 12 of compounds A-L-B, preferably from 10 of compounds A-L-B. In another preferred embodiment, unit A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and the nanoparticle is formed from 10 or 11 or 12 of compounds A- L-B. In another preferred embodiment, unit A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 9 and the nanoparticle is formed from 8 of compounds A-L-B. In another preferred embodiment, unit A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and the nanoparticle is formed from 10 or 11 or 12 of compounds A-L- B, preferably from 10 of compounds A-L-B. In another preferred embodiment, unit A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 and the nanoparticle is formed from 10 or 11 or 12 of compounds A-L-B. In another preferred embodiment, unit A is a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 14 and the nanoparticle is formed from 8 of compounds A-L-B.

[0123] Advantageously, compounds A-L-B of the present invention or nanoparticles formed from a plurality of compounds A-L-B of the present invention may be used as delivery systems for the targeted delivery and controlled release of compounds, such as, but not limited to, therapeutic agents or other compounds having biological activity.

[0124] The object of the present invention is therefore further solved by a pharmaceutically active composition comprising:

[0125] (i) the compound A-L-B of the present invention and a pharmaceutically acceptable carrier; or

[0126] (ii) the nanoparticle formed from a plurality of compounds A-L-B of the present invention and a pharmaceutically acceptable carrier.

[0127] Suitable pharmaceutically acceptable carriers according to Pharmacopoeia Europaea (Ph. Eur.) are known to the person skilled in the art. The pharmaceutically active composition may further comprise stabilisers and other 251172 WO - 10 September 2025 auxiliaries suitable for pharmaceutical compositions known to the person skilled in the art. The pharmaceutically active composition may be formulated for intravascular, lymphatic, intracardial, parenteral, intravenous, intramuscular, intrathecal, pulmonary, epi-, intra- and sub-cutaneous, intranasal, ophthalmic, auricular, buccal, or intraperitoneal administration, or for local administration to an organ, or for administration into the cerebrospinal fluid space. These routes of administration of pharmaceutical compounds and compositions and their execution are known to the person skilled in the art.

[0128] In a preferred embodiment, the pharmaceutically active composition is a vaccine, preferably a vaccine against Nipah virus infection (Nipah disease).

[0129] The object of the present invention is further solved by a compound A-L-B of the present invention, or the nanoparticle formed from a plurality of compounds A-L-B of the present invention, for use as a medicament.

[0130] In an embodiment, the present invention also relates to the compound A-L-B of the present invention, or the nanoparticle formed from a plurality of compounds A-L-B of the present invention, for use in the prevention and / or treatment of an infectious disease, preferably a viral disease, more preferably Nipah virus infection (Nipah disease). Preferably, the compound A-L-B has the amino acid sequence of SEQ ID NO: 11.

[0131] The object of the present invention is further solved by a polynucleotide encoding the compound A-L-B of the present invention. Further disclosed herein are polynucleotides encoding the linker L and the part A-L. It is to be understood that only linkers L, parts A-L, and compounds A-L-B which are completely peptidic (i.e., peptides, polypeptides, or proteins) may be encoded as polynucleotides. By way of example, a NP-forming unit A being a surfactant is not suitable to be encoded by a polynucleotide. The polynucleotides of the invention are characterised by being constituted of DNA, R.NA, genomic DNA or PNA (peptide nucleic acid). Polynucleotides of the present invention shall be used for recombinant peptide expression in pro- or eukaryotic cells, mutagenesis studies, cloning in vectors of interest, in particular those that can be used for gene transfer approaches.

[0132] The object of the present invention is further solved by a vector containing a polynucleotide of the present invention. Vectors encoding polynucleotide sequences of the invention shall be used for recombinant peptide expression in pro- or eukaryotic cells, mutagenesis studies, or for gene transfer into eukaryotic cells.

[0133] The object of the present invention is further solved by a genetically engineered host cell containing the vector of the present invention. Such genetically engineered host cells may be used to recombinantly express the compound A- L-B of the present invention encoded by the polynucleotide of the present invention. Further, such genetically engineered host cells may be used to recombinantly express the linker L or the part A-L disclosed herein. The genetically engineered host cell may be a pro- or eukaryotic cell. An example for a preferred prokaryotic host cell is E. coli, more preferably E. coli BL21(DE3). Preferably, the genetically engineered host cell is an insect cell, preferably an insect cell derived from Bombyx mori, Mamestra brassicae, Spodoptera 251172 WO - 10 September 2025 frugiperda, Trichoplusia ni, or Drosophila melanogaster. Methods for cultivating such cells and for expressing recombinant proteins in such cells are known to the person skilled in the art.

[0134] The object of the present invention is further solved by a process for the preparation of the compound A-L-B of the present invention, the process comprising the following steps: a) heterologous expression of the compound A-L-B of the present invention; b) optionally subsequent posttranslational or chemical modification of the heterologously expressed compound A-L-B of the present invention.

[0135] The disclosed linker L and the disclosed part A-L may also be produced by the said methods. It is to be understood that only linkers L, parts A-L, and compounds A-L-B which are completely peptidic (i.e., peptides, polypeptides, or proteins) may be heterologously expressed by this process. By way of example, a NP-forming unit A being a surfactant is not suitable to be heterologously expressed by this process. Methods of heterologous expression are known to the person skilled in the art.

[0136] The optional subsequent posttranslational modification may take place directly in the host cell used for the heterologous expression. The posttranslationally modified compound may be isolated and purified afterwards by methods known to the person skilled in the art. The optional subsequent chemical modification requires isolation and optionally purification of the heterologously expressed compound. Further steps of purification known to the person skilled in the art may be necessary after completion of the chemical modification. The optional subsequent chemical modification may comprise protease treatment steps suitable for the removal of purification tags.

[0137] Alternatively, it is also possible to produce the compound A-L-B of the present invention by solid-phase synthesis in terms of a Merrifield synthesis or liquidphase synthesis by methods known to the skilled person using protected amino acids, and its purification. The disclosed linker L and the disclosed part A-L may also be produced by the said methods.

[0138] It is to be understood that preferred embodiments or preferred features mentioned for one embodiment are also to be applied in the same way for all other embodiments mentioned in the application. Each preferred embodiment and each preferred feature can be combined with each other without further limitation.

[0139] Brief description of the Figures

[0140] Fig. 1 shows the domain structure of bacteriophage-derived small terminase protein subunit (TerS). TerS proteins are typically composed of N-terminal domain, which contains DNA-binding elements, a central oligomerisation domain and a C-terminal domain, which is unstructured in monomeric TerS form. After TerS oligomerisation, the C-terminal domain can assume a secondary structure or remain unstructured.

[0141] Fig. 2 A shows an SDS-PAGE analysis of CaPC5 and St80B TerSs after purification with affinity chromatography. Full length CaPC5 TerS is insoluble 251172 WO - 10 September 2025 after bacterial overexpression and was not purified with liquid chromatography. Recombinant TerS were expressed in BL21(DE3) E. coli in autoinduction growth medium overnight at 30 °C and 150 rpm. The cells were harvested and lysed using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). Lysate supernatants were loaded on StrepTrapXT affinity column (Cytva) and eluted with elution buffer containing biotin.

[0142] Fig. 2 B shows an SDS-PAGE analysis of StA411 TerS after purification with affinity chromatography. Recombinant TerS was expressed in BL21(DE3) E. coli in autoinduction growth medium overnight at 30 °C and 150 rpm. The cells were harvested and lysed using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). Lysate supernatants were loaded on StrepTrapXT affinity column (Cytva) and eluted with elution buffer containing biotin.

