Cell-free production method and uses

Abstract

The present disclosure relates to the field of cell-free protein or polypeptide production for the purpose of producing a polypeptide of interest (POI) which comprises a moiety, which is a target polypeptide whereof production is desired. The target polypeptide is linked to a coupling peptide which binds to a complementary peptide which is fused to an autotransporter fusion protein which is displayed on the outer surface of a vesicle such that the synthesized POI is coupled to said vesicle via isopeptide bond. Furthermore, the disclosure relates to compositions comprising such a target polypeptide moiety, which are amenable to use in therapeutic applications and / or biomedical applications.

Description

[0001] CELL-FREE PRODUCTION METHOD AND USES

[0002] Field of the invention

[0003] The present disclosure relates in general to the field of polypeptide and protein expression. More specifically, the disclosure relates to a cell-free production method for the purpose of producing a polypeptide of interest (POI) which comprises a moiety, which is a target polypeptide whereof production is desired. Furthermore, the disclosure relates to compositions comprising such a target polypeptide moiety, which are amenable to use in therapeutic and biomedical applications.

[0004] Background of the invention

[0005] Efficient production of proteins, or polypeptides, has become an essential part of the biomedical and industrial biochemical industries. Such pharmaceutical and industrially used proteins or polypeptides are commonly produced as recombinant proteins in host cells that are recombinantly engineered to overexpress the selected protein or polypeptide (Terpe, K., Appl Microbiol Biotechnol. (2006) 72:211-223). The host cells can for instance be selected from bacterial cells, yeast cells and mammalian cells. Although quite efficient, recombinant production of proteins or polypeptides suffers from some disadvantages. Polypeptides produced in a cellular environment are susceptible to degradation through the action of cellular proteases. Also, heterologous disulphide-containing proteins expressed in the cytoplasm of bacteria, such as E. coli, may be unstable or fold incorrectly since the disulphide bridges, which are often important for proper folding and stability, cannot generally form in this compartment. Some polypeptides are so unstable and prone to degradation that they are impossible or very difficult to recover from the host cell, while others form protein aggregates. Some polypeptides are toxic to the host cell and inhibit host cell growth and / or induce host cell death, leading to reduced or even completely ceased protein production. Also, purification and isolation of some desired polypeptides from the other proteins in the protein rich cytoplasm, and from other cell components, can be difficult and very labor intense (Weikert, MJ. et al., Curr Opin Biotechnol. (1996) 7:494-499; Baneyx, F. and Mujacic, M., Nat Biotechnol. (2004) 22:1399-1408). In addition, it is well established that post-translational modifications (PTMs) are essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Posttranslational modifications (PTMs) are covalent processing events that change the properties of a protein by proteolytic cleavage and adding a modifying group, such as acetyl, phosphoryl, glycosyl and methyl, to one or more amino acids. Thus, it is desirable to be able to provide a system which enables production of proteins with the desired modifications.

[0006] In order to circumvent these obstacles, several different techniques have been developed. The desired polypeptide may for instance be produced such that it is exported to the periplasm, which enables disulphide bond formation and provides a more favorable environment for folding and stability of heterologous proteins (Baneyx, F. and Mujacic, M., Nat Biotechnol. (2004) 22:1399-1408). Another strategy is to produce the desired polypeptide such that it is exported to the extracellular space, where it is more easily isolated (Choi, J.H. and Lee, S.Y., Appl Microbiol Biotechnol. (2004) 25 64(5) :625-35). Although such strategies overcome some of the abovementioned problems, there are still some disadvantages associated with them. Export of the desired polypeptide to the periplasm or extracellular space requires translocation of the protein across one or two membranes. Such translocation is often difficult and inefficient and therefore leads to low protein yields (Sahdev, S. et al., Mol Cell Biochem. (2008) 307:249-264). Furthermore, the desired polypeptide may still be toxic to the host cell, and proteins located in the periplasm may be difficult to isolate and purify (Jana, S. and Deb, J.K., Appl Microbiol Biotechnol. (2005) 67:289-298; Swartz, J.R., Curr Opin Biotechnol . (2001) 12:195- 201). One strategy to avoid or overcome these disadvantages is to produce the desired polypeptide in a cell-free system. Another advantage of using a cell-free system is that it is likely easier to obtain regulatory compliance with a standardized in vitro production method than with a production method based on living cells.

[0007] However, while cell-free polypeptide and protein expression and production methods have emerged as a powerful alternative approach to utilizing living cells for specific applications in polypeptide and protein synthesis, the cell-free approach is not without its disadvantages. Cell-free systems may be more expensive and less scalable than in vivo systems because they may require large amounts of purified cell extracts, energy sources, and amino acids. Cell-free systems generally have lower protein yields and stability than in vivo systems, as they may lack the optimal cellular mechanisms for protein folding, quality control, and degradation. Cell-free systems generally also generally provide lower protein activity and functionality than in vivo systems since they generally do not reproduce the native cellular environment and interactions. Thus, there remains a need for improved cell-free production methods for producing polypeptides and proteins.

[0008] Summary of the invention

[0009] It is an object of the present disclosure to overcome problems of the prior art and provide an efficient system for cell-free production of various proteins, including but not limited to aggregation prone proteins, proteins comprising neoantigen derived epitopes, such as proteins comprising multiple tandem fusions or repeats of neoantigen derived epitopes. It is an object to provide a versatile system that can be employed for various proteins with minimal adaptation of the system. It is an object of the present invention to provide a system which may be used for production of various protein requiring post translational modification.

[0010] It is an object of the present disclosure to overcome above mentioned problems of the prior art. It is an object accomplished by the provision of a cell-free production method for producing a polypeptide of interest (POI) comprising at least one target polypeptide moiety and at least one coupling moiety of a coupling system, said POI being coupled to at least one vesicle displaying at least one complementary moiety of said coupling system, wherein said POI is coupled to said vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system. It is a further object of the disclosure to provide a method for efficiently producing polypeptides or proteins, such that the polypeptides or proteins produced may be easily recovered.

[0011] It is an object of the present disclosure to provide a method for production of polypeptides or proteins which are hard to produce in vitro in an efficient manner.

[0012] It is furthermore an object of the present disclosure, to produce polypeptides or proteins in a form which may immediately be used in medical context and / or biotechnological context, or only requiring minimal modification.

[0013] These and other objects which are evident to the skilled person from the present disclosure are met by different aspects of the invention as claimed in the appended claims and as generally disclosed herein.

[0014] According to a first aspect, these and other objects are achieved by a cell-free production method for producing a target polypeptide moiety, the method comprising the following steps: a. providing at least one nucleotide sequence encoding at least one polypeptide of interest (POI), wherein said POI comprises at least one target polypeptide moiety and at least one coupling moiety of a coupling system; b. providing a polypeptide expression-competent cell-free composition; c. providing at least one vesicle displaying on its outer surface at least one complementary moiety of said coupling system, such as providing at least one vesicle displaying on its outer surface at least one autotransporter (AT) fusion protein, wherein said at least one AT fusion protein comprises an AT protein and comprises at least one complementary moiety of said coupling system; and d. bringing said nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and simultaneously or subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.

[0015] Thus, in step d the method involves either creating a first mixture comprising said at least one nucleotide sequence and said polypeptide expression-competent cell-free composition followed by the subsequent addition of at least one vesicle to create a second mixture; or in step d) the method involves creating a first mixture comprising said at least one nucleotide sequence and said polypeptide expression-competent cell-free composition and simultaneously adding said at least one vesicle to the first mixture to create a second mixture, whereby said three components (said at least one nucleotide sequence , said polypeptide expression-competent cell-free composition and said at least one vesicle) are brought together in one mixture. In other words, in this last mentioned embodiment of the method there is no difference between the first and second mixtures as all three components are brought together in both the first and second mixtures.

[0016] In one embodiment, step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and simultaneously bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system. In one embodiment, said step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and simultanously bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and simultanous coupling, such as simultanous to the expression of said POI, of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system. It is to understood that in this embodiment, the first and second mixtures are the same mixture and thus that step d of bringing said nucleotide sequence into contact with said polypeptide expression-competent cell-free composition and said at least one vesicle results in the creation of one mixture. In other words it may be expressed that according to one embodiment of this aspect, the method comprises the following steps: a. providing at least one nucleotide sequence encoding at least one polypeptide of interest (POI), wherein said POI comprises at least one target polypeptide moiety and at least one coupling moiety of a coupling system; b. providing a polypeptide expression-competent cell-free composition; c. providing at least one vesicle displaying on its outer surface at least one complementary moiety of said coupling system, such as providing at least one vesicle displaying on its outer surface at least one autotransporter (AT) fusion protein, wherein said at least one AT fusion protein comprises an AT protein and comprises at least one complementary moiety of said coupling system; and d. bringing said nucleotide sequence into contact with said polypeptide expression-competent cell-free composition and said at least one vesicle to create a mixture, thereby allowing expression of said at least one POI comprising said target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.

[0017] In another embodiment, step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system. In one embodiment, said step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell- free composition to create a first mixture and subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and subsequent coupling, such as subsequent to the expression of said POI, of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system. In said embodiment, the expression of said at least one POI comprising said at least one target polypeptide moiety may occur already in the first mixture and optionally may also occur after the bringing said first mixture into contact with at least one vesicle to create a second mixture, the coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system may occur as well as continued expression of said at least one POI.

[0018] In other words it may be expressed that according to one embodiment of this aspect, the method comprises the following steps: a. providing at least one nucleotide sequence encoding at least one polypeptide of interest (POI), wherein said POI comprises at least one target polypeptide moiety and at least one coupling moiety of a coupling system; b. providing a polypeptide expression-competent cell-free composition; c. providing at least one vesicle displaying on its outer surface at least one complementary moiety of said coupling system, such as providing at least one vesicle displaying on its outer surface at least one autotransporter (AT) fusion protein, wherein said at least one AT fusion protein comprises an AT protein and comprises at least one complementary moiety of said coupling system; and d. bringing said nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.

[0019] The skilled person will appreciate that the method as disclosed herein requires bringing the components used in the method in contact with each other, in other words bringing the nucleotide sequence encoding a polypeptide of interest (POI); the polypeptide expression-competent cell-free composition; and the vesicle, such as said one vesicle displaying on its outer surface an autotransporter (AT) fusion protein, as defined above, in contact with each other so that the polypeptide expression is enabled and expressed POI is coupled to the vesicle via the formation of an isopeptide bond between the at least one coupling moiety of a coupling system comprised in said POI and at least one complementary moiety of said coupling system displayed on said vesicle. As shown in the appended Examples, the steps of a, b, c and d can be performed in varying orders and still result in the successful expression of said target polypeptide moiety. In Example 2, the step d is performed subsequently to steps a, b and c. Whilst in Example 3, the step d is performed simultaneously to steps a, b and c. Furthermore, it is shown in Example 15 that the addition of OMVs before the onset of polypeptide synthesis is a successful strategy to improve coupling system-based coupling of certain polypeptides in a cell-free system. It is also shown in Example 17 that the addition of OMVs after the onset of polypeptide synthesis is a successful strategy for production of polypeptides and does not interfere with coupling system-based coupling of polypeptides in a cell-free system. Thus, in one embodiment, said method comprises the steps a, b, c and d, wherein the steps a, b and c may be performed in any order. In one embodiment, said method comprises the steps a, b, c and d, wherein the steps a, b, and c may be performed in any order, and wherein step d is performed simultaneously to steps a, b and c. In one embodiment, said method comprises steps a, b, c and d, wherein steps a, b, and c may be performed in any order and wherein step d is performed subsequently to steps a, b, and c. As used herein, the term "polypeptide of interest" or "POI" refers to a polypeptide comprising at least one target polypeptide moiety, which is the polypeptide which is desired to be expressed, and at least one coupling moiety of a coupling system as defined herein. Thus, the term "polypeptide of interest" or "POI" refers to a fusion polypeptide, comprising at least two moieties, namely the at least one target polypeptide moiety and the at least one coupling moiety of a coupling system.

[0020] As used herein, the term "target polypeptide moiety" or "target polypeptide" refers to the polypeptide which is desired to be expressed and said terms are used interchangeably herein. The skilled person appreciates that the location of said at least one coupling moiety in relation to the target polypeptide moiety will be dependent of the identity and properties of the target polypeptide. For some target polypeptides it may be suitable for said at least one coupling moiety to be located N- terminally. For some target polypeptides it may be suitable for said at least one coupling moiety to be located C-terminally. For yet some target polypeptides it may be suitable for said at least one coupling moiety to be located C-terminally and N- terminally, for example when more than one coupling moiety is present. In addition, for some other target polypeptides it may be suitable for said at least one coupling moiety to be located internally in said target polypeptide. Additionally, it is also possible that when more than one coupling moiety is present, the coupling moieties are present both N and / or C-terminally and internally. Thus, in one embodiment, said at least one coupling moiety of a coupling system is located C- or N-terminally in relation to said at least one target polypeptide moiety, such as is located N- terminally in relation to said at least one target polypeptide moiety. Thus, in one embodiment, said at least one coupling moiety of a coupling system is located C- or N-terminally in said POI, such as N-terminally of said at least one target polypeptide moiety comprised in said POI. In one embodiment, said at least one coupling moiety of a coupling system is located C- or N-terminally in said POI, such as N-terminally of said at least one target polypeptide moiety in said POI.

[0021] In another embodiment, said at least one coupling moiety of a coupling system is located at the C- or N-terminus of said at least one target polypeptide moiety, such as is located at the N-terminus of said at least one target polypeptide moiety. As used herein, it is to be understood that when it is written that said at least one coupling moiety is "located C- or N-terminally" said moiety is located near said C- or N-terminus, such as within 30 amino acid residues (aa) of said C- or N-terminus, such as within 20 aa of said C- or N-terminus, such as within 10 aa of said C- or N- terminus, such as within 5 aa of said C- or N-terminus, such as within 4 aa of said C- or N-terminus, such as within 3 aa of said C- or N-terminus, such as within 1 aa of said C- or N-terminus.. The skilled person appreciates that when it is written that said at least one coupling moiety is "located at the C- or N-terminus", is to be interpreted as located directly adjacent to the C- or N-terminus. A person of skill in the art will understand that by using the language "located C- or N-terminally" or "located at the C- or N-terminus" one does not imply a strict interpretation of the location of said moiety in relation to other moieties or structures comprised in a construct. In other words, the skilled person will understand that the expressions "located C- or N-terminally" or to express the location of said at least one coupling moiety of a coupling system in relation to said at least one target polypeptide moiety, may imply that said at least one coupling moiety of a coupling system may be directly at the C- or N-terminus of said at least one target polypeptide moiety, or may be indirectly at the C- or N-terminus of said at least one target polypeptide moiety, such as separated by one or several amino acid residues.

[0022] For Example, said coupling moiety may be separated or flanked by a linker or spacer sequence from the N- and / C -terminus of the target polypeptide. An illustrative nonlimiting example of such arrangement is [target polypeptide moiety]-[spacer or linker]-[coupling moiety] or [coupling moiety]-[spacer or li nker]-[ta rget polypeptide moiety] or [spacer or lin ker]-[coupli ng moiety]-[spacer or li nker]-[ta rget polypeptide moiety. In another embodiment, said at least one coupling moiety of a coupling system is located internally in said at least one target polypeptide moiety. By way of illustration, for example, said at least one target polypeptide moiety may be located between two domains of the target polypeptide moiety or between tandem repeats of the target polypeptide moieties. It is also envisioned that when the target polypeptide moiety comprises a fusion polypeptide, said at least one coupling moiety is located between the polypeptides fused.

[0023] With reference to the above explanation of how the terms "located C- or N- terminally" or "located at the C- or N-terminus" are used herein, the skilled person will also understand that by using the language "located internally in said at least one target polypeptide moiety", it is to be further understood that said at least one coupling moiety of a coupling system may be located within the sequence which comprises said at least one target polypeptide moiety. The skilled person understands that when designing constructs comprising more than one moiety or more than one moiety type, it is possible to structurally place said moieties in varying positions in relation to each other and to other moieties or structures comprised in said construct and still retain the intended functional characteristics of said construct.

[0024] The skilled person is familiar with cell free expression systems as an alternative approach to living cells for protein synthesis. Briefly, cell-free expression harnesses the transcription and translation machinery of living cells to enable protein synthesis in vitro through the expression of natural or synthetic DNA or RNA. The complex parts of the translation machinery can be reconstituted in test tubes, without the requirements of a cell membrane or cell reproduction. Such systems can be generated by the lysis of eukaryotic or bacterial cells and optionally the consequent removal of all components not required for protein expression. Such cell lysates and cell extracts can be managed very precisely and the polypeptide expression can be controlled by the addition of suitable DNA templates in combination with components like amino acids, NTPs, or certain cofactors. Alternatively, cell free expression systems may be reconstituted by the de novo addition of the components of transcription and translation machinery to the composition.

[0025] In one embodiment, said polypeptide expression-competent cell-free composition is selected from the group consisting of prokaryotic cellular extracts, prokaryotic cell-free lysates, eukaryotic cellular extracts, eukaryotic cell-free lysates and reconstituted cell-free synthesis systems. As used herein, the term "lysate" refers to a mixture which comprises all the components of the respective lysed cells. As used herein, the term "extract" refers to a mixture which comprises a part or parts of a lysate. In other words, a cell "extract" is to be understood as a broader term when compared to a cell "lysate", as an "extract" of lysed cells will comprise a select component or components of the more crude mixture which is the cell "lysate", said "lysate" comprising all the components of the respective lysed cells. It is also understood that an "extract" or a "lysate" may further comprise components other than those directly derived from the cell "extract" or cell "lysate", such as supplemented with further components. Such further components may be other heterologous components required to create or which improve a functional polypeptide expression-competent composition. Furthermore, it is also understood that said polypeptide expression-competent composition may also be selected from reconstituted cell-free synthesis systems. Such systems are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. Non-exhaustive exemplification of such reconstituted cell-free synthesis systems is the PURE system, as described by Shimizu, Y., et al., Nat. Biotechnol. (2001) 19, 751-755.

[0026] In a further embodiment said polypeptide expression-competent cell-free composition is selected from the group consisting of prokaryotic cellular extracts and prokaryotic cell-free lysates, such as wherein said polypeptide expression- competent cell-free composition is a prokaryotic cell-free lysate. In a more specific embodiment, said prokaryotic cellular extract or prokaryotic cell-free lysate is selected from the group consisting of bacterial cellular extracts, bacterial cell-free lysates, archaeal prokaryotic cellular extracts and archaeal cell-free lysates. In a specific embodiment, said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell-free lysate selected from the group consisting of cellular extracts or cell-free lysates of Escherichia coli, Streptomyces venezuelae, Streptomyces lividans, Bacillus megaterium, Bacillus subtilis, Corynebacterium glutamicum, Vibrio natriegens, Pseudomonas putida, Clostridium autoethanogenum, Mycoplasms capricolum, Sulfolobus solfataricus, Thermococcus kodakaraensis, Campylobacter jejuni, Neisseria meningitidis, Helicobacter pylori, Streptococus pneumoniae, Streptococcus gordonii, Streptococcus parasanguis, Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, Mycobacterium marinum, Mycobacterium tuberculosis, and Mycobacterium avium, such as wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell- free lysate selected from the group consisting of cellular extracts or cell-free lysates of Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, and Escherichia coli, such as wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell-free lysate of Escherichia coli. In one embodiment, said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell-free lysate selected from cellular extracts or cell-free lysates of Escherichia coli.

[0027] In another embodiment, said polypeptide expression-competent cell-free composition is selected from the group consisting of eukaryotic cellular extracts and eukaryotic cell-free lysates, such as wherein said polypeptide expression-competent cell-free composition is a eukaryotic cell-free lysate. In a further embodiment, said polypeptide expression-competent cell-free composition is selected from the group consisting of protozoan cellular extracts, protozoan cell-free lysates, yeast cellular extracts, yeast cell-free lysates, plant cellular extracts, plant cell-free lysates, insect cellular extracts, insect cell-free lysate, mammalian cellular extracts and mammalian cell-free lysate. In one embodiment, said polypeptide expression-competent cell- free composition is a eukaryotic cellular extract or eukaryotic cell-free lysate selected from the group consisting of extract or lysate of wheat germ cells, Tobacco BY-2 cells, Spodoptera frugiperda, Leishmania tarentolae, Saccharomyces cerevisiae, Rabbit reticulocytes, Chinese hamster ovary (CHO) cells, HEK293 cells and HeLa cells. In one particular embodiment, said eukaryotic cellular extracts and eukaryotic cell- free lysates is a plant cellular extracts and plant cell-free lysate, such as a plant cellular extracts and plant cell-free lysate selected from the group consisting of extract or lysate of wheat germ cells or of Tobacco BY-2 cells.

[0028] In another embodiment, said polypeptide expression-competent cell-free composition is a reconstituted cell-free synthesis system. As explained previously, it is to be understood that said polypeptide expression-competent composition may also be selected from reconstituted cell-free synthesis systems. As described herein, such systems are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. A non-exhaustive example of such reconstituted cell-free synthesis systems is the PURE system. The PURE system is a reconstituted cell-free protein synthesis system composed of individually prepared components required for gene expression in Escherichia coli, as described by Shimizu, Y., et al., Nat. Biotechnol. (2001) 19, 751-755. For example, the PURE system contains neither nucleases nor proteases, both of which degrade DNA or mRNA templates and proteins. The skilled person is familiar with other reconstituted cell-free protein synthesis system suitable in the context of the present disclosure.

[0029] The present inventors consider that the provision of the POI, and thus of the target polypeptide moiety, displayed on at least one vesicle may be beneficial for the purpose of purification thereof via means of vesicles as discussed in more detail below. It is to be understood that the display of the POI, and thus of the target polypeptide moiety on said at least one vesicle is dependent on the formation of an isopeptide bond between the respective moieties of the coupling system present on the POI and said at least one vesicle. Said at least one complementary moiety of the coupling system which said at least one vesicle comprises is accessible for binding to the coupling moiety of the POI, thus it is understood to be a present on the outer surface of said at least one vesicle. It may be directly or indirectly coupled to said at least one vesicle, for example via means of membrane bound protein or via a protein anchored in the membrane (such as a protein comprising a transmembrane domain). It is to be understood, that under step (c) of the method as described herein, several different types of vesicles can be provided. For example, it is possible to provide at least one vesicle displaying a first autotransporter fusion protein and provide at least one other vesicle displaying a second autotransporter fusion protein which may be the same as the first autotransporter fusion protein or may be different from the first autotransporter fusion protein. It is also to be understood, it is also possible to provide at least one vesicle displaying more than one type of autotransporter fusion protein, such as displaying a first autotransporter fusion protein and a second autotransporter fusion protein, said first autotransporter fusion protein being different from said second autotransporter fusion protein, said difference may be in the identity of the coupling system or in the identity of the autotransporter itself, or both.

[0030] It is also considered, that in the event the target polypeptide moiety is an antigen moiety, it may be particularly useful to display said target polypeptide moiety on outer membrane vesicles (OMVs) as the OMVs may be used to deliver the antigen to the subject and at the same time make use of the immune system stimulating properties of said vesicles. Outer membrane vesicles (OMVs) are ubiquitously released from the outer membrane (OM) of Gram-negative bacteria. OMVs comprise intrinsic adjuvant activity provided by the presence of various pathogen recognition receptor ligands, such as lipopolysaccharide and / or immunogenic surface proteins. This provides an attractive combination with the non-living, particulate nature of the OMVs. OMVs have been described to protect animals and humans against various pathogens. Thus, OMVs are considered suitable for use in vaccines as well as in targeted drug delivery.

[0031] Thus in one embodiment of the method of present disclosure, said at least one vesicle is a self-adjuvating vesicle. As described herein, a "self-adjuvating vesicle" is a vesicle for which there is no need for a further separate adjuvant in order to elicit an immune response, such as a therapeutically effective immune response, to an antigen in the context of a composition, such as a vaccine. It is to be understood that the adjuvant function is provided by the self-adjuvating vesicle itself, for example via means of lipopolysaccharide and immunogenic surface proteins present thereon. In one embodiment, said at least one vesicle is an Outer Membrane Vesicle (OMV). In one embodiment, said at least one vesicle is derived from a bacterium. As used herein, the term "derived from" is to be interpreted to encompass "originated from", "obtained from", or "isolated from". In one embodiment, said at least one vesicle is derived from a gram-negative bacterium. In another embodiment, said at least one vesicle is selected from the group consisting of vesicles derived from Escherichia coli, Neisseria meningitidis, Neisseria gonorrhea and Salmonella spp, such as selected from the group consisting of vesicles derived from Escherichia coli and Salmonella spp, such as wherein said at least one vesicle is derived from Salmonella spp. In one embodiment, said at least one vesicle is selected from the group consisting of vesicles derived from Escherichia coli and Salmonella spp. In a further embodiment, said at least one vesicle is a vesicle derived from Escherichia coli. In another further embodiment, said at least one vesicle is a vesicle derived from Salmonella spp. In one embodiment, said at least one vesicle is derived from a subspecies of 5. enterica subsp. enterica, such as from Salmonella enterica subsp. enterica serovar Typhimurium (5. Typhimurium).

[0032] In Gram-negative bacteria, secretion and surface display of heterologous proteins are difficult to achieve due to the presence of a complex, multi-layered cell envelope consisting of an inner membrane, an outer membrane, and a gel like substance between the membranes called the periplasm. Several secretion systems have evolved to overcome this barrier and deliver proteins in the extracellular environment. Of these the Autotransporter (AT) pathway (also known as the type V secretion system), is suitable for piggy-back transport of cargo proteins, because it combines simplicity with a high transport capacity. Autotransporter proteins are large proteins that are secreted by Gram-negative bacteria, such as E. coli, and are synthesized as large precursor proteins that contain three domains: an N-terminal signal peptide that targets the protein to the Sec translocon and initiates transfer across the inner membrane; a passenger domain, which comprises the "cargo" protein that is to be secreted; and a C-terminal beta -domain) comprising a betabarrel structure that integrates into the outer membrane and plays an important role in translocation of the passenger domain across the outer membrane into extracellular space. After translocation, the passenger domain is cleaved from the translocator domain and is released into the extracellular environment. Cleavage may be the action of an (external) protease or a protease motif situated between the translocator domain and the passenger domain. Alternatively, cleavage takes place through an intramolecular autocatalytic event at a specific site between the translocator domain and the passenger domain. Autotransporter proteins may be modified by mutations which prevent said cleavage, such that the passenger domain is not released to the extracellular environment and instead remains as part of the autotransporter protein anchored into the membrane (in other words is displayed on the membrane for example the membrane of an OMV). The skilled person appreciates that OMVs with ATs may be derived from host cells producing the AT or, alternatively, ATs can be brought into the membrane of an OMV or lipid vesicle by reconstitution. It is possible to replace the passenger domain, or a part thereof, with a desired polypeptide such that the autotransporter thereby displays a desired polypeptide on the membrane it is anchored into, for example the membrane of an OMV. Alternatively, it is possible to fuse desired polypeptide to said autotransporter such that said autotransporter thereby displays a desired polypeptide on the membrane it is anchored into, for example the membrane of an OMV.

[0033] Suitable autotransporter proteins that can be used to obtain the bacterial cells and OMVs of the present invention are proteins that belong to the pfam autotransporter family ('Autotransporter' PF03797). Non-limiting examples of suitable AT classes include: the classical ATs (Va), where the translocator that forms a 12-stranded p-barrel in the outer membrane, and a mostly p-helical passenger, are part of one polypeptide (e.g. Hbp, Tsh, EspC, IgA protease, Pet, EspP, EstA, Ag43, AIDA-I, IcsA, VacA); trimeric ATs (Vc), which require three polypeptides to constitute a full 12-stranded -barrel translocator to secrete the passengers which includes a coiled-coil stalk and p-helica I head regions (YadA, BadA, and Hia).; patatin-like Ats (Vd), with similar domain architecture to Va but where the translocator is a 16- stranded p-barrel that contains a POTRA domain (e.g. PIpD); inverse Ats (Ve), which comprise an inverted domain organization with an N-terminal signal sequence followed by the translocator, then the linker and a C-terminal passenger Intimin, Invasin); and Hop-family Ats (Vf) possessing an interrupted p-barrel translocator where the passenger is inserted in the loop joining the 1st and second p-strands, and therefore resembling a prolonged loop protruding from the 8-stranded p-barrel (see Clarke et al. Front Immunol. 2022 Jul 1;13:921272). Particularly useful the autotransporter proteins in the present context are classical autotransporters, such as serine protease autotransporters of the Enterobacteriacea ('SPATE') such as described in Yihfen et al., Trends in Microbiol. (2008) 16(8): 370-9. The skilled person appreciates that it would be possible to utilize N-terminally truncated autotransporters, such that at least part of the passenger domain is truncated, such as the entire passenger domain is deleted. To such a truncated autotransporter the complementary moiety can be fused. It is also possible that the entire passenger domain can be replaced by a heterologous moiety and still accomplish display. The skilled person appreciate that the complementary moiety may be fused to such a heterologous moiety. It may be preferred to keep at least the linker domain of said autotransporter intact in order to realize display.

