Peptide tags and binding partners

Abstract

Providing peptide tags and binding partners that can interact via the spontaneous formation of isopeptide bonds. [Solution] They can bind to each other via the spontaneous formation of an isopeptide bond between one reactive residue contained in the binding partner and another reactive residue contained in the peptide tag. A binding partner selected from MoonCake, KatI, or QueenCatcher, or a variant thereof having at least 70% homology or identity; and A peptide pair selected from the following: RumtrunkD9N tag, Rum7 tag, Rum3 tag, Rum2 tag, Rum4 tag, Rum5 tag, Rum6 tag, RumTrunk tag, Spy tag, Sdy tag, Rum tag, Bac tag, Bac2 tag, Bac3 tag, Bac4 tag, Bac5 tag, or a peptide tag selected from Clib9, or a variant thereof having at least 70% homology or identity.

Description

Technical Field

[0001] The present invention relates to peptide tags and binding partners that can interact via the spontaneous formation of isopeptide bonds, as well as related peptide pairs and methods for designing peptide tags, binding partners, and peptide pairs having improved properties.

Background Art

[0002] The use of peptides and peptidomimetic molecules as tags for attachment to proteins and other substances is an important tool in molecular biology. Such peptide tags can enable the detection, purification, and analysis of specific proteins or substances, or can be used for the specific targeting of tagged proteins or substances. They can also be used, for example, in the generation of nanoparticles or virus-like particles, as described in WO 2016 / 112921.

[0003] Peptide tags that can be attached to a protein of interest using recombinant DNA methods (e.g., by ligating a nucleotide sequence encoding the peptide tag in an operable manner to the gene encoding the protein of interest and expressing the protein product) typically have the ability to bind to a binding partner. This binding can enable the detection of the protein of interest if the binding partner is detectable, e.g., an antibody or conjugated to a detectable substance, or can enable the purification of the protein of interest if the binding partner is immobilized on, for example, a solid support. Alternatively, peptide tags and their binding partners can be used to present antigens on the surface of a molecule of interest, e.g., a virus-like particle (VLP) or nanoparticle.

[0004] Thus, the use of peptide tags that can bind to a binding partner has significant applications and can provide a means for manipulating or analyzing a target protein or substance for the generation of bispecific molecules for cancer immunotherapy, for CAR T cell therapy, or for, e.g., VLP-based vaccine production.

[0005] Several peptide tag / binding partner systems have been described, which offer high affinity or irreversible binding and may be useful for the above applications.

[0006] In particular, systems in which peptide tags and binding partners interact via isopeptide bonds are useful. Such pairs have stable or irreversible interactions due to the spontaneous formation of isopeptide bonds between the peptide tag and its binding partner.

[0007] An isopeptide bond is an amide bond formed between a carboxyl / carboxamide and an amino group, where at least one of the carboxyl or amino group exists detached from the protein backbone (protein skeleton). Such bonds are chemically irreversible under biological conditions and are resistant to most proteases. [Overview of the project]

[0008] The present invention is defined in the claims. The present invention therefore uses proteins that can or readily form spontaneous isopeptide bonds to develop improved peptide tag / binding partner pairs that covalently bond to each other and thus result in improved irreversible interactions.

[0009] In this regard, proteins capable of spontaneously forming isopeptide bonds are expressed as separate fragments, providing a peptide tag and a binding partner for the peptide tag, and the two fragments can covalently reconstitute the protein by isopeptide bond formation. This covalent reaction via isopeptide bonds stabilizes peptide-protein interactions under conditions where non-covalent interactions would normally dissociate rapidly.

[0010] As will be discussed in detail below, the peptide tag preferably contains one of the isopeptide-involved residues in the original protein, and the binding partner preferably contains the other residue in the original protein involved in the isopeptide bond. Thus, the protein coding sequence can be cleaved to form fragments encoding the peptide tag and binding partner pair.

[0011] This specification provides a method for producing a modified binding partner that can bind to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag. The above method includes the following steps: i) A step of selecting at least a first peptide pair consisting of a first peptide tag and a first binding partner, and a second peptide pair consisting of a second peptide tag and a second binding partner, wherein for each peptide pair, the peptide tag and the binding partner can or are considered to be able to bind to each other by spontaneously forming an isopeptide bond; ii) A step of identifying the position of the isopeptide bond with respect to a first peptide pair and / or a second peptide pair, thereby identifying a first reactive fragment of the first binding partner and / or a second reactive fragment of the second binding partner, and the remaining fragment of the first binding partner and / or the remaining fragment of the second binding partner, wherein the first and / or second reactive fragments include reactive residues involved in the isopeptide bond; iii) A step of designing a modified binding partner, wherein the modified binding partner comprises or consists of i) a first reactive fragment or a homolog thereof having at least 70% homology, and a second remaining fragment or a homolog thereof having at least 70% homology, wherein the first reactive fragment is preferably upstream of the second remaining fragment, and the modified binding partner does not contain both reactive residues involved in the formation of the isopeptide bond; iv) A step of producing a modified binding partner.

[0012] Also provided herein are modified bonding partners obtained by the methods disclosed herein. Also provided herein are modified binding partners capable of binding to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag, wherein the modified binding partner does not contain both reactive residues involved in the formation of the isopeptide bond, and comprises or consists of a first reactive fragment of a first binding partner containing one reactive residue capable of interacting with a first peptide tag containing another reactive residue via the formation of an isopeptide bond between reactive residues, or a homolog thereof having at least 70% homology, and a second remaining fragment of a second binding partner, wherein the second binding partner can interact with a second peptide tag containing another reactive residue via the formation of an isopeptide bond between reactive residues, and the second remaining fragment does not contain a reactive residue or a homolog thereof having at least 70% homology, preferably the first reactive fragment is upstream of the second remaining fragment.

[0013] Furthermore, this specification provides a method for producing a peptide tag that can bind to a binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained within the peptide tag and another reactive residue contained within the binding partner, preferably the binding partner being a modified binding partner as disclosed herein. The above method includes the following steps: a) A step of identifying candidate peptide tags having at least 60% similarity to a reference peptide tag, wherein the reference peptide tag can spontaneously form an isopeptide bond with at least one reference binding partner, and preferably the reference peptide tag includes a reference binding motif; b) A step of selecting a peptide tag from the candidate peptide tags identified in a), wherein the selected peptide tag comprises at least one reactive residue likely involved in the formation of an isopeptide bond; c) A step of designing and producing peptide tags from selected peptide tags, wherein each peptide tag comprises or consists of a fragment of the selected peptide tag, ranging from 4 to 24 amino acids upstream of a reactive residue likely involved in the formation of an isopeptide bond to 2 to 22 amino acids downstream, or a homolog thereof having at least 70% homology, provided that the homolog contains a reactive residue.

[0014] Also provided herein are peptide tags comprising or consisting of a protein fragment containing at least one reactive residue involved in the formation of an isopeptide bond between the peptide tag and its binding partner, wherein the peptide tag comprises the protein fragment extending from 4 to 24 amino acids upstream of the reactive residue to 2 to 22 amino acids downstream, or a homolog thereof having at least 70% homology, provided that the homolog contains the reactive residue, preferably asparagine or aspartic acid.

[0015] Also provided herein are a modified binding partner and a peptide tag, or a method for producing a peptide pair comprising a modified binding partner and a peptide tag, wherein the modified binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained in the modified binding partner and another reactive residue contained in the peptide tag. The above method includes the following steps: i) the step of producing a modified binding partner by the method disclosed herein; and / or ii) A step of producing a peptide tag by a method disclosed herein.

[0016] Furthermore, provided herein are peptide pairs comprising, or consisting of, a peptide tag as defined in any one of the preceding sections and a modified binding partner as defined herein.

[0017] Furthermore, this specification provides modified bond partners having one or more improved properties compared to the reference bond partner, the one or more improved properties being independently selected from one or more of the following: a) Increased binding efficacy of a modified binding partner to a peptide tag compared to the binding of a reference binding partner to the peptide tag, wherein the modified binding partner and optionally the reference binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained in or within the modified binding partner and another reactive residue contained in the peptide tag, and the increased binding efficacy is at least one of the total binding amount and the binding rate; b) An increased ability to form particles that present the peptide of the target, such as virus-like particles that present the peptide of the target as virus-like particles, compared to the ability of a reference binding partner to form particles under similar conditions, wherein the particles present the peptide of the target, and the particles include a particle-forming protein such as a virus-like particle-forming protein fused to a modified binding partner and the peptide of the target fused to a peptide tag, or the particles include a particle-forming protein fused to a peptide tag and the peptide of the target fused to a modified binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the modified binding partner and the peptide tag; c) Increased ability to present the peptide of interest on particles such as virus-like particles, compared to the ability of a reference binding partner to present the peptide of interest under similar conditions, wherein the particles comprise a particle-forming protein such as a virus-like particle-forming protein fused to a modified binding partner and the peptide of interest fused to a peptide tag, or the particles comprise a particle-forming protein fused to a peptide tag and the peptide of interest fused to a modified binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the modified binding partner and the peptide tag.

[0018] Furthermore, the peptide tags provided herein have one or more improved properties compared to a reference peptide tag, and one or more improved properties are independently selected from one or more of the following: a) Increased binding efficacy of a peptide tag to a reference binding partner compared to the binding of a reference peptide tag to a reference binding partner, wherein the peptide tag and the reference peptide tag can bind to the reference binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained in the peptide tag or within the reference peptide tag and another reactive residue contained in the reference binding partner, and the binding efficacy is increased if at least one of the total binding amount and binding rate is increased; b) An increased ability to form particles such as virus-like particles compared to the ability of a reference peptide tag to form particles under similar conditions, wherein the particles present the peptide of interest, and the particles contain a particle-forming protein such as a virus-like particle-forming protein fused to a reference binding partner and the peptide of interest fused to a peptide tag, or the particles contain a particle-forming protein fused to a peptide tag and the peptide of interest fused to a reference binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the reference binding partner and the peptide tag; c) Increased ability to present a compound of interest, such as a peptide on a particle, such as a virus-like particle, compared to the ability of a reference peptide tag to present the compound of interest under similar conditions, wherein the particle comprises a particle-forming protein, such as a virus-like particle-forming protein, fused to the peptide tag and the compound of interest fused to a binding partner, or the particle comprises a particle-forming protein fused to a binding partner and the compound of interest fused to the peptide tag, and the particle is formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag.

[0019] Furthermore, this specification provides peptide pairs comprising or consisting of a peptide tag and a binding partner, wherein the peptide pairs have one or more improved properties compared to reference peptide pairs comprising a reference peptide tag and a reference binding partner. The binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag. The reference binding partner can bind to the reference peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the reference binding partner and another reactive residue contained within the reference peptide tag. One or more improved characteristics are independently selected from one or more of the following: a) An increased binding potency of a binding partner to a peptide tag as compared to the binding of a reference binding partner to a reference peptide tag, wherein the binding potency is increased when at least one of the total binding amount and the binding rate is increased; b) An increased ability to form particles presenting a peptide of interest, such as virus-like particles presenting the peptide of interest, as compared to the ability of a reference peptide pair to form particles under similar conditions, wherein the particles comprise a particle-forming protein such as a virus-like particle-forming protein fused to a binding partner and a compound of interest fused to a peptide tag, or the particles comprise a virus-like particle-forming protein fused to a peptide tag and a compound of interest fused to a binding partner, and the particles are formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag; and / or c) An increased ability to present a compound of interest, such as a peptide on a particle, such as a virus-like particle, as compared to the ability of a reference peptide pair to present a peptide of interest under similar conditions, wherein the particles comprise a particle-forming protein such as a virus-like particle-forming protein fused to a binding partner and a compound of interest fused to a peptide tag, or the particles comprise a particle-forming protein fused to a peptide tag and a compound of interest fused to a binding partner, and the particles are formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag.

[0020] Also disclosed herein are polynucleotides encoding the modified binding partners and / or peptide tags disclosed herein.

[0021] Vectors containing the polynucleotides described herein are also provided.

[0022] Host cells expressing the modified binding partners and / or peptide tags disclosed herein are also provided. Also disclosed herein is i) Proteins fused to the modified binding partners described herein, and peptides fused to the peptide tags described herein, for example, compounds of interest such as antigens; or ii) Proteins fused to peptide tags described herein, and peptides fused to modified binding partners described herein, for example, compounds of interest such as antigens. The composition contains, and the modified binding partner and peptide tag can interact by the spontaneous formation of isopeptide bonds. The target compound and protein are linked via an isopeptide bond between the modified binding partner and the peptide tag.

[0023] Also provided is a method for producing the pharmaceutical compositions disclosed herein, the method comprising the following steps: i) obtain a first polypeptide comprising or comprising a modified binding partner as disclosed herein fused to a protein, and a second polypeptide comprising or comprising a peptide tag as disclosed herein fused to a compound of interest; or The steps of obtaining a first polypeptide comprising or comprising a peptide tag disclosed herein fused to a protein, and a second polypeptide comprising or comprising a modified binding partner disclosed herein fused to a compound of interest; A step of contacting a first polypeptide with a second polypeptide, thereby enabling the formation of an isopeptide bond between a peptide tag and a modified binding partner, thereby producing the pharmaceutical composition disclosed herein. [Brief explanation of the drawing]

[0024] [Figure 1]Individual catcher domains (i.e., SdyCatcher (SdyC), SpyCatcher (SpyC), Mooncake, and KatI) were mixed with individual tagged VLPs (i.e., SdyT-Ap205, SpyT-AP205, RumTrunk-tagged-AP205, RumTrunkD9N-tagged-Ap205) to obtain a final concentration of 5 μM for each binding partner. Each of these mixed samples was then incubated at 37°C for 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 1 hour, 1 hour 30 minutes, or 3 hours. After incubation, individual samples were electrophoresed on SDS gels containing DTT. The percentage of reconstitution was calculated based on the concentration measurements. [Figure 2A] Solutions (PBS) containing 10 μM of individual soluble catcher domains (Mooncake, KatI) or catcher-VLPs (Mooncake-AP205, KatI-AP205, SpyC-AP205) were mixed in a 1:1 ratio with solutions (PBS) containing 10 μM of soluble tags (RumtrunkD9N tags) (final concentration of each tag / catcher binding partner = 5 μM). These mixed samples were incubated at 37°C for 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 1 hour, 1 hour 30 minutes, or 3 hours. After incubation, the samples were electrophoresed on SDS gels containing DTT. The percentage of reconstitution was calculated based on concentration measurements. [Figure 3] This describes one possible embodiment of presenting a compound of interest on the surface of a particle, such as a virus-like particle, by utilizing spontaneously occurring isopeptide bonds. A particle-forming protein (black line) capable of forming a particle is fused to a peptide tag (black circle). The compound of interest, such as a peptide (dark gray droplet), is fused to a binding partner (light gray semicircle). Upon contact, an isopeptide bond spontaneously forms between the peptide tag and the binding partner, resulting in the presentation of the compound of interest on the surface of the particle. [Figure 4]The mouse groups (n=6) were immunized with the same dose (6mcg) of MoonCake-HER2 virus-like particles (VLP) (LCG), SpyCatcher-HER2 VLP (SPY), or PBS (n=5 in the PBS group), respectively, via prime boost. Serum was collected 2 weeks after each immunization, and antigen-specific IgM and IgG (subclasses 1, 2a, 2b, and 3) levels were measured by ELISA. Immunization with MoonCake-HER2 VLP induced significantly higher levels of antigen-specific total Ig compared to SpyCatcher-HER2 VLP. [Figure 5] Mice were immunized by prime boosting using either MoonCake-HER2 VLP or SpyCatcher-HER2 VLP at the same dose (6 mcg). Serum was collected two weeks after each immunization, and antigen-specific IgM and IgG (subclasses 1, 2a, 2b, and 3) levels were measured by ELISA. Immunization with MoonCake-HER2 VLP induced significantly higher levels of IgM, IgG2a, and IgG2b compared to immunization with SpyCatcher-HER2 VLP. [Modes for carrying out the invention]

[0025] definition As used herein, the term “isopeptide bond” refers to an amide bond between a carboxyl group and an amino group, where at least one of their groups is not derived from the protein backbone or is not considered part of the protein backbone. Isopeptide bonds may form within a single protein, between two peptides, or between a peptide and a protein. Thus, isopeptides may form intramolecularly within a single protein, or intermolecularly, i.e., between two peptide / protein molecules. Typically, an isopeptide bond can occur intramolecularly between two reactive amino acids: lysine and asparagine or aspartic acid. For the process to occur, the two reactive amino acids must be in close proximity in a hydrophobic environment, often containing aromatic residues. Ultimately, the autocatalytic process may be facilitated by a catalytic aspartic or glutamic acid residue, which does not participate in the isopeptide bond formation itself.

[0026] In the case of intermolecular isopeptide bonds, bonding typically occurs between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group or peptide chain of a protein, or between the α-amino terminus or peptide chain of a protein and asparagine, aspartic acid, glutamine, or glutamic acid. Each residue in a pair involved in an isopeptide bond is called a reactive residue. Thus, isopeptide bonds can be formed between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. In particular, isopeptide bonds can occur between the side-chain amine of lysine and the carboxamide group of asparagine.

[0027] The peptide tag and binding partner pairs discussed herein refer to a binding partner (peptide / protein) and a peptide tag that binds to it via an isopeptide bond, preferably a spontaneously occurring isopeptide bond. The peptide tag and binding partner pair are covalently bonded to each other via an isopeptide bond, and therefore preferably the peptide tag contains one reactive residue involved in one isopeptide bond used to design the binding partner, and the binding partner contains the other reactive residue involved in the isopeptide bond. These terms are commonly used in the art; the word “catcher” may also be used instead of “binding partner.”

[0028] As used herein, the term “spontaneous” refers to a bond, particularly an isopeptide bond, which can be formed in a protein or between peptides or between proteins (e.g., between two peptides or between one peptide and one protein) without the presence of any other agent (e.g., an enzyme catalyst) and / or without chemical modification of the protein or peptide, for example, without native chemical ligation or chemical coupling. Spontaneous isopeptide bonds can therefore form spontaneously in the absence of enzymes or other exogenous substances, or without chemical modification. However, spontaneous isopeptides or covalent bonds, in particular, may require the presence of glutamic acid or aspartic acid residues in the proteins involved in the bond, or in one peptide / protein, to enable the formation of the bond.

[0029] In this specification, the term “binding properties” refers to the binding properties of one peptide to another peptide, for example, the binding properties of a binding partner to a peptide tag as defined herein, and vice versa. The most important binding properties from the perspective of this disclosure are the total binding amount and the binding rate.

[0030] The term “total binding amount” (as used herein synonymously with the term “reconstitution rate”) refers to the percentage of molecules of a given binding partner that form an isopeptide bond with a given peptide tag over time. The total binding amount is determined as is known in the art, for example, when the binding partner and peptide tag are mixed in equimolar amounts, and the resulting complex of the binding partner and peptide tag formed from the formation of an isopeptide bond is determined as is known in the art, for example, at a given time point and expressed in comparison to the starting amount of the binding partner.

[0031] The term "binding rate" refers to the rate or percentage of isopeptide bond formation between a given binding partner and a given peptide tag, as a function of time.

[0032] The term "binding efficacy" is a comprehensive term that encompasses both the binding rate and the total amount of binding.

[0033] As used herein, the term “variant” refers to a functional variant of a parent molecule, such as a binding partner or peptide tag, that retains the same function as the parent molecule. A variant binding partner thus retains the ability to spontaneously form an isopeptide bond with a peptide tag, and a variant of a peptide tag thus retains the ability to spontaneously form an isopeptide bond with a binding partner. Throughout this disclosure, it will be understood that a variant having at least 70% homology or identity with a given sequence may have at least 71%, for example at least 72%, for example at least 73%, for example at least 74%, for example at least 75%, for example at least 76%, for example at least 77%, for example at least 78%, for example at least 79%, for example at least 80%, for example at least 81%, for example at least 82%, for example at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% homology or identity with respect to the sequence, for example at least 71%, for example at least 72%, for example at least 73%, for example at least 74%, for example at least 75%, for example at least 76%, for example at least 97%, for example at least 98%, for example at least 99%.