[0143] Fig. 3 shows an SDS-PAGE analysis of SfSh6-TerS and Ae44RR2-TerS samples after anion exchange chromatography. Elutions from StrepTrapXT affinity column were loaded on TOYOPEARL GigaCap Q-650M (TOSOH) column to further purify isolated proteins in ascending salt gradient, collecting samples E4- 5 and E2-3 for Ae44RR2 and ShSf6 TerS, respectively. CIP: cleaning-in-place.

[0144] Fig. 4 shows TerS separation on native PAGE. Purified TerS were loaded on native PAGE to preserve oligomeric state. It was demonstrated that SfSh6 and Ae44RR2 TerS form oligomers, while St80B and StA411 TerS remain in monomeric state.

[0145] Fig. 5 shows an overlay of the AlphaFold2 predictions of CaPC5 TerS monomeric structure (black) and central helical domain of Ae44RR2 TerS crystal structure (light grey; PDB ID 3TXS). The oligomerising central domains of TerS structures closely overlap.

[0146] Fig. 6 shows the domain structure of full length AeRR442 and CaPC5 TerSs with indicated peptide linker (flexible linker of Example 1). cdCaPC5 TerS (also designated as NCACaPC5 TerS herein) is a minimised version of CaPC5 TerS with N- and C-terminal domain deletions. cdCaPC5 TerS (without the linker) has the amino acid sequence of SEQ ID NO: 12. cdCaPC5 TerS with the linker has the amino acid sequence of SEQ ID NO: 16.

[0147] Fig. 7 A shows an SDS PAGE analysis of full-length CaPC5 TerS (see Example 3). Recombinant TerS was expressed in in BL21(DE3) E. coli in autoinduction growth medium overnight at 30 °C and 150 rpm. The cells were harvested and lysed using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). Lysate supernatants were loaded on StrepTrapXT affinity column (Cytiva) and eluted with elution buffer containing biotin.

[0148] Fig. 7 B shows an SDS PAGE analysis of N-terminal deletion mutant of CaPC5 TerS (see Example 3). Recombinant TerS was expressed in in BL21(DE3) E. coli in autoinduction growth medium overnight at 30 °C and 150 rpm. The cells were harvested and lysed using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). Lysate supernatants were loaded on StrepTrapXT affinity column (Cytiva) and eluted with elution buffer containing biotin.

[0149] Fig. 7 C shows an SDS PAGE analysis of C-terminal deletion mutant of CaPC5 TerS (see Example 3). Recombinant TerS was expressed in in BL21(DE3) E. coli 251172 WO - 10 September 2025 in autoinduction growth medium overnight at 30 °C and 150 rpm. The cells were harvested and lysed using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). Lysate supernatants were loaded on StrepTrapXT affinity column (Cytiva) and eluted with elution buffer containing biotin.

[0150] Fig. 7 D shows an SDS PAGE analysis of minimised central domain CaPC5 TerS (N- and C-deletion) (see Example 3). Recombinant TerS was expressed in in BL21(DE3) E. coli in autoinduction growth medium overnight at 30 °C and 150 rpm. The cells were harvested and lysed using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). Lysate supernatants were loaded on StrepTrapXT affinity column (Cytiva) and eluted with elution buffer containing biotin.

[0151] Fig. 8 shows the native PAGE analysis of minimised central domain CaPC5 oligomers (cdCaPC5 TerS; also designated as NCACaPC5 TerS herein) (see Example 3). Purified elution fractions from anion exchange column (El, E2) were loaded on native PAGE. Arrows indicate size corresponding to monomeric and oligomeric states.

[0152] Fig. 9 A shows a TEM micrograph of CaPC5 TerS C-terminal deletion mutant (CACaPC5 TerS) of Example 4. The expressed TerS was purified with affinity and anion exchange chromatography and TEM analysis was performed. TerS sample was stained with uranyl acetate.

[0153] Fig. 9 B shows a TEM micrograph of CaPC5 TerS N-and C-terminal deletion mutant (cdCaPC5 TerS; also designated as NCACaPC5 TerS herein) of Example 4. The expressed TerS was purified with affinity and anion exchange chromatography and TEM analysis was performed. TerS sample was stained with uranyl acetate.

[0154] Fig. 10 shows the results of TEM-diameter measurements for the CaPC5 TerS deletion mutants according to Example 4.

[0155] Fig. 11 shows ShSf6-TerS nanoparticle analysis with mass photometry. The molecular weight distribution is presented as histogram with 5 kDa bin width and fitted gaussian normal distribution curve.

[0156] Fig. 12 shows cdCaPC5-TerS nanoparticle analysis with mass photometry. The molecular weight distribution is presented as histogram with 5 kDa bin width and fitted gaussian normal distribution curve.

[0157] Fig. 13 shows a TEM micrograph of purified ShSf6 TerS-L-Gecd Nipah virus vaccine candidate of Example 5. Scale bar is 100 nm.

[0158] Fig. 14 A shows the structures of soluble monomeric NiV Gecd and oligomeric SfSh6 TerS-L-Gecd nanoparticle used in Example 5.

[0159] Fig. 14 B shows the schedule of the preclinical immunogenicity study in mice of Example 5.

[0160] Fig. 15 shows the results of the immunogenicity study of ShSf6 TerS-L-Gecd vaccine candidate in comparison with recombinant Gecd of Example 5. Endpoint titers were determined with indirect ELISA assay.

[0161] Fig. 16 shows the design and schedule of the preclinical immunogenicity study in hamsters of Example 6. 251172 WO - 10 September 2025

[0162] Fig. 17 shows the results of the prime-and-pull immunogenicity study of ShSf6 TerS-L-Gecd (presented as nSf-NiVac) vaccine candidate in comparison with Nipah virus-like particles (NiVLPs) of Example 6. Area under the curve (AUC) titers were determined with indirect ELISA assay. ADX: AddaVax™ adjuvant. * p < 0.05, ** p < 0.01. For statistical comparison, log-transformed AUC titers were evaluated using a Kruskal-Wallis test corrected with Dunn's multiple comparisons test.

[0163] In the following examples, the present invention is disclosed but without limiting the scope of protection to these specific examples.

[0164] Examples

[0165] Materials and Methods

[0166] The following general methods have been used in the examples described hereinbelow:

[0167] Bacterial expression

[0168] Selected phage TerS candidate genes were codon optimised for expression in E. coll. Synthetic optimised gene sequence DNA fragments were cloned into linearised pET28b expression vector using Gibson assembly (Gibson DG, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature methods, 6(5), 343-345). Constructs for Staphylococcus phage St880B and St411A TerS, Campylobacter phage CaPC5 TerS, Aeromonas phage Ae44RR2 TerS and Shigella phage ShSf6 TerS were prepared. To facilitate purification, a TwinStrep purification sequence (Schmidt TGM, et al. (2013) Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants, Protein Expression and Purification, 92(1), 54-61) with HRV 3C protease cleavage site was added to the TerS N-terminus. Additionally, fusion proteins were prepared with selected TerSs and Nipah virus G extracellular domain. The two sequences were joined with the designed 13 AA linker (TerS-L-Gecd). The sequence of assembled constructs was confirmed with control restriction digestion and Sanger sequencing. The recombinant proteins were expressed in BL21(DE3) E. coli strain. The cells were grown in autoinduction growth medium (Studier FW (2005) Protein production by auto-induction in high-density shaking cultures, Protein Expression and Purification , 41(1), 207-234) overnight at 30 °C and 150 rpm. Cell pellets were harvested by centrifugation at 9000 ref for 10 min at 4 °C and stored at -75 °C until use.