[0034] Thus in one embodiment, the AT protein is an N-terminally truncated AT protein. In one embodiment, the AT fusion protein comprises an N-terminally truncated AT protein and at least one complementary moiety of the coupling system as defined herein.

[0035] In one embodiment, said autotransporter protein is a serine protease autotransporter of the Enterobacteriaceae (SPATE), such as a SPATE protein from Escherichia coli. SPATE proteins contain a highly conserved channel-forming C- terminal domain, which facilitates secretion of the passenger domain to the cell surface and autoproteolytic cleavage, which releases the passenger from the bacterial cell. The passenger folds into a characteristic beta stem structure with an N-terminal globular domain that performs serine proteolytic activity and on the cell surface, the passenger of a SPATE is cleaved from the translocator and released into the extracellular environment. Cleavage occurs in a conserved site located between two consecutive asparagine residues in the linker region joining the passenger and the translocator domains.

[0036] In one embodiment as disclosed herein, said SPATE is selected from the group consisting of hemoglobin-binding protease (Hbp) from E. coli, EspP from E. coli, Pet from E. coli, EspC from E. coli, Tsh from E. coli, Hap from Haemophilus influenzae, Hap from Neisseria gonorrhoeae, Hap from Neisseria meningitidis, IgA protease from Haemophilus influenzae, IgA protease from Neisseria gonorrhoeae, IgA protease from Neisseria meningitidis and SepA from Shigella flexneri, any N-terminally truncated variant thereof retaining ability to translocate the complementary moiety to the vesicle outer surface and polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto; such as the group consisting of hemoglobin-binding protease (Hbp) from E. coli, EspP from E. coli, Pet from E. coli, EspC from E. coli, Tsh from E. coli, Hap from Haemophilus influenzae, Hap from Neisseria gonorrhoeae, Hap from Neisseria meningitidis, IgA protease from Haemophilus influenzae, IgA protease from Neisseria gonorrhoeae, IgA protease from Neisseria meningitidis and SepA from Shigella flexneri, any N- terminally truncated variant thereof retaining ability to translocate the complementary moiety to the vesicle outer surface.

[0037] In one embodiment, said SPATE protein is selected from the group consisting of hemoglobin-binding protease (Hbp) comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60, extracellular serine protease (EspC) comprising or consisting of the amino acid sequence as defined in SEQ ID NO:65 and temperature-sensitive hemagglutinin (Tsh) comprising or consisting of the amino acid sequence as defined in SEQ ID NO:66, any N-terminally truncated variant of SEQ ID NQ:60 retaining ability to translocate the complementary moiety to the vesicle outer surface, any N-terminally truncated variant of SEQ ID NO:65 retaining ability to translocate the complementary moiety to the vesicle outer surface, any N-terminally truncated variant of SEQ ID NO:66 retaining ability to translocate the complementary moiety to the vesicle outer surface, and any proteins exhibiting at least such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 any one of said SEQ ID NQ:60, SEQ ID NO:65,SEQ ID NO:66 and said variants thereof; such as wherein the SPATE protein is selected from the group consisting of Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NQ:60, EspC comprising or consisting of the amino acid sequence as defined in SEQ ID NO:65 and Tsh comprising or consisting of the amino acid sequence as defined in SEQ ID NO:66. In one embodiment, said SPATE protein is EspC comprising or consisting of the amino acid sequence as defined in SEQ ID NO:65. In another embodiment, said SPATE protein is Tsh comprising or consisting of the amino acid sequence as defined in SEQ ID NO:66. In a further embodiment, said SPATE protein is selected from the group consisting of Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NQ:60 and any proteins exhibiting at least such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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; such as wherein the SPATE protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60. In one embodiment, said autotransporter protein comprises at least one mutation that prevents cleavage at the autocatalytic cleavage site.

[0038] The term " percent identity", as used throughout the disclosure, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al., (1994) Nucleic Acids Research, 22: 4673-4680). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as percent identity. As the skilled person will realize, the function of any polypeptide, such as of the autotransporter of the present disclosure, is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the disclosure encompasses modified variants of the autotransporter polypeptide, which have retained functional characteristics thereof. In this way, encompassed by the present disclosure is an autotransporter as defined herein comprising an amino acid sequence with 80 percent or greater identity to a polypeptide as defined in the context of the disclosure. For example, it is possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc.) could be exchanged for another amino acid residue from the same functional group. It will be understood the above reasoning is equally valid in the context of other proteins and components, such as moieties of the coupling system as disclosed herein and solubility enhancing moieties as disclosed herein, of said method and related aspects, and will not be repeated for the mere sake of brevity. In another embodiment said autotransporter protein comprises a mutation that prevents cleavage at the autocatalytic cleavage site. In another embodiment, said autotransporter protein comprises a mutation that disrupts the autocatalytic cleavage site such that cleavage at the autocatalytic cleavage site is prevented. It is known in the art that said autocatalytic cleavage site of SPATE proteins is part of an invariant motif ("linker domain") in SPATES that attains an alpha helical conformation (said linker domain comprising the amino acid sequence "EVNNLNKRMGDLRD", SEQ ID NO:195), as described by Kostakioti, M. and Stathopoulos, C. Infect. Immun. (2006) 74(9): 4961-4969. In the SPATE protein Hbp said autocatalytic cleavage site is specifically between the two Asparagine residues of the aforementioned motif, namely Asnl048 and Asnl049. In Hbp, Asnl048 and Aspll45 form an unusual catalytic dyad that mediates the autocatalytic cleavage event through the cyclization of the asparagine. A person skilled in the art will understand that mutations, in particular non-conservative, in the linker domain motif or outside this motif that affect, directly or indirectly, the formation of the catalytic dyad between Asnl048 and Aspll45, may also prevent cleavage. For example, mutations affecting the alpha-helical conformation of the linker domain or the positioning of the cleavage site residues in the beta-domain pore may prevent cleavage. In one embodiment, said autotransporter protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60 and further comprises at least one mutation in a position selected from the group consisting of N1048, N1049, K1052, R1053, G1055, L1057, R1069, D1145, K1149, E1179, and E1197, which at least one mutation results in prevention of cleavage at the autocatalytic cleavage site, such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and further comprises at least one mutation in a position selected from the group consisting of N1048G, N1048A, N1048D, N1048S, N1049S, K1052A, R1053A, G1055R, L1057R, and D1145X wherein X is any amino acid other than Glu (E), such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and further comprises at least one mutation in a position selected from the group consisting of N1048G and N1049S, such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NO:60 and further comprises mutations in positions N1048G and N1049S. In one preferred embodiment, said autotransporter protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60 and further comprises mutations in positions N1048G and N1049S. This mutated Hbp corresponds to SEQ ID NO:61 as defined herein. In a further embodiment, said Hbp comprises or consists of the amino acid sequence as defined in SEQ ID NO:61 and any proteins exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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, provided said proteins comprise an autocatalytic cleavage site which has been mutated such that cleavage is prevented.

[0039] As it known to the person skilled in the art, an alternative way to removing of the autocatalytic function by introducing at least one mutation that prevents cleavage at the autocatalytic cleavage site, is the partial or complete deletion of the autocatalytic site. Thus, in one embodiment, the SPATE protein comprises a deletion mutation of the autocatalytic cleavage site, such that the autocatalytic cleavage site is partially or completely deleted. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:62-64 and any proteins exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 any one of said sequences SEQ ID NO:62-64. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence according to SEQ ID NO:62. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence according to SEQ ID NO:63. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence according to SEQ ID NO:64.

[0040] As explained above, the present inventors envision that said at least one complementary binding moiety of said coupling system may be incorporated into the AT protein by one or several of N-terminal fusion to said AT protein; internal incorporation of the said at least one complementary binding moiety into the AT protein; and the replacement of part of the AT protein by said at least one complementary binding moiety of said coupling system. For example, complementary binding moiety of said coupling system may be incorporated at the location of one of the side domains of the passenger domain of the AT protein.

[0041] In one embodiment, said AT fusion protein comprises at least one complementary binding moiety of said coupling system located N-terminally in said AT protein, such as located at the N-terminus of the said AT protein. In one embodiment, said AT fusion protein comprises at least one complementary binding moiety of said coupling system located internally in said AT protein, such as internally in the portion of said AT protein displayed on said vesicle. It is possible to insert or completely or partially replace one or more of the side domains of the AT protein, such as of the SPATE protein, with said at least one complementary binding moiety of said coupling system. Thus, in one embodiment, there is provided a method as disclosed herein, wherein said at least one complementary binding moiety of said coupling system is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein.

[0042] For the sake of clarity, the skilled person is aware of that the above discussion of the in relation to the N-terminal location of said at least one complementary binding moiety of said coupling system, such as located N-terminally or located at the N- terminus of the AT protein, is to be interpreted as relating to the location in the AT protein as displayed on the outer membrane (in other words, relating to located N- terminally of or at the N-terminus of the passenger domain, since the signal peptide is not part of the passenger domain). The skilled person is aware that all AT proteins are synthesized with an N-terminal signal peptide that is cleaved off when passing the inner membrane. In the present context, the displayed AT fusion protein does not comprise the signal peptide as it has been cleaved of during the processing thereof.

[0043] In particular, the Hbp comprises at least five side domains located at amino acid positions 53-308 (domain dl), 533-608 (domain d2), 657-697 (domain d3), 735-766 (domain d4) and 898-992 (domain d5) of said Hbp as defined in SEQ ID NO126:, thus comprising the signal peptide corresponding to amino acid residues 1-52.

[0044] As displayed on the outer membrane, the five side domains are located at amino acid positions 1-256 (domain dl), 481-556 (domain d2), 605-645 (domain d3), 683- 714 (domain d4) and 846-940 (domain d5) of Hbp (SEQ ID NO:60) or HbpD (SEQ ID NO:61) (as described in WQ2019081685). The AT fusion protein used in the present method may comprise a complementary moiety of the coupling system at the location of one of the side domains of said passenger domain.

[0045] The Hbp comprising at least one moiety of a coupling system can be obtained by insertion into at least one side domain or by replacing the whole or part of at least one side domain in the passenger domain with a moiety of the coupling system. Thus, in one embodiment, said at least one complementary binding moiety of said coupling system is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein, such as replaces the whole of at least one of the side domains of the AT protein. Each of the side domains dl, d2, d3, d4 or d5 of the Hbp as defined herein, such as wild-type Hbp (SEQ ID NQ:60) or HbpD (SEQ ID NO:61), or other loops projecting from the beta-stem domain may be used for inserting one of the moieties of the coupling system. In one embodiment, said AT fusion protein is selected from the group consisting of AT fusion proteins comprising or consisting of an amino acid sequence selected from SEQ ID NO:60, SEQ ID NO:61 and any proteins exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 and wherein said at least side domain is selected from side domains located at amino acid positions 1-256 (domain dl), 481-556 (domain d2), 605-645 (domain d3), 683-714 (domain d4) and 846-940 (domain d5), such as wherein said at least side domain is selected from side domains located at amino acid positions 1-256 (domain dl), amino acid positions 481-556 (domain d2) and amino acid positions 683-714 (domain d4), such as wherein said side domain is located at amino acid positions 1-256 (domain dl).

[0046] In one particular embodiment, said AT protein fusion is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 1-256 (domain dl) are replaced by at least one said at least one complementary binding moiety of said coupling system. In one particular embodiment, said AT protein fusion is HbpD comprising the amino acid sequence as defined in SEQ ID NO:61, wherein positions 1-255 of positions 1-256 (domain dl) are replaced by at least one said at least one complementary binding moiety of said coupling system. Preferably a moiety of the coupling system is inserted into or replaces the whole of, such as essentially the whole of, or part of side domain 1, side domain 2 or side domain 4, more preferably side domain 1. Thus, in one embodiment, said AT fusion protein comprises or consist of Hbp as defined herein, wherein a moiety of the coupling system is inserted into or replaces the whole or a part of said at least side domain is selected from the group consisting of a side domain located at amino acid positions 1-256 (domain dl) of SEQ ID NO:61, a domain located at amino acid positions 481-556 (domain d2) of SEQ ID NO:61, a domain located at amino acid positions 605-645 (domain d3) of SEQ ID NO:61, a domain located at amino acid positions 683-714 (domain d4) of SEQ ID NO:61 and a domain located at amino acid positions 846-940 (domain d5) of SEQ ID NO:61.

[0047] In one embodiment, said AT fusion protein comprises or consist of Hbp as defined herein, wherein a moiety of the coupling system is inserted into or replaces the whole or a part of said at least side domain is selected from the group consisting of a side domain located at amino acid positions 1-256 (domain dl) of SEQ ID NO:61, a domain located at amino acid positions 481-556 (domain d2) of SEQ ID NO:61, and a domain located at amino acid positions 683-714 (domain d4) of SEQ ID NO:61.

[0048] In one embodiment, said AT fusion protein comprises or consist of Hbp as defined herein, wherein a moiety of the coupling system is inserted into or replaces the whole or a part of the side domain located at amino acid positions 1-256 (domain dl) of SEQ ID NO:61.

[0049] Thus, in one embodiment, said AT fusion protein comprises or consist of Hbp as defined herein, wherein a moiety of the coupling system is inserted into or replaces the whole or a part of said at least side domain is selected from the group consisting of a side domain located at amino acid positions 1-256 (domain dl) of SEQ ID NQ:60, a domain located at amino acid positions 481-556 (domain d2) of SEQ ID NQ:60, a domain located at amino acid positions 605-645 (domain d3) of SEQ ID NQ:60, a domain located at amino acid positions 683-714 (domain d4) of SEQ ID NQ:60 and a domain located at amino acid positions 846-940 (domain d5) of SEQ ID NQ:60.

[0050] In one embodiment, said AT fusion protein comprises or consist of Hbp as defined herein, wherein a moiety of the coupling system is inserted into or replaces the whole or a part of said at least side domain is selected from the group consisting of a side domain located at amino acid positions 1-256 (domain dl) of SEQ ID NQ:60, a domain located at amino acid positions 481-556 (domain d2) of SEQ ID NQ:60, and a domain located at amino acid positions 683-714 (domain d4) of SEQ ID NQ:60. In one embodiment, said AT fusion protein comprises or consist of Hbp as defined herein, wherein a moiety of the coupling system is inserted into or replaces the whole or a part of the side domain located at amino acid positions 1-256 (domain dl) of SEQ ID NO:60. In one embodiment, SEQ ID NO:60 further comprises at least one mutation preventing cleavage at the autocatalytic cleavage site thereof.

[0051] In one embodiment, said at least one complementary binding moiety of said coupling system replaces amino acid residues 1-255 of SEQ ID NO:61. In other words, said at least one complementary binding moiety replaces part of domain dl. In other words, said at least one complementary binding moiety replaces essentially whole domain dl.

[0052] In one embodiment, said at least one complementary binding moiety of said coupling system replaces amino acid residues 482-555 of SEQ ID NO:61. In other words, said at least one complementary binding moiety replaces part of domain d2. In other words, said at least one complementary binding moiety replaces essentially whole domain d2.

[0053] In one embodiment, said at least one complementary binding moiety of said coupling system replaces amino acid residues 708-712 of SEQ ID NO:61. In other words, said at least one complementary binding moiety replaces part of domain d4. In one embodiment, said AT fusion protein comprises two complementary moieties, which may be the same or different. In one embodiment, said AT fusion protein comprises two complementary moieties inserted into or replacing the whole of or part of domain 1 and domain 2 as defined herein. In one embodiment, said AT fusion protein comprises two complementary moieties inserted into or replacing the whole of or part of domain 1 and domain 4 as defined herein.

[0054] In one particular embodiment, said one complementary moiety is flanked by linker or spacers. Without being bound, it is considered that linkers aid in providing optimal exposure of the complementary moiety improving accessibility for binding of the coupling moiety thereto. In one particular embodiment, said one complementary moiety is flanked by linkers or spacers having a length of 3 to 10 amino acid residues. In one embodiment, said linkers or spacers have a length of 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. In one embodiment, said linkers or spacers have a length of 5 amino acid residues. In one embodiment, said linkers or spacers have a length of 10 amino acid residues. In one embodiment, said linkers or spacers are selected from the group consisting of GSS, GGGGS (SEQ ID NO:175), QQQQG (SEQ ID NO:176), GSGSS (SEQ ID NO:177), GSGSG (SEQ ID NO:178), SSSSG (SEQ ID NO:179), GS15G (SEQ ID NQ:180), GSGSSGSASG (SEQ ID NO:181), GEGTGGSGSG (SEQ ID NO:182), GSGSSGSGTS (SEQ ID NO:183), GSSGSGSGSG (SEQ ID NO:184), (G3S)3(SEQ ID NO:185), (G3S)4(SEQ ID NO:186), (G3S)5(SEQ ID NO:187), (G3S)6(SEQ ID NO:188), (G4S)3(SEQ ID NO:189), (G4S)4(SEQ ID NQ:190), (G4S)5(SEQ ID NO:191), (G5S)2(SEQ ID NO:192), (G5S)3(SEQ ID NO:193), (G5S)4(SEQ ID NO:194), (G4S)2(SEQ ID NO:234) and GTGGSG (SEQ ID NO:325). ). In particular, in one embodiment, said linkers or spacers are selected from the group consisting of GSGSS (SEQ ID NO:177), GSGSG (SEQ ID NO:178), GSS, GSGSSGSASG (SEQ ID NO:181), GEGTGGSGSG (SEQ ID NO:182), GSGSSGSGTS (SEQ ID NO:183), GSSGSGSGSG (SEQ ID NO:184) ), (G4S)2(SEQ ID NO:234) and GTGGSG (SEQ ID NO:325). In particular, in one embodiment, said linkers or spacers are selected from the group consisting of GSGSS (SEQ ID NO:177), GSGSG (SEQ ID NO:178), GSS, GSGSSGSASG (SEQ ID NO:181), GEGTGGSGSG (SEQ ID NO:182), GSGSSGSGTS (SEQ ID NO:183) and GSSGSGSGSG (SEQ ID NO:184).

[0055] In one embodiment, said AT fusion protein is selected from the group consisting of AT fusion proteins comprising or consisting of SEQ ID NO:68, 119-125 and 150-157, such as the group consisting of SEQ ID NO:68 and 119, or the group consisting of SEQ ID NQ:120 and 121, or the group consisting of SEQ ID NO: 122 and 123. In one embodiment, said AT fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:68, 120 and 122. In one embodiment, said AT fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:119, 121 and 123. For example, an AT protein comprising SEQ ID NO:67 is a Hbp protein wherein positions corresponding to positions in 1-255, such as in 2-255, of SEQ ID NO:61 have been deleted. In one embodiment, said AT fusion proteins comprises or consists of amino acid SEQ ID NO:68. In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 683-714 (domain d4) are replaced, such as partially or completely replaced, by at least one said at least one complementary binding moiety of said coupling system. In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein said at least one complementary binding moiety of said coupling system has been inserted into domain d4 as defined herein. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence has been inserted into domain d4 as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NO:124 or SEQ ID NO:155 or SEQ ID NO:157.

[0056] In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 481-556 (domain d2) are replaced, such as partially or completely replaced, by at least one said at least one complementary binding moiety of said coupling system. In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein said at least one complementary binding moiety of said coupling system has been inserted into domain d2 as defined herein. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence, such as two flanking linker sequences, have been inserted into domain d2 as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NQ:150 or SEQ ID NO:156.

[0057] In one embodiment, said AT fusion protein comprises a plurality of complementary moieties of said coupling system, such as at least two, such at least three, such as at least four, such as at least five, complementary moieties of said coupling system. Said plurality of complementary moieties may be inserted into or replace, partially or completely, any one of or all of side domains dl, d2, d3, d4 or d5 of Hbp as discussed above. In particular embodiments, wherein said at least two, such as at least three, such as at least four, such as at least five, such as all, of said plurality of complementary moieties of said coupling system comprise the different amino acid sequences.

[0058] In one embodiment, said AT fusion protein comprises a plurality of complementary moieties of said coupling system, such as at least two, such at least three, such as at least five, complementary moieties of said coupling system. In one particular embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 1-256 (domain dl) and positions 683-714 (domain d4) are partially or completely replaced by at least one complementary binding moiety of said coupling system. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence, such as two flanking linker sequences, have been inserted into said domains as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NO:125 or SEQ ID NO:151 or SEQ ID NO:154.

[0059] In one particular embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 1-256 (domain dl) and positions 481-556 (domain d2) are partially or completely replaced by at least one complementary binding moiety of said coupling system. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence, such as two flanking linker sequences, have been inserted into said domains as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NO:152 or SEQ ID NO:153.

[0060] Utilizing a coupling system during polypeptide or protein synthesis to fuse various moieties and parts together to form a final complex or product can be advantageous when compared to, for example, designing a polypeptide or protein which is synthesized as one contiguous sequence, or as "one unit". A coupling system, comprising at least a first and a second molecular part or "moiety" which have affinity for each other and between which a bond may be formed allows for great flexibility when synthesizing the final complex or product. Utilizing a coupling system can allow the user to keep synthesized parts of a final complex separate until it is desired that they are combined and bonded to each other. For example, a protein of interest (POI) comprising a first molecular part or "moiety" of the coupling system, can be synthesized separately from a vesicle comprising the second molecular part or "moiety" of the coupling system, to be later combined and allowed to interact and bond via the respective coupling moieties and form the final complex or product. Particularly, this could allow for mass-production of certain parts while other parts can be tailored, for example in the case of personalized medicine. In context of the present disclosure, the vesicle comprising the autotransporter fusion protein with one moiety of the coupling system, could be mass produced while the POI, comprising the other moiety of the coupling system and the target polypeptide moiety, could be tailored for individual patients, for example in the case of cancer vaccines or the like. In the context of the present method, the vesicles provided in step c) can be prepared in advance and stored until needed. The opposite could also be true, that it is desired that the various parts which make up the final complex or product are synthesized and coupled in realtime. This might be advantageous in cases where one part of the final complex, for example a vesicle comprising the autotransporter fusion protein with one part of the coupling system, acts as a stabilizing platform for the other part of the final complex, like the POI, comprising the other part of the coupling system and the target polypeptide moiety. In cases where the target polypeptide moiety is unstable, toxic, aggregation-prone, or the like, synthesizing the POI while also coupling it to the vesicle may prevent aggregation of the POI, allowing for successful synthesis of the final product. In other words, the utilization of a coupling system, such as in accordance with the method as disclosed herein, can have many and varying advantages depending on the nature of the final product to be created. The present inventors utilize such a coupling system to enable successful and efficient product of desired polypeptides, referred to herein as target polypeptides. In particular, such target polypeptides which are known in the field to be difficult to produce by methods known in the art.

[0061] As explained in more detail below, the term "coupling system" used herein refers to a system comprising at least two, such as two or three, polypeptide moieties (or units) which are capable of forming at least one isopeptide bond between each other. As used herein the terms "coupling moiety" and "complementary moiety", refer individually to the polypeptide moieties (or units) which together are able to form an isopeptide bond between each other.

[0062] For the sake of clarity, the coupling system comprises at least one coupling moiety and a complementary moiety. The designation "coupling moiety" and "complementary moiety" as used herein are to be understood to be merely for the purpose of indicating the interrelation between said moieties, such that the coupling moiety is compatible with its complementary moiety for the formation of an isopeptide bond between said moieties. For example, if a specific coupling system comprises the moieties A and B, then the specific moiety, herein referred to A may be equally well designated a coupling moiety as a complementary moiety and will be compatible with B, which is its complementary moiety or coupling moiety, respectively (depending on the designation chosen for A). In the present context, the designation coupling moiety or complementary moiety may merely be due which of the coupling moiety and complementary moiety is first mentioned, for example in the context of the method of the first aspect as disclosed herein. For example, in the context of reciting specific coupling systems, such as the system C-D, if C is denoted coupling moiety, then D is denoted the complementary moiety and inversely if D is denoted coupling moiety, then C is the denoted complementary moiety. As illustrated by the specific example of SpyTag-SpyCatcher (SEQ ID NO:10- SEQ ID NO:11), herein SpyTag is denoted coupling moiety, then SpyCatcher is the complementary moiety and inversely if SpyCatcher is denoted coupling moiety, then SpyTag is the complementary moiety. In one embodiment, said coupling system is derived from Gram positive bacterial pilus proteins, such as derived from Gram positive bacterial pilus proteins selected from the group consisting of Lactiplantibacillus, Bacillus, Ruminococcus, and Streptococcus, such as preferably derived from Gram positive Streptococcal pilus proteins. In one embodiment, said coupling system is derived from a Gram positive Streptococcal pilus protein selected from the group consisting of the major pilin protein Spy0128 of Streptococcus pyogenes , the fibronectin binding protein FbaB of Streptococcus pyogenes, the fibronectin-binding protein from Streptococcus dysgalactiae, the pilus-subunit RrgA from Streptococcus pneumoniae, and the pilus subunit RrgC from Streptococcus pneumoniae.

[0063] In one embodiment, said coupling moiety and complementary moiety of the coupling system are selected from the group consisting of SnoopTag2 (SEQ ID NO:7), DogTag2 (SEQ ID NO:8), SnoopLigase2 (SEQ ID NO:9), SpyTag (SEQ ID NQ:10), SpyCatcher (SEQ ID NO:11), SnoopTag (SEQ ID NO:12), SnoopCatcher (SEQ ID NO:13), SpyTag002 (SEQ ID NO:14), SypCatcher002 (SEQ ID NO:15), SpyTag003 (SEQ ID NO:16), SpyCatcher003 (SEQ ID NO:17), SdyTag (SEQ ID NO:18), SdyCatcher (SEQ ID NO:19), SilkTag (SEQ ID NQ:20), SilkCatcher (SEQ ID NO:21), DogTag (SEQ ID NO:22), DogCatcher (SEQ ID NO:23), SnoopTagJr (SEQ ID NO:24), SnoopLigase (SEQ ID NO:25), Jo (SEQ ID NO:26), In (SEQ ID NO:27), NGTag (SEQ ID NO:28), NGCatcher (SEQ ID NO:29), SpyCatcher-N (SEQ ID NQ:30), SpyCatcher-21 (SEQ ID NO:31), SpyStapler (SEQ ID NO:32), SpyLigase (SEQ ID NO:33), MoonCake (SEQ ID NO:34), Katl (SEQ ID NO:35), QueenCatcher (SEQ ID NO:36), PsCsCatcher (SEQ ID NO:37), Ktag (SEQ ID NO:38), BDTag (SEQ ID NO:39), RumTrunkTagD9N (SEQ ID NQ:40), RumTrunkTag (SEQ ID NO:41), RumTag (SEQ ID NO:42), Rum2Tag (SEQ ID NO:43), Rum3Tag (SEQ ID NO:44), Rum4Tag (SEQ ID NO:45), Rum5Tag (SEQ ID NO:46), Rum6Tag (SEQ ID NO:47), Rum7Tag (SEQ ID NO:48), BacTag (SEQ ID NO:49), Bac2Tag (SEQ ID NQ:50), Bac3Tag (SEQ ID NO:51), Bac4Tag (SEQ ID NO:52), Bac5Tag (SEQ ID NO:53), Clib9 (SEQ ID NO:54), PhoTag (SEQ ID NO:55), PsCsTag (SEQ ID NO:56), and SpyCatcher (SEQ ID NO:57), and variants thereof exhibiting at least 70 % identity thereto, wherein said moieties are selected such that said moieties have the capacity of forming an isopeptide bond between each other, and wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70 % identity thereto. In one embodiment said variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively. In one embodiment, said a variant of a coupling moiety or of a complementary moiety exhibits at least 71 %, such as at least 72 %, such as at least 73 %, such as at least 74 %, such as at least 75 %, such as at least 76 %, such as at least 77 %, such as at least 78 %, such as at least 79 %, such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 said coupling moiety or said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 71 %, such as at least 72 %, such as at least 73 %, such as at least 74 %, such as at least 75 %, such as at least 76 %, such as at least 77 %, such as at least 78 %, such as at least 79 %, such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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. In one embodiment, said a variant of a coupling moiety or of a complementary moiety exhibits at least 71 %, such as at least 72 %, such as at least 73 %, such as at least 74 %, such as at least 75 %, such as at least 76 %, such as at least 77 %, such as at least 78 %, such as at least 79 %, such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 said coupling moiety or said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively.