[0034] The inventors have developed a method to improve the properties, particularly the binding properties, of a binding partner to a peptide tag (and vice versa). In short, the inventors have found that one or more properties of such a modified binding partner and / or such a modified peptide tag are improved compared to the starting binding partner and / or peptide tag. The general principles of how such modified binding partners can be designed are described, for example, in Example 1.

[0035] Modified coupling partners with improved properties Method for producing modified binding partners This disclosure provides a method for producing a modified binding partner that can bind to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag, the method comprising the following steps: i) A step of selecting at least a first peptide pair consisting of a first peptide tag and a first binding partner, and a second peptide pair consisting of a second peptide tag and a second binding partner, wherein the peptide tag and binding partner for each peptide pair can or are considered to be able to bind to each other by spontaneously forming an isopeptide bond; ii) A step of identifying the position of the isopeptide bond to a first peptide pair and / or a second peptide pair, thereby identifying a first reactive fragment of the first binding partner and / or a second reactive fragment of the second binding partner, and the remaining fragment of the first binding partner and / or the remaining fragment of the second binding partner, wherein the first and / or second reactive fragments include reactive residues involved in the isopeptide bond; iii) A step of designing a modified binding partner, wherein the modified binding partner comprises or consists of a first reactive fragment or a homolog thereof having at least 70% homology thereto, and a second remaining fragment or a homolog thereof having at least 70% homology thereto, wherein the first reactive fragment is upstream of the second remaining fragment, the second reactive fragment is upstream of the first remaining fragment, and the modified binding partner does not contain both reactive residues involved in the formation of the isopeptide bond; iv) A step of producing a modified binding partner.

[0036] In the first step, two peptide pairs are selected: the first peptide pair consists of a first peptide tag and a first binding partner, and the second peptide pair consists of a second peptide tag and a second binding partner. The peptide tags and binding partners of a given peptide pair can (or are thought to be able to) bind to each other by the spontaneous formation of an isopeptide bond between the peptide tag and the binding partner. Such peptide pairs are known in the art and are described further herein. The method may be applied to peptides or polypeptides that are thought to be able to bind to each other via the spontaneous formation of isopeptide bonds.

[0037] For each peptide pair, the location of the isopeptide bond (or the assumed location of the isopeptide bond) is determined within that pair. Each of the binding partner and peptide tag contains one of the reactive residues involved in the isopeptide bond. For many peptide pairs, the location of the isopeptide bond is known and can be obtained from the literature. In other cases, candidate locations that may be involved in the formation of the isopeptide bond can be determined, as is known in the art, particularly by searching for the sequences of the peptide tag and / or binding partner for known motifs, for example, by sequence mining.

[0038] Identifying the location of the isopeptide bond means identifying the locations of the two residues involved in the isopeptide bond. One of these two residues is present in the binding partner, and the other is present in the peptide tag. Once the location of the residue involved in the isopeptide bond is determined in the binding partner, the fragment containing this residue is identified, which is also referred to herein as the reactive fragment. The remaining portion of the binding partner is referred to as the rest fragment.

[0039] The first reactive fragment is therefore identified with respect to the first binding partner, which contains one residue involved in the first isopeptide bond, while the first peptide tag contains the other residue, and the remainder of the first binding partner is the remaining fragment of the first. The second reactive fragment is therefore identified with respect to the second binding partner, which contains one residue involved in the second isopeptide bond, while the second peptide tag contains the other residue, and the remainder of the second binding partner is the remaining fragment of the second. The first isopeptide bond refers to the isopeptide bond between the first binding partner and the first peptide tag, and the second isopeptide bond refers to the isopeptide bond between the second binding partner and the second peptide tag.

[0040] In the next step of the method, a modified coupling partner is designed. In some embodiments, this modified coupling partner is i) A first reactive fragment of a first binding partner, or a homolog thereof having at least 70% homology thereto, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and ii) A second remaining fragment of a second binding partner, or its homologue having at least 70% homology, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology, including or consisting of Preferably, the first reactive fragment is located upstream of the second remaining fragment. In the final step, a modified bonding partner is produced. This is carried out, for example, as is known in the art.

[0041] The bond partner having the highest homology or identity with the modified bond partner is preferably used as a reference for determining whether the modified bond partner has improved properties, as described below herein. In some embodiments, the modified bond partner has higher homology, identity or similarity with respect to the first bond partner than to the second bond partner, and the first bond partner is used as a reference. In other embodiments, the modified bond partner has higher homology with respect to the second bond partner than to the first bond partner, and the second bond partner is used as a reference.

[0042] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 1 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 1 or its homologue as described above.

[0043] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 3 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 3 or its homologue as described above.

[0044] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 9 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 9 or its homologue as described above.

[0045] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 13 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 13 or its homologue as described above.

[0046] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 15 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 15 or its homologue as described above.

[0047] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 17 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 17 or its homologue as described above.

[0048] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 19 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 19 or its homologue as described above.

[0049] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 23 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 23 or its homologue as described above.

[0050] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 25 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 25 or its homologue as described above.

[0051] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 27 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 27 or its homologue as described above.

[0052] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 29 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 29 or its homologue as described above.

[0053] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 30 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 30 or its homologue as described above.

[0054] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 31 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 31 or its homologue as described above.

[0055] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 37 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 37 or its homologue as described above.

[0056] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 39 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 39 or its homologue as described above.

[0057] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 41 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 41 or its homologue as described above.

[0058] Bonding characteristics Once a modified binding partner is obtained by any of the methods described herein, its binding properties can be measured. Accordingly, in some embodiments, the method further includes the step of measuring one or more binding properties of the modified binding partner, wherein the one or more properties are preferably i) total binding amount and ii) binding rate to one or more of the first, second, or third peptide tags of the modified binding partner. Measuring the corresponding one or more binding properties of the first and / or second binding partner to one or more of the first, second, or third peptide tags allows for a comparison of the binding properties of the modified binding partner to one or more of the first, second, or third peptide tags, as well as the binding properties of the first and / or second binding partner. An increase in at least one of the total binding amount and binding rate indicates increased binding efficacy or improved binding properties.

[0059] To measure the binding properties of the modified binding partners according to this disclosure, a peptide tag may be required to which the modified binding partner binds or is expected to bind. This peptide tag may be a first peptide tag of a first peptide pair, a second peptide tag of a second peptide pair, or a third peptide tag, as further described below herein.

[0060] Measuring the increase in the binding rate of a modified binding partner to at least one of the first, second, and third peptide tags compared to the binding rate of the first or second binding partner to at least one of the same peptide tags is an indicator of a modified binding partner having increased binding efficacy. Preferably, the binding rate of the modified binding partner to the peptide tag is measured and compared to the binding rate of the first binding partner to the same peptide tag, and in some embodiments, the binding rate is the binding rate of the modified or first binding partner to the first peptide tag.

[0061] Preferably, the above-mentioned increase in binding rate is at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the binding rate measured for the first and / or second binding partners to the same peptide tag.

[0062] In some embodiments, the binding rate of a modified binding partner to a first peptide tag is measured and compared to the binding rate of a first binding partner to the first peptide tag. In other embodiments, the binding rate of a modified binding partner to a second peptide tag is measured. In other embodiments, the binding rate of a modified binding partner to a third peptide tag is measured. The method may also include measuring the binding rate of a modified partner to two or all of the first, second, or third peptide tags. Preferably, the binding rate of a modified binding partner to at least the first peptide tag is measured and compared to the binding rate of a first binding partner to the first peptide tag.

[0063] Integration Partners Specific binding partners that can be used as starting points in this method, i.e., binding partners that may be first and / or second binding partners, include binding partners that form, or are thought to be able to form, an isopeptide bond with a peptide tag. Certain modified binding partners disclosed herein can also be used as starting points.

[0064] Accordingly, the first and / or second binding partner can be independently selected from SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33, and its homologues having at least 60% homology to it, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology to it.

[0065] In some embodiments, the first binding partner, i.e., the binding partner modified or improved using the Method, is SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, or its homologue having at least 60% homology, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology.

[0066] In some embodiments, the second binding partner is sequence number 1 (SpyCatcher), sequence number 3 (SdyCatcher), sequence number 9 (SnoopCatcher), sequence number 13, sequence number 15, sequence number 17, sequence number 19, sequence number 23, sequence number 25, sequence number 27, sequence number 29, sequence number 30, sequence number 31, and sequence number 33, or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology thereto.

[0067] The modified binding partner preferably includes one of the reactive residues that is or is thought to be involved in the formation of an isopeptide bond with a given peptide tag, the peptide tag including the other reactive residue. This means that the modified binding partner preferably includes the reactive residue of the first binding partner—if homologues of the first binding partner are used, they preferably still include this reactive residue.

[0068] Preferably, the reactive residue present in the modified binding partner and derived from the first binding partner is typically a lysine residue, but it may, in some cases, be an asparagine residue. Preferably, the reactive residue present in the peptide tag is an asparagine or aspartic acid residue. Together, these residues form an isopeptide bond.

[0069] While not theoretically bound, the third residue may be involved in the formation of an isopeptide bond. Although not directly involved in the binding, this third residue may mediate the formation of the bond. Typically, the third residue is a glutamate residue. The modified binding partner preferably contains this third residue. In other words, the first reactive fragment of the first binding partner preferably contains this third residue, which is also present in the modified binding partner.

[0070] Typically, the binding partner is larger than its corresponding peptide tag, and at least, if it is derived from a protein that naturally forms isopeptide bonds, the binding partner contains or consists of a larger fragment or portion of that protein compared to the peptide tag. The binding partner may contain a fragment of the protein that overlaps with the fragment designed to constitute the peptide tag, or it may contain separate, distant fragments of the protein compared to that of the peptide tag.

[0071] In some embodiments, the binding partners (i.e., the first binding partner, the second binding partner, and / or the modified binding partner) are at least 20 amino acids long. Preferably, the binding partners have lengths of 5 amino acids or more, for example 10 amino acids or more, for example 15 amino acids or more, for example 20 amino acids or more, for example 25 amino acids, for example 30 amino acids, for example 35 amino acids, for example 40 amino acids, for example 45 amino acids, for example 50 amino acids, for example 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 amino acids or more. In preferred embodiments, the modified binding partner is at least 20 amino acids long. Preferably, the binding partner has a length of 5 amino acids or more, for example 10 amino acids or more, for example 15 amino acids or more, for example 20 amino acids or more, for example 25 amino acids, for example 30 amino acids, for example 35 amino acids, for example 40 amino acids, for example 45 amino acids, for example 50 amino acids, for example 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 amino acids or more.

[0072] This method can also be used to modify peptides that are thought to be involved in the formation of isopeptide bonds with peptide tags. International Publication No. 2011 / 098772 describes in detail how such potential binding partners and peptide tags can be identified. Using the method described therein, a first and / or second peptide pair and the corresponding first and / or second binding partner and peptide tag can be selected.

[0073] Peptide tags As used herein, the term “peptide tag” generally refers to a small peptide fragment, which may be directly designed or derived from proteins that naturally form intramolecular isopeptide bonds. Peptide tags may also be identified using known binding partners, for example, derived from proteins that naturally form intramolecular isopeptide bonds, for screening peptide libraries.

[0074] In some embodiments, the first peptide tag, second peptide tag, and / or third peptide tag used to measure the binding properties of the modified binding partner in this method are: SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 69 (Snoop tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 50 (Pho tag), SEQ ID NO: 52 (Ent tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag) ), sequence number 35 (Bac3 tag), sequence number 22 (Bac4 tag), sequence number 71 (RumTrunk tag), sequence number 46 (Rum tag), and sequence number 12 (Bac5 tag) or their homologs having at least 60% homology, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, are independently selected from the group consisting of homologs thereof.

[0075] The peptide tag may be 5 to 50 amino acid lengths, for example, 10, 20, 30, or 40 to 50 amino acid lengths, and may be covalently bonded to a binding partner as defined herein via an isopeptide bond. Thus, the peptide tag may contain one reactive residue in the isopeptide protein used to design the binding partner as described above (and the binding partner may contain another reactive residue involved in its binding).

[0076] The peptide tags may be modified; for example, mutations or changes may be introduced into one, two, or three of the first, second, or third peptide tags.

[0077] If the peptide tag is designed using a protein that naturally forms intramolecular isopeptide bonds, the peptide tag may (i) contain or consist of a fragment of the protein having at least 5 amino acid lengths or at least 50% identity with that fragment, for example, at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity, and (ii) may be less than 50 amino acid lengths.

[0078] The peptide tag may contain, or consist of, a fragment of an isopeptide protein having a length of at least 5 amino acids, for example, at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids. As discussed above, a peptide tag can consist of 50 amino acid residues, for example, 50, 40, 30, 20, or 10 amino acid residues. In particular, the peptide tag may contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0079] As previously discussed, peptide tags, i.e., first, second, or third peptide tags, must be able to covalently bond to their corresponding binding partners via spontaneously occurring isopeptide bonds. In this regard, peptide tags preferably contain one of the reactive amino acid residues involved in the formation of the isopeptide bond in the isopeptide protein. Thus, peptide tags contain only one reactive residue derived from the isopeptide bond and do not contain both reactive residues involved. Furthermore, if a peptide tag is modified or mutagenesized, the reactive residues in its fragment preferably remain unchanged. This means that if a homolog of the peptide tag is used, the homolog preferably still contains the reactive residues that were originally present in the peptide tag.

[0080] Preferably, the reactive residue present in the peptide tag is an asparagine or aspartic acid residue, which can form an isopeptide bond with the reactive residue of the binding partner or modified binding partner, as described above.

[0081] While not theoretically bound, the third residue may be involved in the formation of an isopeptide bond. Although not directly involved in the bond, this third residue may mediate the bond formation. Typically, the third residue is a glutamate residue. The modified binding partner preferably contains this third residue. In other words, the peptide tag, i.e., the first, second, or third peptide tag, preferably does not contain this third residue, which is instead present in the modified binding partner.

[0082] In some embodiments of a method comprising measuring the binding properties of one or more modified binding partners to a peptide tag and comparing them with the binding properties of first and / or second binding partners to the same peptide tag, the peptide tag is a first peptide tag (of the first binding pair), a second peptide tag (of the second binding pair), or a third peptide tag different from the first and second peptide tags.

[0083] The third peptide tag may be a known peptide tag. It may also be a computer-designed peptide tag. It may also be a peptide present in a peptide library, which can then be screened for novel binding pairs—this method effectively enables the identification of modified binding partners and candidate peptide tags with improved binding properties. The third peptide tag may also be designed by the method described below in the section “Methods for Producing Peptide Tags” of this specification.

[0084] bond pair The methods described herein are particularly useful for identifying modified binding partners having improved binding properties, especially those having improved binding properties to a given peptide tag. Preferably, the methods are useful for improving the binding of a binding partner (i.e., a first binding partner) to its peptide tag (i.e., a first peptide tag), thereby obtaining an improved peptide pair. The term “improved properties” here refers to any desirable properties, such as binding rate or total binding amount as detailed herein, but also to modified specificity to a given peptide tag—in some cases, it may be desirable to reduce specificity to a peptide tag, while specificity to another peptide tag remains unchanged or is increased.

[0085] Suitable peptide pairs that can be used in this method as starting peptide pairs to be improved include, for example: a) A binding partner for SEQ ID NO: 1 (SpyCatcher) and a peptide tag for SEQ ID NO: 5 (Spy Tag), or a variant thereof having at least 70% homology or identity; b) A binding partner for SEQ ID NO: 3 (SdyCatcher) and a peptide tag for SEQ ID NO: 7 (SdyTag), or a variant thereof having at least 70% homology or identity; c) A binding partner for SEQ ID NO: 9 (SnoopCatcher) and a peptide tag for SEQ ID NO: 69 (Snoop Tag), or a variant thereof having at least 70% homology or identity; d) A binding partner for SEQ ID NO: 39 (MoonCake) and a peptide tag for SEQ ID NO: 47 (RumTrunkD9N tag), or a variant thereof having at least 70% homology or identity; e) A binding partner for SEQ ID NO: 41 (KatI) and a peptide tag for SEQ ID NO: 47 (RumtrunkD9N tag), or a variant thereof having at least 70% homology or identity; f) A binding partner for SEQ ID NO: 39 (MoonCake) and a peptide tag for SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology or identity; g) A binding partner for SEQ ID NO: 41 (KatI) and a peptide tag for SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology or identity; h) A binding partner for SEQ ID NO: 29 (PsCsCatcher) and a peptide tag for SEQ ID NO: 75 (PsCs tag), or a variant thereof having at least 70% homology or identity; And, A variant of a binding partner or peptide tag having at least 70% homology or identity with the binding partner or peptide tag retains the ability to form isopeptide bonds with the corresponding peptide tag or binding partner, and has at least 70%, at least 71%, for example at least 72%, for example at least 73%, for example at least 74%, for example at least 75%, for example at least 76%, for example at least 77%, for example at least 78%, for example at least 79%, for example at least 80%, for example at least 81%, for example at least 82%, for example at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% homology or identity with the binding partner or peptide tag.

[0086] Modified coupling partners Also provided herein are modified bonding partners obtained by the methods disclosed herein, and / or modified bonding partners having improved properties.

[0087] Also provided herein is a modified binding partner capable of binding to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag, wherein the modified binding partner does not contain both reactive residues involved in the formation of the isopeptide bond, and the modified binding partner is a first reactive fragment of a first binding partner containing one reactive residue capable of interacting with a first peptide tag containing another reactive residue via the formation of an isopeptide bond between reactive residues, or at least 70% homology thereto, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least The first reactive fragment comprises or consists of a second remaining fragment of a second binding partner, the second binding partner being able to interact with a second peptide tag comprising another reactive residue via the formation of an isopeptide bond between reactive residues, the second remaining fragment not comprising a reactive residue or a homolog thereof having at least 70% homology, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and the first reactive fragment is preferably upstream of the second remaining fragment.

[0088] The first and second bonding partners may be as described above in the section “Bundling Partners” of this specification. Modified bonding partners may be as described herein, in particular in the section “Bundling Partners” of this specification.

[0089] Accordingly, in some embodiments, the first and / or second binding partner, preferably at least the first binding partner, can be independently selected from SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 33, and its homologues having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology thereto. Other relevant binding partners are described, for example, in Kang et al. 2007.

[0090] The modified binding partner preferably includes one of the reactive residues that is or is thought to be involved in the formation of an isopeptide bond with a given peptide tag, and the peptide tag includes the other reactive residue. This means that the modified binding partner preferably includes the reactive residue of the first binding partner—if homologues of the first binding partner are used, they preferably still include this reactive residue.

[0091] Preferably, the reactive residue present in the modified binding partner and derived from the first binding partner is typically a lysine residue, but it may also be an asparagine residue. Preferably, the reactive residue present in the peptide tag is an asparagine or aspartic acid residue. These residues together form an isopeptide bond.

[0092] The first peptide tag and the second peptide tag may be as described above in the section “Peptide Tags” of this specification.

[0093] In some embodiments, the peptide tags, i.e., a first peptide tag and / or a second peptide tag, and preferably at least the first peptide tag, are: SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 69 (Snoop tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 50 (Pho tag), SEQ ID NO: 52 (Ent tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag) Independently selected from the group consisting of homologs having at least 60% homology to sequence number 35 (Bac3 tag), sequence number 22 (Bac4 tag), sequence number 71 (RumTrunk tag), and sequence number 12 (Bac5 tag) or at least 99% homology to it.

[0094] As previously discussed, peptide tags preferably contain one of the reactive amino acid residues involved in the formation of the isopeptide bond in the isopeptide protein. Therefore, peptide tags contain only one reactive residue derived from the isopeptide bond and do not contain both reactive residues involved. Furthermore, if a peptide tag is modified or mutagenesized, the reactive residues in its fragment preferably remain unchanged. This means that if a homolog of the peptide tag is used, the homolog preferably still contains the reactive residues that were originally present in the peptide tag.

[0095] Using the above method, the inventors designed, produced, and tested several modified bonding partners having improved properties, as described in the following examples.

[0096] These tags can be further improved through mutation or rational design. For example, a tag can be further improved by mutating a reactive residue; for instance, in a peptide tag where the reactive residue is D, mutating this residue to N improves the properties of the peptide tag—and this can further improve the properties of the peptide tag. This may result in increased specificity to the modified binding partner and, in some cases, a decrease in specificity to the first or second binding partner.