[0169] Insect cell expression

[0170] SfSh6 TerS and cdCaPC5 TerS (also designated as NCACaPC5 TerS herein) were selected for expression in insect cells. The synthetic genes were cloned into pFastBacl (Thermo Scientific) shuttle vector backbone using Gibson assembly (Gibson DG, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature methods, 6(5), 343-345). To facilitate purification, a TwinStrep purification sequence (Schmidt TGM, et al. (2013) Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants, Protein Expression and Purification, 92(1), 54- 61) with HRV 3C protease cleavage site was added to the TerS N-terminus. 251172 WO - 10 September 2025

[0171] Additionally, fusion proteins with selected TerSs and Nipah virus G extracellular domain (TerS-L-Gecd) were prepared. The two sequences were joined with the designed 13 AA linker. The sequence of assembled constructs was confirmed with control restriction digestion and Sanger sequencing. The recombinant baculovirus vectors were prepared according to the Bac-to-Bac expression system. First, chemically competent DHIOBac E. coli cells (Thermo Scientific) were transformed with pFastBacl constructs harbouring TerS and TerS-Gecd fusions with TwinStrep tag. DHIOBac clones with recombinant bacmid DNA were selected and the recombinant bacmid DNA was purified. Next, Sf9 cells in ESF 921 medium (Oxford Expression Technologies) were transfected with isolated recombinant bacmid DNA with ExpiFectamine Sf transfection reagent (Thermo Scientific) to produce recombinant baculovirus vectors. After five days, cell media was collected and recombinant baculoviruses were amplified. Recombinant proteins were expressed in Sf9 cells (Oxford Expression Technologies) at 27 °C and 127 rpm for three days after recombinant baculovirus infection at MOI 1. Cell pellets were harvested by centrifugation at 2000 ref for 10 min at 4 °C and stored at -75 °C until use.

[0172] Liquid chromatography

[0173] Bacterially expressed small terminase constructs were purified from thawed cell pellets. Cell lysates were prepared on ice with B-PER bacterial protein extraction reagent (Thermo Scientific), supplemented with lysozyme (Thermo Scientific), 5 mM MgC , DENARASE endonuclease (c-LEcta) and EDTA-free complete™ Protease Inhibitor Cocktail (Roche). After 20 min incubation, lysates were centrifuged for 20 min at 10000 ref at 4 °C and supernatant was collected and loaded on 1 mL StrepTrapXT column (Cytiva), equilibrated with 100 mM Tris- HCI pH 8.0 and 150 mM NaCI. Bound recombinant TerS constructs were eluted in 100 mM Tris-HCI pH 8.0, 150 mM NaCI and 50 mM biotin. The collected elution fractions were diluted 1 :2 in dilution buffer (5 mM Tris-HCI, pH 7.5) and loaded on 1 mL TOYOPEARL GigaCap Q-650M column (Tosoh) that was equilibrated with 25 mM Tris-HCI pH 7.5 and 50 mM NaCI running buffer. Elution fractions were collected in three wash steps of 8% - 2% - 100% of elution buffer 25 mM Tris-HCI pH 7.5, 2 M NaCI. Pure TerS constructs are concentrated in elution fraction between 8% and 28% of elution buffer. Finally, TerS constructs were buffer exchanged to lx PBS (Corning) and concentrated using Vivaspin Turbo 4 ultrafiltration device with 100 kDa molecular weight cutoff (Sartorius).

[0174] TerS constructs that were expressed in insect cells were purified from frozen cell pellets. Thawed pellets on ice were resuspended in 25 mM Tris-HCI pH 8.0, 300 mM NaCI, 10% glycerol and 0.5% Triton X-100 (Merck), which was supplemented with EDTA-free complete™ Protease Inhibitor Cocktail (Roche), 5 mM MgC and DENARASE endonuclease (c-LEcta). Insect cells were physically lysed by douncing. Cell lysates were pelleted by centrifugation for 60 min at 9000 ref and 4 °C. The collected supernatants were filtered through Sartopore 2 300 (0.8 + 0.45 pm) capsule filter (Sartorius) before loading on 1 mL StrepTrapXT column (Cytiva), equilibrated with 25 mM Tris-HCI pH 8.0, 300 mM NaCI and 10% glycerol. Bound recombinant TerS constructs were eluted in 25 mM Tris-HCI pH 8.0, 300 mM NaCI, 10% glycerol and 50 mM biotin. Then, the TwinStrep affinity tag was cleaved with HRV 3C protease (Sigma Aldrich) at 4 °C for 1 h, which was removed by passing the collected fractions through 1 mL 251172 WO - 10 September 2025

[0175] HisTrapFF column (Cytiva). The TerS constructs were polished on 1 mL TOYOPEARL GigaCap Q-650M column (Tosoh) that was equilibrated with 25 mM Tris-HCI pH 8.0, 50 mM NaCI and 10% glycerol. Final fractions were collected in ascending elution gradient to 25 mM Tris-HCI pH 8.0, 2 M NaCI and 10% glycerol. The TerS construct are eluted at buffer conductivity between 15 mS / cm and 30 mS / cm. Finally, TerS constructs were buffer exchanged to lx PBS (Corning) and concentrated using Vivaspin Turbo 4 ultrafiltration device with 100 kDa molecular weight cutoff (Sartorius). If needed, the final product was sterile filtered through 0.22 pm filter (TPP).

[0176] PAGE

[0177] Recombinant TerS constructs were analysed with polyacrylamide gel electrophoresis (PAGE). Under denaturing conditions, samples were mixed with 4x Laemmli sample loading buffer. Protein samples were denatured by heat at 70 °C for 10 min and loaded on mPAGE™ Bis-Tris 4-20% precast gels (Merck) and separated at 200 for 35 min in Millipore MOPS SDS running buffer (Merck). Gels were stained with InstantBlue Coomassie protein stain (Abeam).

[0178] For native PAGE, protein samples were mixed with 3x native loading dye and loaded on Mini Protean TGX precast gel 4-20% (BioRad). The gel was run at 150 V for 5 h in lx Native running buffer. Gels were stained with InstantBlue Coomassie protein stain (Abeam).

[0179] TEM

[0180] Formvar-coated grids were placed on drops of different phage suspensions for 5 min and negative staining was performed using 2% uranyl acetate. The grids were examined using a transmission electron microscope JEOL JEM-1400 Plus (Tokyo, Japan) at 60-120 kV.

[0181] Mass photometry

[0182] Mass photometry analysis was performed using a TwoMP instrument (Refeyn). 10 pL of TerS sample in PBS at 200-300 nM was applied and 2845 frames were recorded. A gaussian curve was fitted to data histogram peaks to determine mean molecular weight values and standard deviation.

[0183] Mice immunogenicity study

[0184] The objective was to evaluate the immunogenicity of ShSf6 TerS-L-Gecd vaccine candidate in BALB / cOlaHsd mouse model in comparison with monomeric NiV Gecd. All animals were treated in accordance with the EC Directive 86 / 609 / EEC for animal experiments, using approved protocols. Female 6-8-week-old mice acclimatised in the animal facility for 1 week before the start of the experiment. On day 0 (dO) before the immunisation, 200 pL of blood was collected from tail snip from all animals. Plasma was isolated and frozen. 3.33 pg of vaccine agents were mixed 1 : 1 with adjuvant (AddaVax adjuvant), incubated 15 minutes at room temperature and then used immediately. All mice received two intramuscular ( / .m.) injections of 50 pl volume in each thigh muscle (100 pl in total) on day 0. Vaccination was repeated on day 21 (d21). Half of the animals in each group were immunised with a second dose on day 21, while other half of the mice were sacrificed on day 21, around 500 pl of blood was collected and plasma was isolated and frozen. The study was terminated with all remaining 251172 WO - 10 September 2025 mice being sacrificed on day 42 (d42). Around 500 |jl of blood was collected, and plasma was isolated and frozen. Blood was collected in tubes containing EDTA or EDTA-coated microvette.