[0064] A "coupling system" is a system comprising at least a first and a second molecular part or "moiety" which have affinity for each other and between which a bond of some sort may be formed. A coupling system may for example consist of two moieties between which a covalent bond may form spontaneously or with the assistance of a moiety with catalytic activity, such as an enzyme, or where chemical crosslinking means are used to link different molecules together. As used in the context of the present diclosure, the term "coupling system" refers to a system comprising at least two, such as two or three, polypeptide moieties (or units) which are capable of forming at least one isopeptide bond between each other. Said polypeptide moieties (or units) are referred to as "coupling moiety" and "complementary moiety". Thus, a "coupling moiety" and "complementary moiety" are capable of forming at least one isopeptide bond between each other. An isopeptide bond is a type of amide bond formed between a carboxyl group of one amino acid and an amino group of another. An isopeptide bond is the linkage between the side chain amino or carboxyl group of one amino acid to the a- carboxyl, a-amino group, or the side chain of another amino acid. Thus, as used herein the terms "coupling moiety" and "complementary moiety", refer individually to the two polypeptide moieties (or units) which together are able to form an isopeptide bond between each other. In the present disclosure, a coupling system specifically refers to a coupling moiety and a complementary moiety that have the functionality to spontaneously form an isopeptide bond or that are able to form an isopeptide bond in the presence of a moiety with catalytic activity, such as an enzyme (e.g. a ligase, such as SpyLigase). It is further possible to link additional molecules to each of the two moieties, creating a complex wherein it is the two moieties with affinity for each other which constitute the ligate of the coupling system.

[0065] In the current disclosure, one of the two moieties of the coupling system is referred "coupling moiety" and the other moiety of the coupling system is referred to as "complementary moiety", wherein the coupling moiety is comprised in a polypeptide of interest (POI) and the complementary moiety may optionally link an additional molecule, such as a vesicle, to the entire complex. Thus, the term "complex" is used herein to describe a POI comprising a coupling moiety and coupled to the complementary moiety via a covalent isopeptide bond between the coupling moiety and the complementary moiety. It should be noted that equally well, the two moieties of the coupling system may be referred inversely; such that one of the two moieties of the coupling system is referred "coupling moiety" and the other moiety of the coupling system is referred to as "complementary moiety", wherein the complementary moiety is comprised in a polypeptide of interest (POI) and the coupling moiety may optionally link an additional molecule, such as a vesicle, to the entire complex. As used herein, the term "complementary coupling moiety" and "complementary moiety" are used interchangeably in the context of the coupling system as discussed above.

[0066] An isopeptide bond is a very robust covalent bond, providing a strong connection between the parts of the coupling system, and anything else linked to the respective moieties.

[0067] As mentioned, the term "coupling moiety" is used herein to describe one of the two moieties constituting the coupling system. More specifically, a coupling moiety is a polymer of amino acids having the distinct functionality of binding the other moiety of the coupling system, i.e. the "complementary moiety". In the context of the invention, the coupling moiety is comprised in a POL In other words, said POI construct further comprising at least one target polypeptide moiety. For the purpose of expression of said POI, a construct encoding said POI may be incorporated into a vector and subsequently expressed to produce a recombinant polypeptide comprising a coupling moiety accessible to the complementary moiety of the coupling system in a cell-free production method as disclosed herein.

[0068] Typically, the coupling moiety comprises one residue involved in the isopeptide bond while the complementary moiety comprises the other residue involved in the isopeptide bond. For example, when the coupling moiety comprises a reactive lysine residue, the complementary moiety comprises a reactive asparagine, aspartic acid, glutamine or glutamic acid residue, or when the coupling moiety comprises a reactive asparagine, aspartic acid, glutamine or glutamic acid residue, the complementary moiety comprises a reactive lysine residue or a reactive alphaamino terminus. Thus, the coupling moiety may comprise a reactive asparagine residue while the complementary moiety may comprise a reactive lysine residue, or the coupling moiety may comprise a reactive lysine residue while the complementary moiety may comprise a reactive asparagine residue.

[0069] Hence, by utilizing a coupling system by designing a POI comprising a coupling moiety with affinity for a complementary moiety, wherein said complementary moiety is in turn displayed on the surface of a vesicle, optionally via means of an autotransporter fusion, the inventors have created a novel and inventive method for the cell-free production method for producing a target polypeptide moiety as disclosed herein.

[0070] The term "complementary moiety" refers to the other moiety of the coupling system as defined in the context of the invention. It is a polymer of amino acids having the distinct functionality of binding the first part of the coupling system, i.e. the "coupling moiety". Unlike the coupling moiety, the complementary moiety is typically produced unattached to a POI. Optionally, it may be expressed in fusion to or in a complex with an additional molecule. While the parts of the coupling systems described herein are referred to as "peptides", it will be appreciated that they may, in some embodiments, comprise more than 50 amino acids.

[0071] The terms "SpyTag-SpyCatcher coupling system", "SpyTag" and "SpyCatcher" describe the first part (coupling moiety) and second part (complementary moiety), respectively, of a particular coupling system which may be used in the current disclosure. The coupling system is derived from a CnaB domain present in the Streptococcus pyogenes fibronectin-binding protein FbaB. Within the hydrophobic core of this domain, a triad of amino acids (lysine, aspartate and a catalytic glutamate) spontaneously form an isopeptide bond (Hagan et al. 2010 Angew Chem Int Ed Engl Nov 2;49(45):8421-5). The isolated CnaB domain was converted into a protein coupling system by splitting it into a peptide, the so called "SpyTag", sometimes abbreviated "SpT", and the remaining protein partner called the "SpyCatcher", sometimes abbreviated "SpC" (Zakeri et al., Proc Natl Acad Sci USA (2012) 109(12):E690-7). The two peptides possess the ability to spontaneously form an isopeptide bond between each other. In the context of the invention, the SpyTag (coupling moiety) and the target polypeptide moiety comprised in a POI may form a recombinant polypeptide wherein the SpyTag acts as the coupling moiety and is accessible to the complementary moiety. The SpyCatcher acts as the complementary moiety and may form a robust bond to the SpyTag.

[0072] The term "SnoopTag-SnoopCatcher coupling system", "SnoopTag" and "SnoopCatcher" refer to the first part (coupling moiety) and second part complementary moiety) respectively of another coupling system, which has been derived from the D4 Ig-like domain of the adhesin RrgA from Streptococcus pneumoniae (Veggiani et al. Proc Natl Acad Sci USA (2016) 113(5):1202-7). To create this system, the D4 Ig-like domain was cleaved to create a coupling moiety called "SnoopTag", sometimes abbreviated "SnT", and a remaining complementary moiety "SnoopCatcher", sometimes abbreviated "SnC". In the context of the invention, the SnoopTag (coupling moiety) and the target polypeptide moiety comprised in a POI may form a recombinant polypeptide, wherein the SnoopTag is accessible to the complementary moiety. The terms "KTag" and "SpyLigase" refer to a peptide tag and an enzyme, respectively. After the successful adaptation of the CnaB domain into the SpyTag / SpyCatcher protein coupling system, the SpyCatcher was further split up into the "KTag", sometimes abbreviated "KT", and "SpyLigase" (Fierer et al., Proc Natl Acad Sci USA (2014) 111(13):E1176-81). KTag acts as a coupling moiety which may form a covalent bond with a complementary moiety in the presence of SpyLigase, which is needed to catalyze the bond formation. It has also been discovered that, in the presence of SpyLigase, both KTag and SpyTag may alternate between acting as a coupling moiety and acting as a complementary moiety. However, SpyLigase remains a polypeptide separate from the coupling system or may remain associated with the complex in non-covalent fashion upon bond formation. This type of coupling system, wherein there are more than two moieties (such as a coupling moiety and a complementary moiety) are referred to as a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first (the coupling moiety) and second (the complementary moiety) moieties are capable of being isopeptide bonded by catalytic means of the third moiety (such as SpyLigase; SEQ ID NO:33). Other tripartite coupling systems exist, wherein the same logic is applied. Examples of such tripartite coupling systems are KTag-SpyTag (SEQ ID NO:38-SEQ ID NO:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SpyTag- BDTag (SEQ ID NQ:10-SEQ ID NO:39), DogTag2-SnoopTagJr (SEQ ID NO:8-SEQ ID NO:24), and wherein the tripartite system further comprises a third moiety which is a ligase. Examples of such ligases are as mentioned above SpyLigase (SEQ ID NO:33), but also SpyStapler (SEQ ID NO:32), SnoopLigase (SEQ ID NO:25), and SnoopLigase2 (SEQ ID NO:9).

[0073] In one embodiment, said coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11), SpyTag- SpyCatcher002 (SEQ ID NQ:10-SEQ ID NO:15), SpyTag-SpyCatcher003 (SEQ ID NQ:10- SEQ ID NO:16), SpyTag-SpyCatcher-N (SEQ ID NQ:10-SEQ ID NQ:30), SpyTag- SpyCatcher-21 (SEQ ID NQ:10-SEQ ID NO:31), SpyTag-SdyCatcher (SEQ ID NQ:10-SEQ ID NO:19), SpyTag002-SpyCatcher002 (SEQ ID NO:14-SEQ ID NO:15), SpyTag002- SpyCatcher (SEQ ID NO:14-SEQ ID NO:11), SpyTag002-SpyCatcher003 (SEQ ID NO:14- SEQ ID NO:17), SpyTag002-SdyCatcher (SEQ ID NO:14-SEQ ID NO:19), SpyTag003- SpyCatcher003 (SEQ ID NO:16-SEQ ID NO:17), SpyTag003-SpyCatcher (SEQ ID NO:16- SEQ ID NO:11), SpyTag003-SpyCatcher002 (SEQ ID NO:16-SEQ ID NO:15), SpyTag003- SdyCatcher (SEQ ID NO:16-SEQ ID NO:19), SnoopTag-SnoopCatcher (SEQ ID NO:12- SEQ ID NO:13), Sil kTag-Sil kCatcher (SEQ ID NQ:20-SEQ ID NO:21), DogTag- DogCatcher (SEQ ID NO:22-SEQ ID NO:23), DogTag2-DogCatcher (SEQ ID NO:8-SEQ ID NO:23), Jo-In (SEQ ID NO:26-SEQ ID NO:27), NGTag-NGCatcher (SEQ ID NO:28-SEQ ID NO:29), SdyTag-SdyCatcher (SEQ ID NO:18-SEQ ID NO:19), SdyTag-SpyCatcher (SEQ ID NO:18-SEQ ID NO:11), SdyTag-SpyCatcher002 (SEQ ID NO:18-SEQ ID NO:15), SdyTag-SpyCatcher003 (SEQ ID NO:18-SEQ ID NO:17), KTag-SpyTag (SEQ ID NO:38- SEQ ID NQ:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SnoopTag2- SnoopCatcher (SEQ ID NO:7-SEQ ID NO:13), DogTag2-SnoopCatcher (SEQ ID NO:8- SEQ ID NO:13), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), MoonCake- RumTrunkTagD9N (SEQ ID NO:34-SEQ ID NQ:40), MoonCake-RumTag (SEQ ID NO:34- SEQ ID NO:42), MoonCake-SpyTag (SEQ ID NO:34-SEQ ID NQ:10), MoonCake-SdyTag (SEQ ID NO:34-SEQ ID NO:18), Katl-SpyTag (SEQ ID NO:35-SEQ ID NQ:10), Katl- SdyTag (SEQ ID NO:35-SEQ ID NO:18), Katl-RumTrunkTagD9N (SEQ ID NO:35-SEQ ID NQ:40), Katl-RumTag (SEQ ID NO:35-SEQ ID NO:42), and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, and exhibits at least 70 % identity thereto, such as a variant selected from the group consisting of SEQ ID NQ:10-19, 22-25, 34 and 38-40 and variants exhibits at least 70 % identity thereto. In one embodiment, said at least 70 % identity is as at least 71 %, such as at least 72 %, such as at least 73 %, such as at least 74 %, such as at least 75 %, such as at least 76 %, such as at least 77 %, such as at least 78 %, such as at least 79 %, such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %.

[0074] A coupling system may also be a tripartite system and such tripartite system may be used in the context of the present invention as discussed above. Thus, in one embodiment, said coupling system is a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first and second moieties are capable of being isopeptide bonded by means of the third moiety. In another embodiment, said tripartite system comprises a first and second moiety selected from the group consisting of KTag-SpyTag (SEQ ID NO:38-SEQ ID NO:10), DogTag- SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), DogTag2-SnoopTagJr (SEQ ID NO:8-SEQ ID NO:24), and wherein the tripartite system further comprises a third moiety which is a catalytic moiety. In one embodiment, said catalytic moiety is a ligase. Suitable ligases which can act as a third moiety that catalyzes isopeptide bonding between the first and the second moiety of the tripartite coupling system are ligases such as SpyLigase (SEQ ID NO:33), but also SpyStapler (SEQ ID NO:32), SnoopLigase (SEQ ID NO:25), and SnoopLigase2 (SEQ ID NO:9).

[0075] In one embodiment, said coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70 % identity thereto, such as wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively.

[0076] In one embodiment, said at least 70 % identity is as at least 71 %, such as at least 72 %, such as at least 73 %, such as at least 74 %, such as at least 75 %, such as at least 76 %, such as at least 77 %, such as at least 78 %, such as at least 79 %, such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 % .

[0077] In one embodiment, said coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70 % identity thereto, such as wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively. In one embodiment, said coupling system is SpyTag- SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11).

[0078] In one embodiment, said coupling system is selected from the group consisting of SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70 % identity thereto, such as wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively. In one said coupling system is SnoopTag- SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13).

[0079] As discussed above, the present disclosure provides a method for the cell free production of various polypeptides, including such polypeptides whereof production is connected with difficulties known in the art. Solubility of many proteins is low and affects recombinant production of proteins, formulation of products and their stability. Two processes are related to soluble and solid phase relations. Solubility refers to the process where proteins have correctly folded structure, whereas aggregation is related to the formation of fibrils, oligomers or amorphous particles. When producing a recombinant protein, several problems can occur, including proteolysis, incorrect folding, formation of inclusion bodies, or protein aggregation, whereby the protein loses or does not attain its natural structure. In particular such difficulties may be associated with the formation of protein aggregates of the desired proteins, for example aggregates that result in precipitation of the desired proteins.

[0080] Typically, it is desirable to obtain the desired protein at a high rate synthesis, high host cell density and, most importantly, high product quality. Reducing the cost of protein production is an important goal in the development of pharmacotherapy and bio-industrial processes. The present inventors have surprisingly found that the cell-free production method as disclosed herein, offers solutions for many of said problems for a wide variety of proteins and polypeptides, which pose challenges in production. As shown in the appended Examples, and as generally disclosed herein, the present method provides for example for the successful protein production, for example production of neoepitopes in tandem fusions and even production of aggregation prone proteins. As used herein, the terms "polypeptide" in general and "target polypeptide moiety" in the context of a protein of interest (POI) as defined herein refer to a molecule comprising at least 3 amino acid residue coupled via means of peptide bonds. Thus, the term encompasses oligopeptides comprising three or more amino acid residues, polypeptides as well as proteins. Non-limiting examples of target polypeptide moieties that can be successfully produced by the present method include aggregation-prone polypeptides and proteins; insoluble polypeptides and proteins; disordered proteins; misfolded proteins; hydrophobic polypeptides and proteins; and self-associating polypeptides and proteins. It is known in the art, that such polypeptides and proteins are difficult to produce in vitro. In the context of the inventive method as disclosed herein, the coupling of the POI comprising the target polypeptide moiety to the vesicle, prevents one or several of unwanted aggregation, misfolding and / or self-association of the target polypeptide moieties.

[0081] Thus, in one embodiment of the present aspect, there is provided a method as disclosed herein, wherein said at least one target polypeptide moiety is selected from the group consisting of aggregation-prone polypeptides and proteins; insoluble polypeptides and proteins; disordered proteins; misfolded proteins; hydrophobic polypeptides and proteins; and self-associating polypeptides and proteins; such as the group consisting of aggregation-prone polypeptides and proteins; and insoluble polypeptides and proteins.

[0082] As used herein, the term "aggregation-prone polypeptides and proteins" refers to proteins which have a tendency to form aggregates in vivo or in vitro, such as during standard protein production conditions. Protein aggregation is a phenomenon in which intrinsically-disordered or mis-folded proteins aggregate. Many protein sequences contain one or several short aggregation prone regions (APR) that can nucleate protein aggregation. Under normal in vivo conditions these APRs are protected from aggregation by protein interactions or because they are buried in the hydrophobic core of native protein domains. However, during recombinant protein production such APRs may be exposed either due to production conditions or due to lack of interaction partner, resulting in unwanted aggregate formation. The skilled person is familiar with the concept aggregation prone proteins.

[0083] Many proteins are insoluble in common solvents, for example membrane proteins which represent 20-30% of the proteins encoded by the human genome. Another example of insoluble proteins are inclusion body forming proteins and polypeptides. In one embodiment, said aggregation-prone polypeptides and proteins are amyloid- forming polypeptides and proteins. Amyloids are aggregates of proteins characterized by a fibrillar morphology of typically 7-13 nm in diameter and a |3- sheet secondary structure. Proteins may form amyloids when a segment exposes its backbone amide N-H groups and C=O groups, permitting them to couple into hydrogen bonds with other protein chains. Several conditions result in exposed backbone amide groups, for example the denaturation of a normally folded proteins, over expression of a protein that overwhelms cellular chaperones and drives it into inclusion bodies, cleavage of a peptide (such as of amyloid |3) from a folded protein; or over production of a natively disordered protein (such as tau). Thus, production of amyloid-forming polypeptides and proteins is challenging using methods known in the art.

[0084] Recent advances in cancer therapy have focused on the identification of neoantigens, which are tumor-specific antigens derived from cancer-specific somatic mutations or gene fusions that have been shown to be crucial regulators of the clinical response to immunotherapy. Examples of neoantigen-targeting therapies include cancer vaccines and T-cell therapies. However, the production of neoantigens for the purpose of therapy may also be challenging, in particular wherein it is desirable to produce several neoantigen, or neoantigen epitopes, in one fusion polypeptide. Neoantigen epitopes may become insoluble when expressed in isolation, out of the context of the native protein. In particular this is a problem when multiple such epitopes are fused in tandem, without any native context, which in turn leads to a disorder. Moreover, there is in some instances a bias toward hydrophobic amino acids within immunogenic epitopes, which may lead to aggregation, particularly when fused in tandem.

[0085] As illustrated in the appended Examples, the present invention allows for the successful production target polypeptide moieties comprising multiple tandem- fused neoantigen epitopes. In another embodiment, said target polypeptide moiety is a polypeptide comprising at least one neoantigen derived epitope, such as wherein the target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes, such as multiple tandem-fused neoantigen derived epitopes. In particular embodiments, said target polypeptide moiety is a polypeptide comprising multiple tandem-fused neoantigen derived epitopes. Said neoantigen derived epitopes may be directly fused together, or all or a subset thereof may comprise additional amino acid residues between the individual neoantigen derived epitope units. Said additional amino acid residues may be spacers or linkers as defined herein. For example, said at least two, such as at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight, such as at least nine, such as at least ten or more of the neoantigen derived epitopes may be separated by spacers, such as wherein all of said neoantigen derived epitopes are separated by spacers. It may also be that said at least two, such as at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight, such as at least nine, such as at least ten or more of the neoantigen derived epitopes are directly fused to each other, such as wherein all of said neoantigen derived epitopes are directly fused to each other. In some embodiment, a subset of said neoantigen derived epitopes of the target polypeptide moiety are separated by spacers or linkers and a subset of said neoantigen derived epitopes of the target polypeptide moiety are directly fused to each other. It is furthermore possible that the target polypeptide moiety comprises a unique neoantigen derived epitope. It is also possible that the target polypeptide moiety comprises several identical neoantigen derived epitopes. Thus, in one embodiment, said target polypeptide moiety comprises two or more identical neoantigen derived epitopes. In another embodiment, said target polypeptide moiety does not comprise more than one copy of each neoantigen derived epitope. It is considered that it may be beneficial to include multiple neoantigen derived epitopes into the target polypeptide moiety. Thus, in one embodiment, said target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes and comprises at least two neoantigen derived epitopes, such as at least three neoantigen derived epitopes, such as at least four neoantigen derived epitopes, such as at least seven neoantigen derived epitopes, such as at least ten neoantigen derived epitopes, such as at least 15 neoantigen derived epitopes, such as at least 20 neoantigen derived epitopes, such as at least 25 neoantigen derived epitopes, such as at least 50 neoantigen derived epitopes, such as at least 60 neoantigen derived epitopes, such as at least 70 neoantigen derived epitopes, such as at least 80 neoantigen derived epitopes, such as at least 90 neoantigen derived epitopes, such as at least 100 neoantigen derived epitopes. In one embodiment, said target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes and comprises 5-150 of neoantigen derived epitopes, such as 5-125 neoantigen derived epitopes, such as 5-100 neoantigen derived epitopes, such as 5-90 neoantigen derived epitopes, such as 5- 80 neoantigen derived epitopes, such as at 5-75 neoantigen derived epitopes, such as 5-60 neoantigen derived epitopes, such as 5-50 neoantigen derived epitopes, such as 5-25 neoantigen derived epitopes, such as 5-20 neoantigen derived epitopes, such as at 5-15 neoantigen derived epitopes, such as 7-12 neoantigen derived epitopes, such as about ten neoantigen derived epitopes.

[0086] It is furthermore possible to combine several target polypeptide moieties into a POI as defined herein. Thus, in one embodiment, said POI comprises a plurality of target polypeptide moieties, such as at least two target polypeptide moieties, such as at least three target polypeptide moieties, such as at least four target polypeptide moieties, such as at least five target polypeptide moieties, such as at least ten target polypeptide moieties. In this context, the target polypeptide moieties in the POI may be similar or different. Thus, in one embodiment, said plurality of target polypeptide moieties are characterized in that at least two, such as at least three, such as at least five, such as at least ten, of said target polypeptide moieties have the same amino acid sequence, such as wherein all target polypeptide moieties have the same amino acid sequence. In another embodiment, said plurality of target polypeptide moieties are characterized in that at least two, such as at least three, such as at least five, such as at least ten, of said target polypeptide moieties have different amino acid sequences, such as wherein all target polypeptide moieties have different amino acid sequence.

[0087] The target polypeptide moieties in the POI may be directly linked to each other. Alternatively, they may be indirectly linked, such as liked via linkers or spacers. In one embodiment of the method as disclosed herein, said plurality of target polypeptide moieties comprises target polypeptide moieties which are linked directly or indirectly to each other. In this context, as subset of the plurality or all of the plurality of target polypeptide moieties may be the same or different and maybe linked via linkers or spacers that are the same or different. Said plurality of target polypeptide moieties in a POI may comprise target polypeptide moieties that are directly linked to each other and target polypeptide moieties which are indirectly linked to each other.

[0088] The term "linker " or "linker polypeptide" as used herein denotes peptide linkers of natural and / or synthetic origin. Such linkers consist of a linear amino acid chain wherein the 20 naturally occurring amino acids are the monomeric building blocks. The chain has a length of from 1 to 50 amino acids, in one embodiment between 1 and 28 amino acids, in one embodiment between 3 and 25 amino acids. The linker may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides, such as polypeptides with a hinge-function. The linker has the function to ensure that the individual entities of a fusion polypeptide can perform their biological activity by allowing the entities to fold correctly and to be presented properly. In one embodiment the linker polypeptide is a "synthetic linker polypeptide" that is designated to be rich in glycine, threonine, glutamine, and / or serine residues. These residues are arranged e.g. in small repetitive units of up to five amino acids, such as GSS, GGGGS (SEQ ID NO:175), QQQQG (SEQ ID NO:176), GSGSS (SEQ ID NO:177), GSGSG (SEQ ID NO:178), or SSSSG (SEQ ID NO:179). This small repetitive unit may be repeated for two to five times to form a multimeric unit. At the amino- and / or carboxy-terminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acids may be added.

[0089] Other synthetic peptidic linkers are composed of a single amino acid, which is repeated between 10 to 20 times and may comprise at the amino- and / or carboxy- terminal end up to six additional arbitrary, naturally occurring amino acids, such as e.g. serine in the linker GS15G (SEQ ID NQ:180). All linker polypeptides can be encoded by a nucleic acid molecule and therefore can be recombinantly expressed. As the linker polypeptides are themselves polypeptides, the entities of the fusion polypeptide are connected to the linker via a peptide bond that is formed between two amino acids. In one embodiment the polypeptide linker has an acid sequence selected from the group consisting of GSGSSGSASG (SEQ ID NO:181), GEGTGGSGSG (SEQ ID NO:182), GSGSSGSGTS (SEQ ID NO:183), GSSGSGSGSG (SEQ ID NO:184), (G3S)3(SEQ ID NO:185), (G3S)4(SEQ ID NO:186), (G3S)5(SEQ ID NO:187), (G3S)6(SEQ ID NO:188), (G4S)3(SEQ ID NO:189), (G4S)4(SEQ ID NQ:190), (G4S)5(SEQ ID NO:191), (G5S)2(SEQ ID NO:192), (G5S)3(SEQ ID NO:193), (G5S)4(SEQ ID NO:194), (G4S)2(SEQ ID NO:234) and GTGGSG (SEQ ID NO:325), such as the group consisting of GSGSSGSASG (SEQ ID NO:181), GEGTGGSGSG (SEQ ID NO:182), GSGSSGSGTS (SEQ ID NO:183), GSSGSGSGSG (SEQ ID NO:184), (G3S)3(SEQ ID NO:185), (G3S)4(SEQ ID NO:186), (G3S)5(SEQ ID NO:187), (G3S)6(SEQ ID NO:188), (G4S)3(SEQ ID NO:189), (G4S)4(SEQ ID NQ:190), (G4S)5(SEQ ID NO:191), (G5S)2(SEQ ID NO:192), (G5S)3(SEQ ID NO:193), and (G5S)4(SEQ ID NO:194).

[0090] As used herein, the term "spacer" refers to shorter amino acid stretch, comprising one or several amino acid residues, such as 1, 2 or 3 amino acid residues and can be inserted between different parts in fusion polypeptides.

[0091] Suitable linkers are known in the art and are for example described in the review article entitled "Fusion protein linkers: Property, design and functionality." by Chen et al., Adv Drug Deliv Rev. (2013) 65(10): 1357-1369.

[0092] The present inventors have found that for the successful production of some, but not all proteins, according to the method as disclosed herein, it may be useful to include a moiety that enhances the solubility of the target polypeptide moiety. As shown in the appended Examples 13-15, the presence of solubility enhancing moiety in particular when the target polypeptide moiety is aggregation prone, aids in production said target polypeptide moiety. As used herein, the term "solubility enhancing moiety" refers to a moiety, for example a polypeptide, which is capable of enhancing the solubility of the target polypeptide moiety. The solubility enhancing moiety may enhance the solubility of the target polypeptide moiety when expressed in fusion with said target polypeptide moiety, such as in direct fusion or in indirect fusion (in other words linked directly or indirectly), but within the same POL For example, said target polypeptide moiety may be connected via a linker or spacer to said solubility enhancing moiety. Thus, in one embodiment of the method as disclosed herein, said POI further comprises at least one solubility-enhancing moiety. As shown in the appended Examples, the effect of the solubility enhancing moiety is achieved independently of its positioning in relation to the target polypeptide moiety. In one embodiment, said at least one solubility-enhancing moiety is linked directly or indirectly to said at least one target polypeptide moiety. In one embodiment, said at least one solubility-enhancing moiety is located between at least two target polypeptide moieties. Alternatively, said at least one solubility-enhancing moiety within a target polypeptide moiety, such as for example in the case wherein the target polypeptide moiety comprises a plurality of neoantigen derived epitope said at least one solubility-enhancing moiety may be located between the neoantigen derived epitopes in the target polypeptide moiety. In another embodiment, said at least one solubility-enhancing moiety is linked, such as linked directly or indirectly, to the N-terminus or C-terminus of said at least one target polypeptide moiety. In one embodiment, said at least one solubilityenhancing moiety is linked, such as linked directly or indirectly, to said at least one complementary binding moiety of said coupling system. In one embodiment, said at least one solubility-enhancing moiety is linked, such as linked directly or indirectly, to the N-terminus or C-terminus of at least one complementary binding moiety of said coupling system.