[0097] In some embodiments, the modified binding partner includes or comprises SEQ ID NO: 37 (QueenCatcher), SEQ ID NO: 39 (MoonCake), or SEQ ID NO: 41 (KatI), a fragment or homologue thereof having at least 60% homology or identity with it, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% homology or identity with it.

[0098] In one embodiment, the modified binding partner includes or consists of SEQ ID NO: 37, or a fragment or homolog thereof having at least 60% homology or identity therewith, provided that the active residue corresponding to residue 31 of SEQ ID NO: 37 is not modified.

[0099] In another embodiment, the modified binding partner includes or consists of SEQ ID NO: 39, or a fragment or homolog thereof having at least 60% homology or identity therewith, provided that the active residue corresponding to residue 31 of SEQ ID NO: 39 is not modified.

[0100] In another embodiment, the modified binding partner includes or consists of SEQ ID NO: 41, or a fragment or homolog thereof having at least 60% homology or identity therewith, provided that the active residue corresponding to residue 31 of SEQ ID NO: 41 is not modified.

[0101] In another embodiment, the modified binding partner includes or consists of SEQ ID NO: 29, or a fragment or homolog thereof having at least 60% homology or identity therewith, provided that the active residue corresponding to residue 8 of SEQ ID NO: 29 is not modified.

[0102] In another embodiment, the binding partner includes or consists of SEQ ID NO: 9, or a fragment or homolog thereof having at least 60% homology or identity therewith, provided that the active residue corresponding to residue 117 of SEQ ID NO: 9 is not modified.

[0103] The modified binding partner shown in SEQ ID NO: 37 was obtained starting from SEQ ID NO: 3. The reactive residue of SEQ ID NO: 3 is located at position 31 in SEQ ID NO: 3 and is retained in the modified binding partner which contains or consists of SEQ ID NO: 37 or its homolog having at least 70% identity or homology thereto. The first reactive fragment derived from SEQ ID NO: 3 extends from positions 1 to 93 in SEQ ID NO: 3. The modified binding partner of SEQ ID NO: 37 further contains a fragment (the remaining fragment) extending from positions 97 to 116 in SEQ ID NO: 1. The reactive residue of SEQ ID NO: 37 is located at position 31.

[0104] The modified binding partners shown in SEQ ID NO: 39 and SEQ ID NO: 41 were both obtained starting from SEQ ID NO: 37. The modified binding partners shown in SEQ ID NO: 39 and SEQ ID NO: 41 were designed by introducing mutations into SEQ ID NO: 37 through computer structural modeling and rational design. The reactive residues in SEQ ID NO: 39 and SEQ ID NO: 41 are located at position 31 in these sequences.

[0105] In one embodiment, the modified binding partner includes or consists of SEQ ID NO: 29, or a fragment or homolog thereof, provided that the residue corresponding to residue 8 of SEQ ID NO: 29, which is the active residue, is not modified.

[0106] Improved characteristics This specification discloses methods for modifying the aforementioned binding partners, as well as the modified binding partners. Such modified partners are of particular interest if they exhibit improved properties compared to the reference binding partner. The improved properties may be improved binding properties, as described in detail below in this specification, or they may be other properties, such as modified specificity to a given partner, as described above. For example, modified binding partners described herein or obtained by the methods described herein may be particularly useful for other applications, such as applications related to peptide presentation on particles, or may have increased stability.

[0107] Accordingly, this disclosure also provides a modified coupling partner having one or more improved properties compared to the reference coupling partner, the one or more improved properties being independently selected from one or more of the following: a) Increased binding efficacy of a modified binding partner to a peptide tag compared to the binding of a reference binding partner to the peptide tag, wherein the modified binding partner and optionally the reference binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained in the modified binding partner or within the reference binding partner and another reactive residue contained in the peptide tag, and the increased binding efficacy is at least one of the total binding amount and the binding rate; b) An increased ability to form particles that present the peptide of the target, such as virus-like particles that present the peptide of the target, compared to the ability of a reference binding partner to form particles under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein, fused to a modified binding partner and the peptide of the target fused to a peptide tag, or the particles comprise a particle-forming protein fused to a peptide tag and the peptide of the target fused to a modified binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the modified binding partner and the peptide tag; c) Increased ability to present the peptide of interest on particles such as virus-like particles, compared to the ability of a reference binding partner to present the peptide of interest under similar conditions, wherein the particles comprise a particle-forming protein such as a virus-like particle-forming protein fused to a modified binding partner and the peptide of interest fused to a peptide tag, or the particles comprise a particle-forming protein fused to a peptide tag and the peptide of interest fused to a modified binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the modified binding partner and the peptide tag.

[0108] The term "under similar conditions" in this specification refers to the formation of the same particles that present the peptide of the same purpose, but here the presentation is achieved by the fusion of a reference binding partner instead of by the fusion of a modified binding partner.

[0109] Typically, the binding properties of a modified binding partner are improved compared to at least the binding properties of a first binding partner, i.e., a binding partner containing one reactive residue still present in the modified binding partner. It may also be interesting to compare the properties of the modified binding partner to those of a binding partner (first or second) having the highest homology, similarity, or identity with respect to it. In other words, throughout this disclosure, the reference binding partner is preferably one of the first or second binding partners having the highest homology, similarity, or identity with respect to the modified binding partner. Thus, in a preferred embodiment, the modified binding partner has higher homology, similarity, or identity with respect to the first binding partner than with respect to the second binding partner, and the reference binding partner is the first binding partner; or the modified binding partner has higher homology, similarity, or identity with respect to the second binding partner than with respect to the first binding partner, and the reference binding partner is the second binding partner.

[0110] However, it will be understood that the bonding properties of the modified bonding partner may also be improved compared to the bonding properties of the third bonding partner, as described above herein.

[0111] The binding properties of the modified binding partner can be determined in relation to binding to one or more of the first peptide tag, the second peptide tag, or the third peptide tag, as described above in the section “Peptide Tags” of this specification, wherein the first peptide tag is a peptide tag that can interact with the first binding partner via isopeptide bond formation, and the second peptide tag is a peptide tag that can interact with the second binding partner via isopeptide bond formation.

[0112] Improved binding properties, as described herein, generally refer to the increased binding efficacy of a modified binding partner to a reference peptide tag, where the reference peptide tag is a first peptide tag, a second peptide tag, or a third peptide tag, and is compared to the binding efficacy of the reference binding partner to the same peptide tag, where the reference binding partner is either the first or second binding partner, or a binding partner capable of interacting with the third peptide tag.

[0113] The modified binding partners disclosed herein are therefore preferably having increased binding efficacy to a given peptide tag compared to a reference binding partner that binds to the same peptide tag; the increased binding efficacy may be an increased binding rate and / or an increased total binding amount of the modified binding partner to the peptide tag.

[0114] In some embodiments, the binding rate of the modified peptide partner is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference binding partner. Preferably, the reference binding partner is one binding partner (first or second) with which the modified binding partner has the highest homology, similarity, or identity, and the binding rate is determined in relation to the binding of the binding partner to the first peptide tag.

[0115] In some embodiments, the total binding amount of the modified peptide partner is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference binding partner. Preferably, the reference binding partner is one binding partner (first or second) with which the modified binding partner has the highest homology, similarity, or identity, and the total binding amount is determined in relation to the binding of the binding partner to the first peptide tag.

[0116] In some embodiments, the binding rate of the modified peptide partner is increased by at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the reference binding partner, and the total binding amount of the modified peptide partner is increased by at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the reference binding partner. Preferably, the reference binding partner is one binding partner (first or second) with which the modified binding partner has the highest homology, similarity, or identity, and the total binding amount is determined in relation to the binding of the binding partner to the first peptide tag.

[0117] Another property of a modified binding partner that can be improved compared to a reference binding partner is the ability of the particle to present the peptide of interest. Such particles can be formed via particle-forming proteins known in the art, e.g., virus-like particle-forming proteins, for example, but not limited to these. By fusing one member of a peptide pair to such a protein and the other member of the peptide pair to the peptide to be presented on the particle, a peptide-presenting particle can be obtained; Figure 3 shows the general principle of one such embodiment. For example, a particle-forming protein is fused to a peptide tag, and the peptide to be presented is fused to the corresponding binding partner. The spontaneous formation of an isopeptide bond between the peptide tag and its binding partner allows the peptide to be presented on the surface of the particle. Using this technique, virus-like particles (VLPs) that present antigenic peptides, e.g., peptides associated with abnormal physiological responses such as diseases, as described in detail in International Publication No. 2016 / 112921, have been generated. Capsid proteins are an example of a suitable particle-forming protein that can be used to generate such VLPs. For example, AP205, Qβ, MS2, HBc, and phage fr, P22, cowpea mosaic virus (CPMV), bromosaic virus (BMV), cowpea chlorotic mottle virus (CCMV), bacteriophage lambda human adenovirus (AdV), and vault particles (PDB:4V60) can be used. Other suitable proteins are known in the art and, for example, the proteins listed in Table 1 of Lieknina et al., 2019 can be used.

[0118] In some embodiments, when the binding partner obtained by this method is used to present a compound of interest, such as an antigen, in virus-like particles or particles as described below, it can produce an increased immune response when administered to a target requiring it, compared to the immune response obtained from a reference binding partner. The reference binding partner may be as described elsewhere in this specification. In some embodiments, the reference binding partner is SpyCatcher (SEQ ID NO: 1).

[0119] Accordingly, the Specified also discloses methods for inducing an immune response in subjects requiring it, the methods comprising administering to subjects particles such as virus-like particles containing or obtained by the methods described herein, preferably the immune response being amplified compared to the immune response obtained after administration of particles such as virus-like particles containing, in particular, SpyCatcher (SEQ ID NO: 1), but otherwise identical, such as any of the reference binding partners described herein.

[0120] In some embodiments, the increased immune response is an increased IgM response and / or an increased IgG2 response, such as an increased IgG2a and / or IgG2b response. In some embodiments, the increased immune response is an increased IgG1 response. In some embodiments, the increased immune response is an increased IgG3 response. Preferably, at least one of the IgM, IgG2a, or IgG2b responses is increased.

[0121] However, the method can generally be applied to generate particles that are not virus-like particles to present target compounds such as peptides. Other examples of such particle-forming proteins include small heat shock proteins (HSPs) (PDB:1SHS), apoferritin (PDB:1DAT), pyruvate dehydrogenase enzyme complex (PDB:1EAA), thermosomes (THS), and i301 (designed from 2-keto-3-deoxyphosphogluconate (KDPG) aldolase derived from the Entner-Doidorov pathway of the hyperthermophilic bacterium Thermotoga maritima).

[0122] This method allows proteins capable of self-assembling into nanoparticles to be genetically modified by fusion with a peptide tag. The assembled nanoparticles (i.e., those displaying the reactive peptide tag) can then be linked to a genetically fused peptide that, upon contact with the peptide tag, can interact with the peptide tag via an isopeptide bond.

[0123] Therefore, particles thus obtained using modified binding partners obtained or obtainable by the disclosed method may have improved properties, for example, in terms of peptide presentation density (i.e., how many peptide molecules are presented on the surface of the particle), uniformity of presentation (i.e., regular and uniform spacing of peptides on the surface of the particle), and immunogenicity when presenting antigenic peptides.

[0124] In other embodiments, the modified binding partner may be used to detect a compound of interest, particularly a protein of interest, if the modified binding partner is detectable, or it may enable the purification of the protein of interest if the binding partner is immobilized, for example, on a solid support.

[0125] Peptide tags Methods for producing peptide tags This disclosure also provides a method for producing a peptide tag that can bind to a binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding peptide tag and another reactive residue contained within the binding partner, the method comprising the following steps: a) A step of identifying candidate peptide tags having at least 60% similarity to a reference peptide tag, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology thereto, wherein the reference peptide tag can spontaneously form an isopeptide bond with at least one reference binding partner; b) A step of selecting a peptide tag from the candidate peptide tags identified in a), wherein the selected peptide tag comprises at least one reactive residue likely involved in the formation of an isopeptide bond; c) A step of designing and producing peptide tags from selected peptide tags, wherein each peptide tag comprises or consists of a fragment of the selected peptide tag spanning 4 to 24 amino acids upstream of the reactive residue involved in the formation of an isopeptide bond to 2 to 22 amino acids downstream, or a homolog thereof having at least 70% homology, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, provided that the homolog thereof contains a reactive residue.

[0126] Preferably, the candidate peptide tag is part of a peptide pair, and the candidate is found based on aligning the peptide pair (which may be a single protein consisting of two domains that interact or are thought to interact intramolecularly via an isopeptide bond, i.e., the peptide tag and its binding partner) with a known peptide pair. In some embodiments, the peptide tag comprises 3, 4, 5, 6, 7, or 8 amino acids or more, instead of having at least 60% similarity to a reference peptide tag, or in addition to that, the portion comprising reactive residues and sharing at least 60% homology, similarity, or identity with a sequence of amino acids in the reference peptide tag, e.g., 3, 4, 5, 6, 7, or 8 amino acids.

[0127] In a preferred embodiment, the binding partner is a modified binding partner as described herein, and the method thus enables the design of a peptide tag capable of forming an isopeptide bond with the modified binding partner as described herein.

[0128] In the first step, candidate peptide tags are identified. This can be done by identifying proteins that are thought to be isopeptide proteins, i.e., proteins in which two domains interact via an intramolecular isopeptide bond. Such proteins have, for example, a fragment or domain, typically a C-terminal fragment or domain, which shares at least 60% similarity, homology, or identity with a reference peptide tag known to interact with at least one reference binding partner via the spontaneous formation of an isopeptide bond. Thus, starting from known peptide tags, candidate proteins can be identified, and from there, candidate peptide tags can be identified. Throughout this disclosure, candidate proteins are known, or at least thought to contain reactive residues capable of forming an isopeptide bond either intramolecularly or intermolecularly. Candidate peptide tags and candidate binding partners are therefore preferably limited to the domains of the candidate protein, as previously defined. Candidate peptide tags may be derived from a library, for example, a peptide library, which can be screened for candidate peptide tags.

[0129] Candidate peptide tags containing at least one reactive residue involved in or potentially involved in the formation of an isopeptide bond are then selected. The peptide tag is then designed from the selected peptide tag.

[0130] The peptide tag designed in the final step contains or consists of a fragment of the selected peptide tag, which spans from an upstream position to a downstream position of the reactive residue. The upstream position is located 4 to 24 residues or amino acids upstream of the reactive residue, and the downstream position is located 2 to 22 amino acids downstream of the reactive residue.

[0131] In some embodiments, the upstream position is located 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 residues upstream of the reactive residue, and the downstream position is located 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 residues downstream of the reactive residue. The peptide tag therefore has a length of 7-47 amino acids, for example 8-46 amino acids, for example 9-45 amino acids, for example 10-44 amino acids, for example 11-43 amino acids, for example 12-42 amino acids, for example 13-41 amino acids, for example 14-40 amino acids, for example 15-39 amino acids, for example 16-38 amino acids, for example 17-37 amino acids, for example 18-36 amino acids, for example 19-35 amino acids, for example 20-34 amino acids, for example 21-33 amino acids, for example 22-32 amino acids, for example 23-31 amino acids, for example 24-30 amino acids, for example 25-29 amino acids, for example 26-28 amino acids, for example 27 amino acids in some embodiments. In some embodiments, the peptide tag has a length of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids.

[0132] Homogenetics or fragments of peptide tags designed in this way and possessing at least 70% homology may also be useful, provided they contain reactive residues.

[0133] Some known peptide tags contain a binding motif, which is referred to herein as a reference binding motif or reference sequence motif, and which can be used to identify candidate peptide tags. Candidate peptide tags may be peptide tags (or proteins) that contain a sequence motif, e.g., a reference sequence motif, within their C-terminal region, for example, within 50 amino acids at their C-terminus. When the peptide tag is in the form of a “free” peptide, i.e., an independent peptide, the term “C-terminus” refers to the C-terminus of that peptide. However, the peptide tag may be part of a protein, as detailed herein, in which case the term “C-terminus” refers to the C-terminal portion of the domain corresponding to the peptide tag in the protein—the C-terminus (of the peptide tag) may therefore be located inside the protein.

[0134] A sequence motif may be a motif known or thought to be characteristic of a peptide tag capable of forming an isopeptide bond with a binding partner. For example, a sequence motif may include or consist of GX1X2X3IVMX4DX5 shown in SEQ ID NO: 73; GX1X2X3YVMX4DX5 shown in SEQ ID NO: 74; GX1X2X3FVMX4DX5 shown in SEQ ID NO: 43; or GX1X2X3WVMX4DX5 shown in SEQ ID NO: 44; X1, X2, X3, X4, and X5 are independently selected from any amino acids.

[0135] The sequence motif may be present in a candidate peptide tag, or it may be present in a protein, for example, an isopeptide protein containing an intramolecular isopeptide bond, from which the candidate peptide tag can subsequently be derived, which are fragments of the protein containing one reactive residue as described herein. The sequence motif may therefore be present within 50 amino acids from the C-terminus of the protein or from the candidate peptide tag, for example, within 45, 40, 35, 30, or 25 amino acids from the C-terminus.

[0136] The method may further include the step of determining the location (or assumed location of the isopeptide bond) of the isopeptide bond within the candidate peptide tag (or isopeptide protein). The candidate peptide tag selected for designing the peptide tag by the above method contains one of two reactive residues involved in the formation of the isopeptide bond, the other of the two reactive residues present in the binding partner.

[0137] Finally, the designed peptide tag is produced. This is carried out, for example, as is known in the art.

[0138] In a preferred embodiment, the designed peptide tag can interact with a modified binding partner via the spontaneous formation of an isopeptide bond, as described herein.

[0139] To determine whether the peptide tag obtained by this method has improved properties, its interaction with a reference binding partner is examined. The reference binding partner is preferably a binding partner known to interact with the reference peptide tag. In other words, to determine whether the peptide tag obtained by this method has improved properties, the properties of the peptide tag in relation to the reference binding partner are preferably compared to the properties of the reference peptide tag in relation to the reference binding partner; binding pairs used as appropriate for reference are described herein in the section “Binding Pairs”. The reference partner may be any of the binding partners described herein, particularly in relation to the first binding partner and the second binding partner. In some embodiments, the reference binding partner is a modified binding partner described herein.

[0140] In some embodiments, the reference join partner is selected from the group consisting of SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 71, and SEQ ID NO: 33, and homologs thereof having at least 60% homology to them, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology to them. In some embodiments, the reference join partner is the join partner to which the modified join partner has the highest homology, similarity, or identity.

[0141] In some embodiments, the reference peptide tag is sequence number 5 (Spy tag), sequence number 7 (Sdy tag), sequence number 69 (Snoop tag), sequence number 46 (Rum tag), sequence number 47 (RumTrunkD9N tag), sequence number 50 (Pho tag), sequence number 52 (Ent tag), sequence number 54 (Rum7 tag), sequence number 56 (Rum3 tag), sequence number 58 (Rum2 tag), sequence number 60 (Rum4 tag), sequence number 62 (Rum5 tag), sequence number 64 (Rum6 tag), sequence number 66 (Bac tag), sequence number 68 (Bac2 tag), sequence number 35 (Bac3 tag), sequence Selected from the group consisting of homologs of number 22 (Bac4 tag), sequence number 31, and sequence number 12 (Bac5 tag) or having at least 60% homology thereto, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0142] For example, the following are reference peptide pairs that can be used in this method to determine whether the peptide tags designed and fabricated by the above method have improved properties: a) Sequence ID 1 and Sequence ID 5 b) Sequence IDs 3 and 7 c) Sequence ID 9 and Sequence ID 69 d) Sequence IDs 47 and 39 e) Sequence IDs 47 and 41 f) Sequence IDs 46 and 39; g) Sequence IDs 46 and 41 Preferably, the reference peptide pair is selected from Spy tag / SpyCatcher (SEQ ID NO: 5 and SEQ ID NO: 1, respectively) and Sdy tag / SdyCatcher (SEQ ID NO: 7 and SEQ ID NO: 3, respectively).