[0185] Hamster immunogenicity study

[0186] The objective was to evaluate the immunogenicity of ShSf6 TerS-L-Gecd vaccine candidate in comparison to Nipah virus-like particles (NiVLPs) in golden Syrian hamsters in a prime-and-pull study. Animal experiments were conducted in compliance with local and European ethical and regulatory guidelines for animal research. Groups of 6 animals were assigned to either PBS-treated control, 25 pG NiVLP-only, 25 pg NiVLP + 25 ug AddaVax™ (Invivogen), 10 pg ShSf6 TerS-L-Gecd, and 10 pg ShSf6 TerS-L-Gecd + 25 ug AddaVax™ (Invivogen) experimental groups. Before the prime immunisation on day 0, blood samples were collected. 50 pL of vaccine antigens were mixed 1 : 1 with AddaVax™ adjuvant or PBS and incubated for 15 minutes at room temperature and then used immediately. All hamsters received an intramuscular prime (i.m.) injection of 100 pL on day 0. Vaccine candidates for pull vaccination were prepared as described before. Immunisation was performed intranasally (i.n.) with 50 pL per nostril on day 21 post-prime, when blood was collected and sera stored. Serum samples were also collected on day 28 and the study finished on day 50 postprime. On day 50, the hamsters were euthanised and a final blood sample was collected.

[0187] ELISA

[0188] The IgG titers against NiV Gecd were determined using an indirect ELISA protocol. Nunc-Immuno™ MicroWell™ 96 well plates (Sigma Aldrich) were coated with 2 pg of pure NiV Gecd in lx PBS (Corning) overnight at 4 °C. Next, the plate was washed with wash buffer (0.01 M PBS pH 7.2, Tween-20 0.1% v / v) and performed blocking with 5% skim milk suspension for 1 h at room temperature. For mice samples, blood plasma samples were added to the plates at 1 : 50 initial dilution after washing. 4-fold serial dilutions were performed and plates were incubated at 4 °C overnight. Next day, plates were washed and HRP-conjugated secondary Anti-mouse IgG (Fab specific) antibodies produced in goat (Sigma Aldrich, A9917) at 1 :20000 dilution were added and incubated for 1 h at room temperature. After washing, plates were incubated with TMB substrate (Sigma Aldrich, T4444) for 100 min. After, 2 M HCI stop solution was added and absorbance at 450 nm and 650 nm was determined. Endpoint anti-Gecd IgG titres were calculated based on reciprocal dilutions above control mice group threshold. The threshold was calculated from control group mean values plus 2.631 standard deviations.

[0189] For hamster samples, 1 :50 initial dilutions were added to the plates after washing. Next, 5-fold serial dilutions were performed, and plates were incubated at room temperature for 2 h. After washing, HRP-conjugated secondary goat anti-Syrian Hamster IgG H&L antibodies (Abeam, ab6892) at 1 : 10,000 dilution were added and incubated for 1 h at room temperature. The microtiter plates were incubated with TMB substrate (Sigma Aldrich, T4444) for 10 min. After, 2 M HCI stop solution was added and absorbance at 450 nm and 650 nm was determined. For area under the curve (AUC) titre calculation, the baseline from mean blank values plus 5 standard deviations (SD) was determined. Corrected 251172 WO - 10 September 2025 values below 0.02 were set to 0. The AUC values were determined by integrating the serial dilution data with trapezoidal rule. For statistical comparison, log- transformed AUC titres were evaluated using a Kruskal-Wallis test corrected with Dunn's multiple comparisons test.

[0190] Example 1 - Flexible linker design

[0191] In order to provide alternative nanoparticle-based delivery systems which can, e.g., be used to present antigens in vaccines against infection diseases, for enzyme immobilisation or as experimental tools for peptide oligomerisation and diagnosis, a linker useful in nanoparticle-based delivery systems was designed. The linker is a polypeptide consisting of 13 amino acids, having the following amino acid sequence:

[0192] GLEGSGFQGPGSG (SEQ ID NO: 3)

[0193] The glycine residues at positions 1, 4, 6, 9,11, and 13 of the polypeptide are important to ensure flexibility and solubility of the linker. The other amino acids can be, independently of each other, any amino acid except histidine (H), cysteine (C) and tryptophan (W).

[0194] The linker can connect two units, e.g., a NP-forming unit and a biologically active compound. Advantageously, it can provide stability, solubility and flexibility between the two connected units.

[0195] Example 2 - Nanoparticle-formino unit discovery

[0196] A NP-forming unit useful in a nanoparticle-based delivery system was discovered by the inventors. The NP-forming unit is based on bacteriophage- derived small terminase protein subunits (TerS). TerS are involved in bacteriophage genome delivery into empty capsids during infection of host bacteria. Crystal structures of isolated subunits reveal ring-like nanoparticle structure, composed of TerS homo-oligomers. Determined structures include Aeromonas phage 44RR2 (Ae44RR2 TerS) (Sun S, et al. (2012) Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages, Proceedings of the National Academy of Sciences, 109(3), 817-822), Bacillus phage SF6 (Buttner CR, et al. (2012) Structural basis for DNA recognition and loading into a viral packaging motor, Proceedings of the National Academy of Sciences, 109(3), 811-816) and Shigella phage Sf6 (ShSf6) TerS (Zhao H, et al. (2010) Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine, Proceedings of the National Academy of Sciences, 107(5), 1971-1976). TerS have very little sequence conservation with diverse structure and oligomerisation patterns across phage families. For example, eleven to twelve TerS monomers form the quaternary ring structure in Ae44RR2 TerS, while eight ShSf6 TerS monomers associate in the final oligomer (Lokareddy RK, et al. (2022) Viral Small Terminase: A Divergent Structural Framework for a Conserved Biological Function, Viruses, 14(10), 2215).

[0197] To identify the best TerS candidates and demonstrate broad usability, Ae44RR2 TerS and ShSf6 TerS with known crystal structures were tested. In addition, phage TerS with unknown structural properties, which were isolated at COBIK, Ajdovscina, Slovenia (Janez N, et al. (2014) Identification and characterisation 251172 WO - 10 September 2025 of new Campylobacter group III phages of animal origin, FEMS Microbiology Leters, 359(1), 64-71; Strancar V, et al. (2023) Isolation and in vitro characterization of novel S. epidermidis phages for therapeutic applications, Frontiers in Cellular and Infection Microbiology, 13, 1169135) were tested : Staphylococcus phage 80 B (St80B TerS), Staphylococcus phage A411 (StA411 TerS) and Campylobacter phage PC5 TerS (CaPC5 TerS).

[0198] The discovery of CaPC5 TerS was totally unexpected, because initial genomic analysis of Campylobacter phages did not detect any TerS candidate sequences. To identify Campylobacter TerS, it was necessary to search for homology with related Cyanobacteria phages, which revealed a new hypothetical protein, which was included a phylogenetic TerS analysis. Further, the monomeric structure of this new hypothetical protein - like the monomeric structures of the other TerS proteins mentioned before - was predicted.

[0199] Comparing the predicted monomer structures, CaPC5 TerS was identified as a promising candidate for spontaneous oligomerisation, which is critical for nanoparticle formation.

[0200] In order to evaluate the oligomerisation properties of the selected TerS polypeptides CaPC5 TerS, Ae44RR2 TerS, St80B TerS, StA411 TerS and ShSf6 TerS, recombinant expression in bacterial culture and liquid chromatography purification was performed. TwinStep tag-TerS fusion proteins were constructed and purified by affinity purification, which was followed by an anion exchange chromatography polishing step. TerS expression was evaluated on polyacrylamide gel electrophoresis (PAGE) under native and denaturing conditions. It was observed that full length CaPC5 TerS remains in insoluble fraction after overexpression in E. coll (Fig. 2 A), while a fraction of Staphylococcus St80B (Fig. 2 A) and StA411 (Fig. 2 B), Ae44RR2 and SfSh6 TerSs can be purified (Fig. 3). Despite, the majority of expressed TerSs remains insoluble after overexpression in bacteria.