[0093] As discussed above, in the context of target polypeptide moieties comprised in the POI, a linkage between two parts of a polypeptide may be direct or indirect. If said linkage is indirect, it may be via the means of a linker or spacer. In particular embodiments, said indirect linkage is via a linker amino acid sequence, such as wherein the linker amino acid sequence comprises at least one amino acid residue. The skilled person appreciates that the linkers and spacer as discussed above are equally relevant for the present context and will not be repeated here merely for the sake of brevity.

[0094] Known in the art are various solubility enhancing moieties, such as for example, SEP- tags (solubility enhancement peptide tags) containing, one, three, and five lysines or arginines (or six arginines) as well as larger polypeptide units. Such larger polypeptide units for improving recombinant protein solubility may be used as tags fused to target polypeptide moieties and non-limiting examples thereof are thioredoxin, maltose-binding protein (MBP), glutathione S-transferase and stress- responsive proteins such as RpoS, SlyD, and PotD and Crr. Thus, in one embodiment, there is provided a method as described herein, wherein said solubility enhancing moiety is selected from the group consisting of MBP (SEQ ID NO:76), PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA(SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), Fh8 (SEQ ID NO:197), H tag (SEQ ID NO:198), Ppi B (SEQ ID NO:199), Bfr (SEQ ID N0:200), YjdC (SEQ ID NQ:201), FolA (SEQ ID NO:202), CheZ (SEQ ID NQ:203), GshB (SEQ ID NQ:204), ArsC (SEQ ID NQ:205), N-ePGK (SEQ ID NQ:206), KDPG aldolase (SEQ ID NQ:207), Tsf (SEQ ID NQ:208), MsyB (SEQ ID NQ:209), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), Z-tag (SEQ ID NQ:210), ZZ- tag (SEQ ID NO:211), GB1 (SEQ ID NO:212), DsbA (SEQ ID NO:213), DsbAmut(SEQ ID NO:214), IF2 domain I, CaBP (SEQ ID NO:215), FTN-H (SEQ ID NO:216), Skp (SEQ ID NO:217), T7PK (SEQ ID NO:218), Ecotin (SEQ ID NO:219), Halo Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88), s / GFP (SEQ ID NQ:220), SNUT (SEQ ID NO:222), EspA (SEQ ID NO:221), BLA, / n / Bl-21 (SEQ ID NO:223), SmbP (SEQ ID NO:224), Ffu, TDX (SEQ ID NO:225), HE-MBP(Pyr), N-terminal domain of rpoD (SEQ ID NO:226), yjgD (SEQ ID NO:227), (Arg)i-3, (Arg)4(SEQ ID NO:58), (Arg)5(SEQ ID NO:59), (His)5(SEQ ID NQ:108), (Arg)w (SEQ ID NQ:109), (Arg-Gly-Gly)3-Gly (SEQ ID NO:117), Poly(Arg), (Gly-Arg)3-(Arg)3(SEQ ID NO:118), (Gly-Arg)4(SEQ ID NO:158), Gly-(Arg)5(SEQ ID NO:159), Gly(Arg-Gly-Gly)3(SEQ ID NQ:160), Gly(Lys-Gly)6(SEQ ID NO:161), (Gly)2- (Arg)2-Gly-Arg (SEQ ID NO:162), Gly-Lys-Gly-(Lys)2(SEQ ID NO:163), (Gly)2-(Lys)4(SEQ ID NO:164), (Lys)i-3, (Lys)4(SEQ ID NO:165), (Lys)5(SEQ ID NO:166), (Lys)6(SEQ ID NO:167), (Lys)w (SEQ ID NO:168), (Asp)5(SEQ ID NO:169), (Glu)5(SEQ ID NQ:170), [Gly]-(Asp)3]3(SEQ ID NO:171), (Asn)5(SEQ ID NO:172), (Gln)5(SEQ ID NO:173), (Ser)5(SEQ ID NO:174) and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto; ; or selected from the group consisting of PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA(SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), Fh8 (SEQ ID NO:197), H tag (SEQ ID NO:198), PpiB (SEQ ID NO:199), Bfr (SEQ ID N0:200), YjdC (SEQ ID NO:201), FolA (SEQ ID NO:202), CheZ (SEQ ID NQ:203), GshB (SEQ ID NO:204), ArsC (SEQ ID NQ:205), N-ePGK (SEQ ID NQ:206), KDPG aldolase (SEQ ID NQ:207), Tsf (SEQ ID NQ:208), MsyB (SEQ ID NQ:209), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), Z-tag (SEQ ID NQ:210), ZZ-tag (SEQ ID NO:211), GB1 (SEQ ID NO:212), DsbA (SEQ ID NO:213), DsbAmut(SEQ ID NO:214), IF2 domain I, CaBP (SEQ ID NO:215), FTN- H (SEQ ID NO:216), Skp (SEQ ID NO:217), T7PK (SEQ ID NO:218), Ecotin (SEQ ID NO:219), Halo Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88), s / GFP (SEQ ID NQ:220), SNUT (SEQ ID NO:222), EspA (SEQ ID NO:221), BLA, / n / Bl-21 (SEQ ID NO:223), SmbP (SEQ ID NO:224), Ffu, TDX (SEQ ID NO:225), HE-MBP(Pyr), N-terminal domain of rpoD (SEQ ID NO:226), yjgD (SEQ ID NO:227), (Arg)i-3, (Arg)4(SEQ ID NO:58), (Arg)5(SEQ ID NO:59), (His)5(SEQ ID NQ:108), (Arg)io (SEQ ID NQ:109), (Arg-Gly-Gly)3-Gly (SEQ ID NO:117), Poly(Arg), (Gly-Arg)3-(Arg)3(SEQ ID NO:118), (Gly-Arg)4(SEQ ID NO:158), Gly-(Arg)5(SEQ ID NO:159), Gly(Arg-Gly-Gly)3(SEQ ID NQ:160), Gly(Lys- Gly)6(SEQ ID NO:161), (Gly)2-(Arg)2-Gly-Arg (SEQ ID NO:162), Gly-Lys-Gly-(Lys)2(SEQ ID NO:163), (Gly)2-(Lys)4(SEQ ID NO:164), (Lys)i-3, (Lys)4(SEQ ID NO:165), (Lys)5(SEQ ID NO:166), (Lys)6(SEQ ID NO:167), (Lys)io (SEQ ID NO:168), (Asp)5(SEQ ID NO:169), (Glu)5(SEQ ID NQ:170), [Gly]-(Asp)3]3(SEQ ID NO:171), (Asn)5(SEQ ID NO:172), (Gln)5(SEQ ID NO:173), (Ser)s (SEQ ID NO:174) and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto; or selected from the group consisting of SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), Fh8 (SEQ ID NO:197), H tag (SEQ ID NO:198), Ppi B (SEQ ID NO:199), Bfr (SEQ ID NQ:200), YjdC (SEQ ID NQ:201), FolA (SEQ ID NQ:202), CheZ (SEQ ID NQ:203), GshB (SEQ ID NQ:204), ArsC (SEQ ID NQ:205), N-ePGK (SEQ ID NQ:206), KDPG aldolase (SEQ ID NQ:207), Tsf (SEQ ID NQ:208), MsyB (SEQ ID NQ:209), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), Z-tag (SEQ ID NQ:210), ZZ-tag (SEQ ID NO:211), GB1 (SEQ ID NO:212), DsbA (SEQ ID NO:213), DsbAmut (SEQ ID NO:214), IF2 domain I, CaBP (SEQ ID NO:215), FTN-H (SEQ ID NO:216), Skp (SEQ ID NO:217), T7PK (SEQ ID NO:218), Ecotin (SEQ ID NO:219), Halo Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88), s / GFP (SEQ ID NQ:220), SNUT (SEQ ID NO:222), EspA (SEQ ID NO:221), BLA, / n / Bl-21 (SEQ ID NO:223), SmbP (SEQ ID NO:224), Ffu, TDX (SEQ ID NO:225), HE-MBP(Pyr), N-terminal domain of rpoD (SEQ ID NO:226), yjgD (SEQ ID NO:227), (Arg)i-3, (Arg)4(SEQ ID NO:58), (Arg)5(SEQ ID NO:59), (His)s (SEQ ID NQ:108), (Arg)io (SEQ ID NQ:109), (Arg-Gly-Gly)3-Gly (SEQ ID NO:117), Poly(Arg), (Gly-Arg)3-(Arg)3(SEQ ID NO:118), (Gly-Arg)4(SEQ ID NO:158), Gly-(Arg)5(SEQ ID NO:159), Gly(Arg-Gly-Gly)3(SEQ ID NQ:160), Gly(Lys-Gly)6(SEQ ID NO:161), (Gly)2-(Arg)2-Gly-Arg (SEQ ID NO:162), Gly-Lys-Gly-(Lys)2(SEQ ID NO:163), (Gly)2-(Lys)4(SEQ ID NO:164), (Lys)i-3, (Lys)4(SEQ ID NO:165), (Lys)5(SEQ ID NO:166), (Lys)6(SEQ ID NO:167), (Lys)io (SEQ ID NO:168), (Asp)5(SEQ ID NO:169), (Glu)5(SEQ ID NQ:170), [Gly]-(Asp)3]3(SEQ ID NO:171), (Asn)5(SEQ ID NO:172), (Gln)5(SEQ ID NO:173), (Ser)s (SEQ ID NO:174) and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto.

[0095] In one embodiment, there is provided a method as described herein, wherein said solubility enhancing moiety is selected from the group consisting of selected from the group consisting of MBP (SEQ ID NO:76), PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA(SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), HALO Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88); and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto. In one particular embodiment, said solubility enhancing moiety is selected from the group consisting of PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA(SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), HALO Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88); and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto. In one particular embodiment, said solubility enhancing moiety is PotD. In one particular embodiment, said PotD is derived from Streptococcus suis. In one embodiment, said PotD is selected from group consisting of PotD comprising or consisting of an amino acid sequence as defined in SEQ ID NQ:70 and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto, such as wherein said PotD is as defined in SEQ ID NQ:70. The present inventors consider that any polypeptide exhibiting at least 80 % identity to SEQ ID NQ:70 and retaining the functional properties of enhancing solubility is useful in the present context.

[0096] It may be beneficial for some target polypeptides, that the POI and thereby the target polypeptide, is subjected to post-translational modification(s). Post- translational modification is well known to the skilled person and is not discuss here in extensive detail as it forms part of the common general knowledge in the field. The term "post-translational modification" refers to covalent process of changing proteins following protein biosynthesis. Posttranslational modifications are covalent processing events that change the properties of a protein for example by proteolytic cleavage and adding a modifying group, such as acetyl, phosphoryl, glycosyl and methyl, to one or more amino acids. Post-translational modifications can occur on the amino acid side chains or at the C- and / or N- terminus of a polypeptide. There are more than 400 different types of PTMs affecting many aspects of protein functions. Phosphorylation is highly effective for controlling the activity of a protein and is the most common change after translation. As it well known, many eukaryotic and prokaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation, which can promote protein folding and improve stability as well as serving regulatory functions. Glycosylation occurs in multiple subcellular locations, such as endoplasmic reticulum, the Golgi apparatus, cytosol and the sarcolemma membrane. Attachment of lipid molecules, known as lipidation, often targets a protein or part of a protein attached to the cell membrane. Other forms of post-translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue. It may be beneficial in the present context to target the POI to a component of the polypeptide expression-competent cell-free composition which allows for desired post translation modification to occur. For example, such components may be microsomes. For example, signaling sequences may be used to promote translocation of a polypeptide to microsomes for post translational modification. Non-limiting examples of such signaling sequences are signaling sequences of honeybee melittin, VSV-G, mouse Ig kappa, mouse Ig heavy, human IgKVIll, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, gaussia luc, albumin (HSA), influenza haemagglutinin, human insulin, silkworm fibroin LC, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and signaling sequences of medicago sativa protein disulphide isomerase (ssPDI).

[0097] Thus, in one embodiment, there provided a method as disclosed herein, wherein said at least one POI further comprises at least one moiety promoting translocation of POI into microsomes, such as wherein said at least one moiety promoting translocation of POI into the microsomes is a signaling sequence.

[0098] In one embodiment, said signaling sequence selected from the group consisting of signaling sequences of honeybee melittin, VSV-G, mouse Ig kappa, mouse Ig heavy, human IgKVIll, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, gaussia luc, albumin (HSA), influenza haemagglutinin, human insulin, silkworm fibroin LC, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and signaling sequences of medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is selected from the group consisting of SEQ ID NO:229 and SEQ ID NO:237-256. In a specific embodiment, said signaling sequence is a signaling sequence of honeybee melittin, such as is SEQ ID NO:229. In another embodiment, said signaling sequence is selected from the group consisting of signaling sequences of honeybee melittin, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is selected from the group consisting of SEQ ID NO:229 and SEQ ID NO:251-256. In a specific embodiment, said signaling sequence is a signaling sequence of honeybee melittin, such as is SEQ ID NO:229. In another embodiment, said signaling sequence is selected from the group consisting of signaling sequences of nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is selected from the group consisting of SEQ ID NO:251-256. The skilled person is aware of additional alternative signaling sequences besides the selection previously listed which would be appropriate for the described context.

[0099] In one embodiment, said at least one POI is provided as a nucleic acid encoding said POI in vector comprising at least one moiety promoting translocation of said POI into the microsomes, such as wherein said at least one moiety promoting translocation of said POI into the microsomes is a signaling sequence.

[0100] In one embodiment, said signaling sequence selected from the group consisting of signaling sequences of honeybee melittin, VSV-G, mouse Ig kappa, mouse Ig heavy, human IgKVIll, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, gaussia luc, albumin (HSA), influenza haemagglutinin, human insulin, silkworm fibroin LC, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and signaling sequences of medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is selected from the group consisting of SEQ ID NO:229 and SEQ ID NO:237-256. In a specific embodiment, said signaling sequence is a signaling sequence of honeybee melittin, such as is SEQ ID NO:229. In another embodiment, said signaling sequence is selected from the group consisting of signaling sequences of honeybee melittin, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is selected from the group consisting of SEQ ID NO:229 and SEQ ID NO:251-256. In a specific embodiment, said signaling sequence is a signaling sequence of honeybee melittin, such as is SEQ ID NO:229. In another embodiment, said signaling sequence is selected from the group consisting of signaling sequences of nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is selected from the group consisting of SEQ ID NO:251-256. The skilled person is aware of additional alternative signaling sequences besides the selection previously listed which would be appropriate for the described context.

[0101] The skilled person appreciates that the signal sequence is cleaved off in the microsomes by the system, whereby the POI displayed on the vesicles as described herein does not contain said signal sequence. However, the skilled person also appreciated that cleavage may not be 100 percent effective and that a minority of POIs may comprise the signal sequences.

[0102] As discussed in the context of the present aspect, the method as disclosed herein may be useful for aiding the purification of target polypeptide moieties, such as target polypeptide moieties comprised in POIs, which POIs are coupled to the vesicles via means of the coupling system as disclosed herein. Thus, in one embodiment, there is provided a method as disclosed herein, wherein the method further comprises the step of recovering the at least one vesicle coupled to said at least one polypeptide of interest. In one embodiment, said step of recovering the at least one vesicle coupled to said at least one polypeptide of interest is performed by a method selected from the group consisting to tangential flow filtration, cross flow filtration, ultracentrifugation and size-exclusion chromatography. The skilled person knows which method for recovery to employ in which context and the advantages and disadvantages of each method. Thus a vesicle which on its surface displays a POI as defined herein, and said POI is coupled to the vesicle via means of the coupling system as disclosed herein can be recovered.

[0103] In a second aspect of the present disclosure there is provided a vesicle, such as an outer membrane vesicle, which on its surface displays a POI as defined herein, and said POI is coupled to the vesicle via means of the coupling system as disclosed herein. It is to be understood that such vesicles may have uses for example in biotechnology and or medicine.

[0104] Thus, in one embodiment of the second aspect as disclosed herein, there is provided a vesicle coupled to a polypeptide of interest (POI), wherein said vesicle on its outer surface displays at least one complementary moiety of a coupling system and a polypeptide of interest (POI); wherein said POI comprises at least one target polypeptide moiety, at least one solubility enhancing moiety and at least one coupling moiety of said coupling system; and wherein said vesicle is coupled to said POI via an isopeptide bond formed between the respective moieties of the coupling system. In other words, the vesicle exhibits on its membrane surface at least one complementary moiety of a coupling system. This coupling moiety forms an isopeptide bond between it and its coupling moiety comprised in the POI. This way the POI is displayed on the surface of the vesicle.

[0105] As explained in the context of the first aspect of the present disclosure, the coupling system as disclosed herein comprises at least one coupling moiety and at least one complementary moiety. It is to be understood that the reverse location of the coupling moieties is possible, such that wherein said vesicle on its outer surface POI comprises at least one coupling moiety. The terms "complementary coupling moiety" and "coupling moiety" are not to be viewed as limiting and indicate that the moieties together have the ability to be connected via an isopeptide bond. It will be appreciated that in one context, a specific moiety may be referred to as a "complementary moiety" and in a different context the same moiety may be referred to as a coupling moiety. In one embodiment, said vesicle as disclosed herein further displays on its outer surface an autotransporter (AT) fusion protein and wherein the AT fusion protein comprises an AT protein and the complementary moiety of said coupling system; and wherein said AT fusion protein is coupled to said POI via an isopeptide bond formed between the moieties of the coupling system on the AT fusion protein and said POI. , The vesicle coupled to a POI may be a vesicle as described in the context of the first aspect. The description of vesicles is not repeated here for the sake of brevity alone, but is to be understood as equally relevant in this context. In particular, said vesicle may be an OMV derived from Salmonella spp.

[0106] In this context, the AT may be a serine protease autotransporter of the Enterobacteriaceae (SPATE), for example a SPATE is selected from the group consisting of hemoglobin-binding protease (Hbp) from E. coli, EspP from E. coli, Pet from E. coli, Tsh from E. coli, EspC from E. coli, Hap from Haemophilus influenzae, Hap from Neisseria gonorrhoeae, Hap from Neisseria meningitidis, IgA protease from Haemophilus influenzae, IgA protease from Neisseria gonorrhoeae, IgA protease from Neisseria meningitidis and SepA from Shigella flexneri. In particular said SPATE protein may be selected from the group consisting of hemoglobin-binding protease (Hbp) from E.coli, mutants thereof, such as mutants having least one mutation that prevents cleavage at the autocatalytic cleavage site. It is to be understood that the AT protein and AT fusion proteins as referred to therein are described in the context of the first aspect of the present disclosure and said description is equally relevant for this second aspect. It is not repeated there merely for the sake of brevity.

[0107] In particular, in one embodiment said autotransporter protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60 and further comprises mutations in positions N1048G and N1049S, thus corresponding to SEQ ID NO:61, and any proteins exhibiting at least 80 % thereto provided said proteins comprise an autocatalytic cleavage site which has been mutated such that cleavage is prevented. As explained in the context of the first aspect as defined above, complementary binding moiety of said coupling system may be incorporated into the AT protein by one or several of N-terminal fusion to said AT protein; internal incorporation of the said at least one complementary binding moiety into the AT protein; and the replacement of part of the AT protein by said at least one complementary binding moiety of said coupling system. For example, said least one complementary binding moiety may be present at the location of one of the side domains of the passenger domain of the AT protein. For example, said at least one coupling moiety may replace the complete or part of the side domain 1 of the passenger domain of the Hbp protein, which Hbp protein comprises an autocatalytic cleavage site which has been mutated such that cleavage is prevented.

[0108] Likewise, the description of the coupling system in the context of the first aspect is equally applicable to this second aspect and is not repeated herein for the same of brevity. In particular, the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13) and variants thereof exhibiting at least 70 % identity thereto.

[0109] In addition, the vesicle as disclosed herein is coupled to a POI as explained above. This POI comprises at least one target polypeptide moiety and a complementary moiety of the coupling system, wherein said at least one target polypeptide moiety may be a defined in the context of the first aspect. Thus, the target polypeptide moiety may be an aggregation prone protein, a polypeptide comprising multiple neoantigen derived epitopes or any other the target polypeptide moiety discussed above. Said POI also comprises a solubility enhancing moiety as defined in connection with the first aspect. For example, said solubility enhancing moiety comprise or consist of PotD is derived from Streptococcus suis. In one embodiment, said PotD is selected from group consisting of PotD comprising or consisting of an amino acid sequence as defined in SEQ ID NO:70 and any polypeptide exhibiting at least 80 %.

[0110] In third aspect as defined herein, there is provided a vesicle coupled to a POI obtainable by the method as disclosed herein. In particular, said vesicle may be an OMV and the POI may comprise a POI, which in turn comprises target polypeptide moiety and solubility enhancing moiety. The POI and the vesicle are coupled via means of a coupling systems as defined herein. In particular, said vesicle coupled to a POI obtainable by the method is obtainable by a method utilizing a POI which comprises a solubility enhancing moiety as disclosed herein.

[0111] It will be understood that the vesicles as disclosed in the context of the second and / or third aspect herein, may be useful in their own right in the field of medicine. The skilled person will appreciate that the vesicles comprising POIs described herein may be useful as medicaments, and the usefulness in the medical context will be dependent on the identity of the target polypeptide moiety comprised in the POI. Thus, in a fourth aspect there is provided a vesicle coupled to a POI as defined herein, for use a medicament. In one embodiment, said POI comprises a target polypeptide moiety having therapeutic activity. Said the therapeutic activity may a curative activity, such as concerned with bringing the body from a pathological state back into its normal, healthy state, and / or concerned with ameliorating the symptoms of disease. Said the therapeutic activity may a prophylactic or preventive activity, such as concerned with prevention of a pathological state.

[0112] In one particular embodiment, said medicament is a vaccine. In this context, said target polypeptide moiety is an antigen or an immunogenic fragment thereof.

[0113] In a related fifth aspect of the present disclosure, there is provided a vesicle coupled to a polypeptide of interest (POI) as defined herein for use in the treatment and / or prevention of a disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease, infectious disease and transplant rejection, such as a disease selected from the group consisting of infectious disease and cancer. In one particular embodiment there is provided a vesicle coupled to a polypeptide of interest (POI) as defined herein, for use in the in the treatment of an infectious disease, wherein the target polypeptide moiety is an antigen or an immunogenic fragment thereof. It will be understood that the usefulness of the present vesicle is not limited to a particular infectious disease. Rather it extends to any type of infectious disease and will be dependent on that the target polypeptide moiety comprises an antigen or an immunogenic fragment thereof, which antigen and / or fragment are capable of eliciting a protective immune response to said disease in a subject.

[0114] In one particular embodiment there is provided a vesicle coupled to a polypeptide of interest (POI) as defined herein, for use in the treatment of cancer, wherein the target polypeptide moiety comprises at least one neoantigen derived epitope as described herein. It will be understood that the usefulness thereof is not limited to a particular form of cancer, rather it extends to any type of cancer from which neoantigens have been isolated.

[0115] In one particular embodiment the vesicle is an OMV. It may be beneficial that OMVs are used in this context due to their self-adjuvating properties.

[0116] In sixth aspect of the present disclosure, there is provided a pharmaceutical composition comprising a vesicle coupled to a polypeptide of interest (POI) as defined herein and at least one pharmaceutically acceptable excipient, wherein said POI comprises a target polypeptide moiety having therapeutic activity. In one embodiment, said therapeutic activity is prophylactic activity. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, in Alfonso R Gennaro, Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472). It should be understood that in addition to the ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for intranasal or intramuscular administration.

[0117] In one particular embodiment, there is provided a pharmaceutical composition as described herein, wherein said POI comprises a target polypeptide moiety which is an antigen or an immunogenic fragment thereof.

[0118] In a closely related aspect, there is provided a vaccine composition comprising a vesicle coupled to a POI as defined herein and at least one pharmaceutically acceptable excipient, wherein said POI comprises a target polypeptide moiety which is an antigen or an immunogenic fragment thereof and wherein said composition comprises an additional agent with adjuvant effect. The vaccine composition disclosed herein may comprise an agent with adjuvant effect in an amount that is im mu no-effective. Suitably, said adjuvant stimulates systemic or mucosal immunity. The skilled person is aware of suitable adjuvants. Non-limiting examples of suitable adjuvant in the context of the present disclosure include polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, immunostimulating sequences (ISS), an oil in water emulsion, cation lipids containing a quaternary ammonium salt, cytokines, aluminum hydroxide or aluminum phosphate, saponin or nanoparticles or any combinations or mixtures thereof.

[0119] In one embodiment, said additional agent with adjuvant effect is present in an immune-effective amount in said composition. As used herein, the term "immune- effective" refers a sufficient amount of an adjuvant to increase the vaccine's immunogenicity to a level high enough to effectively vaccinate a typical patient. In one particular embodiment, there is provided a vaccine composition comprising a vesicle coupled to a POI as defined herein, and at least one pharmaceutically acceptable excipient, wherein said POI comprises a target polypeptide moiety which is an antigen or an immunogenic fragment thereof and wherein said composition does not comprise any additional agent with adjuvant effect.

[0120] It is envisioned that said vesicles may be useful in several contexts in medicine, prognosis, diagnosis and / or imaging, depending on the choice of the target polypeptide moiety used. Thus, in an eight aspect of the present disclosure there is provided a vesicle coupled to a POI as defined herein, a pharmaceutical composition as defined herein or a vaccine composition as defined herein for use as an agent select from the group consisting of diagnostic agent, prognostic agent, prophylactic agent, therapeutic agent and imaging agent, such as the group consisting of diagnostic agent, prognostic agent and imaging agent.

[0121] It is furthermore envisioned that said vesicles as disclosed herein may be used as a drug delivery platform, wherein the drug is the target polypeptide and the vesicle platform is used to expression of said drug and subsequent administration to a patient in need thereof. Thus, in a related aspect is provided a use of a vesicle coupled to a POI as defined herein as a display platform for delivery of an antigen or an immunogenic fragment thereof. In a related aspect is provided a drug delivery platform comprising a vesicle coupled to a polypeptide of interest (POI) as defined herein, wherein said POI comprises a target polypeptide moiety having therapeutic activity.

[0122] In a related nineth aspect there is provided a method of treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a vesicle coupled to a POI as defined herein, or a pharmaceutical composition as defined herein, wherein said disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease and transplant rejection, such as a disease selected from the group consisting of infectious disease and cancer. In one embodiment, said disease is cancer.

[0123] In one embodiment, there is provided a method of treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a vesicle coupled to a POI as defined herein, a pharmaceutical composition as defined herein, or a vaccine composition as defined herein, wherein said disease is an infectious disease. In a tenth aspect, there is provided a use of a vesicle coupled to a POI as defined herein for use in the manufacture of a medicament, wherein said POI comprises a target polypeptide moiety, which target polypeptide moiety has therapeutical activity, for example prophylactic activity. In one embodiment said use is in the manufacture of a medicament for the treatment of a disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease and transplant rejection, such as a disease selected from the group consisting of infectious disease and cancer, wherein said POI comprises a target polypeptide moiety, which target polypeptide moiety has therapeutical activity.

[0124] In particular, said use may be use in the manufacture of a medicament for the prophylactic treatment of an infectious disease, wherein said POI comprises a target polypeptide moiety, which target polypeptide moiety is an antigen or an immunogenic fragment thereof.

[0125] It is to be understood that the disclosure and embodiments discussed in relation to the fourth and fifth aspects herein, are equally relevant for this nineth and tenth aspects and are not repeated here merely for the sake of brevity.

[0126] In yet additional aspects of the present disclosure as outlined below there are provided diagnostic and / or prognostic uses of the vesicles coupled to said POI as defined herein. As explained above, the usefulness of said methods is dictated by the identity of the target polypeptide moiety / moieties comprised in the POI. The skilled person will appreciate that depending on the choice of target polypeptide moiety / moieties said use may be related to diagnosis or prognosis of various diseases and / conditions.

[0127] Thus, in one aspect there is provided a method of diagnosis in vitro, comprising the steps:

[0128] - providing a vesicle coupled to said polypeptide of interest (POI) as defined herein, wherein the POI is capable of binding to a target of interest (TOI);

[0129] - contacting a sample suspected to contain said TOI with the vesicle coupled to said POI, thereby allowing said POI to bind to said TOI;

[0130] - detecting the binding of said POI to indicate the presence of said TOI in the sample; and

[0131] - using the information obtained to establish a diagnosis.

[0132] In the context of any one of said last three mentioned aspects, a TOI is the target of interest which is to be detected and / or quantified in order to obtain information for establishing a diagnosis. In this context, the POI comprises a target polypeptide moiety which has affinity for and is capable of binding said TOI. Said TOI may be a protein or polypeptide.