[0143] Bonding characteristics Once a peptide tag is obtained by any of the methods described herein, its binding properties can be measured. Accordingly, in some embodiments, the method further includes the step of measuring one or more binding properties of the peptide tag, wherein the one or more properties are preferably i) total binding amount and ii) binding rate of the peptide tag to a reference binding partner, preferably a modified binding partner. Determining the corresponding one or more binding properties of the reference peptide tag to the same binding partner (i.e., a reference binding partner, preferably a modified binding partner) allows for a comparison of the binding properties of the peptide tag to the binding partner. An increase in at least one of the total binding amount and binding rate indicates an increase in binding efficacy or an improvement in binding properties.

[0144] Measuring the increased binding rate of a peptide tag to a reference binding partner is an indicator of a peptide tag having increased binding efficacy, compared to the binding rate of the reference peptide tag to the reference binding partner. Preferably, the binding rate of a peptide tag to a reference binding partner is measured and compared to the binding rate of the reference peptide tag to the same binding partner, where the reference peptide tag and the reference binding partner (i.e., the reference binding pair) can interact via the spontaneous formation of an isopeptide bond, and in some embodiments, the binding rate is the binding rate of the modified or first binding partner to the first peptide tag. In some embodiments, the reference peptide tag is a Spy tag (SEQ ID NO: 5) or a Sdy tag (SEQ ID NO: 7), and the reference binding partner is a SpyCatcher (SEQ ID NO: 1) or a SdyCatcher (SEQ ID NO: 3).

[0145] Preferably, the above increase in the bond ratio is at least 5%, for example at least 10%, for example at least 15%, for example at least 20%, for example at least 25%, for example at least 30%, for example at least 40%, for example at least 50%, for example at least 60%, for example at least 70%, for example at least 80%, for example at least 90%, for example at least 100%, or more, compared to the bond ratio measured for the reference bond pair.

[0146] In some embodiments, the binding rate of the peptide tag to the first reference binding partner is measured and compared to the binding rate of the reference peptide tag to the reference binding partner.

[0147] Integration Partners Certain binding partners that can be used as reference binding partners in this method include binding partners that are known to or are thought to be able to form an isopeptide bond with the (reference) peptide tag. Certain modified binding partners disclosed herein can also be used as reference binding partners.

[0148] Therefore, the reference join partner can be selected from SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, as well as their homologs having at least 60% homology, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homology. Preferably, the reference join partner is SpyCatcher (SEQ ID NO: 1) or SdyCatcher (SEQ ID NO: 3).

[0149] In some embodiments, the reference binding partner for which the binding of the peptide tag obtained by this method is measured is SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, or its homologue having at least 60% homology, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology.

[0150] The reference binding partner preferably comprises one of the reactive residues that is or is thought to be involved in the formation of an isopeptide bond with a given reference peptide tag, and the reference peptide tag or the peptide tag produced by the above method comprises the other reactive residue. The reactive residues are discussed herein. The reference binding partner is preferably one of the first or second binding partners that has the highest homology, similarity, or identity with the modified binding partner. Thus, in a preferred embodiment, the modified binding partner has higher homology, similarity, or identity with respect to the first binding partner than with respect to the second binding partner, and the reference binding partner is the first binding partner; or the modified binding partner has higher homology, similarity, or identity with respect to the second binding partner than with respect to the first binding partner, and the reference binding partner is the second binding partner.

[0151] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 1 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 1 or its homologue as described above.

[0152] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 3 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 3 or its homologue as described above.

[0153] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 9 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 9 or its homologue as described above.

[0154] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 13 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 13 or its homologue as described above.

[0155] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 15 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 15 or its homologue as described above.

[0156] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 17 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 17 or its homologue as described above.

[0157] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 19 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 19 or its homologue as described above.

[0158] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 23 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 23 or its homologue as described above.

[0159] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 25 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 25 or its homologue as described above.

[0160] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 27 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 27 or its homologue as described above.

[0161] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 29 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 29 or its homologue as described above.

[0162] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 30 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 30 or its homologue as described above.

[0163] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 31 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 31 or its homologue as described above.

[0164] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 37 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 37 or its homologue as described above.

[0165] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 39 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 39 or its homologue as described above.

[0166] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 41 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 41 or its homologue as described above.

[0167] In some embodiments, the first or second bonding partner having the highest homology, similarity, or identity with respect to the modified bonding partner is Sequence ID No. 71 or its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, and the reference bonding partner is Sequence ID No. 71 or its homologue as described above.

[0168] Peptide tags As used herein, the term “peptide tag” generally refers to a small peptide fragment, which can be designed or derived directly from proteins that naturally form intramolecular isopeptide bonds—such proteins are referred herein as “isopeptide proteins.” Peptide tags can also be identified for screening peptide libraries by using known binding partners, for example, derived from proteins that naturally form intramolecular isopeptide bonds.

[0169] In some embodiments, the reference peptide tags used to measure the binding properties of peptide tags designed by the methods described herein are: SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 69 (Snoop tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 50 (Pho tag), SEQ ID NO: 52 (Ent tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 Selected from the group consisting of the following: the reference peptide tag, SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 31, and SEQ ID NO: 12 (Bac5 tag), or homologs thereof having at least 60% homology, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology. Preferably, the reference peptide tag is the Spy tag (SEQ ID NO: 5) or the Sdy tag (SEQ ID NO: 7).

[0170] Peptide tags are 5 to 50 amino acid lengths, e.g., 10, 20, 30, 40 to 50 amino acid lengths, and may be covalently bonded to a binding partner as defined herein via an isopeptide bond. Thus, a peptide tag may contain one reactive residue in the isopeptide protein used to design the binding partner as described above (and the binding partner may contain another reactive residue involved in its binding).

[0171] If the peptide tag is designed directly using a protein that naturally forms intramolecular isopeptide bonds, the peptide tag may (i) contain or consist of a fragment of the protein having at least 5 amino acid lengths or at least 50% identity with that fragment, for example, at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity, and (ii) may be less than 50 amino acid lengths.

[0172] The peptide tag may contain, or consist of, a fragment of an isopeptide protein having a length of at least 5 amino acids, for example, at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids.

[0173] As discussed above, a peptide tag can consist of 50 amino acid residues, for example, 50, 40, 30, 20, or 10 amino acid residues. In particular, peptide tags can consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0174] As previously discussed, peptide tags, i.e., reference peptide tags or peptide tags obtained by the methods described herein, must be able to covalently bond to their corresponding binding partners via naturally occurring isopeptide bonds. In this regard, peptide tags preferably contain one of the reactive amino acid residues involved in the formation of an isopeptide bond in an isopeptide protein. Thus, peptide tags contain a reactive residue derived from only one isopeptide bond and do not contain both reactive residues involved. Furthermore, if a peptide tag is modified or mutagenesized, the reactive residues in its fragment are preferably left unchanged. This means that if a homologue of a peptide tag is used, the homologue still contains the reactive residues that were originally present in the peptide tag.

[0175] bond pair The methods described herein are particularly useful for identifying peptide tags having improved properties, especially improved binding properties to a given binding partner. Preferably, the methods are used to improve the binding of a peptide tag to a reference binding partner (e.g., one of the modified binding partners described herein), thereby obtaining an improved peptide pair. Suitable peptide pairs that can be used in this method as starting peptide pairs to be improved include, for example, the following: a) Sequence ID 1 and Sequence ID 5 b) Sequence IDs 3 and 7 c) Sequence ID 9 and Sequence ID 69 d) Sequence IDs 47 and 39 e) Sequence IDs 47 and 41 f) Sequence IDs 46 and 39 g) Sequence ID 46 and Sequence ID 41, Or a variant thereof that has at least 70% homology or identity with them.

[0176] In some embodiments, the starting peptide pair is a) A binding partner for SEQ ID NO: 1 (SpyCatcher) and a peptide tag for SEQ ID NO: 5 (Spy Tag), or a variant thereof having at least 70% homology or identity; b) A binding partner for SEQ ID NO: 3 (SdyCatcher) and a peptide tag for SEQ ID NO: 7 (SdyTag), or a variant thereof having at least 70% homology or identity; c) A binding partner for SEQ ID NO: 9 (SnoopCatcher) and a peptide tag for SEQ ID NO: 69 (Snoop Tag), or a variant thereof having at least 70% homology or identity; d) A binding partner for SEQ ID NO: 39 (MoonCake) and a peptide tag for SEQ ID NO: 47 (RumtrunkD9N tag), or a variant thereof having at least 70% homology or identity; e) A binding partner for SEQ ID NO: 41 (KatI) and a peptide tag for SEQ ID NO: 47 (RumtrunkD9N tag), or a variant thereof having at least 70% homology or identity; f) A binding partner for SEQ ID NO: 39 (MoonCake) and a peptide tag for SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology or identity; g) A binding partner for SEQ ID NO: 41 (KatI) and a peptide tag for SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology or identity; h) A binding partner for SEQ ID NO: 29 (PsCsCatcher) and a peptide tag for SEQ ID NO: 75 (PsCs tag), or a variant thereof having at least 70% homology or identity; And, Each variant of the binding partner or peptide tag having at least 70% homology or identity with the binding partner or peptide tag retains the ability to form an isopeptide bond with the corresponding peptide tag or binding partner, and has at least 70%, at least 71%, for example at least 72%, for example at least 73%, for example at least 74%, for example at least 75%, for example at least 76%, for example at least 77%, for example at least 78%, for example at least 79%, for example at least 80%, for example at least 81%, for example at least 82%, for example at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% homology or identity with the binding partner or peptide tag.

[0177] Designed peptide tags Also provided herein are peptide tags and binding partners obtained by the methods disclosed herein, and / or peptide tags having improved properties.

[0178] Furthermore, provided herein are peptide tags that can bind to a reference binding partner, preferably a modified binding partner as described herein, via the spontaneous formation of an isopeptide bond between one reactive residue contained in the reference binding partner and another reactive residue contained in the peptide tag, wherein neither the reference binding partner nor the peptide tag (or reference peptide tag) contains any reactive residues involved in the formation of an isopeptide bond. The reference binding partner may be a modified binding partner as described herein above, particularly in the sections “Binding Partner” and “Method for Producing a Modified Binding Partner.”

[0179] Also provided herein are peptide tags comprising or consisting of a protein fragment containing at least one reactive residue involved in the formation of an isopeptide bond between the peptide tag and its binding partner, wherein the peptide tag comprises the protein fragment extending from 4 to 24 amino acids upstream of the reactive residue to 2 to 22 amino acids downstream, or a homolog thereof having at least 70% homology, provided that the homolog contains the reactive residue, preferably asparagine or aspartic acid.

[0180] Peptide tags may include the binding motifs described herein. Sequence motifs may be motifs known or considered to be characteristic of peptide tags capable of forming isopeptide bonds with their binding partners. For example, sequence motifs may include or consist of GX1X2X3IVMX4DX5 shown in SEQ ID NO: 73; GX1X2X3YVMX4DX5 shown in SEQ ID NO: 74; GX1X2X3FVMX4DX5 shown in SEQ ID NO: 43; or GX1X2X3WVMX4DX5 shown in SEQ ID NO: 44; X1, X2, X3, X4, and X5 may be independently selected from any amino acids.

[0181] Useful peptide tags are not limited to, but include: SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 69 (Snoop tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 75 (PsCs tag), SEQ ID NO: 50 (Pho tag), SEQ ID NO: 52 (Ent tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: Examples of peptide tags include 22 (Bac4 tag), SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 12 (Bac5 tag), and homologs thereof that have at least 60% homology, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homology. In a preferred embodiment, the peptide tag is selected from the group consisting of SEQ ID NO: 46 (Rum tag); SEQ ID NO: 71 (RumTrunk tag); SEQ ID NO: 58 (Rum2 tag); SEQ ID NO: 56 (Rum3 tag); SEQ ID NO: 60 (Rum4 tag); SEQ ID NO: 62 (Rum5 tag); SEQ ID NO: 64 (Rum6 tag); SEQ ID NO: 54 (Rum7 tag); SEQ ID NO: 69 (Snoop tag); SEQ ID NO: 66 (Bac tag); SEQ ID NO: 68 (Bac2 tag); SEQ ID NO: 35 (Bac3 tag); SEQ ID NO: 22 (Bac4 tag); SEQ ID NO: 12 (Bac5 tag); and SEQ ID NO: 75 (PsCs tag) and its homologs having at least 60% homology. In a more preferred embodiment, the peptide tag is selected from SEQ ID NO: 46 (Rum tag) and SEQ ID NO: 71 (RumTrunk tag) or its homologs having at least 60% homology.

[0182] As previously discussed, peptide tags preferably contain one of the reactive amino acid residues involved in the formation of an isopeptide bond in an isopeptide protein. Therefore, peptide tags contain only one reactive residue derived from an isopeptide bond and do not contain both reactive residues involved. Furthermore, if a peptide tag is modified or mutagenesized, the reactive residues in its fragment preferably remain unchanged. This means that if a homolog of a peptide tag is used, the homolog preferably still contains the reactive residues that were originally present in the peptide tag.

[0183] However, as will be detailed below, the inventors found that mutating the reactive residues could conveniently be used to further improve the properties of the designed peptide tag.

[0184] Using the above method, the inventors designed, produced, and tested several peptide tags having improved properties, as described in the following examples.

[0185] In some embodiments, the peptide tag includes, or has improved binding properties to a binding partner or modified binding partner comprising, SEQ ID NO: 37 (QueenCatcher), SEQ ID NO: 39 (MoonCake), SEQ ID NO: 29 (PsCsCatcher), or SEQ ID NO: 41 (KatI), or a fragment or homolog thereof having at least 60% homology, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology. Preferably, the peptide tag includes, or has improved binding properties to a binding partner comprising, SEQ ID NO: 37 (QueenCatcher), SEQ ID NO: 39 (MoonCake), or SEQ ID NO: 41 (KatI), or a fragment or homolog thereof.

[0186] In one embodiment, the peptide tag has improved binding properties to a modified binding partner comprising or containing SEQ ID NO: 37 (QueenCatcher) or a fragment or homolog thereof, provided that the active residue, corresponding to residue 31 of SEQ ID NO: 37, is not modified.

[0187] In another embodiment, the peptide tag has improved binding properties to a modified binding partner comprising or consisting of SEQ ID NO: 39 (MoonCake) or a fragment or homolog thereof, provided that the active residue, corresponding to residue 31 of SEQ ID NO: 39, is not modified. This residue corresponds to residue 35 of SEQ ID NO: 79, which, compared to SEQ ID NO: 39, contains four residual N-terminal amino acids derived from the cloning procedure.

[0188] In another embodiment, the peptide tag has improved binding properties to a modified binding partner comprising or consisting of SEQ ID NO: 41 (KatI) or a fragment or homolog thereof, provided that the active residue, corresponding to residue 31 of SEQ ID NO: 41, is not modified. This residue corresponds to residue 35 of SEQ ID NO: 80, which, compared to SEQ ID NO: 41, contains four residual N-terminal amino acids derived from the cloning procedure.

[0189] In another embodiment, the peptide tag has improved binding properties to a binding partner comprising or consisting of SEQ ID NO: 29 (PsCsCatcher) or a fragment or homolog thereof, provided that the active residue, corresponding to residue 8 of SEQ ID NO: 29, remains unmodified.

[0190] Accordingly, also provided herein are modified binding partners as described herein, in particular the binding partner shown in SEQ ID NO: 39 (MoonCake), or its variant having at least 70% homology or identity; the binding partner shown in SEQ ID NO: 41 (KatI), or its variant having at least 70% homology or identity; the binding partner shown in SEQ ID NO: 37 (QueenCatcher), or its variant having at least 70% homology or identity; the binding partner shown in SEQ ID NO: 9 (SnoopCatcher), or its variant having at least 70% homology or identity; the binding partner shown in SEQ ID NO: 29 (PsCsCatcher), or its variant having at least 70% homology or identity; or the binding partner shown in SEQ ID NO: 37 (QueenCatcher), or its variant having at least 70% homology or identity. Preferably, the modified binding partner is the binding partner shown in SEQ ID NO: 39 (MoonCake), or a variant thereof having at least 70% homology or identity; the binding partner shown in SEQ ID NO: 41 (KatI), or a variant thereof having at least 70% homology or identity; or the binding partner shown in SEQ ID NO: 37 (QueenCatcher), or a variant thereof having at least 70% homology or identity.

[0191] The modified binding partner shown in SEQ ID NO: 37 was obtained starting from SEQ ID NO: 3. The reactive residue of SEQ ID NO: 3 is located at position 31 in SEQ ID NO: 3 and is retained in the modified binding partner which contains or consists of SEQ ID NO: 37 or its homolog having at least 70% identity or homology thereto. The first reactive fragment derived from SEQ ID NO: 3 extends from positions 1 to 93 in SEQ ID NO: 3. The modified binding partner of SEQ ID NO: 37 further contains a fragment (the remaining fragment) extending from positions 97 to 116 in SEQ ID NO: 1. The reactive residue of SEQ ID NO: 37 is located at position 31.

[0192] The modified binding partners shown in SEQ ID NO: 39 and SEQ ID NO: 41 were both obtained starting from SEQ ID NO: 37. The modified binding partners shown in SEQ ID NO: 39 and SEQ ID NO: 41 were designed by computer structural modeling and rational design, introducing mutations into SEQ ID NO: 37. The reactive residues in SEQ ID NO: 39 and SEQ ID NO: 41 are located at position 31 in these sequences.

[0193] The binding partner indicated by SEQ ID NO: 39 (MoonCake), or its variant having at least 70% homology or identity, can spontaneously form isopeptide bonds with peptide tags selected from SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), and SEQ ID NO: 12 (Bac5 tag); or its variant having at least 70% homology or identity. In a preferred embodiment, the binding partner represented by SEQ ID NO: 39 (MoonCake), or a variant thereof having at least 70% homology or identity thereto, can spontaneously form an isopeptide bond with the peptide tag represented by SEQ ID NO: 47 (RumtrunkD9N tag), or a variant thereof having at least 70% homology thereto. In another embodiment, the binding partner represented by SEQ ID NO: 39 (MoonCake), or a variant thereof having at least 70% homology or identity thereto, can spontaneously form an isopeptide bond with the peptide tag represented by SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology thereto.

[0194] The binding partner indicated by SEQ ID NO: 41(KatI), or its variant having at least 70% homology or identity, can spontaneously form isopeptide bonds with peptide tags selected from SEQ ID NO: 47(RumtrunkD9N tag), SEQ ID NO: 54(Rum7 tag), SEQ ID NO: 56(Rum3 tag), SEQ ID NO: 58(Rum2 tag), SEQ ID NO: 60(Rum4 tag), SEQ ID NO: 62(Rum5 tag), SEQ ID NO: 64(Rum6 tag), SEQ ID NO: 71(RumTrunk tag), SEQ ID NO: 5(Spy tag), SEQ ID NO: 7(Sdy tag), SEQ ID NO: 46(Rum tag), SEQ ID NO: 66(Bac tag), SEQ ID NO: 68(Bac2 tag), SEQ ID NO: 35(Bac3 tag), SEQ ID NO: 22(Bac4 tag), and SEQ ID NO: 12(Bac5 tag); or its variant having at least 70% homology or identity. In a preferred embodiment, the binding partner represented by SEQ ID NO: 41 (KatI), or a variant thereof having at least 70% homology or identity thereto, can spontaneously form an isopeptide bond with the peptide tag represented by SEQ ID NO: 47 (RumtrunkD9N tag), or a variant thereof having at least 70% homology thereto. In another embodiment, the binding partner represented by SEQ ID NO: 41 (KatI), or a variant thereof having at least 70% homology or identity thereto, can spontaneously form an isopeptide bond with the peptide tag represented by SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology thereto.

[0195] The binding partner indicated by SEQ ID NO: 9 (SnoopCatcher), or a variant thereof having at least 70% homology or identity with it, can spontaneously form an isopeptide bond with the peptide tag indicated by SEQ ID NO: 69 (Snoop Tag), or a variant thereof having at least 70% homology with it.