[0201] Importantly, native PAGE size separation identified Ae44RR2 and ShSf6 TerS oligomers, while Staphylococcus St80B and StA411 TerS remain in monomeric form (Fig. 4). Likely, some phage TerS (e.g. St80B and StA411 TerS) form higher-order structures only in presence of other phage proteins, e.g. TerL.

[0202] Surprisingly, despite low sequence similarity, the predicted CaPC5 TerS structure adopts a similar central domain helical fold to T4-like Ae44RR2 TerS (Fig. 5) with known crystal structure and oligomerisation properties (Sun S, et al. (2012) Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages, Proceedings of the National Academy of Sciences, 109(3), 817-822). This allowed identification of conserved amino acids of the central CaPC5 TerS oligomerisation domain which are necessary for spontaneous oligomerisation (X denotes any amino acid) :

[0203] LKXXFXXMRXXLVXXIXXGQXXLXXISXXIXXXXXXXXXXXVXXYXXLVXXIXXSTXXLX XIYXXIXXI (SEQ ID NO: 4)

[0204] For Ae44RR2 TerS the sequence showing the conserved amino acids is (X denotes any amino acid): 251172 WO - 10 September 2025

[0205] YXXVRXXMXXQSXXLLXXAXXALXXAXXXXXXXXXXXFXXLMXXMXXTNXXMXXMHX XM (SEQ ID NO: 5)

[0206] For ShSf6 TerS the sequence showing the conserved amino acids is (X denotes any amino acid):

[0207] PDAXEVAKARLRVDTXKWXLARMNPRKYGDXVTNELXXXXXXAIQIETS (SEQ ID NO: 6)

[0208] Example 3 - Design of minimised TerS

[0209] Interestingly, the predicted CaPC5 TerS monomer structure is similar to Ae44RR2 TerS, which successfully produced oligomers in the above experiments. However, the full-length CaPC5 TerS protein remained insoluble after expression (Fig. 2 A). To improve the solubility of recombinantly expressed TerS, N- and C-terminal CaPC5 TerS deletion mutants (minimised CaPC5 TerS, see Fig. 6) based on AlphaFold2 structure prediction and comparison with known Ae44RR2 TerS mutants were produced.

[0210] The full-length CaPC5 TerS and deletion mutants of CaPC5 TerS were expressed in E. coli. The resulting recombinant proteins were purified with TwinStrep tag affinity chromatography. Based on SDS-PAGE analysis, it was concluded that full-length and N-terminal deletion TerS mutants are poorly soluble and overwhelmingly remain in bacterial cell pellet after lysis (Fig. 7 A & B). In contrast, the C-deletion mutant and minimised central domain (N- and C- deletion) CaPC5 TerS are soluble and readily purified with affinity chromatography (Fig. 7 C & D). This suggests that C-terminal TerL-binding domain interferes with TerS solubility when overexpressed. Furthermore, oligomer self-assembly of the minimised CaPC5 TerS central domain (cdCaPC5 TerS) was confirmed with native PAGE (Fig. 8; cdCaPC5 TerS). Thus, the central domain was identified as minimal oligomerisation motif. Advantageously, a shorter nanoparticle-forming domain can reduce complexity and prevent undesirable biological interactions.

[0211] Example 4 - Structural characterisation of minimised TerS oligomers

[0212] To further characterise the structural properties of purified TerS deletion mutants, transmission electron microscopy (TEM) was performed on purified C- deletion mutant (CACaPC5 TerS) (Fig. 9 A) and the minimised CaPC5 TerS central domain (cdCaPC5 TerS) (Fig. 9 B). Both TerS deletion mutants display characteristic rings with 6 nm mean diameter (Fig. 10; C-deletion mutant designated as CdelCaPC5-TerS).

[0213] Additionally, the nanoparticle molecular weights were analysed with mass photometry for ShSf-TerS and the minimised CaPC5 TerS central domain (cdCaPC5; also designated as NCACaPC5 TerS herein). The ShSf6 TerS forms particles with 153 kDa mean molecular weight (Fig. 11). With a predicted monomer size of 19 kDa for ShSf6 TerS fusion with Twin-Strep-tag, this corresponds to a nanoparticle with 8 subunits, as previously reported in literature (Zhao H, et al. (2010) Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine, Proceedings of the National Academy of Sciences, 107(5), 1971-1976). In contrast, cdCaPC5 TerS forms particles with 142 kDa, corresponding to a decamer (10-mer) 251172 WO - 10 September 2025 structure (Fig. 12). This is a new and surprising finding since structurally similar Ae44RR2 TerS forms 11- and 12-member rings (Sun S, et al. (2012) Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages, Proceedings of the National Academy of Sciences, 109(3), 817-822).

[0214] Example 5 - Immunogenicity of Nioah virus vaccine candidate in mice

[0215] A small terminase-based vaccine candidate against Nipah virus infection was prepared. A fusion protein of ShSf6 TerS and the Nipah virus attachment glycoprotein G ectodomain (NiV Gecd) was prepared. NiV Gecd is exposed on the Nipah virus surface and is the dominant viral antigen for vaccines. Furthermore, the designed flexible peptide linker (SEQ ID NO: 3) was used to connect the TerS domain and NiV Gecd (ShSf6 TerS-L-Gecd; SEQ ID NO: 11). The vaccine candidate can spontaneously oligomerise to form a ring-like nanoparticle with eight subunits. There, the Nipah Gecd antigens are exposed to the outside to boost immunogenicity. Periodic antigen structure may boost antibody production by stimulating B cell leukocytes (Kelly HG, et al. (2019) Immunological basis for enhanced immunity of nanoparticle vaccines, Expert Review of Vaccines, 18(3), 269-280).

[0216] The ShSf6 TerS-L-Gecd vaccine candidate was produced in insect cell culture and purified with affinity and anion exchange chromatography. The pure vaccine candidate was analysed with TEM and the ring-like oligomeric structure of ShSf6 TerS-L-Gecd was confirmed (Fig. 13). Next, vaccine candidate was formulated in physiological phosphate buffered saline (PBS) with AddaVax (Invivogen; ADX) adjuvant and a preclinical immunogenicity trial on mice was performed. The immunogenicity of oligomeric ShSf6 TerS-L-Gecd nanoparticles (designated as "ShSf6 TerS-Gecd" in Fig. 14 A) was compared with soluble recombinant NiV Gecd monomer (ProteoGenix PX-P6271) (Fig. 12 A). A similar HeV Gecd subunit vaccine (Equivac® HeV) is available for horse immunisation against related Hendra virus since 2012 (Middleton D, et al. (2014) Hendra Virus Vaccine, a One Health Approach to Protecting Horse, Human, and Environmental Health, Emerging Infectious Diseases, 20(3), 372-379).