[0133] In a related aspect there is provided a method of prognosis in vitro, comprising the steps:

[0134] - providing a vesicle coupled to said polypeptide of interest (POI) as defined herein, wherein the POI is capable of binding to a target of interest (TOI);

[0135] - contacting a sample suspected to contain said TOI with the vesicle coupled to said POI, thereby allowing said POI to bind to said TOI;

[0136] - detecting the binding of said POI to indicate the presence of said TOI in the sample; and

[0137] - using the information obtained to establish a prognosis.

[0138] In a related aspect there is provided a method for detection of a target of interest (TOI) in a sample, comprising the steps:

[0139] - providing a vesicle coupled to said polypeptide of interest (POI) as defined herein, wherein the POI is capable of binding to said TOI;

[0140] - contacting a sample suspected to contain said TOI with the vesicle coupled to said POI, thereby allowing said POI to bind to said TOI;

[0141] - detecting the binding of said POI to indicate the presence of said TOI in the sample. In the context of any one of said last three mentioned aspects, a TOI is the target of interest which is to be detected and / or quantified in order to obtain information for establishing a diagnosis. The presence, absence or level of said TOI may be indicative of a disease or conditioned to be diagnosed. Alternatively, the absence, presence or level of said TOI may be indicative of a disease or conditioned to be diagnosed. The skilled person will appreciate that the interpretation of the method of diagnosis, will be dependent of the identity of the TOI. In this context, the POI comprises a target polypeptide moiety which has affinity for and is capable of binding said TOL Said TOI may be a protein or polypeptide.

[0142] It will be understood that any one of said method may be repeated several times for example for the purpose of monitoring of the subject before, during, or after treatment. Said method may comprise only a qualitative assessment or it may comprise a quantitative assessment of the presence of TOI in the sample. Thus, said in one embodiment of said method of diagnosis in vitro, method of prognosis, or method of detection, the method further comprises the steps:

[0143] - repeating the steps of detection, wherein said detection is performed at several time points at intervals in the same provided sample or a different provided sample, for example as part of a monitoring of the subject before, during, or after treatment.

[0144] In one embodiment, there is provided a method of diagnosis, a method of prognosis or a method of detection as defined herein, further comprising a step of obtaining a value corresponding to the amount of the vesicle coupled to a POI that has bound to said TOI in said sample.

[0145] In one embodiment, there is provided a method of diagnosis, a method of prognosis or a method of detection as defined herein, further comprising a step of comparing said value to a reference.

[0146] Thus, said in one embodiment said method of diagnosis in vitro or method of prognosis may be useful in the diagnosis and / or prognosis of an infectious disease or cancer. Thus, in one embodiment, said method may be in relation to an infectious disease. In one embodiment, said method may be in relation to a cancer. In one embodiment, there is provided a method of diagnosis in vitro or a method of prognosis in vitro , wherein said diagnosis or prognosis is in relation to an infectious disease or cancer. As used herein, the term "patient" or "subject" refers to an individual who exhibits or is at risk of exhibiting symptom(s) of the disease at hand. Said subject can be an animal or a human subject.

[0147] As used herein, the terms "a" or "an" when referring to entities such as protein of interest (POI), target polypeptide moiety, autotransporter fusion protein, autotransporter protein, solubility enhancing moiety, vesicle, coupling system, coupling moiety, and / or complementary moiety, it is to be interpreted as referring to at least one of said entity. It will be appreciated that the interpretation of the terms also extends to not here explicitly exemplified entities.

[0148] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the scope of the invention. Therefore, it is intended that the invention is not limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims. The invention will be further illustrated by the following non-limiting Examples.

[0149] References

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[0165] Thompson et al., Nucleic Acids Research (1994) 22: 4673-4680.

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[0168] Weikert, MJ. et al., Curr Opin Biotechnol. (1996) 7:494-499.

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[0171] Brief Description of the Figures

[0172] Figure 1 shows a Coomassie stained SDS-PAGE gel demonstrating successful expression of target polypeptide moieties in fusion with a coupling moiety as described in Example 1 is shown in lanes 1-5. The respective synthesized target polypeptide moiety is indicated with arrowheads, and Molecular mass (kDa) markers are indicated at the left side of the panel.

[0173] Figure 2 shows a Coomassie stained SDS-PAGE gel (A) and a Western Blot (B) as described in Example 2, demonstrating successful synthesis of multiple tandem- fused neoepitopes (SEQ ID NO:2) fused to a coupling moiety comprised in the POI (SEQ ID NO: 93) in the absence (lane 2, Figure 2A) and presence (lane 2, Figure 2A) of an autotransporter fusion protein and a complementary moiety as displayed on the OMV. The adduct comprising the POI and autotransporter fusion protein and complementary moiety as displayed on the OMV is indicated with an arrowhead in lane 3 in Figure 2A and identity of the adduct was confirmed by Western blot as shown in Figure 2B as described in Example 2.

[0174] Figure 3 shows a Coomassie stained SDS-PAGE gel (A) and a Western Blot (B) as described in Example 3, demonstrating successful synthesis of six target polypeptide moieties (lanes 1-6) employing co-translational administration of OMVs in the presence or absence of the solubility enhancer PotD. Molecular mass (kDa) markers are indicated at the left side of the panel. Adducts are indicated with black arrowheads, while open arrowheads indicate HbpD-SpC as displayed on the OMVs. The identity of the adducts was confirmed by Western blot as shown in Figure 3B as described in Example 3.

[0175] Figure 4A shows a Ponceau-S dye stained nitrocellulose membrane demonstrating equal protein transfer for all total reaction mixture (Tot) samples and the supernatant samples SI and S2 as described in Example 4. Figure 4B shows a Western Blot of the Tot, SI and S2 samples for target polypeptide moiety PC (lanes 1-3), target polypeptide moiety PC in fusion with a solubility enhancing moiety (lanes 4-6) and target polypeptide moiety PnrA (lanes 7-9).

[0176] Figure 5 shows a Coomassie stained SDS-PAGE gel showing protein content of fusion protein comprising IB formation tags ssTorA(3X) or TrM in fusion with MBP (lanes 1-3), PotD (lanes 4-5), Spike SI (lanes 4-8) and hisGFP (lanes 9-11) as indicated in the Figure. Shown are the urea solubilized IBs (input), and supernatant and pellet fractions after dialysis as described in Example 5. Molecular mass (kDa) markers are indicated at the left side of the panel.

[0177] Figure 6 shows a Coomassie stained SDS-PAGE gel showing protein content of fusion protein comprising IB formation tags ssTorA(3X) or TrM in fusion with multiple tandem-fused epitopes "NC" (SEQ ID NO:1) or neoepitopes "PC" (SEQ ID NO:2) in pellet and supernatant fractions as indicated in the Figure and described in Example 6.

[0178] Figure 7 shows Coomassie stained SDS-PAGE gel showing protein content of POIs comprising IB formation tag TrM in fusion with multiple tandem-fused neoepitopes "PC" (SEQ ID NO:2), SpyTag (SEQ ID: NQ:10) and either MBP (lane 1) or PotD (lane 2) coupled HbpD-SpC (SEQ ID NO:68) as displayed on the OMVs and as described in Example 7. HbpD-SpyCatcher (HbpD-SpC) as displayed on the OMVs and the adduct comprising the POI and HbpD-SpyCatcher as displayed on the OMVs are indicated with arrowheads. Molecular mass (kDa) markers are indicated at the righthand side of the panel.

[0179] Figure 8 shows a Coomassie stained SDS-PAGE gel showing protein content of POIs comprising a target protein moiety (PD, MN1 or RvOlll), IB formation tag TrM, SpyTag, and either PotD from 5. suis or PotD from E. col'i in IB starting material (IB), and the pellet (P) and supernatant (S) after dialysis of urea solubilized IBs as described in Example 8.

[0180] Figure 9 shows a Coomassie stained SDS-PAGE gel showing protein content of adducts which comprise POIs comprising a target protein moiety PC, IB formation tag TrM, SpyTag, and either PotD from 5. suis or PotD from E. coli coupled autotransporter protein in fusion with SpyCatcher (HbpD-SpC) as displayed on OMVs, in lanes 2 and 3, respectively, as described in Example 9. Arrowheads indicate adduct and HbpD-SpyCatcher (HbpD-SpC) as displayed on the OMVs. Molecular mass (kDa) markers are indicated at the left side of the panel.

[0181] Figure 10 shows an alignment of the amino acid sequences of PotD from the Gram-positive bacterium 5. suis (SEQ ID NO:70) and the variant from the Gramnegative bacterium E. coli (SEQ ID NO:71).

[0182] Figure 11 shows a Western blot of SpT-PotD-NC-HA produced in a cell free system in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of complete™ protease inhibitor as described in Example 11. OMVs displaying HbpD-SpyCatcher (HbpD-SpC) were added to mixtures shown in lanes 3 and 4. Open arrowheads indicate SpT-PotD-NC-HA and the adducts formed. Molecular mass (kDa) markers are indicated at the left side of the panels.

[0183] Figure 12 shows a Coomassie blue stained SDS-PAGE gel showing the protein content in input (cell lysate), supernatant and pellet of RvOlll in fusion with SpyTag (as indicated by empty arrowhead as H6-SpT-Rv0111) as described in Example 12. Molecular mass (kDa) markers are indicated at the left side of the panels. Figure 13 shows a Western blot showing samples of POIs as indicated in lanes 1-12, wherein samples in lanes 1-6 were mixed with OMVs displaying the autotransporter fusion protein HbpD-SpyCatcher (HbpD-SpC) as described in Example 13. Molecular mass (kDa) markers are indicated at the left side of the panels. Black arrowheads indicated adducts formed, open arrowheads indicate synthesized polypeptide and * indicated aggregates formed.

[0184] Figure 14 A and Figure 14 B show a Coomassie stained SDS-PAGE gel and Western blot with POI samples in lanes 4-8 as indicated. Lane 1 and 2 comprise control (cell free reaction mixture) alone or with "HbpD-SpC" as displayed on OMVs, respectively and lane 3 contains "HbpD-SpC", as displayed on OMVs, alone. OMVs displaying "HbpD-SpC" were added to the POI samples in lanes 4-8. Black arrowhead indicates adduct and * indicates aggregated. In Figure 14 A, open arrowhead indicates OMVs displaying "HbpD-SpC" and # indicates truncate. In Figure 14 B, open arrowhead indicates synthesized polypeptide (POI). Molecular mass (kDa) markers are indicated at the left side of the panels.

[0185] Figure 15 shows a Coomassie stained SDS-PAGE gel showing POI samples in lanes 1-8, wherein OMVs displaying "HbpD-SpC" were added from the start of the synthesis reaction (0 hours; lanes 2, 4, 6, 8) and wherein OMVs displaying "HbpD- SpC" (SEQ ID NO:68) were added 3.5 hours after the onset of polypeptide synthesis (3.5 hours; lanes 1, 3, 5, 7) as described in Example 15. Molecular mass (kDa) markers are indicated at the left side of the panel. Black arrowhead indicates adduct, * indicates aggregated and # indicates potential adduct truncate.

[0186] Figure 16 shows a Coomassie stained SDS-PAGE gel showing POI samples in lanes 1-4 synthesized in temperature conditions as indicated and described in Example 16. OMVs displaying "HbpD-SpC" were added to the POI samples in lanes 2 and 4. Molecular mass (kDa) markers are indicated at the left side of the panel. Black arrowhead indicates adduct, open arrowhead indicates "HbpD-SpC" as displayed on OMVs.

[0187] Figure 17A shows a Western blot showing POI samples in lanes 2-3 synthesized using a lysate of N. tabacum c.v. BY-2 cells as described in Example 17. It is shown that the POI hemagglutinin (HA) was obtained expression in different cellular compartments, as shown in lane 01 for cytosolic compartment and in lane 02 for the microsomal compartment detected using the a-HA antibody. Black arrowheads indicate the HA (glycosylated) and HA (not glycosylated). Molecular mass (kDa) markers are indicated at the left side of the panel. Figure 17B show a Coomassie stained SDS-PAGE gel and Western blot with samples in lanes 3, 4 and 5 and 7-8. OMVs displaying "HbpD-SpC" were added to the samples of the POI hemagglutinin (HA) in lanes 4 and 7. Lanes 2 and 8 contain OMVs displaying "HbpD- SpC" alone. Molecular mass (kDa) markers are indicated at the left side of the panel and are used in lanes 1, 5 and 9. Black arrowheads indicate the HbpD-HA adduct, HbpD-SpC, HA (glycosylated) and HA (not glycosylated).

[0188] Examples

[0189] The following examples disclose the development of a cell-free production method for producing a target polypeptide moiety. The examples further describe and demonstrate that the cell-free production method is efficient in producing a variety of polypeptides of interest (POIs) comprising various target polypeptide moieties, with or without additional moieties, such as at least one solubility enhancing moiety comprised in the protein of interest (POI). Provided below is a list of selected materials and methods generally applied in the following examples, more detailed lists of materials and methods can be found in each respective example text:

[0190] General procedures

[0191] Host cell strains

[0192] E. coli strains TOP1OF', NEB5a or DH5a was used as the host strain for cloning procedures. E. coli strains TOP1OF' or BL21(DE3) were used for expression and isolation of inclusion bodies. TOP1OF' was purchased from Life Technologies. Strain NEB5a was purchased from New England Biolabs. Strain DH5a was obtained from lab stock. Strain BL21 (DE3) was purchased from Thermo Scientific. Outer membrane vesicles (OMVs) were produced using strain Salmonella Typhimurium

[0193] SL3261 AtoIRA AmsbB (Kuipers et al, Infect Immun. 2017 Sep 20; 85(10)).

[0194] Plasmids

[0195] In Examples 1-4 and 11, 13-16 polypeptides were expressed from the expression vector plVEX2.3d backbone. The vector is optimized for protein synthesis in bacterial cell-free systems under control of bacteriophage T7 transcription elements.

[0196] In Examples 5-9 fusion proteins were expressed from the expression vector pASK-IBA3 (IBA GmbH, Germany). In this high copy number vector, constructs were under the control of the anhydrotetracycline-inducible TetA / TetR promoter.

[0197] In Example 12 the H6-SpT-Rv0111 polypeptide was expressed from a pET28 expression vector derivative under control of a T7 promoter.

[0198] In Example 17 HA-TFD-SpT polypeptide was expressed from a pALiCEOl expression vector (LenioBio, Germany) and from a pALiCE02 expression vector, also comprising the melittin signal peptide (SEQ ID NO:229) (LenioBio, Germany).

[0199] In Examples 2-3, 7 and 9, the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:69), comprising an autotransporter protein in the form of HbpD (a double substitution mutant, N1048G and N1049S, also with domain 1 partially deleted, Adi) (SEQ ID NO:133) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was expressed from the mediumcopy pEH3 vector backbone (described in Hashemzadeh-Bonehi et al. Mol Microbiol. (1998) 30(3):676-8) that drives expression under control of a isopropyl 0-D-1- thiogalactopyranoside-inducible LacUV5 promoter. A person skilled in the art will understand that the autotransporter fusion protein is initially expressed with a signal peptide (SEQ ID NO:69), but is presented on the surface of OMVs without a signal peptide (SEQ ID NO:68), see the following section "Autotransporter fusion protein production" for more information. Autotransporter fusion protein production

[0200] As described in the section above, the autotransporter fusion protein "HbpD- SpC" SEQ ID NO:69), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:133) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was expressed from vector pEH3. Following expression, the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:67) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was displayed on the surface of OMVs in Examples 2-3 and 7, 9, 11, 13-16. However, before being displayed on the surface of OMVs, the fusion protein "HbpD- SpC" was first expressed from the plasmid "pEH3-HbpD-SpC". The expressed version of "HbpD-SpC" comprised a cleavable signal peptide (SEQ ID NO:69). When the autotransporter fusion protein was displayed on the surface of OMVs, the signal peptide was cleaved, resulting in an autotransporter fusion protein sequence without the signal peptide displayed on the OMV membrane (SEQ ID NO:68). Herein, whenever reference is made to OMVs displaying autotransporter fusion proteins, it should be assumed that the autotransporter fusion protein sequence does not comprise a signal peptide sequence. Whenever reference is made to "expressed" autotransporter fusion proteins which have yet to be displayed on the surface of OMVs, it should be assumed that the autotransporter fusion protein sequence further comprises a signal peptide sequence.

[0201] Outer Membrane Vesicle (OMV) production

[0202] OMVs displaying the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) were produced using Salmonella Typhimurium SL3261 AtoIRA AmsbB transformed with plasmid pEH3-HbpD-SpC. Batch fermentation was carried out using an animal component-free and fermentation-optimized version of TYMC-medium at 30 °C (Kuipers et al, Infect Immun. 2017 Sep 20; 85(10)). To harvest OMVs, cultures of "HbpD-SpC" (SEQ ID NO:68) expressing Salmonella were initially subjected to low- speed centrifugation to remove most cells. The supernatant was then filtered, on a filter with a pore size of 0.45 .m, to remove residual bacteria. The resulting filtered OMV solution was concentrated and diafi Itrated (PBS) by tangential flow filtration (TFF) with a molecular weight cutoff (MWCO) of 500 kDa to obtain the OMV suspension used in Examples 2-3, 7, 9, 11, and 13-16.

[0203] OMVs used in Example 3, 11, and 13-16 were subjected to an additional concentration and diafi Itration TFF-step, with a MWCO of 500 kD replacing PBS for a 50 mM HEPES, 8 mM Mg-glutamate, 29 mM KOH, pH 7.5 OMV suspension buffer.

[0204] SDS PAGE and Western blot

[0205] SDS-PAGE was performed using standard 10%, 12%, 14% or 15% SDS-PAGE gels. Protein samples were solubilized in SDS-PAGE sample buffer (50 mM TrisHCI pH 6.8, 2% w / v SDS, 10% glycerol, 0.01 % w / v bromophenol blue, 50 mM DTT) and boiled for 10 min. Gels were stained with Coomassie Brilliant Blue G-250 and captured using a Molecular Imager GS-800 Calibrated Densitometer (BioRad). Western bloting was performed using anti HA Tag mouse monoclonal antibody (2- 2.2.14) (Invitrogen, Catalog # 26183) and a goat anti-mouse IgG (H + L)-HRP Conjugate (BioRad, Catalog #170-6515) or using an anti-HA(PR8) (Influenza A H1N1 HA (A / Puerto Rico / 8 / 34) Polyclonal Antibody, Invitrogen Catalog # PA5-81670) and a goat anti-rabbit IgG ( H+L)-H RP (Rockland, item # 611-1302) or using an anti-HpdD antibody (raised in rabbits against Hbp at Davids Biotechnologie GmbH, ProtA- purified from serum) and a goat anti-rabbit IgG ( H+L)-H RP (Rockland, item # 611- 1302).

[0206] Example 1

[0207] Cell-free synthesis of neoepitope constructs carrying a SpyTag in a bacterial extract This Example demonstrates successful cell-free synthesis of a polypeptide of interest (POI) comprising at least one target polypeptide moiety, a moiety of a coupling system in the form of SpyTag ("SpT", SEQ ID NO:10), with or without further comprising a solubility enhancing moiety ("PotD", SEQ ID NO:70), in a bacterial extract.

[0208] Materials and Methods

[0209] The POIs in Example 1 comprised at least one target polypeptide moiety in the form of multiple tandem-fused epitopes (multiple tandem-fused epitopes "NC", SEQ ID NO: 1 or multiple tandem-fused neoepitopes "PC", SEQ ID NO: 2) or a pneumococcal antigen ("PnrA", SEQ ID NO:3). All expressed POIs further comprised an N-terminal SpyTag ("SpT", SEQ ID NQ:10), and a C-terminal hemagglutinin (HA) tag for detection purposes ("HA-tag", SEQ ID NO:4), and were cloned into pIVEX plasmid vector backbones under control of a T7 promoter, see Table 1 for a summary of the tested POIs and their respective SEQ ID NO's. The resulting constructs were isolated from Escherichia coli and used as template in a NEBExpress® Cell-free Protein Synthesis reaction (New England Biolabs). The NEBExpress® Cell-free protein synthesis system is an E. coli extract-based coupled transcription / translation system for synthesis of proteins encoded by DNA templates under the control of a T7 RNA Polymerase promoter. Reactions were carried out following manufacturer's recommendations using 50 ng of DNA template per 10 pl reaction volume and incubation at 37 °C for 4 hours. A 2 pl sample of the reaction mixtures was analyzed by SDS-PAGE and Coomassie staining.

[0210] Successful synthesis of the polypeptides was concluded based on the appearance of specific bands on the Coomassie stained gel corresponding to theoretical molecular mass of the respective POIs as indicated below the gel image of Figure 1.

[0211] Table 1: Design of POIs synthesized in Example 1

[0212] Results

[0213] Successful synthesis of the target polypeptide moiety comprised in each respective POI was achieved regardless of the further presence of a solubility enhancing moiety comprised in the POI. Herein the term "solubility enhancing moiety" is used interchangeably with the term "solubility enhancer". The respective synthesized target polypeptide moiety is indicated with arrowheads, and Molecular mass (kDa) markers are indicated at the left side of the panel (Figure 1).

[0214] Successful synthesis was observed for varying target polypeptide moieties, such a POI comprising a pneumococcal antigen PnrA or POIs comprising multiple tandem- fused epitopes, demonstrating the system's versatility towards the nature and origin of the expressed target polypeptide moiety.

[0215] Example 2

[0216] Coupling of a neoepitope construct upon post-translational administration of OMVs This Example demonstrates successful cell-free synthesis of a polypeptide of interest (POI) (SEQ ID NO:93) comprising at least one target polypeptide moiety, a moiety of a coupling system in the form of SpyTag ("SpT", SEQ ID NO:10), further comprising a solubility enhancing moiety ("PotD", SEQ ID NO:70), in a bacterial extract. Moreover, the example demonstrates successful coupling of the POI to bacterial outer membrane vesicles (OMVs) displaying a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), added after the onset of cell-free polypeptide synthesis.

[0217] Materials and Methods

[0218] A nucleotide segment encoding a POI (SEQ ID NO:93), comprising an N- terminal SpT (SEQ ID NQ:10) to allow coupling and a C-terminal HA-tag (SEQ ID NO:4) for detection purposes, was cloned under T7 promoter control. The POI construct was expressed in duplo in the NEBExpress® Cell-free Protein Synthesis system for 4 hours at 37 °C as described in Example 1. Three hours after the onset of polypeptide synthesis, 27 g of Salmonella OMVs displaying an autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:67) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was added to one of the sample reactions, and incubation was continued for one hour at 37 °C. Samples (Figure 2) comprising the same amount of cell-extract, with (Figure 2, lane 3) or without (Figure 2, lane 2) added OMVs, were analyzed by SDS-PAGE and Coomassie staining (A), as well as by Western bloting using HA-antiserum (B). For comparison, a Cell- free Protein Synthesis sample containing OMVs but lacking expressed POI was also analyzed (Figure 2, lane 1).

[0219] Results

[0220] Successful synthesis of the target polypeptide moiety, multiple tandem-fused epitopes (SEQ ID NO:2) comprised in the POI (SEQ ID NO: 93), was achieved and was observed via the emergence of a ~ 54 kDa band (see Fig. 2) on Coomassie stained gel in samples allowing synthesis of the target polypeptide moiety (Figure 2, lanes 2 and 3). Spy-based coupling to OMVs was demonstrated by the formation of a ~180 kDa adduct comprising sequence with SEQ ID NO:93 and SEQ ID NO:68 in samples containing both the POI and the autotransporter fusion protein as displayed on OMVs (Figure 2, lane 3). For comparison, in the absence of POI (SEQ ID NO: 93), only uncoupled autotransporter fusion protein as displayed on OMVs (SEQ ID NO:68) can be observed and no adduct (Figure 2A, lane 1). The identity of the adduct could be confirmed by Western bloting detecting the HA-tag at the C-terminus of POI (SEQ ID NO:93) (Figure 2B, lane 3). Molecular mass (kDa) markers are indicated at the left side of the panels.

[0221] Example 3

[0222] Coupling of neoepitopes and antigens upon co-translational administration of OMVs in the presence and absence of a solubility enhancer

[0223] This Example demonstrates successful and efficient Spy-based coupling of polypeptides of interest (POI) comprising a coupling moiety SpyTag ("SpT"; SEQ ID NQ:10) and at least one target polypeptide moiety, such as either multiple tandem- fused epitopes (multiple tandem-fused epitopes "NC", SEQ ID NO: 1 or multiple tandem-fused neoepitopes "PC", SEQ ID NO: 2) or a pneumococcal antigen ("PnrA", SEQ ID NO:3), to Salmonella-derived bacterial outer membrane vesicles (OMVs). The OMVs, displaying a complementary moiety of the Spy-based coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), are present at the onset of the cell-free protein synthesis. Moreover, the example demonstrates that a solubility-enhancing sequence (PotD; SEQ ID NQ:70) comprised in the POI is a viable strategy to improve coupling of POIs that otherwise atach to OMVs with low efficiency.

[0224] Materials and Methods

[0225] Nucleotide sequences encoding a POI comprising an N-terminal SpT (SEQ ID NQ:10), to allow coupling, and a C-terminal HA-tag (SEQ ID NO:4), for detection purposes, were cloned under T7 promoter control for cell-free synthesis in the NEBExpress® system as described for Example 1. The POIs comprised at least one target polypeptide moiety in the form of either multiple tandem-fused epitopes (multiple tandem-fused epitopes "NC", SEQ ID NO: 1 or multiple tandem-fused neoepitopes "PC", SEQ ID NO: 2) or a pneumococcal antigen ("PnrA", SEQ ID NO:3). Furthermore, the POIs did or did not comprise a solubility-enhancing sequence (PotD; SEQ ID NO:70). The POI constructs used in Example 3 are outlined in Table 2 below.

[0226] Table 2: Design of POIs synthesized in Example 3

[0227] Cell-free synthesis reaction mixtures also comprised Salmonella-derived OMVs (3.5 p.g / p.1) at the start of the synthesis, the OMVs displaying an autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:67) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11). Synthesis proceeded at 37 °C for 4 hours after which the reaction mixtures were incubated overnight at 4 °C to allow optimal coupling of POIs to the OMVs (Figure 3, lanes 1-5). One additional reaction mixture comprising a POI (SEQ ID NO:93) and OMVs was transferred directly to -20 °C after cell-free protein synthesis to analyze the efficiency of coupling during this short 4-hour period (lane 6). Samples containing equal amounts of cell-free translation reaction mixture were analyzed by SDS-PAGE and Coomassie staining (Figure 3A) and by Western bloting (Figure 3B).

[0228] Results

[0229] Efficient Spy-based coupling to OMVs decorated with an autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) was observed for the following POIs upon overnight incubation: SpT-NC-HA (Figure 3, lane 1; SEQ ID NO:90), SpT-PotD-NC-HA (Figure 3, lane 3; SEQ ID NO:92), SpT-PotD-PC-HA (Figure 3, lane 4; SEQ ID NO:93) and SpT-PnrA-HA (Figure 3, lane 5; SEQ ID NO:94), demonstrated by Coomassie staining (Figure 3, panel A). This follows from the efficient formation of adducts corresponding to covalent linkage between SpyCatcher comprised in the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) to the respective coupling moiety SpyTag, comprised in the POIs (Figure 3A, adducts indicated with black arrowheads). Furthermore, low to non-existing amounts of uncoupled autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) were observed in these samples (Figure 3A, uncoupled HbpD-SpC indicated with "<"). In the case of POI construct comprising "PotD-PC" (SEQ ID NO:93), similar amounts of adduct are observed when analyzing the sample directly after the synthesis period (Figure 3A, compare lanes 6 and 4), indicating time-efficient OMV-conjugate formation. In turn, recovery of similar amounts of "PotD-PC" (SEQ ID NO:93) containing adduct in sample lanes 4 and 6 demonstrates the stability of coupled vesicle-POl constructs in bacterial extract despite an extended overnight incubation period.

[0230] Relatively low efficiency coupling was observed for the POI construct comprising "SpT-PC-HA" (SEQ ID NO:91), as shown by low detected amounts of adduct and high amounts of uncoupled sequence with SEQ ID NO:91 (Figure 3 A, lane 2, "<"). In contrast, upon inclusion of a solubility enhancing moiety "PotD" in the POI construct "SpT-PotD-NC-HA" (SEQ ID NO:93), for the same target polypeptide moiety "PC", high amounts of adduct was observed and while uncoupled "HbpD- SpC" (SEQ ID NO:68) was virtually absent (Figure 3 A, lane 4). This demonstrates that the inclusion of solubility enhancing moieties in synthesized POIs comprising at least one target polypeptide moiety can improve the efficiency of POI-coupling to OMVs when using a cell-free synthesis approach. The identity of adducts was confirmed by Western bloting detecting the HA-tag at the C-terminus of synthesized POIs (Figure 3B). Molecular mass (kDa) markers are indicated at the left side of the panels. Example 4

[0231] Solubility of neoepitope construct in bacterial extract in the presence and absence of a solubility enhancer

[0232] This Example demonstrates improved solubility of an aggregation-prone neoepitope-comprising polypeptide of interest (POI) in a cell-free translation system upon inclusion of solubility enhancing moiety ("PotD", SEQ ID NO:70) to the POI construct.