[0196] The binding partner indicated by SEQ ID NO: 37 (QueenCatcher), or a variant thereof having at least 70% homology or identity, can spontaneously form isopeptide bonds with peptide tags selected from SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 66 (Bac tag); SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 12 (Bac5 tag), and SEQ ID NO: 76 (Clib9), or variants thereof having at least 70% homology or identity. In a preferred embodiment, the binding partner represented by SEQ ID NO: 37 (QueenCatcher), or a variant thereof having at least 70% homology or identity thereto, can spontaneously form an isopeptide bond with the peptide tag represented by SEQ ID NO: 47 (RumtrunkD9N tag), or a variant thereof having at least 70% homology thereto. In another embodiment, the binding partner represented by SEQ ID NO: 37 (QueenCatcher), or a variant thereof having at least 70% homology or identity thereto, can spontaneously form an isopeptide bond with the peptide tag represented by SEQ ID NO: 46 (Rum tag), or a variant thereof having at least 70% homology thereto.

[0197] The binding partner indicated by Sequence ID No. 29 (PsCsCatcher), or a variant thereof having at least 70% homology or identity with it, can spontaneously form an isopeptide bond with the peptide tag indicated by Sequence ID No. 75 (PsCs tag), or a variant thereof having at least 70% homology with it.

[0198] Methods for improving the properties of peptide tags Any of the tags obtained by this method can be further improved by mutation or rational design. For example, a tag can be further improved by mutating the reactive residue D to N.

[0199] Accordingly, also provided herein are peptide tags, and in particular methods for improving the properties of peptide tags obtained by the methods disclosed herein. In some embodiments, the peptide tag used as a starting point includes a certain reactive residue, which is still present in the peptide tag obtained by the above methods. Muting this reactive residue can further improve any of the properties of the peptide tag, as detailed below herein. In some embodiments, the method for designing a peptide tag described herein therefore further includes the step of muting a reactive residue in the designed peptide tag. In some embodiments, the reactive residue is D and is mutated to N.

[0200] Accordingly, also provided herein are methods for improving the properties of peptide tags containing the reactive residues described herein, in particular any of the peptide tags described herein, by mutating at least the reactive residues.

[0201] Improved characteristics Peptide tags described herein are of particular interest if they exhibit improved properties compared to their reference binding partner, or if they exhibit improved binding to a given binding partner compared to a reference peptide tag. The improved properties may be improved binding properties, as detailed above or below herein, or they may be other properties. For example, peptide tags described herein or obtained by the methods described herein may be particularly useful for other applications, such as applications related to peptide presentation on particles.

[0202] This disclosure therefore also provides peptide tags having one or more improved properties compared to a reference peptide tag, the one or more improved properties being independently selected from one or more of the following: a) Increased binding efficacy of a peptide tag to a reference binding partner, particularly a modified binding partner, compared to the binding of a reference peptide tag to the reference binding partner, wherein the peptide tag and optionally the reference peptide tag can bind to the reference binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained in the peptide tag or the reference binding partner and another reactive residue contained in the peptide tag, and the increased binding efficacy is at least one of the total binding amount and the binding rate; b) An increased ability to form particles that present the target peptide, such as virus-like particles that present the target peptide, compared to the ability to form particles of a reference peptide tag under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein fused to a reference binding partner, and the target peptide fused to a peptide tag, or the particles comprise a particle-forming protein fused to a peptide tag and the target peptide fused to a reference binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the reference binding partner and the peptide tag; c) Increased ability to present a target peptide on a particle, such as a virus-like particle, compared to the ability of a reference peptide tag to present the target peptide under similar conditions, wherein the particle comprises a particle-forming protein, such as a virus-like particle-forming protein fused to the peptide tag, or the particle comprises a particle-forming protein fused to the peptide tag and a target peptide fused to a reference binding partner, and the particle is formed by the spontaneous formation of an isopeptide bond between the reference binding partner and the peptide tag.

[0203] The binding properties of a peptide tag can be determined in relation to its binding to one or more reference binding partners. Reference peptide tags may be as described herein. In some embodiments, the peptide tag is obtained by further modification of a peptide tag obtained, for example, by mutation of a reactive residue, using the methods disclosed herein, in which case it may be advantageous to compare its properties to those of a peptide tag still containing the original reactive residue. The binding partner having the highest homology or identity with the modified binding partner is preferably used as a reference to determine whether the modified binding partner has improved properties, as described below herein. In some embodiments, the modified binding partner has higher homology or identity or similarity to the second binding partner with respect to the first binding partner, and the first binding partner is used as a reference. In other embodiments, the modified binding partner has higher homology to the first binding partner with respect to the second binding partner, and the second binding partner is used as a reference.

[0204] Improved binding properties are described herein and generally refer to increased binding efficacy between the binding partner and the peptide tag.

[0205] The peptide tags disclosed herein are therefore preferably having increased binding efficacy to a given binding partner compared to a reference peptide tag that binds to the same peptide binding partner; the increased binding efficacy may be an increased binding rate and / or an increased total binding amount.

[0206] In some embodiments, the binding rate of the peptide tag is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference peptide tag.

[0207] In some embodiments, the total binding amount of the peptide tag is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference peptide tag.

[0208] In some embodiments, the binding rate is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference peptide tag, and the total binding amount of the peptide tag is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference peptide tag.

[0209] Another property of peptide tags that can be improved compared to reference peptide tags is the ability of particles to present the peptide of interest. Such particles can be formed via particle-forming proteins, such as, but not exclusively, virus-like particle-forming proteins, as is known in the art, and the advantages of binding pairs containing the peptide tags obtained by this method can be taken advantage of, as described herein for the modified binding partners in the section "Improved Properties".

[0210] In some embodiments, when the peptide tags obtained by this method are used to present a target compound, such as an antigen, in, for example, virus-like particles or particles as described below, they can produce an increased immune response upon administration to a target that requires the peptide tag, compared to the immune response obtained from the particle in which the reference peptide tag is used.

[0211] This specification also discloses methods for inducing an immune response in a subject requiring such response, the methods comprising administering to a subject particles, such as virus-like particles, which include or are obtained by the methods described herein, preferably the immune response being amplified compared to the immune response obtained after administration of particles, such as virus-like particles, which include a reference peptide tag, such as one of the reference peptide tags described herein, but are otherwise identical.

[0212] In some embodiments, the increased immune response is an increased IgM response and / or an increased IgG2 response such as an increased IgG2a and / or IgG2b. In some embodiments, the increased immune response is an increased IgG1 response. In some embodiments, the increased immune response is an increased IgG3 response. Preferably, at least one of the IgM, IgG2a, or IgG2b responses is increased.

[0213] In some embodiments, the reference peptide tag is a Spy tag (SEQ ID NO: 5) or an Sdy tag (SEQ ID NO: 7), and the improved binding properties relate to binding to the corresponding binding partner, namely, a SpyCatcher (SEQ ID NO: 1) or an SdyCatcher (SEQ ID NO: 3).

[0214] Therefore, particles obtained in this manner using peptide tags obtained or obtainable by the disclosed method may have improved properties, for example, in terms of peptide presentation density (i.e., how many peptide molecules are presented on the surface of the particle), uniformity of presentation (i.e., regular and uniform spacing of peptides on the surface of the particle), and immunogenicity when presenting antigenic peptides.

[0215] Peptide pairs Also provided herein are a modified binding partner and a peptide tag, or a method for producing a peptide pair comprising a modified binding partner and a peptide tag, wherein the modified binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained in the modified binding partner and another reactive residue contained in the peptide tag. The above method includes the following steps: i) the step of producing a modified bonding partner by any of the methods described herein; and / or ii) A step of producing a peptide tag by any of the methods described herein. In this way, the following peptide pairs, consisting of a binding partner and a peptide tag, which can interact via the spontaneous formation of isopeptide bonds, can be obtained: • Known binding partners and peptide tags designed by any of the above methods; • Modified binding partners that can be designed by any of the above methods, and known peptide tags; A modified binding partner that can be designed by any of the above methods, and a peptide tag that can be designed by any of the above methods. The binding partner and peptide tag may be any of the binding partners and peptide tags described herein.

[0216] Accordingly, the method of the present invention may be used to identify peptide pairs, which preferably have improved properties compared to known peptide pairs. In particular, the properties of peptide pairs obtained by this method may be improved compared to the properties of peptide pairs containing the same binding partner (when a known binding partner is included), or, in the case of pairs containing a modified binding partner, compared to the properties of peptide pairs containing the binding partner used in the starting point (first binding partner) and the same peptide tag.

[0217] Therefore, also provided herein are peptide pairs comprising or consisting of a peptide tag and a binding partner, wherein the peptide pair has one or more improved properties compared to a reference peptide pair comprising a reference peptide tag and a reference binding partner. The binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag. The reference binding partner can bind to the reference peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the reference binding partner and another reactive residue contained within the reference peptide tag. One or more improved characteristics are independently selected from one or more of the following: a) Increased binding efficacy of a binding partner to a peptide tag compared to the binding of a reference binding partner to a reference peptide tag, wherein the binding efficacy is increased when at least one of the total binding amount and the binding rate is increased; b) an increased ability to form particles that present the target peptide, such as virus-like particles that present the target peptide, compared to the ability to form particles of a reference peptide pair under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein fused to a binding partner, and the target peptide fused to a peptide tag, or the particles comprise a virus protein fused to a peptide tag and the target peptide fused to a binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the binding partner and the peptide tag; and c) Increased ability to present a target peptide on a particle, such as a virus-like particle, compared to the ability to present a target peptide on a reference peptide pair under similar conditions, wherein the particle comprises a particle-forming protein, such as a virus-like particle-forming protein, fused to a binding partner and the target peptide fused to a peptide tag, or the particle comprises a particle-forming protein fused to a peptide tag and the target peptide fused to a binding partner, and the particle is formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag.

[0218] In some embodiments, the bonding partner is the modified bonding partner described above. The bonding partner having the highest homology or identity with the modified bonding partner is preferably used as a reference to determine whether the modified bonding partner has improved properties, as described below herein. In some embodiments, the modified bonding partner has higher homology, identity or similarity with respect to the second bonding partner than to the first bonding partner, and the first bonding partner is used as a reference. In other embodiments, the modified bonding partner has higher homology with respect to the first bonding partner than to the second bonding partner, and the second bonding partner is used as a reference.

[0219] In some embodiments in which the modified binding partner comprises or consists of a first reactive fragment of the first binding partner and a second remaining fragment of the second binding partner as defined herein, the reference binding partner is preferably the second binding partner.

[0220] In some embodiments in which the modified binding partner comprises or consists of a second reactive fragment of the second binding partner as defined above herein and a first remaining fragment of the first binding partner, the reference binding partner is preferably the first binding partner.

[0221] The reference bond partner is preferably one of the first or second bond partners having the highest homology, similarity, or identity with the modified bond partner. Thus, in a preferred embodiment, the modified bond partner has higher homology, similarity, or identity with respect to the first bond partner than with respect to the second bond partner, and the reference bond partner is the first bond partner; or the modified bond partner has higher homology, similarity, or identity with respect to the second bond partner than with respect to the first bond partner, and the reference bond partner is the second bond partner.

[0222] In some embodiments, the peptide pair is selected from the following: a) Sequence ID 9 and Sequence ID 69 b) Sequence IDs 47 and 39; c) Sequence IDs 47 and 41; d) Sequence IDs 46 and 39; and e) Sequence IDs 46 and 41.

[0223] In some embodiments, the peptide pair is: a) A binding partner for SEQ ID NO: 39 (MoonCake) and SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), and SEQ ID NO: 12 (Bac5 tag); or a peptide tag selected from its variants having at least 70% homology or identity; b) A binding partner for SEQ ID NO: 41 (KatI) and a peptide tag selected from SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), and SEQ ID NO: 12 (Bac5 tag); or a peptide tag selected from its variants having at least 70% homology or identity; c) A binding partner for SEQ ID NO: 37 (QueenCatcher) and a peptide tag selected from SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 12 (Bac5 tag), and SEQ ID NO: 76 (Clib9); or a variant thereof having at least 70% homology or identity. including or consisting of A variant of the binding partner or peptide tag having at least 70% homology or identity with the binding partner or peptide tag is a functional variant that retains the ability to form an isopeptide bond with the corresponding peptide tag or binding partner, and has at least 70%, at least 71%, and so on, for example, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology or identity with the binding partner or peptide tag.

[0224] binding effect The peptide tags, modified binding partners, and binding peptide pairs described herein or obtained by the methods described herein preferably have one or more improved properties compared to the reference material.

[0225] One such objective characteristic, as discussed above, is binding efficacy, and the peptide tags and binding partners obtained by this method preferably have increased binding efficacy. The peptide pairs described herein also have increased binding efficacy, i.e., at least one of the total binding amount and binding rate of the peptide pair and its binding partner is increased compared to the reference peptide pair. The reference peptide pair preferably includes either the same peptide tag or the same binding partner. The reference peptide pair is therefore a pair that includes a binding partner (first or second binding partner) that has the highest homology with the modified binding partner. Thus, in a preferred embodiment, the modified binding partner has higher homology with the first binding partner than with the second binding partner, and the reference binding partner is the first binding partner; or the modified binding partner has higher homology with the second binding partner than with the first binding partner, and the reference binding partner is the second binding partner.

[0226] In some embodiments, the binding rate of the peptide pair is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the reference peptide pair.

[0227] In some embodiments, the total amount of the peptide pair bound is increased by at least 5%, for example, at least 10%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, or more, compared to the peptide pair.

[0228] In some embodiments, the binding rate of the peptide pair is increased by at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the reference peptide pair, and the total binding amount of the modified peptide partner is increased by at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the reference binding partner.

[0229] particle formation The peptide pairs described herein may also, instead of or in addition to, have an increased ability to form particles presenting the peptide of interest, such as virus-like particles presenting the peptide of interest, compared to the ability of a reference peptide pair to form particles under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein, fused or bound to a binding partner and the compound of interest fused to a peptide tag, or the particles comprise a virus-like particle-forming protein fused or bound to a peptide tag and the compound of interest fused to a binding partner, and the particles are formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag.

[0230] Such particles can be formed via particle-forming proteins known in the art, such as, for example, virus-like particle-forming proteins, for example, but not limited to, these. Particles that present peptides can be obtained by fusing one member of a peptide pair to such a protein and the other member of the peptide pair to a compound to be presented on the particle, such as a peptide. For example, a particle-forming protein is fused to a peptide tag, and the peptide to be presented is fused to its corresponding binding partner. The spontaneous formation of an isopeptide bond between the peptide tag and its binding partner allows the peptide to be presented on the surface of the particle. Using this technique, virus-like particles (VLPs) presenting antigenic peptides, such as peptides associated with abnormal physiological responses such as diseases, as described in detail in International Publication No. 2016 / 112921, have been generated. Capsid proteins are an example of a suitable particle-forming protein that can be used to generate such VLPs, as detailed herein. For example, AP205, Qβ, MS2, HBc, and phage fr, P22, cowpea mosaic virus (CPMV), bromosaic virus (BMV), cowpea chlorotic mottle virus (CCMV), bacteriophage lambda human adenovirus (AdV), and vault particles (PDB:4V60) can be used. Other suitable proteins are known in the art and can be used, for example, the proteins listed in Table 1 of Lieknina et al., 2019.

[0231] The method, however, can generally be applied to generate particles that are not virus-like particles. Other examples of such particle-forming proteins include small heat shock proteins (HSPs) (PDB:1SHS), apoferritin (PDB:1DAT), pyruvate dehydrogenase enzyme complex (PDB:1EAA), thermosomes (THS), and i301 (designed from 2-keto-3-deoxyphosphogluconate (KDPG) aldolase derived from the Entner-Doidorov pathway of the hyperthermophilic bacterium Thermotoga maritima).

[0232] This method allows for the genetic modification of proteins capable of self-assembling into nanoparticles through the fusion of peptide tags. The constructed nanoparticles (i.e., those presenting the reactive peptide tag) can then be linked to a genetically fused peptide that, upon contact with the peptide tag, can interact with the peptide tag via isopeptide bonds.

[0233] Any of the peptide tags, modified binding partners, and peptide pairs described herein can be advantageously used to generate self-assembling nanoparticles.

[0234] In some embodiments, when the peptide pairs obtained by this method are used to present a target compound, such as an antigen, in, for example, virus-like particles or particles as described below, they can produce an increased immune response when administered to a target in need, compared to the immune response obtained from particles using a reference peptide pair, and compared to the immune response obtained using, for example, SpyCatcher / Spy tags (SEQ ID NO: 1 and SEQ ID NO: 5, respectively).

[0235] In some embodiments, the peptide pair consists of a modified binding partner as described herein, for example, the binding partner of SEQ ID NO: 39 (MoonCake), the binding partner of SEQ ID NO: 41 (KatI), or the binding partner of SEQ ID NO: 37 (QueenCatcher), and a preferred peptide tag as described herein. In such embodiments, the reference peptide pair may consist of the same peptide tag and a reference binding partner as described herein, for example, SpyCatcher (SEQ ID NO: 1). In some embodiments, the reference peptide pair may consist of SpyCatcher / Spy tag (SEQ ID NO: 1 and SEQ ID NO: 5, respectively).

[0236] This specification also discloses methods for inducing an immune response in subjects requiring it, the methods comprising administering to subjects particles, such as virus-like particles, containing the peptide pairs described herein or obtained by the methods described herein, preferably the immune response being amplified compared to the immune response obtained after administration of particles, such as virus-like particles, which contain a reference peptide pair but are otherwise identical, such as one of the reference peptide pairs described herein.

[0237] In some embodiments, the enhanced immune response is an enhanced IgM response and / or an enhanced IgG2 response such as enhanced IgG2a and / or IgG2b. In some embodiments, the enhanced immune response is an enhanced IgG1 response. In some embodiments, the enhanced immune response is an enhanced IgG3 response. Preferably, at least one of the IgM, IgG2a, or IgG2b responses is enhanced.

[0238] Presentation of compounds on particles The peptide pairs described herein may also have an increased ability to present a compound of interest, such as a peptide, on particles, such as virus-like particles, compared to the ability of a reference peptide pair to present the peptide of interest under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein, fused or bound to a binding partner and the compound of interest fused or bound to a peptide tag, or the particles comprise a particle-forming protein fused or bound to a peptide tag and the compound of interest fused or bound to a binding partner, and the particles are formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag.

[0239] Any of the peptide tags, modified binding partners, and peptide pairs described herein can be advantageously used to generate self-assembling nanoparticles capable of presenting the compound of interest.

[0240] Examples of particle-forming proteins include capsid proteins such as viral capsid proteins. For example, AP205, Qβ, MS2, HBc, and phage fr, P22, cowpea mosaic virus (CPMV), bromosaic virus (BMV), cowpea chlorotic mottle virus (CCMV), bacteriophage lambda human adenovirus (AdV), and vault particles (PDB:4V60) can be used. Other suitable proteins are known in the art and can be used, for example, the proteins listed in Table 1 of Lieknina et al., 2019.

[0241] Examples of particle-forming proteins other than viral capsid proteins include small heat shock proteins (HSPs) (PDB:1SHS), apoferritin (PDB:1DAT), pyruvate dehydrogenase enzyme complex (PDB:1EAA), thermosomes (THS), and i301 (designed from 2-keto-3-deoxyphosphogluconate (KDPG) aldolase derived from the Entner-Doidorov pathway of the hyperthermophilic bacterium Thermotoga maritima).

[0242] Examples of the compound of interest presented on the surface of the particle (which may be the inner or outer surface) include the peptide of interest, such as an antigenic peptide. In some embodiments, the antigenic peptide can induce an immune response in animals such as cows, pigs, horses, sheep, goats, llamas, mice, rats, and monkeys, most preferably humans, or birds such as chickens, or fish such as salmon.

[0243] In some embodiments, the compound of interest is an antigen associated with a disease or disorder, such as abnormal physiological reactions, e.g., cancers such as breast cancer, gastric cancer, ovarian cancer, and serous adenocarcinoma of the uterine body; cardiovascular diseases such as dyslipidemia, atherosclerosis, and / or hypercholesterolemia; immunoinflammatory or chronic diseases such as eosinophilic asthma, allergies, nasal polyps, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome, and Churg-Strauss syndrome; neurological diseases such as Alzheimer's disease; infectious diseases such as infectious diseases selected from the group consisting of diseases caused by viruses such as coronaviruses, e.g., SARS-CoV-2, malaria, tuberculosis, HIV, and influenza; dyslipidemia such as hyperlipidemia, type I, II, III, IV, or V hyperlipidemia, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatous disease, cholesterol acetyltransferase deficiency; arteriosclerotic conditions such as atherosclerosis; or coronary artery disease.