[0217] Eight BALB / c female mice per experimental group were included in the study (Fig. 14 B). On day 0, mice received prime immunisation dose of 3.33 pg of either recombinant soluble NiV Gecd or ShSf6 TerS-L-Gecd in combination with AddaVax adjuvant (ADX). After three weeks, four mice were euthanised to collect plasma samples. Also, the remaining mice received a booster dose of 3.33 pg per vaccine candidate. The study concluded 42 days post-prime (dpp). To determine vaccine immunogenicity, an indirect ELISA and endpoint anti-G antibody titre calculations (Frey A, et al. (1998) A statistically defined endpoint titer determination method for immunoassays, Journal of Immunological Methods, 221(1), 35-41) with reciprocal plasma sample dilutions (Fig. 15) were performed. Surprisingly, substantially higher titres against NiV G were observed in mice that were immunised with ShSf6 TerS-L-Gecd (see "ShSf6 TerS-Gecd + ADX" in Fig. 15). Particularly, while there were no detectable anti-G antibodies 3 weeks after prime dose for Gecd-immunised mice, there was already robust immune response present in ShSf6 TerS-L-Gecd experimental group (dark grey bars in Fig. 15). In line with expectations, the antibody titres further increased 251172 WO - 10 September 2025 after booster immunisation for both groups. The anti-G antibody titres remained higher for ShSf6 TerS-L-Gecd-immunised mice also at the completion of the study, 42 days post-prime. This demonstrates favourable use of oligomeric small terminase subunits with flexible linker peptides as a nanoparticle vaccine vehicle in comparison with traditional soluble subunit vaccines.

[0218] Example 6 - Immunogenicity of Nipah virus vaccine candidate in hamsters

[0219] To further explore the immunogenicity of the ShSf6 TerS-L-Gec vaccine candidate, a prime-and-pull immunisation study was performed in Syrian golden hamsters and the immunogenicity was compared to insect cell-derived Nipah virus-like particles (NiVLPs). The prime-and-pull immunisation strategy in hamsters for vaccine development involves an initial systemic "prime" intramuscular immunisation to activate immune cells, followed by a mucosal "pull" booster with intranasal administration that recruits the primed immune cells to mucosal sites. This approach aims to induce both systemic and mucosal immunity, which together can enhance protection against viral infections and reduce transmission (Bernstein, D I, et al. (2019). Successful application of prime and pull strategy for a therapeutic HSV vaccine. Npj Vaccines, 4(1), 33).

[0220] As described in Example 5, the ShSf6 TerS-L-Gecd vaccine candidate was produced in an insect cell culture and purified with liquid chromatography. Syrian golden hamsters were assigned to groups of six animals, which received prime intramuscular dose of PBS (control), 25 pg NiVLP, 25 pg NiVLP and 25 pg AddaVax adjuvant (ADX; Invivogen), 10 pg ShSf6 TerS-L-Gecd (designated nSf- NiVac), and 10 pg ShSf6 TerS-L-Gecd (designated nSf-NiVac) with 25 pg ADX. The booster pull immunisation followed after 21 days and was administered intranasally. The study completed 50 days post-prime and blood samples were collected on days 0, 14, 28 and 50 for antibody titre determination (Fig. 16). The anti-NiV Gecd hamster antibody titres were calculated as area under the curve (AUC). The AUC values were determined by integrating the serial dilution data from an indirect ELISA assay with trapezoidal rule. For statistical significance calculation, log-transformed AUC titres were evaluated using a Kruskal-Wallis test corrected with Dunn's multiple comparisons test.

[0221] Superior immunogenicity of adjuvanted ShSf6 TerS-L-Gecd (designated nSf- NiVac) was observed compared to other vaccine candidate formulations for every assayed timepoint after day 0. The unadjuvanted ShSf6 TerS-L-Gecd (nSf- NiVac) was also more immunogenic than NiVLPs with or without the ADX adjuvant throughout the study, reaching statistical significance above the control group on day 28. Conversely, NiVLP formulations with or without ADX were poorly immunogenic and did not generate statistical significance above the PBS control group (Fig. 17). This study demonstrates that the TerS-L-based Nipah virus vaccine platform is superior to insect cell NiVLPs. The adjuvanted ShSf6 TerS-L-Gecd was the best performer, which induced rapid systemic antibody immunity after prime that was further amplified after intranasal pull booster. Likewise, the unadjuvanted ShSf6 TerS-L-Gecd intranasal boost increased the anti-NiV Gecd titres for 15-fold. This demonstrates the potential of a TerS-L-based vaccine platform against the high-risk Nipah virus when administered in prime-and-pull strategy. 251172 WO - 10 September 2025

[0222] Together, Example 5 and Example 6 demonstrate the versatility and increased immunogenicity of TerS-L-based vaccine candidates in mice and hamster preclinical models when compared to traditional vaccine platforms. The modular TerS platform can be adopted to other emerging viral targets or therapeutic applications, e.g., cancer vaccines.

[0223] 251172 WO - 10 September 2025

[0224] Sequence listing

[0225] SEO ID NO: 1 :

[0226] GXXGXGXXGXGXG (wherein X in positions 2, 3, 5, 7, 8, 10 and 12 = any amino acid except H, C, and W)

[0227] SEO ID NO: 2:

[0228] GXXGXGXXGXGXG (wherein :

[0229] X in position 2 = L, I, M, or V

[0230] X in position 3 = E or D

[0231] X in position 5 = S, t, N, or Q

[0232] X in position 7 = F or Y

[0233] X in position 8 = Q, N, S or T

[0234] X in position 10 = P

[0235] X in position 12 = S, T, N, or Q)

[0236] SEO ID NO : 3:

[0237] GLEGSGFQGPGSG

[0238] SEO ID NO: 4:

[0239] LKXXFXXMRXXLVXXIXXGQXXLXXISXXIXXXXXXXXXXXVXXYXXLVXXI

[0240] XXSTXXLXXIYXXIXXI (wherein each X = any amino acid)

[0241] SEO ID NO: 5:

[0242] YXXVRXXMXXQSXXLLXXAXXALXXAXXXXXXXXXXXFXXLMXXMXXTNX

[0243] XMXXMHXXM (wherein each X = any amino acid)

[0244] SEO ID NO: 6:

[0245] PDAXEVAKARLRVDTXKWXLARMNPRKYGDXVTNELXXXXXXAIQIETS (wherein each X = any amino acid)

[0246] SEO ID NO: 7:

[0247] SNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLVDT INNSTKLLTDIYKNIVDIQIKIA

[0248] SEO ID NO: 8:

[0249] NDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNMHY

[0250] QSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGAAT VAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD

[0251] SEO ID NO: 9:

[0252] ATEPKAGR.PSDYMPE ADDICSLLSSGESLLK CKR.PGMPDKST FR.WLAKHEDFR.DK YAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVTNEL VGKDGGAIQIETSPMSTLFGK

[0253] SEO ID NO: 10: 251172 WO - 10 September 2025

[0254] KPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAY SHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEF YYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFALRNIEKGKYDKV MPYGPSGIKQGDTLYFPAVGFLVRTEFTYNDSNCPIAECQYSKPENCRLSMGIRPNSHYI LRSGLLKYNLSDEENSKIVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGD VQTVNPLVVNWRDNTVISRPGQSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFL DSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDN VIRPKLFAVKIPEQCT

[0255] SEO ID NO: 11 :

[0256] GPATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGKGLEGSGFQGPGSGKPQTEGVSNLVGLPNNICLQKT SNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLEKIGSCSRGVSKQRIIGVG EVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEFYYVLCAVSVVGDPILNSTYWS GSLMMTRLAVKPKNNGESYNQHQFALRNIEKGKYDKVMPYGPSGIKQGDTLYFPAVGF LVRTEFTYNDSNCPIAECQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDEENSKIVFIE ISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVQTVNPLVVNWRDNTVISRPG

[0257] QSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEVLYR AQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT

[0258] SEO ID NO: 12:

[0259] GPSNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIA

[0260] SEO ID NO: 13:

[0261] GPNDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD

[0262] SEO ID NO: 14:

[0263] GPATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGK

[0264] SEO ID NO: 15:

[0265] LEVLFQGP

[0266] SEO ID NO: 16:

[0267] GPSNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIAGLEGSGFQGPGSG

[0268] SEO ID NO: 17:

[0269] GPNDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDDGLEGSGFQGPGSG 251172 WO - 10 September 2025

[0270] SEO ID NO: 18:

[0271] GPATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR

[0272] DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT

[0273] NELVGKDGGAIQIETSPMSTLFGKGLEGSGFQGPGSG

[0274] SEO ID NO: 19:

[0275] LPXTG

[0276] SEO ID NO: 20:

[0277] GGGG

[0278] 251172 WO - 10 September 2025

[0279] Preferred Embodiments

[0280] (1) A compound L, wherein L is a polypeptide having the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, wherein

[0281] X1= any amino acid except H, C, and W,

[0282] X2= any amino acid except H, C, and W,

[0283] X3= any amino acid except H, C, and W,

[0284] X4= any amino acid except H, C, and W,

[0285] X5= any amino acid except H, C, and W,

[0286] X6= any amino acid except H, C, and W,

[0287] X7= any amino acid except H, C, and W, z1represents the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids, and z2represents the C-terminus of the polypeptide, or a modification of the C- terminal carboxyl group of the polypeptide, which modification

[0288] (1) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1or -C(O)-NR2R3, wherein R1is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C=CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2and R3is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, -(CH2)n-C=CH, -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or

[0289] (ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or

[0290] (iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond; wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof.