[0233] Materials and Methods

[0234] Nucleotide sequences encoding the POIs "SpT-PC-HA" (SEQ ID NO:91), "SpT- PotD-PC-HA" (SEQ ID NO:93) and "SpT-PnrA-HA" (SEQ ID NO:94) were used for protein synthesis in the NEBExpress® system at 37°C for 4 hours essentially as described in Example 1. Following incubation, the 14.4 p.1 reaction mixtures were diluted with phosphate buffered saline (PBS), pH 7.4, to a total volume of 250 p.1. To separate soluble from insoluble proteins, total reaction mixture (Tot) samples were subjected to centrifugation at 10,000 x g at 4°C for 10 min. The resulting supernatant (SI) was transferred to a fresh tube and subjected to ultracentrifugation at 300,000 x g at 4 °C for 10 min to yield supernatant (S2). Samples of the supernatants, all derived from the same amount of total reaction mixture (Tot), were analyzed by SDS- PAGE and Western bloting using HA-antiserum.

[0235] Results

[0236] Protein staining of the nitrocellulose membrane using Ponceau-S dye confirmed equal protein transfer for all total reaction mixture (Tot) samples and the respective derived supernatant samples SI and S2. Specific detection of respective POIs comprising target polypeptides using HA-antiserum showed significantly decreased amounts of the POI construct "SpT-PC-HA" (SEQ ID NO:91) in the supernatant from the first centrifugation step compared to the total reaction mixture sample (Figure 4, compare lanes 1 and 2), and an almost complete loss of the synthesized POI upon the second centrifugation step (Figure 4, compare lanes 1 and 3). It appeared that the synthesized POI construct "SpT-PC-HA" (SEQ ID NO:91) was removed upon centrifugation, indicative of aggregation of the target polypeptide in the cell-free reaction mixture. In contrast, the POI construct SpT-PotD-PC-HA (SEQ ID NO:93), comprising a solubility enhancing moiety "PotD", was equally detected in all analyzed fractions (Figure 4, lanes 4-6), comparable to the known soluble control POI construct SpT-PnrA-HA (SEQ ID NO:94) (Figure 4, lanes 7-9). Hence, the POI construct "SpT-PotD-PC-HA (SEQ ID NO:93) was soluble upon cell-free synthesis. In conclusion, the inclusion of a solubility enhancing moiety in the POI construct, such as PotD, alleviated the otherwise aggregation-prone properties of the neoepitope target polypeptide moiety "PC" (SEQ ID NO:2) comprised in the POI. Molecular mass (kDa) markers are indicated at the left side of the panels.

[0237] Example 5

[0238] Streptococcus derived PotD has the ability to keep aggregation-prone fusion proteins soluble

[0239] This Example illustrates that PotD derived from Streptococcus suis has the ability to keep aggregation-prone polypeptides carrying an inclusion body (IB) formation tag soluble. PotD derived from Streptococcus suis (SEQ ID NQ:70) outperforms the known solubility enhancer MBP derived from Escherichia coli (SEQ ID NO:76) (as described in Waugh, D.S. Postepy Biochem. (2016) 62(3):377-382).

[0240] Methods and Materials

[0241] Fusion proteins comprising IB formation tags ssTorA(3X) or ssTorA(A29- 3613X) (herein, "ssTorA(A29-3613X" is referred to as "TrM") (Jong et al., Front. Bioeng Biotechnol. (2020) 7:442) were expressed in E. coli and the resulting inclusion bodies were isolated essentially as described in Schetters et al., Cell Mol Immunol. (2020)17(4):415-417. Fusion constructs used in Example 5 are outlined in Table 3 below. Table 3: Design affusion protein constructs in Example 5

[0242] 2 mg of IB material from each sample type was solubilized in 6 M urea and the resulting solutions were dialyzed against 20 mM Tris-CI pH 8.0 using Spectra / POR dialysis membrane (MWCO 3500) overnight. Dialyzed material was then subjected to centrifugation at 15,000 x g for 10 min to separate the insoluble protein from the soluble protein content. Samples of resulting pellet and supernatant, as well as of the corresponding urea solubilized IBs (input), were analyzed by SDS-PAGE and Coomassie staining. Protein bands corresponding to the fusion constructs are indicated with black arrowheads in Figure 5. Molecular mass (kDa) markers are indicated at the left side of the panels.

[0243] Results

[0244] Fusion protein constructs comprising IB formation tags and either SARS-COV- 2 antigen Spike SI ("TrM-Sl-TrM", SEQ ID NO:97; see Figure 5, lanes 6-8) or His- tagged Green Fluorescent Protein ("ssTorA(3X)-hisGFP", SEQ ID NO:98; Figure 5, lanes 9-11) clearly reaggregated upon dialysis. This was observed by their recovery in the pellet after centrifugation (see Figure 5, lanes 8 and 11). Furthermore, a fusion protein construct comprising the well-known solubility enhancer "MBP" was also detected almost entirely in the pellet ("ssTorA(3X)-MBP-SpT", SEQ ID NO:95; Figure 5, lane 3), indicating a high degree of insolubility of the fusion protein upon removal of urea by dialysis. In clear contrast, a fusion protein comprising the solubility enhancer "PotD" ("TrM-PotD-TrM", SEQ ID NO:96) was detected almost entirely in the supernatant after centrifugation, despite the presence of IB formation tags (TrM) at both the N- and the C-terminus (Figure 5, lane 5). This demonstrates the ability of the 5. su / s-derived solubility enhancer "PotD" (SEQ ID NQ:70), when comprised in a fusion construct together with an aggregation-prone protein, to keep the fusion construct in solution. Example 6

[0245] PotD outperforms MBP in retaining solubility of aggregation-prone neoepitopes comprised in POIs

[0246] This Example demonstrates the surprising superiority of the solubility enhancer "PotD" (SEQ ID NO:70) derived from 5. suis, in contrast to the well-known solubility enhancer "MBP" (SEQ ID NO:76) derived from E. coli, in retaining the solubility of polypeptides of interest (POIs) comprising aggregation-prone target polypeptide moieties of various natures, such as either multiple tandem-fused epitopes "NC" (SEQ ID NO: 1) or multiple tandem-fused neoepitopes "PC" (SEQ ID NO: 2).

[0247] Materials and Methods

[0248] POIs comprising an N-terminal IB formation tag ssTorA(A29-3613X) (alternatively called "TrM") (Jong et al, Front. Bioeng Biotechnol. (2020) 7:442), a C- terminal SpT, a solubility enhancing moiety, and a target polypeptide moiety in the form of multiple tandem-fused neoepitopes were expressed in E. coli. The POI constructs used in Example 6 are outlined in Table 4 below.

[0249] Table 4: Design of POIs utilized in Example 6

[0250] Resulting inclusion bodies were isolated essentially as described in Schetters et al., Cell Mol Immunol. (2020) 17(4):415-417. 800 - 2000 g of IB material from each sample type was solubilized in 8 M urea for 30 min at room temperature and the resulting solutions were dialyzed against PBS using Spectra / POR dialysis membrane (MWCO 3500) overnight. Dialyzed material was subjected to centrifugation at 15,000 x g for 10 min to separate the insoluble from the soluble protein content. Samples of resulting pellet (P) and supernatant (S), respectively, were analyzed by SDS-PAGE and Coomassie staining. Protein bands corresponding to the POIs are indicated with arrowheads in Figure 6. Molecular mass (kDa) markers are indicated at the left side of the panels.

[0251] Results

[0252] Dialyzed POIs comprising E. coli- derived "MBP" as a solubility enhancing moiety, as in "TrM -MBP-NC-SpT" (SEQ ID NO:99) or "TrM -MBP-PC-SpT" (SEQ ID NO:101), were detected almost entirely in the centrifugation pellet (see Figure 6, lane 1 and 5). In contrast, when the POI construct comprised 5. su / s-derived "PotD" (SEQ ID NO:70) as a solubility enhancing moiety, as in "TrM -PotD-NC-SpT" (SEQ ID NQ:100) or "TrM -PotD-PC-SpT" (SEQ ID NQ:102), the POI constructs were virtually absent in the pellet (Figure 6, lanes 3 and 7) and abundantly present in the supernatant (Figure 6, lanes 4 and 8), indicating the sample material was in a soluble state. These data demonstrated that 5. su / s-derived PotD is more effective than E. coli-derived MBP in preserving the solubility of neoepitope-comprising POIs that are otherwise prone to aggregation.

[0253] Example 7

[0254] Superior coupling of POIs comprising neoepitope sequences and the solubility enhancer PotD to OMVs

[0255] This Example demonstrates the superior Spy-based coupling of OMVs to POIs comprising an aggregation prone target polypeptide moiety, and further comprising the solubility enhancer 5. su / s-derived PotD, when compared to POIs comprising the same target polypeptide moiety and a different solubility enhancer, E. co / / -derived

[0256] MBP. Materials and Methods

[0257] POI constructs, "TrM-MBP-PC-SpT" (SEQ ID NO:101) and "TrM-PotD-PC-SpT" (SEQ ID NO:102), comprising an N-terminal IB formation tag ssTorA(A29-3613X) (alternatively called TrM) (Jong et al., Front. Bioeng. Biotechnol. (2020) 7:442), a C- terminal SpyTag coupling moiety, a target polypeptide moiety in the form of the aggregation-prone multiple tandem-fused neoepitopes "PC" (SEQ ID NO: 2), and a solubility enhancing moiety, were expressed in E. coli to allow inclusion body formation. IBs were isolated essentially as described in Schetters et al., Cell Mol Immunol. (2020) 17(4):415-417 and solubilized in 8 M urea at room temperature for 30 min. The resulting solutions were dialyzed against PBS using Spectra / POR dialysis membrane (MWCO 3500) overnight. Dialyzed material was subjected to centrifugation at 15,000 x g for 10 min to remove insoluble protein content. The supernatant, comprising soluble proteins, was tested for Spy-coupling competence. To this end, equal volumes of supernatant comprising "TrM-MBP-PC-SpT" or "TrM- PotD-PC-SpT"- were incubated with ~5 pg of Salmonella-derived OMVs displaying an autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:67) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), at 4°C overnight. Samples were analyzed by SDS-PAGE and Coomassie staining.

[0258] Results

[0259] Superior and efficient coupling to OMVs was observed for the POI construct comprising the solubility enhancing moiety PotD, when compared to the POI construct comprising the solubility enhancing moiety MBP. This is observed by the appearance of an intense adduct band (Figure 7, lane 2) representing the Spy-based covalent coupling of "TrM-PotD-PC-SpT" (SEQ ID NQ:102) to "HbpD-SpC " (SEQ ID NO:68). Analysis utilizing supernatant comprising the other POI construct, "TrM- MBP-PC-SpT" (SEQ ID NQ:101), yielded only low amounts of adduct and a significant amount of uncoupled autotransporter fusion protein ("HbpD-SpC"; SEQ ID NO:68; Figure 7, lane 1). Hence, it was demonstrated that the use of PotD as a solubility enhancing moiety comprised in a POI was beneficial for the generation of OMV- neoepitope conjugates, likely due to the solubility-enhancing capacities of PotD comprising POIs as demonstrated in Examples 3-6. In Figure 7 relevant proteins are indicated with arrowheads and Molecular mass (kDa) markers are indicated at the right-hand side of the panel.

[0260] Example 8

[0261] Streptococcus su / 's-derived PotD is superior to Escherichia co / / -de rived PotD in keeping aggregation-prone POIs soluble

[0262] This Example shows that use of Streptococcus su / s-derived PotD (SEQ ID NO:70) as a solubility enhancing moiety is more effective at keeping aggregation- prone POIs soluble compared to Escherichia co / / -de rived PotD (SEQ ID NO:71), a previously described solubility enhancer (Han et al, Biochim Biophys Acta. (2007) 1774(12):1536-43). POIs comprising target polypeptide moieties in the form of either neoepitope stretches of various nature (multiple tandem-fused neoepitope "PC", SEQ ID NO: 2 or multiple tandem-fused neoepitope "MN1", SEQ ID NO: 5) or a Mycobacterium tuberculosis antigen ("RvOlll", SEQ ID NO:6) were tested.

[0263] Materials and Methods

[0264] POIs comprising a N-terminal IB formation tag "TrM" (Jong et al., Front Bioeng Biotechnol. (2020) 7:442), a C-terminal SpyTag coupling moiety, a target polypeptide moiety, and a solubility enhancing moiety, were expressed in E. coli and resulting IBs were isolated essentially as described (Schetters et al., Cell Mol Immunol. (2020) 17(4):415-417). The POI constructs used in Example 8 are outlined in Table 5 below. Table 5: Design of POIs utilized in Example 8

[0265] 2 mg of IB material from each sample type was solubilized in 8M urea at room temperature for 30 min. To remove urea, the resulting solutions were dialyzed against PBS using Spectra / POR dialysis membrane (MWCO 3500) overnight. The solutions were then subjected to centrifugation at 15,000 x g for 5 min to separate insoluble from soluble protein content. Pellet (P) and supernatant (S) samples were analyzed by SDS-PAGE and Coomassie staining in parallel to the IB starting material (IB). All samples are derived from 2.5 g of IB starting material. Only the relevant sections of the respective gels are shown in Figure 8. Results

[0266] POI constructs "TrM-PotD-PC-SpT" (SEQ ID NO :102), "TrM -PotD-MNl-SpT" (SEQ ID NO:103), and "TrM -PotD-RvOlll-SpT" (SEQ ID NO:104) comprising the solubility enhancing moiety 5. su / s-derived PotD (SEQ ID NQ:70) were fully or partially (MN1) recovered from the centrifugation supernatant upon dialysis- mediated removal of urea, demonstrating that the referred to POI constructs remained in a soluble state. In contrast, the POI constructs comprising E. co / / -derived PotD (SEQ ID NO:71) were recovered almost exclusively in the pellet after centrifugation, indicating aggregation of the referred to POI constructs. These data surprisingly indicate that use of Streptococcus suis-de rived PotD (SEQ ID NO:70) as a solubility enhancing moiety is more effective at keeping aggregation-prone POIs soluble compared to the previously described Escherichia co / / '-de rived PotD (SEQ ID NO:71) sequence.

[0267] Example 9

[0268] Streptococcus su / 's-derived PotD allows for more efficient coupling of a POI construct to OMVs compared to Escherichia co / / -de rived PotD

[0269] This Example demonstrates that the inclusion of PotD from Streptococcus suis (SEQ ID NQ:70) in a POI further comprising multiple tandem-fused neoepitope sequences ("PC") is beneficial for Tag / Catcher-based coupling to OMVs when compared to OMV coupling of POIs comprising the same neoepitope sequences and PotD from Escherichia coli ("EcPotD"; SEQ ID NO:71).

[0270] Materials and Methods

[0271] POI constructs "TrM-PotD-PC-SpT" (SEQ ID NO:102) and "TrM-EcPotD-PC- SpT" (SEQ ID NQ:105) comprising an N-terminal IB formation tag "TrM" (Jong et al., Front Bioeng Biotechnol. (2020) 7:442), a C-terminal SpyTag coupling moiety, a target polypeptide moiety, and a solubility enhancing moiety, were expressed in E. coli to allow inclusion body formation. IBs were isolated essentially as described in Schetters et al., Cell Mol Immunol. (2020) 17(4):415-417 and solubilized in 8 M urea at room temperature for 30 min. The resulting solutions were dialyzed against PBS using Spectra / POR dialysis membrane (MWCO 3500) overnight. Dialyzed material was subjected to centrifugation at 15,000 x g for 5 min to remove insoluble protein content. The supernatant, comprising soluble proteins, was tested for Spy-coupling competence. To this end, supernatant comprising "TrM-PotD-PC-SpT" or "TrM- EcPotD-PC-SpT" were incubated with ~5pg of Salmonella-derived OMVs displaying an autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:67) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), at 4°C overnight. Of note, a 5 times excess of supernatant comprising POI construct "TrM-EcPotD-PC-SpT" (SEQ ID NO:105; comprising E. co I i -de rived PotD orEcPotD) was used for coupling compared to the POI construct "TrM-PotD-PC-SpT" (SEQ ID NQ:102; comprising S.su / s-de rived PotD). Samples were analyzed by SDS-PAGE and Coomassie staining.

[0272] Results

[0273] More efficient coupling to OMVs was clearly observed for the POI construct comprising 5. su / s-derived PotD (SEQ ID NQ:102) compared to the POI construct comprising E. coli-derived PotD (SEQ ID NQ:105). This is illustrated by the detection of an intense adduct band representing covalent coupling of "TrM-PotD-PC-SpT" to the OMVs via the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) (Figure 9, lane 2). Of note, uncoupled "HbpD-SpC" as displayed on OMVs, which is normally present (Figure 9, lane 1) is almost completely absent in lane 2, underlining the efficiency of the coupling between the POI construct comprising 5. su / s-derived PotD and OMVs. On the other hand, analysis of supernatant containing "TrM-EcPotD-PC- SpT" yielded only low amounts of adduct and left a considerable amount of OMVs displaying uncoupled autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) (Figure 9, lane 3). Taken together, PotD from Streptococcus suis (SEQ ID NO:70) outperforms PotD from Escherichia coli ("EcPotD"; SEQ ID NO:71) as a solubility enhancing moiety in the generation of OMV-neoepitope conjugates. This may be due to Streptococcus su / s-derived PotD's more favorable solubilization capacities as shown in Example 8. Relevant proteins are indicated with arrowheads. Molecular mass (kDa) markers are indicated at the left side of the panel.

[0274] Example 10

[0275] Alignment of 5. suis PotD and E. coli PotD

[0276] This Example shows relatively low conservation between the amino acid sequences of PotD from the Gram-positive bacterium Streptococcus suis (SEQ ID NO:70) and the variant from the Gram-negative bacterium Escherichia coli (SEQ ID NO:71), to which solubility-enhancing potential has been attributed previously (Han et al., Biochim Biophys Acta. (2007) 1774(12):1536-43). This low conservation provides a possible explanation for the superior solubility enhancing capacities observed with Streptococcal PotD in Examples 8 and 9.

[0277] Materials and Methods

[0278] Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) was used to align the sequence of Streptococcus suis-de rived PotD (SEQ ID NO:70; UniProtKB: A0A4V0E299; residues 28 - 356) and the sequence of Escherichia coli BL21(DE3)- derived PotD (SEQ ID NO:71; UniProtKB: A0A140NCN3; residues 20 - 348) as used in the examples (Figure 10). The latter sequence (SEQ ID NO:71) was also used in Han et al., Biochim Biophys Acta. (2007) 1774(12):1536-43). The aligned sequences were used as input for the Ident and Sim online bioinformatics tool (https: / / www.bioinformatics.org / sms2 / ident_sim.html) to calculate identity and similarity on the amino acid level.

[0279] Results

[0280] Relatively low scores of 39 percent identity and 52 percent similarity were obtained for SEQ ID NQ:70 and SEQ ID NO:71.

[0281] Example 11

[0282] Successful coupling of POI to OMVs in the presence of a broad-spectrum protease inhibitor cocktail

[0283] The addition of a protease inhibitor cocktail to prevent degradation of target polypeptides during cell-free synthesis may be taken as a precautionary measure to secure efficient production of OMV-conjugates. However, these compounds could potentially interfere with cell-free protein synthesis and the coupling system-based, such as Tag / Catcher based, coupling to OMVs. This Example demonstrates that the addition of a broad-spectrum protease inhibitor cocktail does not interfere with cell- free synthesis and subsequent coupling system-based coupling of a POI comprising a polyepitope stretch to OMVs. Materials and Methods

[0284] POI construct "SpT-PotD-NC-HA" (SEQ ID NO:92) was synthesized in the NEBExpress® system at 37 °C as described in Example 1 in the presence (Figure 11, lanes 2 and 4) or absence (Figure 11, lanes 1 and 3) of complete™ (EDTA-free) protease inhibitor cocktail that inhibits serine, cysteine, and metalloproteases (Roche, # 11873580001). The protease inhibitor was used according to the manufacturer's recommended dosing. Salmonella-derived OMVs displaying HbpD- SpC were added to a reaction mixture comprising the synthesized POI construct "SpT-PotD-NC-HA" with (Figure 11, lane 4) and without (Figure 11, lane 3) complete™. After 3.5 hours, a second dose of complete™ was added to the samples that already received the first dose of the protease inhibitor (Figure 11, lanes 2 and 4). All reactions were continued at 37°C for 30 min after which samples were incubated at 4°C overnight. Equal amounts of cell-free translation reaction mixture were analyzed by Western bloting using HA-antibodies.

[0285] Results

[0286] Adduct formation, representing Spy-based covalent bonding between the POI "SpT-PotD-NC-HA" and the autotransporter fusion protein "HbpD-SpC" displayed on OMVs, was observed upon addition of OMVs displaying "HbpD-SpC" to the synthesis reaction mixtures. The presence of complete™ nor did affect synthesis levels of SpT- PotD-NC-HA (Figure 11, compare lanes 1 and 2), neither did it influence the efficiency of adduct formation (Figure 11, compare lanes 3 and 4). These data show the presence of protease inhibitors is compatible with the cell-free production of OMV-polypeptide conjugates. Molecular mass (kDa) markers are indicated at the left side of the panels in Figure 11. Example 12

[0287] Mycobacterial tuberculosis RvOlll antigen is prone to aggregation upon recombinant expression in E. coli

[0288] This Example demonstrates that RvOlll (SEQ ID NO:6) is an aggregation- prone protein, even in low temperature conditions. Full-length RvOlll is a transmembrane protein of Mycobacterium tuberculosis. A truncated form thereof, herein referred to as "RvOlll" (SEQ ID NO:6), lacks all but one of its transmembrane segments and has previously been investigated as an antigen for tuberculosis (TB) vaccine development (Leung-Theung-Long et al., PLoS One. (2015) 10(11)). Here we show that a POI construct comprising the truncate antigen "RvOlll", as well as a SpyTag coupling moiety and a His6-tag ("H6-SpT-Rv0111"; SEQ ID NQ:110), is insoluble when expressed in E. coli despite using low expression temperature, which conditions normally favor soluble expression of recombinant proteins.

[0289] Materials and Methods

[0290] E. coli BL21(DE3) cells were transformed with a pET28 plasmid derivative encoding the 36 kDa POI construct "H6-SpT-Rv0111" (SEQ ID NQ:110) under control of T7 promoter. Cells were grown and induced for expression at 37 °C for 3 h (Figure 12, lanes 7-9). Alternatively, they were grown and induced at lower temperature regimes (30 °C for 3 h, Figure 12, lanes 4-6; 12 °C for 22 h, Figure 12, lanes 1-3) to reduce protein synthesis kinetics and provide more favorable conditions for soluble protein expression. Aggregation of POI construct "H6-SpT-Rv0111" was assayed using a fractionation technique. Induced cells were collected and resuspended in ice cold lysis buffer (100 mM NaCI, 1 mM EDTA, 50 mM Tris-HCL pH 7.6) after which lysozyme (17 ng / ml) was added. After 15 min incubation on ice, the suspensions were subjected to snap freezing and sonification (Branson 15 Sonifier 250) to disrupt the cells. Subsequently, the cell lysates were subjected to centrifugation (4,500 g, 10 min) to pellet aggregated proteins. The resulting supernatant, containing soluble proteins, was TCA precipitated. Corresponding amounts of cell lysate (input), aggregated protein content (pellet) and soluble protein content (sup) were analyzed by SDS-PAGE and Coomassie blue staining.

[0291] Results

[0292] Under all conditions (Figure 12, lanes 1-9) it was observed that the POI construct "H6-SpT-Rv0111" was detected entirely in the pellet fraction, demonstrating the aggregation-prone nature of the RvOlll antigen.

[0293] Example 13

[0294] Successful coupling of POIs comprising various target polypeptide moieties to OMVs

[0295] This Example shows successful coupling, such as Tag / Catcher based coupling, of POIs comprising either target polypeptide moieties comprising multiple tandem- fused epitopes of various nature ("MN1", SEQ ID NO:5; "MN2", SEQ ID NO:89; "NC", SEQ ID NO:1) or an aggregation prone mycobacterial antigen ("RvOlll", SEQ ID NO:6; see Example 12) to Salmonella-derived OMVs. Moreover, it is demonstrated that the presence of a solubility enhancing moiety, 5. su / s-derived "PotD", has a positive effect on adduct formation, even with a POI construct comprising the target polypeptide moiety "MN1", which otherwise forms strong aggregates that resist the denaturing nature of the SDS-PAGE analysis procedure.

[0296] Materials and Methods

[0297] Plasmids encoding the following POIs were used for protein synthesis in the NEBExpress® system at 37 °C for 4 hours essentially as described in Example 1: "SpT- MN1-HA" (SEQ ID NO:111), SpT-MN2-HA (SEQ ID NO:112), SpT-PotD-MNl-HA (SEQ ID NO:113), SpT-PotD-MN2-HA (SEQ ID NO:114), SpT-PotD-NC-HA (SEQ ID NO:92) or SpT-RvOlll-HA (SEQ ID NO:115). After 3.5 hours, OMVs displaying the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) were added to half the samples (Figure 13, lanes 1-6). The reactions were continued at 37 °C for half an hour before shifting the temperature to 4 °C for incubation overnight. Samples containing equal amounts of cell-free translation reaction mixture were analyzed by SDS-PAGE and Western bloting using anti-HA. Results

[0298] Upon addition of OMVs displaying "HbpD-SpC" (SEQ ID NO:68) to the POI synthesis reaction mixture, adducts could be observed for synthesized POI constructs "SpT-RvOlll-HA" (SEQ ID NO:115), "SpT-PotD-MNl-HA" (SEQ ID NO:113), "SpT-PotD- MN2-HA" (SEQ ID NO:114) and "SpT-PotD-NC-HA" (SEQ ID NO:92) (Figure 13, lanes 1 and 4-6), demonstrating successful Spy-based coupling of the above POIs to OMVs displaying "HbpD-SpC". Of note, apparent amounts of high-molecular weight species were detected upon synthesis of POIs comprising "MN1" (marked with "*" in Figure 13). This points to the formation of rather stable aggregates that even survive the denaturative conditions of the SDS-PAGE and Western bloting procedures, which involves sample incubation in 2% of SDS at 96 °C and gel-electrophoresis in the presence of 1% SDS. Interestingly, while "SpT-MNl-HA" (SEQ ID NO:111) did not show successful coupling (Figure 13, lane 2), its counterpart comprising the solubility enhancing moiety 5. su / s-derived PotD did give rise to adduct formation (Figure 13, lane 4). This highlights the beneficial effect of 5. su / s-derived PotD on the coupling of an aggregation prone construct to OMVs.

[0299] Example 14

[0300] Successful coupling of POIs comprising polyepitope sequences regardless of the position of PotD in the POI

[0301] This Example confirms successful coupling of POI constructs "SpT-PotD-MNl- HA" (SEQ ID NO:113), "SpT-PotD-MN2-HA" (SEQ ID NO:114), "SpT-PotD-NC-HA" (SEQ ID NO:92) or "SpT-RvOlll-HA" (SEQ ID NO:115, as shown in Example 13) to OMVs. Moreover, it is demonstrated that the position of PotD with respect to the polyepitope stretch "NC" can be altered within the POI without losing the competency to couple the POI to OMVs.

[0302] Materials and Methods

[0303] POI constructs "SpT-PotD-MNl-HA" (SEQ ID NO:113), "SpT-PotD-MN2-HA" (SEQ ID NO:114), "SpT-PotD-NC-HA" (SEQ ID NO:92), "SpT-NC-PotD-HA" (SEQ ID NO:116) and "SpT-RvOlll-HA" (SEQ ID N0:115) were synthesized in cell-free reactions at 37°C as described for Example 1 (Figure 14, lanes 4-7). To investigate the influence of positioning of 5. su / s-derived PotD on the coupling of POIs to OMVs, a construct with an inverted orientation of the solubility enhancing moiety "PotD" relative to the target polypeptide moiety "NC" was tested in parallel. After 3.5 hours of synthesis, OMVs displaying "HbpD-SpC" (SEQ ID NO:68) were added to the synthesis reaction mixtures and incubations were continued at 37 °C for 30 min (Figure 14, lanes 4-8), before further incubation at 4 °C overnight. Samples comprising equal amounts of cell-free translation reaction mixture were analyzed by SDS-PAGE and Coomassie staining (Figure 14A) or Western bloting using anti-HA (Figure 14B). For comparison, samples comprising reaction mixture lacking both polypeptide-encoding DNA and OMVs (CFP, lane 1 in Figure 14A and 14B), reaction mixture lacking DNA (CFP + OMV, lane 2 in Figure 14A and 14B) or OMVs in the absence of reaction mixture (OMV, lane 3 in Figure 14A and 14B) were analyzed.