[0244] In some embodiments, the antigen is a protein, peptide, and / or antigenic fragment derived from a group consisting of cancer-specific polypeptides, cardiovascular disease-related polypeptides, asthma-related polypeptides, nasal polyp-related polypeptides, atopic dermatitis-related polypeptides, eosinophilic esophagitis-related polypeptides, hypereosinophilic syndrome-related polypeptides, Churg-Strauss syndrome-related polypeptides, and / or polypeptides related to pathogenic organisms.

[0245] This composition may therefore be a vaccine composition having prophylactic use. This composition may therefore be useful for the prevention or treatment of disease or disorder. This composition may also be useful for inducing an immune response in a subject by administering the composition to the subject at least once.

[0246] Polynucleotides Also provided herein are polynucleotides encoding modified binding partners obtained by the methods disclosed herein. Also provided herein are polynucleotides encoding peptide tags obtained by the methods disclosed herein. As used herein, the term polynucleotide refers to a nucleic acid molecule, preferably a DNA molecule, which encodes a given polypeptide, peptide or protein.

[0247] In some embodiments, the polynucleotide encodes a modified binding partner described herein.

[0248] In some embodiments, the polynucleotide encodes a modified binding partner represented by SEQ ID NO: 37, and said polynucleotide comprises, or consists of, a homolog thereof having at least 60% identity, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 38. Preferably, the codon encoding the residue corresponding to residue 31 of SEQ ID NO: 37 is not modified, i.e., the codons at positions 91-93 encode lysine.

[0249] In some embodiments, the polynucleotide encodes a modified binding partner shown in SEQ ID NO: 39, and the polynucleotide includes or comprises SEQ ID NO: 38 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto. Preferably, the codon encoding the residue corresponding to residue 31 of SEQ ID NO: 38 is not modified, i.e., the codons at positions 91-93 encode lysine.

[0250] In some embodiments, the polynucleotide encodes a modified binding partner represented by SEQ ID NO: 41, and the polynucleotide includes or comprises SEQ ID NO: 42 or its homologues having at least 60% identity, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 31 of SEQ ID NO: 42 is not modified, i.e., the codons at positions 91-93 encode lysine.

[0251] In some embodiments, the polynucleotide encodes a modified binding partner shown in SEQ ID NO: 29, or a homolog thereof having at least 60% identity, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 8 in SEQ ID NO: 29 is not modified, i.e., the codons at positions 24-26 encode lysine.

[0252] In some embodiments, the polynucleotide encodes a modified binding partner shown in SEQ ID NO: 9, and the polynucleotide includes or comprises SEQ ID NO: 10 or its homologues having at least 60% identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 31 of SEQ ID NO: 10 is not modified, i.e., the codons at positions 352-354 encode asparagine.

[0253] In some embodiments, the polynucleotide encodes a modified binding partner represented by SEQ ID NO: 31, and the polynucleotide includes or comprises SEQ ID NO: 32 or its homologues having at least 60% identity, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 31 of SEQ ID NO: 32 is not modified, i.e., the codons at positions 91-93 encode aspartic acid.

[0254] In some embodiments, the polynucleotide encodes the peptide tag shown in SEQ ID NO: 46, and the polynucleotide includes or comprises SEQ ID NO: 45 or its homologues having at least 60% identity, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 12 of SEQ ID NO: 46 is not modified, i.e., the codons at positions 37-39 encode aspartic acid.

[0255] In some embodiments, the polynucleotide encodes the peptide tag shown in SEQ ID NO: 48, and the polynucleotide includes or comprises SEQ ID NO: 47 or its homologues having at least 60% identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 9 of SEQ ID NO: 48 is not modified, i.e., the codons at positions 25-27 encode asparagine.

[0256] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 50, and the polynucleotide includes or comprises SEQ ID NO: 49 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0257] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 52, and the polynucleotide includes or comprises SEQ ID NO: 51 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0258] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 54, and the polynucleotide includes or comprises SEQ ID NO: 53 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0259] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 56, and the polynucleotide includes or comprises SEQ ID NO: 55 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0260] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 58, and the polynucleotide includes or comprises SEQ ID NO: 57 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0261] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 60, and the polynucleotide includes or comprises SEQ ID NO: 59 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0262] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 62, and the polynucleotide includes or comprises SEQ ID NO: 61 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0263] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 64, and the polynucleotide includes or comprises SEQ ID NO: 63 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0264] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 66, and the polynucleotide includes or comprises SEQ ID NO: 65 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0265] In some embodiments, the polynucleotide encodes the peptide tag shown in SEQ ID NO: 68, and the polynucleotide includes or comprises SEQ ID NO: 67 or its homologues having at least 60% identity, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 13 of SEQ ID NO: 68 is not modified, i.e., the codons at positions 37-39 encode aspartic acid.

[0266] In some embodiments, the polynucleotide encodes the peptide tag shown in SEQ ID NO: 35, and the polynucleotide includes or comprises SEQ ID NO: 36 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0267] In some embodiments, the polynucleotide encodes the peptide tag shown in SEQ ID NO: 22, and the polynucleotide includes or comprises SEQ ID NO: 21 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0268] In some embodiments, the polynucleotide encodes a peptide tag represented by SEQ ID NO: 12, and the polynucleotide includes or comprises SEQ ID NO: 11 or its homologues having at least 60% identity thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity thereto.

[0269] In some embodiments, the polynucleotide encodes the peptide tag set forth in SEQ ID NO: 71, and the polynucleotide comprises, or consists of, SEQ ID NO: 72 or a homolog thereof having at least 60% identity thereto, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. Preferably, the codon encoding the residue corresponding to residue 9 of SEQ ID NO: 72 is not modified, i.e., the codons at positions 25-27 of SEQ ID NO: 72 encode asparagine.

[0270] In some embodiments, the polynucleotide encodes the peptide tag set forth in SEQ ID NO: 69, and the polynucleotide comprises, or consists of, SEQ ID NO: 70 or a homolog thereof having at least 60% identity thereto, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.

[0271] In some embodiments, the polynucleotide encodes the peptide tag shown in SEQ ID NO: 75, and the polynucleotide includes or comprises SEQ ID NO: 77 or its homologues having at least 60% identity, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% identity. Preferably, the codon encoding the residue corresponding to residue 8 of SEQ ID NO: 75 is not modified, i.e., the codons at positions 22-24 of SEQ ID NO: 77 encode asparagine.

[0272] The polynucleotides mentioned above may be provided within a vector, i.e., within a DNA molecule used as a carrier to artificially transport exogenous genetic material into a cell, where they can be replicated and / or expressed. There are four types of vectors: plasmids, viral vectors, cosmids, and artificial chromosomes. The vector itself is usually a DNA sequence consisting of an insertion (transgene) and a larger sequence that functions as the vector's "skeleton." The purpose of a vector to transfer genetic information to another cell is usually to isolate, grow, or express the insertion fragment in the target cell. Expression vectors (expression constructs) are specifically for the expression of a transgene in a target cell and usually contain a promoter sequence that promotes the expression of the transgene.

[0273] In some embodiments where the polynucleotide is a homolog of SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 36, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 72, or SEQ ID NO: 11, the codon of the polynucleotide encoding the amino acid corresponding to one of the reactive residues involved in the formation of the isopeptide bond is preferably immutable, or the sequence of the polynucleotide is such that the encoded polypeptide still contains the reactive residue.

[0274] Vectors, such as plasmids, containing the polynucleotides described herein are also provided. The vectors may contain several polynucleotides, as is known in the art. Polynucleotide systems are therefore provided herein, such as a system comprising two polynucleotides: a first polynucleotide encoding a binding partner described herein, in particular a modified binding partner described herein, and a second polynucleotide encoding a peptide tag described herein, in particular a peptide tag bound to the modified binding partner. In some embodiments, the system comprises two vectors: a first vector comprising a first polynucleotide encoding a binding partner described herein, in particular a modified binding partner described herein, and a second vector comprising a second polynucleotide encoding a peptide tag described herein, in particular a peptide tag bound to the modified binding partner.

[0275] In some embodiments, the binding pair (i.e., a binding pair consisting of a binding partner and a peptide tag, where the binding partner may be a known binding partner or a modified binding partner as described herein, and the peptide tag may be a known peptide tag or a peptide tag as described herein) is used to link two compounds, in particular two polypeptides. For example, the binding partner is fused to a first polypeptide and the peptide tag is fused to a second polypeptide—those skilled in the art will know how such a fusion is obtained. For the formation of an isopeptide bond between the binding partner and the peptide tag, the two compounds (in our example, the first and second polypeptides) are positioned close to each other. As detailed above, this can be used, for example, to generate a VLP that presents an antigenic peptide by fusioning a capsid protein capable of forming a VLP to one of the binding partners and peptide tags, and by fusioning an antigenic peptide to the other binding partner and peptide tag.

[0276] The fusion can be realized by designing a polynucleotide encoding a first compound fused to a binding partner and another polynucleotide encoding a second compound fused to a peptide tag. Alternatively, the fusion can be realized by designing a polynucleotide encoding a first compound fused to a peptide tag and another polynucleotide encoding a second compound fused to a binding partner. Spacers or linkers, particularly glycine-serine spacers or linkers, may be introduced between the first compound and the binding partner or peptide tag, and / or between the second compound and the binding partner or peptide tag.

[0277] The compound of interest may be fused to a peptide tag or binding partner via an N-terminal fusion, a C-terminal fusion, or, for example, an internal fusion in a loop.

[0278] host cell Also provided herein are host cells expressing the modified binding partners and / or peptide tags disclosed herein. This can be carried out as is known in the art. The host cells contain one or more polynucleotides described herein that encode the modified binding partners or peptide tags. The polynucleotides may be incorporated into the genome of the host cell, or they may be provided as part of one or more vectors that are introduced into the cell by methods known in the art.

[0279] Polynucleotides can be codon-optimized to improve their expression in host cells, as is known in the art.

[0280] The host cell may be a bacterial cell, yeast cell, fungal cell, plant cell, animal cell, mammalian cell, or insect cell.

[0281] In some embodiments, the host cell may belong to one of the following: Escherichia coli, armyworm (sf9), Trichoplasia ni (BTI-TN-5B1-4), Salmonella tiphyllum (salmonella typhimurium), Pichia pastris (methanol-assimilating yeast), Saccharomyces cerevisiae (budding yeast), Schizosaccharomyces pombe (fission yeast), Hanzenula polymorpha (methylotropic yeast), Drosophila schneider 2 (S2); Lactobacillus lactis, African clawed moth L, Chinese hamster ovary (CHO), COS-1, HepG2, HeLA, BHK, human fetal kidney 293, tobacco cultivar Samson tobacco, and potato cultivar Solara, cells of the genus Nicotiana, cells of the genus Solanum (eggplant), cells of the genus Lupinus (legume), lettuce, tomato cells such as Solanum lycopersicum (tomato), Glycyne max (soybean), CPMV cells, PVX cells or Magnicon cells, or Pseudomonas systems. The cells may be transgenic cells.

[0282] composition Furthermore, the following compositions are provided herein: i) A protein fused to a modified binding partner disclosed herein, and a compound of interest such as a peptide, e.g., an antigen, fused to a peptide tag disclosed herein; or ii) A compound of interest, such as a peptide, fused to a peptide tag as disclosed herein, and to a modified binding partner as disclosed herein, e.g., an antigen. Includes, The modified binding partners and peptide tags can interact through the spontaneous formation of isopeptide bonds. The target compound and protein are linked via an isopeptide bond between the modified binding partner and the peptide tag.

[0283] Some specific compositions: a) A particle-forming protein fused to a modified binding partner as described herein, and a compound of interest, such as a peptide, fused to a peptide tag as described herein, e.g., an antigen; or b) A particle-forming protein fused to a peptide tag described herein, and a compound of interest such as a peptide, e.g., an antigen, fused to a modified binding partner described herein. Includes, The modified binding partners and peptide tags can interact through the spontaneous formation of isopeptide bonds. The target compound and particle-forming protein are linked via an isopeptide bond between the modified binding partner and the peptide tag, and The particle-forming protein and the target compound form particles that present the target compound.

[0284] Examples of particle-forming proteins include capsid proteins such as viral capsid proteins. For example, AP205, Qβ, MS2, HBc, and phage fr, P22, cowpea mosaic virus (CPMV), bromosaic virus (BMV), cowpea chlorotic mottle virus (CCMV), bacteriophage lambda human adenovirus (AdV), and vault particles (PDB:4V60) can be used. Other suitable proteins are known in the art and can be used, for example, the proteins listed in Table 1 of Lieknina et al., 2019.

[0285] Examples of particle-forming proteins other than viral capsid proteins include small heat shock proteins (HSPs) (PDB:1SHS), apoferritin (PDB:1DAT), pyruvate dehydrogenase enzyme complex (PDB:1EAA), thermosomes (THS), and i301 (designed from 2-keto-3-deoxyphosphogluconate (KDPG) aldolase derived from the Entner-Doidorov pathway of the hyperthermophilic bacterium Thermotoga maritima).

[0286] Examples of the compound of interest presented on the surface of the particle (which may be the inner or outer surface) include the peptide of interest, such as an antigenic peptide. In some embodiments, the antigenic peptide can induce an immune response in animals such as cows, pigs, horses, sheep, goats, llamas, mice, rats, and monkeys, most preferably humans, or birds such as chickens, or fish such as salmon.

[0287] In some embodiments, the compound of interest is an antigen associated with a disease or disorder, such as abnormal physiological reactions, e.g., cancers such as breast cancer, gastric cancer, ovarian cancer, and serous adenocarcinoma of the uterine body; cardiovascular diseases such as dyslipidemia, atherosclerosis, and / or hypercholesterolemia; immunoinflammatory or chronic diseases such as eosinophilic asthma, allergies, nasal polyps, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome, and Churg-Strauss syndrome; neurological diseases such as Alzheimer's disease; infectious diseases such as infectious diseases selected from the group consisting of diseases caused by viruses such as coronaviruses, e.g., SARS-CoV-2, malaria, tuberculosis, HIV, and influenza; dyslipidemia such as hyperlipidemia, type I, II, III, IV, or V hyperlipidemia, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatous disease, cholesterol acetyltransferase deficiency; arteriosclerotic conditions such as atherosclerosis; or coronary artery disease.

[0288] In some embodiments, the antigen is a protein, peptide, and / or antigenic fragment derived from a group consisting of cancer-specific polypeptides, cardiovascular disease-related polypeptides, asthma-related polypeptides, nasal polyp-related polypeptides, atopic dermatitis-related polypeptides, eosinophilic esophagitis-related polypeptides, hypereosinophilic syndrome-related polypeptides, Churg-Strauss syndrome-related polypeptides, and / or polypeptides related to pathogenic organisms.

[0289] This composition may therefore be a vaccine composition having prophylactic use. This composition may therefore be useful for the prevention or treatment of disease or disorder. This composition may also be useful for inducing an immune response in a subject by administering the composition to the subject at least once.

[0290] Furthermore, provided herein are methods for producing the pharmaceutical compositions described herein, the methods comprising the following steps: i) obtaining a first polypeptide comprising or comprising a modified binding partner as defined herein fused to a protein, and a second polypeptide comprising or comprising a peptide tag as defined herein fused to a compound of interest; or The steps of obtaining a first polypeptide comprising or consisting of a peptide tag described herein fused to a protein, and a second polypeptide comprising or consisting of a modified binding partner described herein fused to a compound of interest; ii) The step of contacting a first polypeptide with a second polypeptide, thereby enabling the formation of an isopeptide bond between the peptide tag and the modified binding partner, thereby producing the pharmaceutical composition described herein.

[0291] In a particular embodiment, the method for producing the composition is: i) obtaining a first polypeptide comprising or comprising a modified binding partner as described herein fused to a particle-forming protein, and a second polypeptide comprising or comprising a peptide tag as described herein fused to a compound of interest, such as a peptide; or The steps of obtaining a first polypeptide comprising or consisting of a peptide tag described herein fused to a particle-forming protein, and a second polypeptide comprising or consisting of a modified binding partner described herein fused to a compound of interest; ii) A step of providing the first polypeptide under conditions that enable particle formation; iii) The step of obtaining particles by forming an isopeptide bond between the particle-forming protein of the second polypeptide and the particle-forming protein of the first polypeptide; and iv) A step of producing a pharmaceutical composition containing the above particles, wherein the pharmaceutical composition is as described herein, Includes, This is used to obtain a pharmaceutical composition. The particle-forming protein may be any of the particle-forming proteins listed above in this specification, for example, a capsid protein.

[0292] Examples Example 1: The modified binding partner shown in SEQ ID NO: 37 was obtained starting from SEQ ID NO: 3. The reactive residue of SEQ ID NO: 3 is located at position 31 in SEQ ID NO: 3 and is retained in the modified binding partner which contains or consists of SEQ ID NO: 37 or its homolog having at least 70% identity or homology thereto. The first reactive fragment derived from SEQ ID NO: 3 extends from positions 1 to 93 in SEQ ID NO: 3. The modified binding partner of SEQ ID NO: 37 further includes a fragment (the remaining fragment) extending from positions 97 to 116 in SEQ ID NO: 1. The reactive residue of SEQ ID NO: 37 is located at position 31.

[0293] Both the modified binding partners shown in SEQ ID NO: 39 and SEQ ID NO: 41 were obtained starting from SEQ ID NO: 37. The modified binding partners shown in SEQ ID NO: 39 and SEQ ID NO: 41 were designed by computer structural modeling and rational design, introducing mutations into SEQ ID NO: 37. The reactive residues in SEQ ID NO: 39 and SEQ ID NO: 41 are located at position 31 in these sequences.

[0294] Example 2: Improved properties of VLPs obtained by isopeptide bond formation between soluble catcher and tag-VLP fusion. To measure the kinetics of coupling (EC50 coupling over time), soluble catchers and VLP tags (fusion of VLP-forming protein and tag) were diluted to 10 μM in PBS, mixed in a 1:1 ratio (final 5 μM), incubated at 37°C for 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 1 hour, 1 hour 30 minutes, or 3 hours, and electrophoresed on SDS gel. Reconstitution percentage was also measured; this was the endpoint binding after 3 hours and was determined here as the average of two experiments. The results are shown in Figure 1 and Table 1. [Table 1]

[0295] As can be seen from the figures and tables, the dynamics of the Mooncake+VLP-RumTrunkD9N tag are improved compared to the SdyCatcher+VLP-Sdy tag, while the dynamics of the KATI+VLPRumTrunkD9N tag are virtually unchanged compared to the SpyCatcher+VLP-Spy tag. The reconstruction percentage is improved compared to the SdyCatcher+VLP-Sdy tag.

[0296] To measure the maximum binding of the above VLP, the soluble catcher and VLP tag were diluted to 20 μM and 10 μM respectively in PBS, mixed in a 1:1 ratio (final 10 μM and 5 μM), incubated at 37°C for 24 hours, and electrophoresed on an SDS gel. To measure the VLP yield, the soluble catcher was expressed in BL21 cells and grown in ampicillin-containing 2*YT medium. Pre-culture was performed overnight, and the cells were transferred to 400 mL of ampicillin-containing 2*YT medium, where expression of our protein was induced with 0.1 μM IPTG. After induction, the culture was centrifuged overnight, the pellet was weighed (mg protein / gr pellet), the protein was purified using IMAC, and the protein concentration (mg / L) was measured. The results are shown in Table 2. [Table 2] The maximum number of tags is increased compared to SdyCatcher+VLP-Sdy tags. In summary, these data demonstrate that the VLP of the present invention has improved characteristics, particularly compared to the SdyCatcher / Sdy tag VLP.

[0297] Example 3: Improved properties of soluble catcher and soluble tag pair The experiment was carried out in the same manner as in Example 1 unless otherwise specified. Soluble tags were used instead of VLP-tag fusions.