[0291] (2) The compound L according to (1), having the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, wherein

[0292] X1= L, I, M, or V,

[0293] X2= E or D,

[0294] X3= S, T, N, or Q, 251172 WO - 10 September 2025

[0295] X4= F or Y,

[0296] X5= Q, N, S, or T,

[0297] X6= P,

[0298] X7= S, T, N, or Q, wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; and wherein z1and z2have the same meaning as in claim 1, preferably wherein z1represents the N-terminus of the polypeptide and / or z2represents the C- terminus of the polypeptide.

[0299] (3) The compound L according to any one of (1) or (2), having the following amino acid sequence written in the single letter code

[0300] GLEGSGFQGPGSG-z2, wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; wherein z2has the same meaning as in claim 1, preferably wherein z2represents the C-terminus of the polypeptide.

[0301] (4) A compound A-L, wherein

[0302] L is the compound according to any one of (1) to (3), with the N-terminus of L being covalently bound to A, preferably via a peptide bond; and

[0303] A is a nanoparticle (NP)-forming unit, preferably a NP-forming polypeptide or a NP-forming protein, preferably a NP-forming polypeptide or a NP-forming protein having its C-terminus covalently bound to the N-terminus of L via a peptide bond.

[0304] (5) The compound A-L according to (4), wherein A is a bacteriophage- derived small terminase protein subunit (TerS) capable of spontaneous oligomerisation or a derivative thereof being capable of spontaneous oligomerisation, preferably a central domain of a bacteriophage-derived TerS capable of spontaneous oligomerisation or a derivative thereof being capable of spontaneous oligomerisation.

[0305] (6) The compound A-L according to (5), wherein the bacteriophage-derived TerS is selected from the group consisting of Aeromonas phage 44R.R.2 TerS, Shigella phage Sf6 TerS, Staphylococcus phage 80B TerS, Staphylococcus phage A411 TerS, and Campylobacter phage PC5 TerS, preferably selected from the group consisting of Aeromonas phage 44R.R.2 TerS, Shigella phage Sf6 TerS, and Campylobacter phage PC5 TerS.

[0306] (7) The compound A-L according to any one of (4) to (6), wherein A is a polypeptide comprising one of the following amino acid sequences of SEQ ID NOs: 4, 5, or 6: i) LKXXFXXMRXXLVXXIXXGQXXLXXISXXIXXXXXXXXXXXVXXYXXLVXXI XXSTXXLXXIYXXIXXI (SEQ ID NO: 4), 251172 WO - 10 September 2025 wherein each X, independently of each other, denotes any amino acid; ii) YXXVRXXMXXQSXXLLXXAXXALXXAXXXXXXXXXXXFXXLMXXMXXTNX XMXXMHXXM (SEQ ID NO: 5), wherein each X, independently of each other, denotes any amino acid; iii) PDAXEVAKARLRVDTXKWXLARMNPRKYGDXVTNELXXXXXXAIQIETS (SEQ ID NO: 6), wherein each X, independently of each other, denotes any amino acid.

[0307] (8) The compound A-L according to any one of (4) to (7), wherein A is a polypeptide comprising or consisting of one of the following amino acid sequences:

[0308] D z3-SNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIA;

[0309] H) z3-NDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYEL\ / RRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD; iii) z3-ATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGK; wherein z3represents the N-terminus of the polypeptide, or an additional N- terminal group consisting of 1 to 10 amino acids, preferably wherein z3= Gly- Pro-.

[0310] (9) A compound A-L-B, wherein

[0311] A-L is the compound according to any one of (4) to (8), with the C-terminus of L being covalently bound to B, preferably via a peptide bond; and

[0312] B is a compound having biological activity, preferably a protein, which preferably has its N-terminus covalently bound to the C-terminus of L via a peptide bond.

[0313] (10) The compound A-L-B according to (9), wherein B is selected from the group consisting of small molecules, small molecule drugs, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, siRNA, miRNA, shRNA, DNA vaccines, or nucleic acid mimetic molecules.

[0314] (11) The compound A-L-B according to any one of (9) or (10), wherein B is a polypeptide or protein selected from the group consisting of virus antigens, enzymes, antibodies, growth factors, tumour suppressors, DNA-binding domains, RNA-binding domains, pilins, adhesins, antigenic peptides, cancer neoantigens, and diagnostic peptides, preferably wherein B is a virus antigen, more preferably Nipah virus receptor binding protein extracellular domain having the following amino acid sequence of SEQ ID NO: 10 :

[0315] KPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAY SHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEF 251172 WO - 10 September 2025

[0316] YYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFALRNIEKGKYDKV MPYGPSGIKQGDTLYFPAVGFLVRTEFTYNDSNCPIAECQYSKPENCRLSMGIRPNSHYI LRSGLLKYNLSDEENSKIVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGD VQTVNPLVVNWRDNTVISRPGQSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFL DSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDN VIRPKLFAVKIPEQCT (SEQ ID NO: 10).

[0317] (12) A nanoparticle, formed

[0318] (i) from a plurality of compounds A-L according to any one of (4) to (8); and / or

[0319] (ii) from a plurality of compounds A-L-B according to any one of (9) to (11).

[0320] (13) A pharmaceutically active composition comprising:

[0321] (i) the compound A-L-B according to any one of (9) to (11) and a pharmaceutically acceptable carrier; or

[0322] (ii) the nanoparticle formed from a plurality of compounds A-L-B according to (12) and a pharmaceutically acceptable carrier.

[0323] (14) A polynucleotide encoding the compound L according to any one of (1) to (3), the compound A-L according to any one of (4) to (8), or the compound A-L-B according to any one of (9) to (11).

[0324] (15) A vector containing a polynucleotide according to (14).

[0325] (16) A genetically engineered host cell containing the vector according to (15).

[0326] (17) The compound A-L-B according to any one of (9) to (11), or the nanoparticle formed from a plurality of compounds A-L-B according to (12), for use as a medicament.

[0327] (18) A process for the preparation of the compound L according to any one of (1) to (3), the compound A-L according to any one of (4) to (8), or the compound A-L-B according to any one of (9) to (11), the process comprising the following steps: a) heterologous expression of the compound L according to any one of (1) to (3), the compound A-L according to any one of (4) to (8), or the compound A- L-B according to any one of (9) to (11); b) optionally subsequent posttranslational or chemical modification of the heterologously expressed compound L according to any one of (1) to (3), the heterologously expressed compound A-L according to any one of (4) to (8), or the heterologously expressed compound A-L-B according to any one of (9) to (11).