[0304] Results

[0305] Adduct formation was observed for aggregation prone POI construct "SpT- PotD-MNl-HA" (SEQ ID NO:113), "SpT-PotD-MN2-HA" (SEQ ID NO:114), "SpT-PotD- NC-HA" (SEQ ID NO:92) and "SpT-RvOlll-HA" (SEQ ID NO:115), confirming the observations of Example 12. Efficient adduct formation was also observed upon reversing the orientation of the target polypeptide moiety "NC" and the solubility enhancing moiety "PotD" comprised in the POI (Figure 14, lanes 7-8), despite the occurrence of a partial truncate of the "HbpD-SpC-SpT-NC-PotD-HA" coupling product (marked with "#" in Figure 14 panel A, lane 8) that migrated somewhat slower on SDS-PAGE compared to non-coupled "HbpD-SpC" (marked with "<" in Figure 14 panel A, lanes 2, 3, 4). These data demonstrate a tolerance regarding the positioning of a solubility enhancing moiety, such as S.su / s-derived "PotD", and the target polypeptide moiety, such as "NC", comprised in POI constructs destined for Tag / Catcher-based coupling to OMVs in a cell-free system. Example 15

[0306] The presence of OMVs at the start of cell-free protein synthesis is beneficial for coupling

[0307] Presence of OMVs displaying "HbpD-SpC" (SEQ ID NO:68) at the onset of a cell-free synthesis reaction may allow coupling of POI constructs comprising a SpyTag before these constructs form aggregates and lose coupling competency. This Example demonstrates that the presence of OMVs from the start of protein synthesis is beneficial for adduct formation compared to addition of OMVs at a later timepoint. The Example further illustrates that swapping the order of at least one target polypeptide moiety and at least one solubility enhancing moiety, such as PotD, does not impair coupling of a polypeptide to OMVs.

[0308] Materials and Methods

[0309] POI constructs "SpT-PotD-MNl-HA" (SEQ ID NO:113), "SpT-PotD-MN2-HA" (SEQ ID NO:114), "SpT-PotD-NC-HA" (SEQ ID NO:92) and "SpT-NC-PotD-HA" (SEQ ID NO:116) were synthesized in cell-free reactions as described for Example 1. Half of the reactions comprised OMVs displaying "HbpD-SpC" (SEQ ID NO:68) from the start of the synthesis reaction (0 hours; Figure 15, lanes 2, 4, 6, 8), whereas the other half was supplemented with OMVs displaying "HbpD-SpC" (SEQ ID NO:68) 3.5 hours after the onset of polypeptide synthesis (3.5 hours; Figure 15, lanes 1, 3, 5, 7). Reaction mixtures were incubated at 37 °C for 4 hours after which the temperature was shifted to 4 °C and incubation was continued overnight to allow optimal Tag / Catcher based coupling. Samples comprising equal amounts of cell-free translation reaction mixture were analyzed by SDS-PAGE and Coomassie staining.

[0310] Results

[0311] Adducts were observed for the POI construct "SpT-PotD-NC-HA" (SEQ ID NO:92) regardless of the addition timepoint of the OMVs (Figure 15, lane 5 and 6). Similarly, efficient adduct formation was observed for the POI variant with an inverted PotD-NC orientation ("SpT-NC-PotD-HA", SEQ ID NO:116) in both samples (Figure 15, lanes 7-8), as well as the emergence of a potential adduct truncate (marked as "#" in Figure 15, lanes 7-8), showing that the timepoint of OMV addition was not critical for a successful coupling reaction (refer also to the "Results" section of Example 14). Surprisingly, direct availability of OMVs at the start of polypeptide synthesis did appear to be beneficial for coupling of aggregation-prone (see Example 13 and 14) POI construct "SpT-PotD-MNl-HA" (SEQ ID NO:113), as a clear adduct could be detected in samples comprising OMVs from the start (Figure 15, lane 1) as opposed to samples comprising OMVs from 3.5 hours onwards (Figure 15, lane 2). Similarly improved coupling was observed for POI construct "SpT-PotD-MN2-HA" (SEQ ID NO:114) when comparing samples comprising OVMs from the start to samples receiving a late addition of OMVs. This was observed from a relative increase of the Coomassie stained band representing the adduct relative to the other proteins bands in the reaction mixture (Figure 15, compare lanes 4 and 3). Taken together, addition of OMVs before the onset of polypeptide synthesis is a successful strategy to improve coupling system-based coupling of certain polypeptides in a cell- free system.

[0312] Example 16

[0313] Successful coupling of a mycobacterial antigen at different reaction temperatures

[0314] This Example shows successful coupling of a POI construct, comprising a target polypeptide moiety in the form of the mycobacterial antigen "RvOlll" (SEQ ID NO:6), to OMVs in a cell-free system under different reaction conditions.

[0315] Materials and Methods

[0316] POI construct "SpT-Rv0111-HA" (SEQ ID NO:115) was synthesized in a cell-free reaction in the presence (Figure 16, lanes 2 and 4) or absence (Figure 16, lanes 1 and 3) of OMVs displaying "HbpD-SpC" (SEQ ID NO:68). Half of the synthesis reactions were incubated at 37 °C for 4 hours, as described for Example 1, after which the temperature was shifted to 4 °C and incubation was continued overnight (Figure 16, lanes 1 and 2). The other synthesis reactions were incubated at 25 °C for 16 hours (Figure 16, lanes 3 and 4). Samples comprising equal amounts of cell-free translation reaction mixture were analyzed by SDS-PAGE and Coomassie staining.

[0317] Results

[0318] Successful formation of adducts representing Spy-based covalent bonding between OMVs decorated with "HbpD-SpC" (SEQ ID NO:68) and the POI construct "SpT-Rv0111-HA" (SEQ ID NO:115) was observed for both incubation regimes (Figure 16, lanes 2 and 4).

[0319] Example 17

[0320] Successful cell free production of influenza antigen in eukaryotic system

[0321] This example shows successful production of protein of interest, namely an antigen hemagglutinin (HA) (SEQ ID NO:228) from influenza A PRP-virus in a eukaryotic plant based system using the method of the present invention.

[0322] Materials and Methods

[0323] A gene encoding the fusion protein of interest (POI) HA-TFD-SpT with a melittin signal peptide (melittin signal sequence_PR8 HA 19-529_Foldon motif_SpyTag (SEQ ID NO:232)) , comprising the melittin signal sequence (SEQ ID NO:229); a linker (SEQ ID NO:234); a fragment of the antigen hemagglutinin from influenza A PRP-virus, corresponding to amino acid residues 19-529 (SEQ ID NQ:230) of said hemagglutinin; a linker (SEQ ID NO:235); a foldon trimerization motif (SEQ ID NO:231) corresponding to the C-terminal domain of T4 fibritin (foldon) (also referred to as TFD herein); a linker (SEQ ID NO:236) and SpyTag (SEQ ID NQ:10), was constructed and expressed from a pALiCE02-based plasmid (LenioBio, Germany). In addition a construct encoding the fusion protein HA-TFD-SpT without the melittin signal peptide (PR8 HA 19-529_Foldon motif_SpyTag (SEQ ID NO:233), comprising said fragment of the antigen hemagglutinin from influenza A PRP-virus (SEQ ID NQ:230); a foldon trimerization motif (SEQ ID NO:231); and SpyTag (SEQ ID NQ:10), was prepared and expressed from pALiCEOl-based plasmid (LenioBio, Germany). TOPlOF'chemically competent E. coli (Thermo Fisher Scientific, USA) were transformed with the plasmid and subsequently the plasmid was isolated and purified. A mini scale (50 pL) ALiCE® cell-free protein expression kit (LenioBio, Germany), containing a lysate derived from N. tabacum c.v. BY-2 cells, was used for protein expression. The reaction was performed following the manufacturer's instructions with the following deviation: a regular 96-well plate was used instead of a half-well plate; empty wells in the 96-well plate were filled with 200 pL of PBS and the space between the wells was also filled with PBS; the plate was closed with a regular cover and sealed with parafilm to prevent evaporation; the plate was incubated for 48 h in a shaker incubator with an orbit of 1.5 mm.

[0324] After the reaction, OMVs containing HbpD (SEQ ID NO:61) in fusion with SpC (SEQ ID NO:11) corresponding to HbpD-SpC (SEQ ID NO:69) produced as described in general procedures were added to part of the reaction mixture and the resulting mixture was incubated for 16 h at 4°C to allow potential bonding of the protein HA- TFD-SpT (SEQ ID NO:233) with HbpD-SpC in the OMVs via the formation of an isopeptide bond. The ALiCE® reaction mixture and the OMVs incubated with the reaction mixture were analyzed by SDS-PAGE followed by Coomassie staining or by Western blot and immunodetection using anti-HA or anti-Hbp antibodies (performed essentially as described in general procedures).

[0325] Results

[0326] Figure 17 A shows that the ALiCE® cell free production system allows expression in different cellular compartments, as shown in lane 01 for cytosolic compartment and in lane 02 for the microsomal compartment detected using the a-HA antibody. Expression from pALiCEOl results in cytoplasmic expression without post- translational modifications (as shown in lane 01), while fusion of the protein of interest with the melittin signal sequence in pALiCE02 targets the protein to the microsomes resulting in post-translational modifications, including N-glycosylation (as shown in lane 02). The melittin signal sequence is automatically cleaved off after expression. The molecular mass of the non-modified fusion protein is predicted to be 63.5 kDa. Expression from plasmid pALiCEOl yielded a fusion protein identified by immunodetection with a-HA antibodies having a mass of approximately 63.5 kDa. It was thus concluded that non-modified fusion protein of interest was obtained. Expression from pALiCE02 yielded a second variant corresponding to glycosylated HA, in addition to the 63.5 kDa also seen in lane 01. It was thus concluded that both non-modified fusion protein of interest as well as post translationally modified fusion protein of interest was obtained.

[0327] Upon addition of OMVs displaying "HbpD-SpC" to the reaction mixtures, adduct formation, representing Spy-based covalent bonding between the post translationally modified POI "HA-TFD-SpT" and the autotransporter fusion protein "HbpD-SpC" displayed on OMVs, was observed. This adduct comprising HbpD and HA was detectable with Coomassie staining and identified by immunodetection with a-HA and a-Hbp antibodies as shown in Figure 17B (see lanes 4 and 7). No such adduct was formed in the absence of OMVs displaying HbpD-SpC (see lanes 3 and 6 which were loaded with only the reaction mixture CFPS) or in the absence of the reaction mixtures (see lanes 2 and 8 which were loaded with only OMVs) in Figure 17B). Molecular mass (kDa) markers are indicated at the left side of the panels and correspond to lane 1 in Figure 17A and to lanes 1, 5 and 9, in Figure 17B. These data show the successful cell-free production of OMV-polypeptide conjugates in a eukaryotic cell free system, wherein the polypeptide has been subjected to post translational modifications.

[0328] ITEMIZED LIST OF EMBODIMENTS

[0329] 1. A cell-free production method for producing a target polypeptide moiety, the method comprising the following steps: a. providing at least one nucleotide sequence encoding at least one polypeptide of interest (POI), wherein said at least one POI comprises at least one target polypeptide moiety and at least one coupling moiety of a coupling system; b. providing a polypeptide expression-competent cell-free composition; c. providing at least one vesicle displaying on its outer surface at least one autotransporter (AT) fusion protein, wherein said at least one AT fusion protein comprises an AT protein and comprises at least one complementary moiety of said coupling system; and d. bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture, and simultaneously or subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.

[0330] 2.The method according to item lwherein step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and simultaneously bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling, such as simultaneous coupling, of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system. 3.The method according to item 1, wherein step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling, such as subsequent coupling, of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.

[0331] 4. The method according to any one of items 1-3, wherein said at least one coupling moiety of a coupling system is located C- or N-terminally in relation to said at least one target polypeptide moiety, such as is located N-terminally in relation to said at least one target polypeptide moiety.

[0332] 5. The method according to any one of items 1-4, wherein said at least one coupling moiety of a coupling system is located at the C- or N-terminus of said at least one target polypeptide moiety, such as is located at the N-terminus of said at least one target polypeptide moiety.

[0333] 6. The method according to any one of items 1-3, wherein said at least one coupling moiety of a coupling system is located internally in said at least one target polypeptide moiety.

[0334] 7. The method according to any one of items 1-6, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of prokaryotic cellular extracts, prokaryotic cell-free lysates, eukaryotic cellular extracts, eukaryotic cell-free lysates and reconstituted cell-free synthesis systems.

[0335] 8. The method according to any one of items 1-7, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of prokaryotic cellular extracts and prokaryotic cell-free lysates, such as wherein said polypeptide expression-competent cell-free composition is a prokaryotic cell-free lysate.

[0336] 9. The method according to any one of items 7-8, wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is selected from the group consisting of bacterial cellular extracts, bacterial cell-free lysates, archaeal prokaryotic cellular extracts and archaeal cell-free lysates.

[0337] 10. The method according to any one of items 7-9, wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell- free lysate selected from the group consisting of cellular extracts or cell-free lysates of Escherichia coli, Streptomyces venezuelae, Streptomyces lividans, Bacillus megaterium, Bacillus subtilis, Corynebacterium glutamicum, Vibrio natriegens, Pseudomonas putida, Clostridium autoethanogenum, Mycoplasms capricolum, Sulfolobus solfataricus, Thermococcus kodakaraensis, Campylobacter jejuni, Neisseria meningitidis, Helicobacter pylori, Streptococus pneumoniae, Streptococcus gordonii, Streptococcus parasanguis, Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, Mycobacterium marinum, Mycobacterium tuberculosis, and Mycobacterium avium, such as wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell-free lysate selected from the group consisting of cellular extracts or cell-free lysates of Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, and Escherichia coli, such as wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell-free lysate of Escherichia coli.

[0338] 11. The method according to any one of items any one of items 1-7, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of eukaryotic cellular extracts and eukaryotic cell-free lysates, such as wherein said polypeptide expression-competent cell-free composition is a eukaryotic cell-free lysate.

[0339] 12. The method according to any one of items 1-7 and 11, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of protozoan cellular extracts, protozoan cell-free lysates, yeast cellular extracts, yeast cell-free lysates, plant cellular extracts, plant cell-free lysates, insect cellular extracts, insect cell-free lysate, mammalian cellular extracts and mammalian cell-free lysate.

[0340] 13. The method according to any one of items 1-7 and 11-12, wherein said polypeptide expression-competent cell-free composition is a eukaryotic cellular extract or eukaryotic cell-free lysate selected from the group consisting of extract or lysate of wheat germ cells, Tobacco BY-2 cells, Spodoptera frugiperda, Leishmania tarentolae, Saccharomyces cerevisiae, Rabbit reticulocytes, Chinese hamster ovary (CHO) cells, HEK293 cells and HeLa cells, such as .from the group consisting of extract or lysate of wheat germ cells and extract or lysate of Tobacco BY-2 cells, such as extract or lysate of Tobacco BY-2 cells.

[0341] 14. The method according to any one of items 1-7, wherein said polypeptide expression-competent cell-free composition is a reconstituted cell-free synthesis system.

[0342] 15. The method according to any one of items 1-14, wherein said at least one vesicle is a self-adjuvating vesicle.

[0343] 16. The method according to any one of items 1-15, wherein said at least one vesicle is an Outer Membrane Vesicle (OMV).

[0344] 17. The method according to any one of items 1-16, wherein said at least one vesicle is derived from a bacterium.

[0345] 18. The method according to any one of items 1-17, wherein said at least one vesicle is derived from a gram-negative bacterium.

[0346] 19. The method according to any one of the preceding items, wherein said at least one vesicle is selected from the group consisting of vesicles derived from Escherichia coli, Neisseria meningitidis, Neisseria gonorrhea and Salmonella spp, such as selected from the group consisting of vesicles derived from Escherichia coli and Salmonella spp, such as wherein said at least one vesicle is derived from Salmonella spp.

[0347] 20. The method according to any one of the preceding items, wherein said at least one vesicle is derived from a subspecies of 5. enterica subsp. enterica, such as from 5. Typhimurium.

[0348] 21. The method according to any one of the preceding items, wherein said autotransporter protein is a serine protease autotransporter of the Enterobacteriaceae (SPATE), such as a SPATE protein from Escherichia coli.

[0349] 22. The method according to item 21, wherein said SPATE protein is selected from the group consisting of hemoglobin-binding protease (Hbp) comprising the amino acid sequence as defined in SEQ ID NO:60, extracellular serine protease (EspC) comprising the amino acid sequence as defined in SEQ ID NO:65 and temperaturesensitive hemagglutinin (Tsh) comprising the amino acid sequence as defined in SEQ ID NO:66 and any proteins exhibiting at least such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 any one of said SEQ ID NQ:60, SEQ ID NO:65 and SEQ ID NO:66; such as wherein the SPATE protein is selected from the group consisting of Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60, EspC comprising the amino acid sequence as defined in SEQ ID NO:65 and Tsh comprising the amino acid sequence as defined in SEQ ID NO:66.

[0350] 23. The method according to item 21 or 22, wherein said SPATE protein is selected from the group consisting of Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and any proteins exhibiting at least such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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; such as wherein the SPATE protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60. 24. The method according to any one of the preceding items, wherein said autotransporter protein comprises at least one mutation that prevents cleavage at the autocatalytic cleavage site, such as at least one mutation which disrupts the autocatalytic cleavage site such that cleavage at the autocatalytic cleavage site is prevented.

[0351] 25. The method according to item 24, wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NO:60 and further comprises at least one mutation in a position selected from the group consisting of N1048, N1049, K1052, R1053, G1055, L1057, R1069, D1145, K1149, E1179, and E1197, which at least one mutation results in prevention of cleavage at the autocatalytic cleavage site, such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NO:60 and further comprises at least one mutation in a position selected from the group consisting of N1048G, N1048A, N1048D, N1048S, N1049S, K1052A, R1053A, G1055R, L1057R, and D1145X wherein X is any amino acid other than Glu (E), such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and further comprises at least one mutation in a position selected from the group consisting of N1048G and N1049S, such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and further comprises mutations in positions N1048G and N1049S.

[0352] 26. The method according to item 24, wherein said Hbp comprises the amino acid sequence as defined in SEQ ID NO:61 and any proteins exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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, provided said proteins comprise an autocatalytic cleavage site which has been mutated such that cleavage is prevented.

[0353] 27. The method according to item 21 or 22, wherein the SPATE protein comprises a deletion mutation of the autocatalytic cleavage site, such that the autocatalytic cleavage site is partially or completely deleted.

[0354] 28. The method according to any one of items 21, 22 and 27, wherein said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises an amino acid sequence as selected from the group consisting of SEQ ID NO:62-64 and any proteins exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 any one of said sequences SEQ ID NO:62-64.

[0355] 29. The method according to any one of the preceding items, wherein said at least one AT fusion protein comprises at least one complementary binding moiety of said coupling system located N-terminally in said AT protein, such as located at the N- terminus of the said AT protein.

[0356] 30. The method according to any one of the preceding items, wherein said at least one AT fusion protein comprises at least one complementary binding moiety of said coupling system located internally in said AT protein, such as internally in the portion of said AT protein displayed on said vesicle.

[0357] 31. The method according to any one of items 29-30, wherein said at least one complementary binding moiety of said coupling system is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein, such as at least two side domains of the AT protein.

[0358] 32. The method according to any one of items 29-30, wherein said at least one complementary binding moiety of said coupling system replaces a part of at least one of the side domains of the AT protein.

[0359] 33. The method according to item 31 or 32, wherein said at least one AT fusion protein comprises or consists of Hbp as defined in any one of items 22-28 and wherein said at least side domain is selected from side domains located at amino acid positions 1-256 (domain dl), 481-556 (domain d2), 605-645 (domain d3), 683- 714 (domain d4) and 846-940 (domain d5), such as wherein said side domain is located at amino acid positions 1-256 (domain dl).

[0360] 34. The method according to any one of items 31-33, wherein said at least one AT fusion protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein amino acid positions 1-255 are replaced by said at least one complementary binding moiety of said coupling system, wherein said at least one complementary binding moiety is optionally flanked with one or two linkers or spacers.

[0361] 35. The method according to any one of items 31-34, wherein said at least one AT fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:68, 119-125 and 150-157, such as the group consisting of SEQ ID NO:68 and 119.

[0362] 36. The method according to any one of the preceding items, wherein said at least one AT fusion protein comprises a plurality of complementary moieties of said coupling system, such as at least two, such at least three, such as at least five, complementary moieties of said coupling system.

[0363] 37. The method according to item 36, wherein said at least two, such at least three, such as at least five, such as all, of said plurality of complementary moieties of said coupling system comprise the same amino acid sequence.

[0364] 38. The method according to item 36, wherein said at least two, such as at least three, such as at least five, such as all, of said plurality of complementary moieties of said coupling system comprise the different amino acid sequences.

[0365] 39. The method according to any one of the preceding items, wherein the coupling system is derived from Gram positive bacterial pilus proteins, such as derived from Gram positive bacterial pilus proteins selected from the group consisting of Lactiplantibacillus, Bacillus, Ruminococcus, and Streptococcus, such as preferably derived from Gram positive Streptococcal pilus proteins.

[0366] 40. The method according to item 39, wherein the coupling system is derived from a Gram positive Streptococcal pilus protein selected from the group consisting of the major pilin protein Spy0128 of Streptococcus pyogenes, the fibronectin binding protein FbaB of Streptococcus pyogenes, the fibronectin-binding protein from Streptococcus dysgalactiae, the pilus-subunit RrgA from Streptococcus pneumoniae, and the pilus subunit RrgC from Streptococcus pneumoniae.

[0367] 41. The method according to item 39 or 40, wherein the coupling moiety and complementary moiety of the coupling system are selected from the group consisting of SnoopTag2 (SEQ ID NO:7), DogTag2 (SEQ ID NO:8), SnoopLigase2 (SEQ ID NO:9), SpyTag (SEQ ID NQ:10), SpyCatcher (SEQ ID NO:11), SnoopTag (SEQ ID NO:12), SnoopCatcher (SEQ ID NO:13), SpyTag002 (SEQ ID NO:14), SypCatcher002 (SEQ ID NO:15), SpyTag003 (SEQ ID NO:16), SpyCatcher003 (SEQ ID NO:17), SdyTag (SEQ ID NO:18), SdyCatcher (SEQ ID NO:19), SilkTag (SEQ ID NQ:20), SilkCatcher (SEQ ID NO:21), DogTag (SEQ ID NO:22), DogCatcher (SEQ ID NO:23), SnoopTagJr (SEQ ID NO:24), SnoopLigase (SEQ ID NO:25), Jo (SEQ ID NO:26), In (SEQ ID NO:27), NGTag (SEQ ID NO:28), NGCatcher (SEQ ID NO:29), SpyCatcher-N (SEQ ID NQ:30), SpyCatcher-21 (SEQ ID NO:31), SpyStapler (SEQ ID NO:32), SpyLigase (SEQ ID NO:33), MoonCake (SEQ ID NO:34), Katl (SEQ ID NO:35), QueenCatcher (SEQ ID NO:36), PsCsCatcher (SEQ ID NO:37), Ktag (SEQ ID NO:38), BDTag (SEQ ID NO:39), RumTrunkTagD9N (SEQ ID NQ:40), RumTrunkTag (SEQ ID NO:41), RumTag (SEQ ID NO:42), Rum2Tag (SEQ ID NO:43), Rum3Tag (SEQ ID NO:44), Rum4Tag (SEQ ID NO:45), Rum5Tag (SEQ ID NO:46), Rum6Tag (SEQ ID NO:47), Rum7Tag (SEQ ID NO:48), BacTag (SEQ ID NO:49), Bac2Tag (SEQ ID NQ:50), Bac3Tag (SEQ ID NO:51), Bac4Tag (SEQ ID NO:52), Bac5Tag (SEQ ID NO:53), Clib9 (SEQ ID NO:54), PhoTag (SEQ ID NO:55), PsCsTag (SEQ ID NO:56), and SpyCatcher (SEQ ID NO:57), and variants thereof exhibiting at least 70 % identity thereto, wherein said moieties are selected such that said moieties have the capacity of forming an isopeptide bond between each other, and wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70 % identity thereto.

[0368] 42. The method according to any one of items 39-41, wherein the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11), SpyTag-SpyCatcher002 (SEQ ID NO:10-SEQ ID NO:15), SpyTag- SpyCatcher003 (SEQ ID NQ:10-SEQ ID NO:16), SpyTag-SpyCatcher-N (SEQ ID NQ:10- SEQ ID NQ:30), SpyTag-SpyCatcher-21 (SEQ ID NQ:10-SEQ ID NO:31), SpyTag- SdyCatcher (SEQ ID NQ:10-SEQ ID NO:19), SpyTag002-SpyCatcher002 (SEQ ID NO:14- SEQ ID NO:15), SpyTag002-SpyCatcher (SEQ ID NO:14-SEQ ID NO:11), SpyTag002- SpyCatcher003 (SEQ ID NO:14-SEQ ID NO:17), SpyTag002-SdyCatcher (SEQ ID NO:14- SEQ ID NO:19), SpyTag003-SpyCatcher003 (SEQ ID NO:16-SEQ ID NO:17), SpyTag003- SpyCatcher (SEQ ID NO:16-SEQ ID NO:11), SpyTag003-SpyCatcher002 (SEQ ID NO:16- SEQ ID NO:15), SpyTag003-SdyCatcher (SEQ ID NO:16-SEQ ID NO:19), SnoopTag- SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13), Si IkTag-Sil kCatcher (SEQ ID NQ:20-SEQ ID NO:21), DogTag-DogCatcher (SEQ ID NO:22-SEQ ID NO:23), DogTag2-DogCatcher (SEQ ID NO:8-SEQ ID NO:23), Jo-In (SEQ ID NO:26-SEQ ID NO:27), NGTag-NGCatcher (SEQ ID NO:28-SEQ ID NO:29), SdyTag-SdyCatcher (SEQ ID NO:18-SEQ ID NO:19), SdyTag-SpyCatcher (SEQ ID NO:18-SEQ ID NO:11), SdyTag-SpyCatcher002 (SEQ ID NO:18-SEQ ID NO:15), SdyTag-SpyCatcher003 (SEQ ID NO:18-SEQ ID NO:17), KTag- SpyTag (SEQ ID NO:38-SEQ ID NQ:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SnoopTag2-SnoopCatcher (SEQ ID NO:7-SEQ ID NO:13), DogTag2- SnoopCatcher (SEQ ID NO:8-SEQ ID NO:13), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), MoonCake-RumTrunkTagD9N (SEQ ID NO:34-SEQ ID NQ:40), MoonCake- RumTag (SEQ ID NO:34-SEQ ID NO:42), MoonCake-SpyTag (SEQ ID NO:34-SEQ ID NQ:10), MoonCake-SdyTag (SEQ ID NO:34-SEQ ID NO:18), Katl-SpyTag (SEQ ID NO:35-SEQ ID NQ:10), Katl-SdyTag (SEQ ID NO:35-SEQ ID NO:18), Katl- RumTrunkTagD9N (SEQ ID NO:35-SEQ ID NQ:40), Katl-RumTag (SEQ ID NO:35-SEQ ID NO:42), and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, and exhibits at least 70 % identity thereto, such as a variant selected from the group consisting of SEQ ID NO:10-19, 22-25, 34 and 38-40 and variants exhibits at least 70 % identity thereto.

[0369] 43. The method according to any one of items 39-40, wherein the coupling system is a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first and second moieties are capable of being isopeptide bonded by means of the third moiety.

[0370] 44. The method according to item 43, wherein said tripartite system comprises a first and second moiety selected from the group consisting of KTag-SpyTag (SEQ ID NO:38-SEQ ID NO:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SpyTag- BDTag (SEQ ID NQ:10-SEQ ID NO:39), DogTag2-SnoopTagJr (SEQ ID NO:8-SEQ ID NO:24), and wherein the tripartite system further comprises a third moiety which is a catalytic moiety, such as wherein said catalytic moiety is a ligase.

[0371] 45. The method according to any one of items 1-37, wherein the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as selected from SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as wherein said coupling system is SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11).