[0298] To measure kinetics, the soluble catcher and soluble tag were diluted to 10 μM in PBS, mixed in a 1:1 ratio (final 5 μM), incubated at 37°C for 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 1 hour, 1 hour 30 minutes, or 3 hours, and electrophoresed on an SDS gel. To measure binding, the soluble catcher and soluble tag were diluted to 10 μM in PBS, mixed in a 1:1 ratio (final 5 μM), incubated at 37°C for 50 minutes, and electrophoresed on an SDS gel. To measure maximum binding, the soluble catcher and soluble tag were diluted to 20 μM and 10 μM respectively in PBS, mixed in a 1:1 ratio (final 10 μM and 5 μM), incubated at 37°C for 24 hours, and electrophoresed on an SDS gel.

[0299] The dynamics, coupling percentages (reconstruction after 50 minutes), and maximum coupling of the SdyC+Sdy tag, Mooncake+MBP-RumTrunkD9N tag, and KATI+MBP-RumTrunkD9N tag are shown in Figures 2A and 2B and Table 3. [Table 3]

[0300] Example 4: Improved VLP presentation To measure kinetics, the VLP catcher and soluble tag were diluted to 10 μM in PBS, mixed in a 1:1 ratio (final 5 μM), incubated at 37°C for 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 1 hour, 1 hour 30 minutes, or 3 hours, and electrophoresed on an SDS gel. To measure reconstitution (binding to RumtrunkD9N tag-MBP at 50 minutes), the soluble catcher and VLP tag were diluted to 10 μM in PBS, mixed in a 1:1 ratio (final 5 μM), incubated at 37°C for 3 hours, and electrophoresed on an SDS gel. To measure maximum binding, the VLP catcher and soluble tag were diluted to 20 μM and 10 μM, respectively, in PBS, mixed in a 1:1 ratio (final 10 μM and 5 μM, respectively), incubated at 37°C for 24 hours, and electrophoresed on an SDS gel. To measure VLP yield, VLP catcher fusions were expressed in BL21 cells and grown in ampicillin-containing 2*YT medium. Pre-culture was performed overnight, and the cells were transferred to 400 mL of ampicillin-containing 2*YT medium. Expression of our protein was induced with 0.1 μM IPTG. After induction, the culture was centrifuged overnight, the pellet was weighed (mg protein / g pellet), and the protein was purified using an OptiPrep gradient and centrifuged again. VLP concentration (mg / L) was measured by BCA assay. The results are shown in Tables 4 and 5. [Table 4] [Table 5]

[0301] Example 5: Increased immune response A group of mice (n=6) were immunized with the same dose (6mcg) of MoonCake-HER2 VLP (LCG), SpyCatcher-HER2 VLP (SPY), or PBS (n=5 in the PBS group) using prime booster. Serum was collected 2 weeks after each immunization, and the levels of antigen-specific IgM and IgG (subclasses 1, 2a, 2b, and 3) were measured by ELISA. Immunization with MoonCake-HER2 VLP induced significantly higher levels of antigen-specific total Ig compared to SpyCatcher-HER2 VLP (Figure 4). Immunization with MoonCake-HER2 VLP induced significantly higher levels of IgM, as well as IgG2a and IgG2b, compared to SpyCatcher-HER2 VLP (Figure 5). The two vaccines exhibited comparable coupling efficiencies (i.e., number of HER2 presentations / VLPs) and monodispersity, respectively, as measured by SDS-PAGE densitometry and dynamic light scattering analysis.

[0302] [Table 6] TIFF2026048917000008.tif242160TIFF2026048917000009.tif242162TIFF20260489170 00010.tif242161TIFF2026048917000011.tif241160TIFF2026048917000012.tif238162

[0303] References Lieknina I, Kalnins G, Akopjana I, Bogans J, Sisovs M, Jansons J, Rumnieks J, Tars K (2019) Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.J Nanobiotechnology 17(1):61.doi:10.1186 / s12951-019-0497-8. Kang et al.Stabilizing isopeptide bonds revealed in gram-positive bacterial pilus structure Science.2007 Dec 7;318(5856):1625-8. Schwarz-Linek et al.Yet more intramolecular cross-links in Gram-positive surface proteins.PNAS January 28,2014 111(4)1229-1230;https: / / doi.org / 10.1073 / pnas.1322482111

[0304] item 1. A method for producing a modified binding partner that can bind to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag, The above method includes the following steps: i) A step of selecting at least a first peptide pair consisting of a first peptide tag and a first binding partner, and a second peptide pair consisting of a second peptide tag and a second binding partner, wherein for each peptide pair, the peptide tag and the binding partner can or are considered to be able to bind to each other by spontaneously forming an isopeptide bond; ii) A step of identifying the position of the isopeptide bond to a first peptide pair and / or a second peptide pair, thereby identifying a first reactive fragment of the first binding partner and / or a second reactive fragment of the second binding partner, and the remaining fragment of the first binding partner and / or the remaining fragment of the second binding partner, wherein the first and / or second reactive fragments include reactive residues involved in the isopeptide bond; iii) A step of designing a modified binding partner, wherein the modified binding partner comprises or consists of i) a first reactive fragment, or a homolog thereof having at least 70% homology thereto, and a second remaining fragment, or a homolog thereof having at least 70% homology thereto, wherein the first reactive fragment is preferably upstream of the second remaining fragment, and the modified binding partner does not contain both reactive residues involved in the formation of the isopeptide bond; iv) A step of producing a modified binding partner. 2. The method described in item 1, further including the following steps: v) A step of measuring one or more binding properties of a modified binding partner, wherein the one or more properties are preferably selected from i) total binding amount and ii) binding rate to one or more of the first peptide tag, second peptide tag or third peptide tag of the modified binding partner; vi) A step of measuring the corresponding binding properties of one or more of the first binding partners and / or second binding partners to one or more of the first peptide tag, the second peptide tag, or the third peptide tag; vii) A step of comparing the binding properties measured in steps e) and f), wherein the increased binding efficacy of the modified partner to one or more of the first peptide tag, the second peptide tag, or the third peptide tag compared to the binding efficacy of the first and / or second binding partner to the same tag is an indicator of the modified binding partner having improved properties compared to the first and / or second binding partner, and the binding efficacy is increased if at least one of the total binding amount and binding rate is increased compared to the binding efficacy of the first and / or second binding partner, preferably compared to the binding efficacy of the first binding partner. 3. The method according to item 1 or 2, wherein an increase in the binding rate of the modified binding partner to at least one of the first, second, and third peptide tags compared to the binding rate of at least one of the first and / or second binding partners to the same peptide tag is an indicator of a modified binding partner having increased binding efficacy compared to the first and / or second binding partner, preferably compared to the binding efficacy of the first binding partner, and preferably such increase is at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more. 4. The method according to any one of items 1 to 3, wherein the increase in the total binding amount of the modified binding partner to at least one of the first, second, and third peptide tags compared to the total binding amount of at least one of the first and / or second binding partners to the same peptide tag is an indicator of a modified binding partner having increased binding efficacy compared to the first and / or second binding partner, preferably compared to the binding efficacy of the first binding partner, and preferably the increase is at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more. 5. The method according to any one of items 1 to 4, wherein the modified binding partner has a length of 5 amino acids or more, e.g., 10 amino acids or more, e.g., 15 amino acids or more, e.g., 20 amino acids or more, e.g., 25 amino acids, e.g., 30 amino acids, e.g., 35 amino acids, e.g., 40 amino acids, e.g., 45 amino acids, e.g., 50 amino acids, e.g., 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 amino acids or more. 6. The method according to any one of items 1 to 5, wherein the first and / or second binding partner is independently selected from SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 71, and SEQ ID NO: 33, and its homologues having at least 60% homology to it, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology to it. 7. The first peptide tag, the second peptide tag, and / or the third peptide tag are sequence numbers 46, 5 (Spy tag), 7 (Sdy tag), 69 (Snoop tag), 46 (Rum tag), 47 (RumTrunkD9N tag), 50 (Pho tag), 52 (Ent tag), 54 (Rum7 tag), 56 (Rum3 tag), 58 (Rum2 tag), 60 (Rum4 tag), 62 (Rum5 tag), 64 (Rum6 tag), 66 (Bac tag), 68 (Bac2 tag), 35 (Bac3 tag) The method according to any one of items 1 to 6, independently selected from the group consisting of homologs having at least 60% homology with sequence number 22 (Bac4 tag), sequence number 31 and sequence number 12 (Bac5 tag) or at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%. 8. The method according to any one of items 1 to 7, wherein the reactive residue in the first, second, or third peptide tag is an aspartic acid or asparagine residue, and / or the reactive residue in the first, second, and / or modified binding partner is a lysine or asparagine residue. 9. Modified binding partners that can be obtained by the method described in any one of items 1 to 8. 10. A modified binding partner capable of binding to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag, wherein the modified binding partner does not contain both reactive residues involved in the formation of the isopeptide bond, and the modified binding partner comprises or consists of a first reactive fragment of the first binding partner containing one reactive residue capable of interacting with a first peptide tag containing another reactive residue via the formation of an isopeptide bond between reactive residues, or a homolog thereof having at least 70% homology thereto, and a second remaining fragment of the second binding partner, wherein the second binding partner can interact with a second peptide tag containing another reactive residue via the formation of an isopeptide bond between reactive residues, and the second remaining fragment does not contain a reactive residue or a homolog thereof having at least 70% homology thereto, preferably the first reactive fragment is upstream of the second remaining fragment. 11. Modified binding partners described in item 9 or 10, which can be obtained by the method described in any one of items 1 through 8. 12. A modified binding partner according to any one of items 9 to 11, wherein the first and second binding partners are independently selected from SEQ ID NO: 1 (SpyCatcher), SEQ ID NO: 3 (SdyCatcher), SEQ ID NO: 9 (SnoopCatcher), SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, and homologs thereof having at least 60% homology, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology. 13. A modified binding partner as described in any one of items 9 to 12, comprising or comprising homologs thereof having at least 60% homology, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology. 14. A modified binding partner according to any one of items 9 to 13, having increased binding efficacy to the same reference peptide tag compared to the binding efficacy of any one or more reference peptide tags of the reference binding partner, wherein the reference peptide tag is a first peptide tag, and the second or third peptide tag is as defined in any one of items 1 to 8, and the binding efficacy is increased when at least one of the total binding amount and binding rate is increased compared to the binding efficacy of the reference binding partner, preferably compared to the binding efficacy of the first binding partner. 15. A modified binding partner according to any one of items 9 to 14, wherein the binding rate of the modified peptide partner is increased by at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the reference binding partner. 16. A modified binding partner according to any one of items 9 to 15, wherein the total binding amount of the modified peptide partner is increased by at least 5%, e.g., at least 10%, e.g., at least 15%, e.g., at least 20%, e.g., at least 25%, e.g., at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., at least 100%, or more, compared to the reference binding partner. 17. The first peptide tag, the second peptide tag, and / or the third peptide tag are at least 60% equivalent to SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 69 (Snoop tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 50 (Pho tag), SEQ ID NO: 52 (Ent tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 46, SEQ ID NO: 31 and SEQ ID NO: 12 (Bac5 tag) or with at least 60% equivalent. A modified binding partner according to any one of items 9 to 15, independently selected from the group consisting of homologs having the same sex, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, and preferably the first peptide tag, the second peptide tag, and / or the third peptide tag are independently selected from the group consisting of SEQ ID NO: 46 (Rum tag), SEQ ID NO: 5 (Spy tag), and SEQ ID NO: 7 (Sdy tag). 18. A method for producing a peptide tag that can bind to a binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained in the peptide tag and another reactive residue contained in the binding partner, wherein the binding partner is a modified binding partner as described in any one of items 9 to 14. a) A step of identifying candidate peptide tags having at least 60% similarity to a reference peptide tag, wherein the reference peptide tag can spontaneously form an isopeptide bond with at least one reference binding partner, and preferably the reference peptide tag includes a reference binding motif; b) A step of selecting a peptide tag from the candidate peptide tags identified in a), wherein the selected peptide tag comprises at least one reactive residue likely involved in the formation of an isopeptide bond; c) A step of designing and producing peptide tags from selected peptide tags, wherein each peptide tag comprises or consists of a fragment of the selected peptide tag, ranging from 4 to 24 amino acids upstream of a reactive residue likely involved in the formation of an isopeptide bond to 2 to 22 amino acids downstream, or a homolog thereof having at least 70% homology, provided that the homolog contains a reactive residue. A method that includes this. 19. The method according to item 18, further comprising step d) of mutating a reactive residue, preferably aspartic acid which is mutated to asparagine, and step d) is carried out after step c). 20. The method according to item 18 or 19, wherein the candidate peptide tag contains a sequence motif within 50 amino acids from its C-terminus, for example, within 45, 40, 35, 30, or 25 amino acids from its C-terminus. 21. The method according to item 20, wherein the sequence motif includes or consists of sequence number 73 (GX1X2X3IVMX4DX5), sequence number 74 (GX1X2X3YVMX4DX5), sequence number 43 (GX1X2X3FVMX4DX5), or sequence number 74 (GX1X2X3WVMX4DX5). 22. A peptide tag comprising or consisting of a protein fragment containing at least one reactive residue involved in the formation of an isopeptide bond between the peptide tag and its binding partner, wherein the peptide tag comprises the protein fragment extending from 4 to 24 amino acids upstream of the reactive residue to 2 to 22 amino acids downstream, or a homolog thereof having at least 70% homology, provided that the homolog contains a reactive residue, preferably the reactive residue being asparagine or aspartic acid. 23. Sequence IDs 46 (Rum tag), 47 (RumTrunkD9N tag), 50 (Pho tag), 52 (Ent tag), 54 (Rum7 tag), 56 (Rum3 tag), 58 (Rum2 tag), 60 (Rum4 tag), 62 (Rum5 tag), 64 (Rum6 tag), 66 (Bac tag), 68 (Bac2 tag), 35 (Bac3 tag), 22 (Bac4 tag), 29, 31, and 12 (Bac5 tag) ) and a homolog thereof having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology thereto, or comprising the peptide tag described in item 22. 24. The peptide tag according to item 22 or 23, wherein the peptide tag comprises SEQ ID NO: 73, preferably within 20 amino acids of its C-terminus, for example, within 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids of its C-terminus, or comprises a sequence motif consisting thereof. 25. A method for producing a peptide pair comprising or consisting of a modified binding partner and a peptide tag, wherein the modified binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained in the modified binding partner and another reactive residue contained in the peptide tag. i) A step of producing a binding partner modified by any one of items 1 to 8, and / or ii) A step of producing a peptide tag by the method described in any one of items 18 to 21, Methods that include... 26. A peptide pair comprising, or consisting of, a peptide tag as defined in any one of items 1 to 25 and a modified binding partner as defined in any one of items 1 to 25. 27. Peptide pairs: a) Sequence ID 9 and Sequence ID 69 b) Sequence IDs 47 and 39; and c) Sequence IDs 47 and 41; d) Sequence IDs 46 and 39; e) Sequence ID 46 and Sequence ID 41, A peptide pair selected from item 26. 28. A modified bond partner having one or more improved properties compared to a reference bond partner, wherein one or more improved properties are a) Increased binding efficacy of a modified binding partner to a peptide tag compared to the binding of a reference binding partner to the peptide tag, wherein the modified binding partner and optionally the reference binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained in the modified binding partner or within the reference binding partner and another reactive residue contained in the peptide tag, and the increased binding efficacy is at least one of the total binding amount and the binding rate; b) An increased ability to form particles presenting the peptide of the target, such as virus-like particles presenting the peptide of the target, compared to the ability to form particles of a reference binding partner under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein, fused to a modified binding partner and the peptide of the target fused to a peptide tag, or the particles comprise a particle-forming protein fused to a peptide tag and the peptide of the target fused to a modified binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the modified binding partner and the peptide tag; c) An increased ability to present the peptide of interest on a particle, such as a virus-like particle, compared to the ability of a reference binding partner to present the peptide of interest under similar conditions, wherein the particle comprises a particle-forming protein, such as a virus-like particle-forming protein, fused to a modified binding partner and the peptide of interest fused to a peptide tag, or the particle comprises a particle-forming protein fused to a peptide tag and the peptide of interest fused to a modified binding partner, and the particle is formed by the spontaneous formation of an isopeptide bond between the modified binding partner and the peptide tag, A modified binding partner, independently selected from one or more of the following. 29. The particle, when administered to a target in need, induces a greater immune response than the particle, which is otherwise identical to the particle, preferably the reference binding partner is SpyCatcher (SEQ ID NO: 1), and optionally the amplified immune response is an amplified IgM response and / or an amplified IgG2 response, such as an amplified IgG2a and / or IgG2b, as described in item 28. 30. Modified binding partners as described in item 28 or 29, comprising or consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 29, SEQ ID NO: 71 or SEQ ID NO: 41, or a fragment thereof, or homologues having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology thereto. 31. A peptide tag having one or more improved properties compared to a reference peptide tag, wherein one or more improved properties are a) Increased binding efficacy of a peptide tag to a reference binding partner compared to the binding of the reference peptide tag to the reference binding partner, wherein the peptide tag and the reference peptide tag can bind to the reference binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained in the peptide tag or within the reference peptide tag and another reactive residue contained in the reference binding partner, and the binding efficacy is increased if at least one of the total binding amount and binding rate is increased; b) An increased ability to form particles such as virus-like particles compared to the ability of a reference peptide tag to form particles under similar conditions, wherein the particles present the peptide of interest, and the particles contain a particle-forming protein such as a virus-like particle-forming protein fused to a reference binding partner and the peptide of interest fused to a peptide tag, or the particles contain a particle-forming protein fused to a peptide tag and the peptide of interest fused to a reference binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the reference binding partner and the peptide tag; c) An increased ability to present a target compound, such as a peptide on a particle, such as a virus-like particle, compared to the ability of a reference peptide tag to present the target compound under similar conditions, wherein the particle comprises a particle-forming protein, such as a virus-like particle-forming protein, fused to the peptide tag and the target compound fused to a binding partner, or the particle comprises a particle-forming protein fused to a binding partner and the target compound fused to the peptide tag, and the particle has an increased ability to be formed by the spontaneous formation of isopeptide bonds between the binding partner and the peptide tag. A peptide tag, independently selected from one or more of the following. 32. When administered to a target in need of it, the particle induces a greater immune response than the particle, which is otherwise identical to the particle formed with the reference peptide tag, and optionally the amplified immune response is an amplified IgM response and / or an amplified IgG2 response, such as an amplified IgG2a and / or IgG2b, as described in item 31 of the peptide tag. 33. Sequence IDs 46 (Rum tag), 47 (RumTrunkD9N tag), 50 (Pho tag), 52 (Ent tag), 54 (Rum7 tag), 56 (Rum3 tag), 58 (Rum2 tag), 60 (Rum4 tag), 62 (Rum5 tag), 64 (Rum6 tag), 66 (Bac tag), 68 (Bac2 tag), 35 (Bac3 tag), 22 (Bac4 tag), 29, 31, and 12 (Bac5 tag) A peptide tag as described in item 31 or 32, comprising or consisting of its homologue having at least 60% homology thereto, e.g., at least 65%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% homology thereto. 34. A peptide pair comprising or consisting of a peptide tag and a binding partner, having one or more improved properties compared to a reference peptide pair comprising a reference peptide tag and a reference binding partner. The binding partner can bind to the peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the modified binding partner and another reactive residue contained within the peptide tag. A reference binding partner can bind to a reference peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the reference binding partner and another reactive residue contained within the reference peptide tag. One or more improved characteristics are: a) Increased binding efficacy of a binding partner to a peptide tag compared to the binding of a reference binding partner to a reference peptide tag, wherein the binding efficacy is increased if at least one of the total binding amount and the binding rate is increased; b) An increased ability to form particles that present the peptide of the target, such as virus-like particles that present the peptide of the target, compared to the ability of a reference peptide pair to form particles under similar conditions, wherein the particles comprise a particle-forming protein, such as a virus-like particle-forming protein fused to a binding partner, and the compound of the target fused to a peptide tag, or the particles comprise a virus-like particle-forming protein fused to a peptide tag and the compound of the target fused to a binding partner, and the particles are formed by the spontaneous formation of isopeptide bonds between the binding partner and the peptide tag; and / or c) An increased ability to present a compound of interest, such as a peptide on a particle, such as a virus-like particle, compared to the ability of a reference peptide pair to present the peptide of interest under similar conditions, wherein the particle comprises a particle-forming protein, such as a virus-like particle-forming protein, fused to a binding partner and the compound of interest fused to a peptide tag, or the particle comprises a particle-forming protein fused to a peptide tag and the compound of interest fused to a binding partner, and the particle is formed by the spontaneous formation of an isopeptide bond between the binding partner and the peptide tag, A peptide pair, independently selected from one or more of the following. 35. When administered to a target in need, the particles induce a greater immune response than the particles, which are otherwise identical to the reference peptide pair, and optionally the amplified immune response is an amplified IgM response and / or an amplified IgG2 response, such as an amplified IgG2a and / or IgG2b, and preferably the reference peptide pair is the peptide pair described in item 34, which includes SpyCatcher (SEQ ID NO: 1). 36. A binding partner is a binding partner whose definition has been modified in any one of items 1 through 35. a) If the modified binding partner includes or consists of the first reactive fragment of the first binding partner and the second remaining fragment of the second binding partner as defined in any one of items 1 to 35, the reference binding partner is the second binding partner, b) If the modified binding partner includes or consists of the second reactive fragment of the second binding partner and the first remaining fragment of the first binding partner as defined in any one of items 1 to 35, the reference binding partner is the first binding partner. The peptide pair described in item 34 or 35. 37. A modified binding partner, peptide tag, or peptide pair described in any one of items 9-17, 22-24, or 26-35, wherein the particle is a virus-like particle. 38. A modified binding partner, peptide tag, or peptide pair as described in any one of items 9-17, 22-24, or 26-35, wherein the particle is a virus-like particle and the particle-forming protein is a viral capsid protein. 39. A modified binding partner, peptide tag, or peptide pair as described in any one of items 9-17, 22-24, or 26-35, wherein the viral capsid protein is selected from the group consisting of AP205 capsid protein, MS2 capsid protein, HBc capsid protein, and phage fr capsid protein. 40. A polynucleotide encoding a modified partner and / or a peptide tag as described in any one of items 9-17, 28, or 29. 41. a) Modified binding partners represented by SEQ ID NO: 37, wherein the above polynucleotide contains or consists of SEQ ID NO: 38; b) A modified mutagenesis partner represented by SEQ ID NO: 39, wherein the polynucleotide contains or consists of SEQ ID NO: 40; c) A modified binding partner represented by SEQ ID NO: 41, wherein the polynucleotide contains or consists of SEQ ID NO: 42; d) Modified binding partners represented by SEQ ID NO: 29, wherein the above polynucleotide contains or consists of SEQ ID NO: 30; e) Modified binding partners represented by SEQ ID NO: 71, wherein the polynucleotide contains or consists of SEQ ID NO: 72; or homologs having at least 60% identity with them, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% identity with them, The polynucleotide described in item 40, which codes for the polynucleotide. 42. a) A peptide tag represented by SEQ ID NO: 46, wherein the polynucleotide described above contains or consists of SEQ ID NO: 45; b) A peptide tag represented by Sequence ID No. 48, wherein the polynucleotide described above contains or consists of Sequence ID No. 47; c) A peptide tag represented by Sequence ID No. 50, wherein the above polynucleotide contains or consists of Sequence ID No. 49; d) A peptide tag represented by SEQ ID NO: 52, wherein the above polynucleotide contains or consists of SEQ ID NO: 51; e) A peptide tag represented by SEQ ID NO: 54, wherein the polynucleotide described above contains or consists of SEQ ID NO: 53; f) A peptide tag represented by SEQ ID NO: 56, wherein the above polynucleotide contains or consists of SEQ ID NO: 55; g) A peptide tag represented by SEQ ID NO: 58, wherein the above polynucleotide contains or consists of SEQ ID NO: 57; h) A peptide tag represented by SEQ ID NO: 60, wherein the above polynucleotide contains or consists of SEQ ID NO: 59; i) A peptide tag represented by Sequence ID No. 62, wherein the above polynucleotide contains or consists of Sequence ID No. 61; j) A peptide tag represented by SEQ ID NO: 64, wherein the above polynucleotide contains or consists of SEQ ID NO: 63; k) A peptide tag represented by Sequence ID No. 66, wherein the above polynucleotide contains or consists of Sequence ID No. 65; l) A peptide tag represented by Sequence ID 68, wherein the above polynucleotide contains or consists of Sequence ID 67; m) A peptide tag represented by SEQ ID NO: 35, wherein the above polynucleotide contains or consists of SEQ ID NO: 36; n) A peptide tag represented by Sequence ID No. 22, wherein the above polynucleotide contains or consists of Sequence ID No. 21; o) A peptide tag represented by Sequence ID No. 12, wherein the above polynucleotide contains or consists of Sequence ID No. 11; p) A peptide tag represented by SEQ ID NO: 31, wherein the above polynucleotide contains or consists of SEQ ID NO: 32; or homologs having at least 60% identity with them, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% identity with them, A polynucleotide as described in item 40 or 41, which codes for a polynucleotide. 43. A vector containing a polynucleotide as described in any one of items 40-42. 44. i) Modified binding partners as described in any one of items 9-17, 28, or 29, and / or ii) Peptide tags as described in any one of items 22-23 or 31, A host cell that expresses [the specified gene]. 45. A host cell as described in item 44, where the cell is a bacterial cell, yeast cell, fungal cell, plant cell, mammalian cell, or insect cell. 46. i) A protein fused to a modified binding partner as described in any one of items 9-17 or 28 or 29, and a compound of interest such as a peptide, e.g., an antigen, fused to a peptide tag as described in any one of items 22 or 23 or 31; or ii) A protein fused to a peptide tag as described in any one of items 22, 23, or 31, and a compound of interest, such as a peptide, fused to a modified binding partner as described in any one of items 9-17, 28, or 29, e.g., an antigen. Includes, The modified binding partners and peptide tags can interact through the spontaneous formation of isopeptide bonds. A composition in which the target compound and protein are linked via an isopeptide bond between a modified binding partner and a peptide tag. 47. The composition according to item 46, wherein the protein fused to the modified binding partner is a particle-forming protein. 48. The composition according to item 47, wherein the particle-forming protein and the compound of interest form particles that present the compound of interest. 49. The composition according to item 48, wherein the particles are virus-like particles and the particle-forming protein is a viral capsid protein. 50. The composition according to any one of items 46 or 47, wherein the target peptide is an antigen, preferably the antigen is a peptide or an antigenic fragment thereof, such as a peptide or an antigenic fragment thereof associated with an abnormal physiological reaction. 51. A composition according to any one of items 46-50, wherein the abnormal physiological response is a disease such as a cardiovascular disease, an infectious disease and / or an allergic reaction / disease. 52. A composition according to any one of items 46 to 51, wherein the disease is cancer, such as breast cancer, gastric cancer, ovarian cancer, and serous adenocarcinoma of the uterine body; cardiovascular disease, such as dyslipidemia, atherosclerosis, and / or hypercholesterolemia; immunoinflammatory or chronic disease, such as eosinophilic asthma, allergy, nasal polyps, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome, and Churg-Strauss syndrome; neurological disease, such as Alzheimer's disease; infectious disease, such as infectious disease, selected from the group consisting of diseases caused by viruses, such as coronavirus, e.g., SARS-CoV-2, malaria, tuberculosis, HIV, and influenza; dyslipidemia, such as hyperlipidemia, type I, II, III, IV, or V hyperlipidemia, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatous disease, cholesterol acetyltransferase deficiency; arteriosclerotic condition, such as atherosclerosis; or coronary artery disease. 53. A composition according to any one of items 46 to 52, wherein the antigen is an antigenic fragment derived from the group consisting of proteins, peptides and / or cancer-specific polypeptides, polypeptides associated with cardiovascular disease, polypeptides associated with asthma, polypeptides associated with nasal polyps, polypeptides associated with atopic dermatitis, polypeptides associated with eosinophilic esophagitis, polypeptides associated with hypereosinophilic syndrome, polypeptides associated with Churg-Strauss syndrome and / or polypeptides associated with pathogenic organisms. 54. A composition described in any one of items 46-53, a polynucleotide described in any one of items 40-42, or a vector described in item 43, for use in the prevention or treatment of disease. 55. Compositions, polynucleotides, or vectors for use as described in item 53, for use in which the disease is cancer, such as breast cancer, gastric cancer, ovarian cancer, and serous adenocarcinoma of the uterine body; cardiovascular diseases, such as dyslipidemia, atherosclerosis, and / or hypercholesterolemia; immunoinflammatory or chronic diseases, such as eosinophilic asthma, allergies, nasal polyps, atopic dermatitis, eosinophilic esophagitis, hypereosinophilic syndrome, and Churg-Strauss syndrome; neurological diseases, such as Alzheimer's disease; infectious diseases, such as infectious diseases selected from the group consisting of diseases caused by viruses, such as coronaviruses, e.g., SARS-CoV-2, malaria, tuberculosis, HIV, and influenza; dyslipidemia, such as hyperlipidemia, type I, II, III, IV, or V hyperlipidemia, secondary hypertriglyceridemia, hypercholesterolemia, familial hypercholesterolemia, xanthomatous disease, cholesterol acetyltransferase deficiency, etc.; arteriosclerotic conditions, such as atherosclerosis, etc.; or coronary artery disease. 56. A composition according to any one of items 46 to 53, a polynucleotide according to any one of items 40 to 42, or a vector according to item 43, for use in a method for inducing an immune response in a subject, comprising the step of administering the composition, polynucleotide, or vector to the subject at least once. 57. i) obtain a first polypeptide containing or comprising a modified binding partner as defined in any one of items 9-17 or 28 or 29 fused to a protein, and obtain a second polypeptide containing or comprising a peptide tag as defined in any one of items 22-24 or 31 fused to a compound of interest; or The steps of obtaining a first polypeptide comprising or consisting of a peptide tag as defined in any one of items 22-24 or 31, and a second polypeptide comprising or consisting of a modified binding partner as defined in any one of items 9-17 or 28 or 29 fused to the compound of interest, ii) A step of contacting a first polypeptide with a second polypeptide, thereby enabling the formation of an isopeptide bond between the peptide tag and the modified binding partner, to produce the pharmaceutical composition described in any one of items 46 to 56. A method for producing a pharmaceutical composition as described in any one of items 46 to 56, including the above. 58. The method described in item 57, wherein the protein is a particle-forming protein.