Claims

251172 WO - 10 September 2025CLAIMS1. A compound A-L-B, whereinA is a nanoparticle (NP)-forming unit, preferably a NP-forming polypeptide or a NP-forming protein, preferably a NP-forming polypeptide or a NP-forming protein having its C-terminus covalently bound to the N-terminus of L via a peptide bond;L is a polypeptide having the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, whereinX1= any amino acid except H, C, and W,X2= any amino acid except H, C, and W,X3= any amino acid except H, C, and W,X4= any amino acid except H, C, and W,X5= any amino acid except H, C, and W,X6= any amino acid except H, C, and W,X7= any amino acid except H, C, and W, z1represents the N-terminus of the polypeptide, or a group consisting of 1 to 10 amino acids, and z2represents the C-terminus of the polypeptide, or a modification of the C- terminal carboxyl group of the polypeptide, which modification(i) forms together with the carboxyl group of the C-terminal amino acid of the polypeptide a moiety having the structure -C(O)-O-R1or -C(O)-NR2R3, wherein R1is a functional group selected from the group consisting of -(CH2)n-N3, - (C H2)n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); and wherein one of R2and R3is H and the other one is a functional group selected from the group consisting of -(CH2)n-N3, - (C H2)n- C = C H , -(CH2)n-difluorooctyne (DIFO), and -(CH2)n-dibenzylcyclooctyne (DIBO); wherein n = 1, 2, 3, 4, or 5; or(ii) is a polypeptidic catcher group which is able to form an isopeptide bond with a polypeptidic tag group in a catcher / tag pair reaction; or(iii) is a Staphylococcus aureus sortase A transpeptidase recognition site consisting of the amino acids LPXTG, being attached to the C-terminus of the polypeptide via a peptide bond; wherein the side chain of each amino acid of the polypeptide L independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; andB is a compound having biological activity, preferably a protein, which preferably has its N-terminus covalently bound to the C-terminus of L via a peptide bond;251172 WO - 10 September 2025 wherein the N-terminus of L is covalently bound to A, preferably via a peptide bond, and wherein the C-terminus of L is covalently bound to B, preferably via a peptide bond.

2. The compound A-L-B according to claim 1, wherein L has the following amino acid sequence written in the single letter code zx-G X1X2G X3G X4X5G X6G X7G-z2, whereinX1= L, I, M, or V,X2= E or D,X3= S, T, N, or Q,X4= F or Y,X5= Q, N, S, or T,X6= P,X7= S, T, N, or Q, wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; and wherein z1and z2have the same meaning as in claim 1, preferably wherein z1represents the N-terminus of the polypeptide and / or z2represents the C- terminus of the polypeptide.

3. The compound A-L-B according to any one of claims 1 or 2, wherein L has the following amino acid sequence written in the single letter codeGLEGSGFQGPGSG-z2, wherein the side chain of each amino acid of the polypeptide independently of each other may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated or combinations thereof; wherein z2has the same meaning as in claim 1, preferably wherein z2represents the C-terminus of the polypeptide.

4. The compound A-L-B according to any one of claims 1 to 3, wherein A is a bacteriophage-derived small terminase protein subunit (TerS) capable of spontaneous oligomerisation or a derivative thereof being capable of spontaneous oligomerisation, preferably a central domain of a bacteriophage- derived TerS capable of spontaneous oligomerisation or a derivative thereof being capable of spontaneous oligomerisation.

5. The compound A-L-B according to claim 4, wherein the bacteriophage- derived TerS is selected from the group consisting of Aeromonas phage 44R.R.2 TerS, Shigella phage Sf6 TerS, Staphylococcus phage 80B TerS, Staphylococcus phage A411 TerS, and Campylobacter phage PC5 TerS, preferably selected from the group consisting of Aeromonas phage 44R.R.2 TerS, Shigella phage Sf6 TerS, and Campylobacter phage PC5 TerS.251172 WO - 10 September 20256. The compound A-L-B according to any one of claims 1 to 5, wherein A is a polypeptide comprising one of the following amino acid sequences of SEQ ID NOs: 4, 5, or 6: i) LKXXFXXMRXXLVXXIXXGQXXLXXISXXIXXXXXXXXXXXVXXYXXLVXXI XXSTXXLXXIYXXIXXI (SEQ ID NO: 4), wherein each X, independently of each other, denotes any amino acid; ii) YXXVRXXMXXQSXXLLXXAXXALXXAXXXXXXXXXXXFXXLMXXMXXTNX XMXXMHXXM (SEQ ID NO: 5), wherein each X, independently of each other, denotes any amino acid; iii) PDAXEVAKARLRVDTXKWXLARMNPRKYGDXVTNELXXXXXXAIQIETS (SEQ ID NO: 6), wherein each X, independently of each other, denotes any amino acid.

7. The compound A-L-B according to any one of claims 1 to 6, wherein A is a polypeptide comprising or consisting of one of the following amino acid sequences:D z3-SNPEENLKFTYLKEDFNLMRESLVNIIKRGQDILEVISNNILADPLSSNQAVMAYSTLV DTINNSTKLLTDIYKNIVDIQIKIA;H) z3-NDVLDFTQLKDLNGIEGIHGEDVQVYAPLVLRDPVSNPNNRKIDQDDDYELVRRNM HYQSQMLLDMAKIALENAKNADSPRHVEVFAQLMGQMTTTNKEMLKMHKEMKDLAGA ATVAIDGQVQKDADGEFIEFEGSPDELLDLELADEDIGDD; iii) z3-ATEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFR DKYAKATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVT NELVGKDGGAIQIETSPMSTLFGK; wherein z3represents the N-terminus of the polypeptide, or an additional N- terminal group consisting of 1 to 10 amino acids, preferably wherein z3= Gly- Pro-.

8. The compound A-L-B according to any one of claims 1 to 7, wherein B is selected from the group consisting of small molecules, small molecule drugs, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, siRNA, miRNA, shRNA, DNA vaccines, or nucleic acid mimetic molecules.

9. The compound A-L-B according to any one of claims 1 to 8, wherein B is a polypeptide or protein selected from the group consisting of virus antigens, enzymes, antibodies, growth factors, tumour suppressors, DNA-binding domains, RNA-binding domains, pilins, adhesins, antigenic peptides, cancer neoantigens, and diagnostic peptides, preferably wherein B is a virus antigen, more preferably Nipah virus receptor binding protein extracellular domain having the following amino acid sequence of SEQ ID NO: 10:KPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAY SHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEF YYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFALRNIEKGKYDKV251172 WO - 10 September 2025MPYGPSGIKQGDTLYFPAVGFLVRTEFTYNDSNCPIAECQYSKPENCRLSMGIRPNSHYI LRSGLLKYNLSDEENSKIVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGD VQTVNPLVVNWRDNTVISRPGQSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFL DSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDN VIRPKLFAVKIPEQCT (SEQ ID NO: 10).

10. A nanoparticle, formed from a plurality of compounds A-L-B according to any one of claims 1 to 9.

11. A pharmaceutically active composition comprising:(i) the compound A-L-B according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier; or(ii) the nanoparticle formed from a plurality of compounds A-L-B according to claim 10 and a pharmaceutically acceptable carrier.

12. A polynucleotide encoding the compound A-L-B according to any one of claims 1 to 9.

13. A vector containing a polynucleotide according to claim 12.

14. A genetically engineered host cell containing the vector according to claim 13.

15. The compound A-L-B according to any one of claims 1 to 9, or the nanoparticle formed from a plurality of compounds A-L-B according to claim 10, for use as a medicament.

16. A process for the preparation of the compound A-L-B according to any one of claims 1 to 9, the process comprising the following steps: a) heterologous expression of the compound A-L-B according to any one of claims 1 to 9; b) optionally subsequent posttranslational or chemical modification of the heterologously expressed compound A-L-B according to any one of claims 1 to 9.

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