Claims

1. A peptide pair comprising a peptide tag and a binding partner, wherein the peptide tag and the binding partner can bind to each other via the spontaneous formation of an isopeptide bond between one reactive residue contained in the binding partner and another reactive residue contained in the peptide tag. The peptide pair is a) A binding partner having the amino acid sequence of SEQ ID NO: 41 (KatI), or an amino acid sequence having at least 90% identity with SEQ ID NO: 41, wherein the residue corresponding to residue 31 in SEQ ID NO: 41 is not modified; and a peptide tag having an amino acid sequence having at least 90% identity with SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), or SEQ ID NO: 12 (Bac5 tag); or b) A binding partner having the amino acid sequence of SEQ ID NO: 37 (QueenCatcher), or an amino acid sequence having at least 90% identity with SEQ ID NO: 37, wherein the residue corresponding to residue 31 in SEQ ID NO: 37 is not modified; and a peptide tag having an amino acid sequence having at least 90% identity with SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 12 (Bac5 tag), or SEQ ID NO: 76 (Clib9); That is, Peptide pairs.

2. A binding partner capable of binding to a peptide tag via the spontaneous formation of an isopeptide bond between one reactive residue contained within the binding partner and another reactive residue contained within the peptide tag, a) The amino acid sequence of SEQ ID NO: 41 (KatI), or an amino acid sequence having at least 90% identity with SEQ ID NO: 41, in which the residue corresponding to residue 31 in SEQ ID NO: 41 has not been modified; or b) The amino acid sequence of SEQ ID NO: 37 (QueenCatcher), or an amino acid sequence having at least 90% identity with SEQ ID NO: 37, in which the residue corresponding to residue 31 in SEQ ID NO: 37 has not been modified; A combining partner having the following characteristics.

3. a) The binding partner has the amino acid sequence of SEQ ID NO: 41 (KatI), or an amino acid sequence having at least 90% identity with SEQ ID NO: 41, wherein the residue corresponding to residue 31 in SEQ ID NO: 41 is not modified and can bind to the peptide tag via an isopeptide bond, wherein the peptide tag has the amino acid sequence of SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumtrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), or SEQ ID NO: 12 (Bac5 tag); or b) The binding partner has the amino acid sequence of SEQ ID NO: 37 (QueenCatcher), or an amino acid sequence that is at least 90% identical to SEQ ID NO: 37, wherein the residue corresponding to residue 31 in SEQ ID NO: 37 is not modified and can bind to the peptide tag via an isopeptide bond, and the peptide tag is SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 47 (RumtrunkD9N tag) , having the amino acid sequence of SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 12 (Bac5 tag), or SEQ ID NO: 76 (Clib9); A peptide pair or binding partner according to any one of claims 1 to 2.

4. A peptide tag that can bind to the binding partner via the spontaneous formation of an isopeptide bond between one reactive residue contained in the binding partner and another reactive residue contained in the peptide tag, a) Sequence ID 47 (RumtrunkD9N tag); b) Sequence ID 46 (Rum tag); c) Sequence ID 71 (RumTrunk tag); d) Sequence ID 58 (Rum2 tag); e) Sequence ID 56 (Rum3 tag); f) Sequence ID 60 (Rum4 tag); g) Sequence ID 62 (Rum5 tag); h) Sequence ID 64 (Rum6 tag); i) Sequence ID 54 (Rum7 tag); j) Sequence ID 66 (Bac tag); k) Sequence ID 68 (Bac2 tag); l) Sequence ID 35 (Bac3 tag); m) Sequence ID 22 (Bac4 tag); n) Sequence ID 12 (Bac5 tag); o) Sequence ID 50 (Pho tag); and p) Sequence ID 52 (Ent tag); A peptide tag having an amino acid sequence selected from; or an amino acid sequence having at least 90% identity therewith, wherein the amino acid sequence having at least 90% identity includes a reactive residue involved in the formation of an isopeptide bond, or, if the reactive residue is aspartic acid (D), the reactive residue is mutated to asparagine (N);

5. A polynucleotide encoding a binding partner according to any one of claims 2 to 3 or a peptide tag according to claim 4.

6. a) Sequence ID 47 (RumtrunkD9N tag); b) Sequence ID 46 (Rum tag); c) Sequence ID 71 (RumTrunk tag); d) Sequence ID 58 (Rum2 tag); e) Sequence ID 56 (Rum3 tag); f) Sequence ID 60 (Rum4 tag); g) Sequence ID 62 (Rum5 tag); h) Sequence ID 64 (Rum6 tag); i) Sequence ID 54 (Rum7 tag); j) Sequence ID 66 (Bac tag); k) Sequence ID 68 (Bac2 tag); l) Sequence ID 35 (Bac3 tag); m) Sequence ID 22 (Bac4 tag); or n) Sequence ID 12 (Bac5 tag); The polynucleotide according to claim 5, which encodes a peptide tag having the amino acid sequence of; or an amino acid sequence having at least 90% identity therewith, wherein the amino acid sequence having at least 90% identity includes a reactive residue involved in the formation of an isopeptide bond, or, if the reactive residue is aspartic acid (D), the reactive residue is mutated to asparagine (N).

7. A system comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a peptide tag, the second polynucleotide encodes a binding partner, and the peptide tag and the binding partner are able to bind to each other via the spontaneous formation of an isopeptide bond between one reactive residue contained in the binding partner and another reactive residue contained in the peptide tag. a) The first polynucleotide codes for a peptide tag having the amino acid sequence of SEQ ID NO: 47 (RumTrunkD9N tag), SEQ ID NO: 54 (Rum7 tag), SEQ ID NO: 56 (Rum3 tag), SEQ ID NO: 58 (Rum2 tag), SEQ ID NO: 60 (Rum4 tag), SEQ ID NO: 62 (Rum5 tag), SEQ ID NO: 64 (Rum6 tag), SEQ ID NO: 71 (RumTrunk tag), SEQ ID NO: 5 (Spy tag), SEQ ID NO: 7 (Sdy tag), SEQ ID NO: 46 (Rum tag), SEQ ID NO: 66 (Bac tag), SEQ ID NO: 68 (Bac2 tag), SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), or SEQ ID NO: 12 (Bac5 tag), and the second polynucleotide codes for a binding partner having the amino acid sequence of SEQ ID NO: 41 (KatI), or an amino acid sequence having at least 90% identity with SEQ ID NO: 41, in which the residue corresponding to residue 31 in SEQ ID NO: 41 is not modified; or b) The first polynucleotide is sequence number 47 (RumTrunkD9N tag), sequence number 54 (Rum7 tag), sequence number 56 (Rum3 tag), sequence number 58 (Rum2 tag), sequence number 60 (Rum4 tag), sequence number 62 (Rum5 tag), sequence number 64 (Rum6 tag), sequence number 71 (RumTrunk tag), sequence number 5 (Spy tag), sequence number 7 (Sdy tag), sequence number 46 (Rum tag), sequence number 66 (Bac tag), sequence number 68 (Bac2 tag) The second polynucleotide encodes a peptide tag having the amino acid sequence of SEQ ID NO: 35 (Bac3 tag), SEQ ID NO: 22 (Bac4 tag), SEQ ID NO: 12 (Bac5 tag), or SEQ ID NO: 76 (Clib9), wherein the second polynucleotide encodes a binding partner having the amino acid sequence of SEQ ID NO: 37 (QueenCatcher), or an amino acid sequence having at least 90% identity with SEQ ID NO: 37, in which the residue corresponding to residue 31 in SEQ ID NO: 37 is not modified; system.

8. The peptide tag is the peptide tag described in Claim 4; and / or The aforementioned bonding partner is the bonding partner described in any one of claims 2 to 3. The system according to claim 7.

9. i) a protein fused to a binding partner according to any one of claims 2 to 3, and the compound of the target fused to a peptide tag according to claim 4; or ii) A protein fused to the peptide tag according to claim 4, and the compound of the target fused to the binding partner according to any one of claims 2 to 3; A composition comprising, The binding partner and peptide tag can interact through the spontaneous formation of isopeptide bonds. The target compound and the protein are linked via an isopeptide bond between the binding partner and the peptide tag. composition.

10. The composition according to claim 9, wherein the protein is a particle-forming protein.

11. The composition according to claim 10, wherein the particle-forming protein and the target compound form particles that present the target compound.

12. The composition according to any one of claims 9 to 11, wherein the compound of interest is an antigen, and the antigen is a protein, peptide and / or antigen fragment thereof derived from the group consisting of cancer-specific polypeptides, polypeptides related to cardiovascular disease, polypeptides related to asthma, polypeptides related to nasal polyps, polypeptides related to atopic dermatitis, polypeptides related to eosinophilic esophagitis, polypeptides related to hypereosinophilic syndrome, polypeptides related to Churg-Strauss syndrome and / or polypeptides related to pathogenic organisms.

13. A pharmaceutical composition for the prevention or treatment of a disease, comprising: i) the composition according to any one of claims 9 to 12; ii) a polynucleotide encoding a binding partner according to any one of claims 2 to 3 and a peptide tag according to claim 4; or iii) the system according to any one of claims 7 to 8.

14. A composition according to any one of claims 9 to 12, a polynucleotide encoding a binding partner according to any one of claims 2 to 3 and a peptide tag according to claim 4, or a system according to any one of claims 7 to 8, for use in a method of inducing an immune response in a subject. The method includes the step of administering the composition, the polynucleotide, or the system to the subject at least once. The composition, the polynucleotide, or the system.

15. A method for producing the composition according to any one of claims 9 to 12, i) the step of obtaining a fusion of a first polypeptide comprising a protein and a binding partner according to any one of claims 2 to 3, and a fusion of a second polypeptide comprising the compound of interest and a peptide tag according to claim 4; or the step of obtaining a fusion of a first polypeptide comprising a protein and a peptide tag according to claim 4, and a fusion of a second polypeptide comprising the compound of interest and a binding partner according to any one of claims 2 to 3; ii) A step of bringing the first polypeptide and the second polypeptide into contact, thereby enabling the formation of an isopeptide bond between the peptide tag and the binding partner, and producing the composition according to any one of claims 9 to 12. including, method.

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