[0372] 46. The method according to any one of the preceding items, wherein said at least one target polypeptide moiety is selected from the group consisting of aggregation- prone polypeptides and proteins; insoluble polypeptides and proteins; disordered proteins; misfolded proteins; hydrophobic polypeptides and proteins; and selfassociating polypeptides and proteins; such as the group consisting of aggregation- prone polypeptides and proteins; and insoluble polypeptides and proteins.

[0373] 47. The method according to item 46, wherein said aggregation-prone polypeptides and proteins are amyloid-forming polypeptides and proteins.

[0374] 48. The method according to any one of items 1-45, wherein said at least one target polypeptide moiety is a polypeptide comprising at least one neoantigen derived epitope, such as wherein said at least one target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes, such as multiple tandem-fused neoantigen derived epitopes.

[0375] 49. The method according to any one of items 1-45 and 48, wherein said at least one target polypeptide moiety is a polypeptide comprising multiple tandem-fused neoantigen derived epitopes.

[0376] 50. The method according to item 48 or 49, wherein said at least one target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes, and wherein at least two of the neoantigen derived epitopes are separated by spacers, such as wherein all of said neoantigen derived epitopes are separated by spacers.

[0377] 51. The method according to any one of item 48-50, wherein said at least one target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes, and wherein at least two of the neoantigen derived epitopes are directly fused to each other, such as wherein all of said neoantigen derived epitopes are directly fused to each other.

[0378] 52. The method according to any one of items 48-51, wherein said at least one target polypeptide moiety comprises two or more identical neoantigen derived epitopes. 53. The method according to any one of items 48-52, wherein said at least one target polypeptide moiety does not comprise more than one copy of each neoantigen derived epitope.

[0379] 54. The method according to any one of items 48-53, wherein said at least one target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes comprises at least two neoantigen derived epitopes, such as at least three neoantigen derived epitopes, such as at least four neoantigen derived epitopes, such as at least seven neoantigen derived epitopes, such as at least ten neoantigen derived epitopes, such as at least 15 neoantigen derived epitopes, such as at least 20 neoantigen derived epitopes, such as at least 25 neoantigen derived epitopes, such as at least 50 neoantigen derived epitopes, such as at least 60 neoantigen derived epitopes, such as at least 70 neoantigen derived epitopes, such as at least 80 neoantigen derived epitopes, such as at least 90 neoantigen derived epitopes, such as at least 100 neoantigen derived epitopes.

[0380] 55. The method according to any one of items 48-53, wherein said at least one target polypeptide moiety is a polypeptide comprising multiple neoantigen derived epitopes and comprises 5-150 of neoantigen derived epitopes, such as 5-125 neoantigen derived epitopes, such as 5-100 neoantigen derived epitopes, such as 5- 90 neoantigen derived epitopes, such as 5- 80 neoantigen derived epitopes, such as at 5-75 neoantigen derived epitopes, such as 5-60 neoantigen derived epitopes, such as 5-50 neoantigen derived epitopes, such as 5-25 neoantigen derived epitopes, such as 5-20 neoantigen derived epitopes, such as at 5-15 neoantigen derived epitopes, such as 7-12 neoantigen derived epitopes, such as about ten neoantigen derived epitopes.

[0381] 56. The method according to any one of items 1-55, wherein said at least one POI comprises a plurality of target polypeptide moieties, such as at least two target polypeptide moieties, such as at least three target polypeptide moieties, such as at least four target polypeptide moieties, such as at least five target polypeptide moieties, such as at least ten target polypeptide moieties.

[0382] 57. The method according to item 56, wherein said plurality of target polypeptide moieties are characterized in that at least two, such as at least three, such as at least five, such as at least ten, of said target polypeptide moieties have the same amino acid sequence, such as wherein all target polypeptide moieties have the same amino acid sequence.

[0383] 58. The method according to item 56, wherein said plurality of target polypeptide moieties are characterized in that at least two, such as at least three, such as at least five, such as at least ten, of said target polypeptide moieties have different amino acid sequences, such as wherein all target polypeptide moieties have different amino acid sequence.

[0384] 59. The method according to any one of items 56-58, wherein said plurality of target polypeptide moieties comprises target polypeptide moieties which are linked directly or indirectly to each other.

[0385] 60. The method according to any one of the preceding items, wherein said at least one POI further comprises at least one solubility-enhancing moiety.

[0386] 61. The method according to item 60, wherein said at least one solubility-enhancing moiety is linked directly or indirectly to said at least one target polypeptide moiety.

[0387] 62. The method according to item 60 or 561, wherein said at least one solubilityenhancing moiety is located between at least two target polypeptide moieties.

[0388] 63. The method according to any one of items 60-62, wherein said at least one solubility-enhancing moiety is linked, such as linked directly or indirectly, to the N- terminus or C-terminus of said at least one target polypeptide moiety.

[0389] 64. The method according to item 60, wherein said at least one solubility-enhancing moiety is linked, such as linked directly or indirectly, to said at least one complementary binding moiety of said coupling system.

[0390] 65. The method according to item 60 or 64, wherein said at least one solubilityenhancing moiety is linked, such as linked directly or indirectly, to the N-terminus or C-terminus of at least one complementary binding moiety of said coupling system.

[0391] 66. The method according to any one of items 61-65, wherein the indirect linkage is via a linker amino acid sequence, such as wherein the linker amino acid sequence comprises at least one amino acid residue. 67. The method according to any one of items 60-66, wherein said at least one solubility enhancing moiety is selected from the group consisting of MBP (SEQ ID NO:76), PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA (SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), TF (SEQ ID NO:196), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), Fh8 (SEQ ID NO:197), H tag (SEQ ID NO:198), Ppi B (SEQ ID NO:199), Bfr (SEQ ID NQ:200), YjdC (SEQ ID NQ:201), FolA (SEQ ID NQ:202), CheZ (SEQ ID NQ:203), GshB (SEQ ID NQ:204), ArsC (SEQ ID NQ:205), N-ePGK (SEQ ID NQ:206), KDPG aldolase (SEQ ID NQ:207), Tsf (SEQ ID NQ:208), MsyB (SEQ ID NQ:209), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), Z-tag (SEQ ID NQ:210), ZZ-tag (SEQ ID NO:211), GB1 (SEQ ID NO:212), DsbA (SEQ ID NO:213), DsbAmut(SEQ ID NO:214), IF2 domain I, CaBP (SEQ ID NO:215), FTN- H (SEQ ID NO:216), Skp (SEQ ID NO:217), T7PK (SEQ ID NO:218), Ecotin (SEQ ID NO:219), Halo Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88), s / GFP (SEQ ID NQ:220), SNUT (SEQ ID NO:222), EspA (SEQ ID NO:221), BLA, / n / Bl-21 (SEQ ID NO:223), SmbP (SEQ ID NO:224), Ffu, TDX (SEQ ID NO:225), HE-MBP(Pyr), N-terminal domain of rpoD (SEQ ID NO:226), yjgD (SEQ ID NO:227), (Arg)i-3, (Arg)4(SEQ ID NO:58), (Arg)5(SEQ ID NO:59), (His)5(SEQ ID NQ:108), (Arg)io (SEQ ID NQ:109), (Arg-Gly-Gly)3-Gly (SEQ ID NO:117), Poly(Arg), (Gly-Arg)3-(Arg)3(SEQ ID NO:118), (Gly-Arg)4(SEQ ID NO:158), Gly-(Arg)5(SEQ ID NO:159), Gly(Arg-Gly-Gly)3(SEQ ID NQ:160), Gly(Lys- Gly)6(SEQ ID NO:161), (Gly)2-(Arg)2-Gly-Arg (SEQ ID NO:162), Gly-Lys-Gly-(Lys)2(SEQ ID NO:163), (Gly)2-(Lys)4(SEQ ID NO:164), (Lys)i-3, (Lys)4(SEQ ID NO:165), (Lys)5(SEQ ID NO:166), (Lys)6(SEQ ID NO:167), (Lys)io (SEQ ID NO:168), (Asp)5(SEQ ID NO:169), (Glu)5(SEQ ID NQ:170), [Gly]-(Asp)3]3(SEQ ID NO:171), (Asn)5(SEQ ID NO:172), (Gln)5(SEQ ID NO:173), and (Ser)s (SEQ ID NO:174), such as selected from the group consisting of MBP (SEQ ID NO:76), PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA(SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), HALO Tag7 (SEQ ID NO:87), and NusA (SEQ ID NO:88).

[0392] 68. The method according to any one of items 60-67, wherein said at least one solubility enhancing moiety is PotD.

[0393] 69. The method according to any one of items 67-68, wherein said PotD is derived from Streptococcus suis.

[0394] 70. The method according to any one of items 67-69, wherein said PotD is selected from group consisting of PotD comprising an amino acid sequence as defined in SEQ ID NQ:70 and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto, such as wherein said PotD is as defined in SEQ ID NQ:70.

[0395] 71. The method according to any one of the preceding items, wherein said at least one POI further comprises at least one moiety promoting translocation of POI into microsomes, such as wherein said at least one moiety promoting translocation of said POI into the microsomes is a signaling sequence.

[0396] 72. The method according to item 71, wherein said signaling sequence selected from the group consisting of signaling sequences of honeybee melittin, VSV-G, mouse Ig kappa, mouse Ig heavy, human IgKVIll, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, gaussia luc, albumin (HSA), influenza haemagglutinin, human insulin, silkworm fibroin LC, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and signaling sequences of medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is SEQ

[0397] ID NO:229. 73. The method according to any one of items 1-70, wherein said at least one POI is provided as a nucleic acid encoding said POI in a vector comprising at least one moiety promoting translocation of POI into the microsomes, such as wherein said at least one moiety promoting translocation of said POI into the microsomes is a signaling sequence.

[0398] 74. The method according to item 73, wherein said signaling sequence selected from the group consisting of signaling sequences of honeybee melittin, VSV-G, mouse Ig kappa, mouse Ig heavy, human IgKVIll, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, human IL-2, gaussia luc, albumin (HSA), influenza haemagglutinin, human insulin, silkworm fibroin LC, nicotiana tabacum PR-1B protein (ssPRIB), nicotiana benthamiana extensin domain (ssExt), oryza sativa alpha-amylase (ssRamy), nicotiana benthamiana pathogenesis related protein 1 (ssPrl), glycine max VspA vegetative storage protein (ssVsp), and signaling sequences of medicago sativa protein disulphide isomerase (ssPDI), such as wherein said signaling sequence is SEQ ID NO:229.

[0399] 75. The method according to any one of the preceding items, wherein the method further comprises the step of recovering the at least one vesicle coupled to said at least one polypeptide of interest.

[0400] 76. The method according to items 75, wherein said the step of recovering the at least one vesicle coupled to said at least one polypeptide of interest is performed by a method selected from the group consisting to tangential flow filtration, cross flow filtration, ultracentrifugation and size-exclusion chromatography.

[0401] 77. A vesicle coupled to at least one polypeptide of interest (POI), wherein said vesicle on its outer surface displays at least one complementary moiety of a coupling system and at least one POI; wherein said at least one POI comprises at least one target polypeptide moiety, at least one solubility enhancing moiety and at least one coupling moiety of said coupling system; and wherein said vesicle is coupled to said at least one POI via an isopeptide bond formed between the respective moieties of the coupling system. 78. The vesicle coupled to at least one POI according to item 77, wherein said vesicle further displays on its outer surface at least one autotransporter (AT) fusion protein and wherein said at least one AT fusion protein comprises an AT protein and at least one complementary moiety of said coupling system; and wherein said at least one AT fusion protein is coupled to said at least one POI via an isopeptide bond formed between the moieties of the coupling system on said at least one AT fusion protein and said at least one POI.

[0402] 79. The vesicle coupled to at least one POI according to item 77 or 78, wherein said vesicle is as defined in any one of items 15-20.

[0403] 80. The vesicle coupled to at least one POI according to any one of items 77-79, wherein said autotransporter (AT) protein is as defined in any one of the items 21-38.

[0404] 81. The vesicle coupled to at least one POI according to any one of items 77-80, wherein said coupling system is as defined in any one of items 39-45.

[0405] 82. The vesicle coupled to at least one POI according to any one of items 77-81, wherein said at least one target polypeptide moiety is as defined in any one of items 44-45 and 55-58.

[0406] 83. The vesicle coupled to at least one POI according to any one of items 77-82, wherein said at least one target polypeptide moiety is as defined in any one of items 46-59.

[0407] 84. The vesicle coupled to at least one POI according to item 77-82, wherein said at least one solubility-enhancing moiety is selected from the group as defined in any one of items 60-70.

[0408] 85. The vesicle coupled to at least one POI obtainable by the method according to any one of items 1-76.

[0409] 86. The vesicle coupled to at least one POI obtainable by the method according to any one of items 60-76.

[0410] 87. A vesicle coupled to at least one POI according to any one of items 77-86, for use as a medicament.

[0411] 88. A vesicle coupled to at least one POI for use according to 87, wherein said at least one POI comprises at least one target polypeptide moiety having therapeutical activity.

[0412] 89. The vesicle coupled to at least one POI to item 87 or 88, wherein said medicament is a vaccine.

[0413] 90. The vesicle coupled to at least one POI according to item 89, wherein said at least one target polypeptide moiety is an antigen or an immunogenic fragment thereof.

[0414] 91. A vesicle coupled to at least one POI according to any one of items 77-90 for use in the treatment of a disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease, infectious disease and transplant rejection, such as a disease selected from the group consisting of infectious disease and cancer.

[0415] 92. A vesicle coupled to at least one POI according to any one of items 77-91 for use in the treatment of a disease selected from the group consisting of infectious disease and cancer.

[0416] 93. A pharmaceutical composition comprising a vesicle coupled to at least one POI according to any one of items 77-86 or a vesicle coupled to at least one POI for use according to any one of times 87-92 and at least one pharmaceutically acceptable excipient, wherein said at least one POI comprises at least one target polypeptide moiety having therapeutical activity.

[0417] 94. A pharmaceutical composition according to item 93, wherein said at least one POI comprises at least one target polypeptide moiety which is an antigen or an immunogenic fragment thereof.

[0418] 95. A vaccine composition comprising a vesicle coupled to at least one POI as defined in any one of items 77-86 and 89-94 or a vesicle coupled to at least one POI for use according to any one of times 87-92, and at least one pharmaceutically acceptable excipient, wherein said at least one POI comprises at least one target polypeptide moiety which is an antigen or an immunogenic fragment thereof and wherein said composition comprises an additional agent with adjuvant effect.

[0419] 96. A vaccine composition comprising a vesicle coupled to at least one POI as defined in any one of items 77-86 and 89-94 or a vesicle coupled to at least one POI for use according to any one of times 87-92, and at least one pharmaceutically acceptable excipient, wherein said at least one POI comprises at least one target polypeptide moiety which is an antigen or an immunogenic fragment thereof and wherein said composition does not comprise any additional agent with adjuvant effect.

[0420] 97. A vesicle coupled to at least one POI according to any one of items 77-86 and 89- 94, a pharmaceutical composition according to any one of items 93-94 or a vaccine composition according to any one of items 95-96 for use as an agent select from the group consisting of diagnostic agent, prognostic agent, prophylactic agent, therapeutic agent and imaging agent, such as the group consisting of diagnostic agent, prognostic agent and imaging agent.

[0421] 98. Use of a vesicle coupled to at least one POI as defined in any one of items 77-86 as a display platform for delivery of an antigen or an immunogenic fragment thereof.

[0422] 99. Drug delivery platform comprising a vesicle coupled to at least one POI as defined in any one of items 77-86 wherein said at least one POI comprises at least one target polypeptide moiety having therapeutic activity.

[0423] 100. A method of treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a vesicle coupled to at least one POI according to any one of items 77-86, or of a pharmaceutical composition according to any one of items 893-894, wherein said disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease and transplant rejection.

[0424] 101. A method of treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a vesicle coupled to at least one POI according to any one of items 88-86, a pharmaceutical composition according to any one of items 93-94, or a vaccine composition according to any one of items 95- 96, wherein said disease is an infectious disease.

[0425] 102. Use of a vesicle coupled to at least one POI according to any one of items 77-86 for use in the manufacture of a medicament, wherein said at least one POI comprises at least one target polypeptide moiety, which target polypeptide moiety has therapeutical activity.

[0426] 103. Use of a vesicle coupled to at least one POI according to any one of items 77-86 for use in the manufacture of a medicament for the treatment of a disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease and transplant rejection, such as a disease selected from the group consisting of infectious disease and cancer, wherein said at least one POI comprises at least one target polypeptide moiety, which target polypeptide moiety has therapeutical activity.

[0427] 104. Use of a vesicle coupled to at least one POI according to any one of items 77-86 for use in the manufacture of a medicament for the prophylactic treatment of an infectious disease, wherein said at least one POI comprises at least one target polypeptide moiety, which target polypeptide moiety is an antigen or an immunogenic fragment thereof.

[0428] 105. A method of diagnosis in vitro, comprising the steps:

[0429] - providing a vesicle coupled to at least one polypeptide of interest (POI) according to any one of items 77-86, wherein said at least one POI is capable of binding to a target of interest (TOI);

[0430] - contacting a sample suspected to contain said TOI with the vesicle coupled to said at least one POI, thereby allowing said at least one POI to bind to said TOI;

[0431] - detecting the binding of said at least one POI to indicate the presence of said TOI in the sample; and

[0432] - using the information obtained to establish a diagnosis.

[0433] 106. A method of prognosis in vitro, comprising the steps:

[0434] - providing a vesicle coupled to at least one polypeptide of interest (POI) according to any one of items 77-86, wherein said at least one POI is capable of binding to a target of interest (TOI);

[0435] - contacting a sample suspected to contain said TOI with the vesicle coupled to said at least one POI, thereby allowing said at least one POI to bind to said TOI;

[0436] - detecting the binding of said at least one POI to indicate the presence of said TOI in the sample; and - using the information obtained to establish a prognosis.

[0437] 107. A method of detection of a target of interest (TOI) in a sample, comprising the steps:

[0438] - providing a vesicle coupled to at least one polypeptide of interest (POI) according to any one of items 77-86, wherein said at least one POI is capable of binding to said TOI;

[0439] - contacting a sample suspected to contain said TOI with the vesicle coupled to said at least one POI, thereby allowing said at least one POI to bind to said TOI;

[0440] - detecting the binding of said at least one POI to indicate the presence of said TOI in the sample.

[0441] 108. Method of diagnosis in vitro according to item 105, method of prognosis in vitro according to item 106, or method of detection according to item 107, further comprising the steps:

[0442] - repeating the steps of detection, wherein said detecting is performed at several time points at intervals in the same provided sample or a different provided sample, for example as part of a monitoring of the subject before, during, or after treatment.

[0443] 109. Method of diagnosis in vitro according to item 105 or 108, method of prognosis in vitro according to item 106 or 108, or method of detection according to item 107 or 108, further comprising a step of obtaining a value corresponding to the amount of the vesicle coupled to at least one POI that has bound to said TOI in said sample.

[0444] 110. Method of diagnosis, method of prognosis or method of detection according to item 109, further comprising a step of comparing said value to a reference.

[0445] 111. Method of diagnosis in vitro according to any one of items 105 or 108-110, method of prognosis in vitro according to any one of items 106 or 108-110, wherein said diagnosis or prognosis is in relation to an infectious disease or cancer.

Claims

CLAIMS1. A cell-free production method for producing a target polypeptide moiety, the method comprising the following steps: a. providing at least one nucleotide sequence encoding at least one polypeptide of interest (POI), wherein said at least one POI comprises at least one target polypeptide moiety and at least one coupling moiety of a coupling system; b. providing a polypeptide expression-competent cell-free composition; c. providing at least one vesicle displaying on its outer surface at least one autotransporter (AT) fusion protein, wherein said at least one AT fusion protein comprises an AT protein and comprises at least one complementary moiety of said coupling system; and d. bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture, and simultaneously or subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.2.The method according to claim 1, wherein step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and simultaneously bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling, such as simultaneous coupling, of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.3.The method according to claim 1, wherein step d comprises bringing said at least one nucleotide sequence into contact with said polypeptide expression-competent cell-free composition to create a first mixture and subsequently bringing said first mixture into contact with at least one vesicle to create a second mixture, thereby allowing expression of said at least one POI comprising said at least one target polypeptide moiety and coupling, such as subsequent coupling, of said at least one POI to said at least one vesicle via the formation of an isopeptide bond between their respective moieties of the coupling system.

4. The method according to any one of claims 1-3, wherein said at least one coupling moiety of a coupling system is located at the C- or N-terminus of said at least one target polypeptide moiety, such as is located at the N-terminus of said at least one target polypeptide moiety.

5. The method according to any one of claims 1-3, wherein said at least one coupling moiety of a coupling system is located internally in said at least one target polypeptide moiety.

6. The method according to any one of claims 1-5, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of prokaryotic cellular extracts, prokaryotic cell-free lysates, eukaryotic cellular extracts, eukaryotic cell-free lysates and reconstituted cell-free synthesis systems.

7. The method according to any one of claims 1-6, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of prokaryotic cellular extracts and prokaryotic cell-free lysates, such as wherein said polypeptide expression-competent cell-free composition is a prokaryotic cell-free lysate.

8. The method according to any one of claims 6-7, wherein said prokaryotic cellular extract or prokaryotic cell-free lysate is a bacterial cellular extract or bacterial cell- free lysate of Escherichia coli.

9. The method according to any one of items any one of claims 1-6, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of eukaryotic cellular extracts and eukaryotic cell-free lysates, such as wherein said polypeptide expression-competent cell-free composition is a eukaryotic cell-free lysate.

10. The method according to any one of claims 1-6 and 9, wherein said polypeptide expression-competent cell-free composition is selected from the group consisting of as extract or lysate of Tobacco BY-2 cells.

11. The method according to any one of claims 1-6, wherein said polypeptide expression-competent cell-free composition is a reconstituted cell-free synthesis system.

12. The method according to any one of claims 1-11, wherein said at least one vesicle is an Outer Membrane Vesicle (OMV) derived form a from a gram-negative bacterium.

13. The method according to any one of the preceding claims, wherein said at least one vesicle is derived from a subspecies of 5. enterica subsp. enterica, such as from 5. Typhimurium.

14. The method according to any one of the preceding claims, wherein said autotransporter protein is a serine protease autotransporter of the Enterobacteriaceae (SPATE), such as a SPATE protein from Escherichia coli.

15. The method according to claim 14, wherein said SPATE protein is selected from the group consisting of hemoglobin-binding protease (Hbp) comprising the amino acid sequence as defined in SEQ ID NO:60, extracellular serine protease (EspC) comprising the amino acid sequence as defined in SEQ ID NO:65 and temperaturesensitive hemagglutinin (Tsh) comprising the amino acid sequence as defined in SEQ ID NO:66 and any proteins exhibiting at least such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 any one of said SEQ ID NQ:60, SEQ ID NO:65 and SEQ ID NO:66.

16. The method according to claim 14 or 15, wherein said SPATE protein is selected from the group consisting of Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and any proteins exhibiting at least such as at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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; such as wherein the SPATE protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60.

17. The method according to any one of the preceding claims, wherein said autotransporter protein comprises at least one mutation that prevents cleavage at the autocatalytic cleavage site, such as at least one mutation which disrupts the autocatalytic cleavage site such that cleavage at the autocatalytic cleavage site is prevented.

18. The method according to claim 17, wherein said Hbp comprises the amino acid sequence as defined in SEQ ID NO:61 and any proteins exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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, provided said proteins comprise an autocatalytic cleavage site which has been mutated such that cleavage is prevented.

19. The method according to any one of the preceding claims, wherein said at least one AT fusion protein comprises at least one complementary binding moiety of said coupling system located N-terminally in said AT protein, such as located at the N- terminus of the said AT protein.

20. The method according to any one of the preceding claims, wherein said at least one AT fusion protein comprises at least one complementary binding moiety of said coupling system located internally in said AT protein, such as internally in the portion of said AT protein displayed on said vesicle.

21. The method according to any one of claims 1920, wherein said at least one complementary binding moiety of said coupling system is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein, such as at least two side domains of the AT protein.

22. The method according to any one of the preceding claims, wherein the coupling system is derived from Gram positive bacterial pilus proteins, such as derived from Gram positive bacterial pilus proteins selected from the group consisting of Lactiplantibacillus, Bacillus, Ruminococcus, and Streptococcus, such as preferably derived from Gram positive Streptococcal pilus proteins.

23. The method according to claim 22, wherein the coupling moiety and complementary moiety of the coupling system are selected from the group consisting of SnoopTag2 (SEQ ID NO:7), DogTag2 (SEQ ID NO:8), SnoopLigase2 (SEQ ID NO:9), SpyTag (SEQ ID NQ:10), SpyCatcher (SEQ ID NO:11), SnoopTag (SEQ ID NO:12), SnoopCatcher (SEQ ID NO:13), SpyTag002 (SEQ ID NO:14), SypCatcher002 (SEQ ID NO:15), SpyTag003 (SEQ ID NO:16), SpyCatcher003 (SEQ ID NO:17), SdyTag (SEQ ID NO:18), SdyCatcher (SEQ ID NO:19), SilkTag (SEQ ID NQ:20), SilkCatcher (SEQ ID NO:21), DogTag (SEQ ID NO:22), DogCatcher (SEQ ID NO:23), SnoopTagJr (SEQ ID NO:24), SnoopLigase (SEQ ID NO:25), Jo (SEQ ID NO:26), In (SEQ ID NO:27), NGTag (SEQ ID NO:28), NGCatcher (SEQ ID NO:29), SpyCatcher-N (SEQ ID NQ:30), SpyCatcher-21 (SEQ ID NO:31), SpyStapler (SEQ ID NO:32), SpyLigase (SEQ ID NO:33), MoonCake (SEQ ID NO:34), Katl (SEQ ID NO:35), QueenCatcher (SEQ ID NO:36), PsCsCatcher (SEQ ID NO:37), Ktag (SEQ ID NO:38), BDTag (SEQ ID NO:39), RumTrunkTagD9N (SEQ ID NQ:40), RumTrunkTag (SEQ ID NO:41), RumTag (SEQ ID NO:42), Rum2Tag (SEQ ID NO:43), Rum3Tag (SEQ ID NO:44), Rum4Tag (SEQ ID NO:45), Rum5Tag (SEQ ID NO:46), Rum6Tag (SEQ ID NO:47), Rum7Tag (SEQ ID NO:48), BacTag (SEQ ID NO:49), Bac2Tag (SEQ ID NQ:50), Bac3Tag (SEQ ID NO:51), Bac4Tag (SEQ ID NO:52), Bac5Tag (SEQ ID NO:53), Clib9 (SEQ ID NO:54), PhoTag (SEQ ID NO:55), PsCsTag (SEQ ID NO:56), and SpyCatcher (SEQ ID NO:57), and variants thereof exhibiting at least 70 % identity thereto, wherein said moieties are selected such that said moieties have the capacity of forming an isopeptide bond between each other, and wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70 % identity thereto.

24. The method according to any one of claims 22-23, wherein the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11), SpyTag-SpyCatcher002 (SEQ ID NO:10-SEQ ID NO:15), SpyTag- SpyCatcher003 (SEQ ID NQ:10-SEQ ID NO:16), SpyTag-SpyCatcher-N (SEQ ID NQ:10- SEQ ID NQ:30), SpyTag-SpyCatcher-21 (SEQ ID NQ:10-SEQ ID NO:31), SpyTag- SdyCatcher (SEQ ID NQ:10-SEQ ID NO:19), SpyTag002-SpyCatcher002 (SEQ ID NO:14- SEQ ID NO:15), SpyTag002-SpyCatcher (SEQ ID NO:14-SEQ ID NO:11), SpyTag002- SpyCatcher003 (SEQ ID NO:14-SEQ ID NO:17), SpyTag002-SdyCatcher (SEQ ID NO:14- SEQ ID NO:19), SpyTag003-SpyCatcher003 (SEQ ID NO:16-SEQ ID NO:17), SpyTag003- SpyCatcher (SEQ ID NO:16-SEQ ID NO:11), SpyTag003-SpyCatcher002 (SEQ ID NO:16- SEQ ID NO:15), SpyTag003-SdyCatcher (SEQ ID NO:16-SEQ ID NO:19), SnoopTag- SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13), Si IkTag-Sil kCatcher (SEQ ID NQ:20-SEQ ID NO:21), DogTag-DogCatcher (SEQ ID NO:22-SEQ ID NO:23), DogTag2-DogCatcher (SEQ ID NO:8-SEQ ID NO:23), Jo-In (SEQ ID NO:26-SEQ ID NO:27), NGTag-NGCatcher (SEQ ID NO:28-SEQ ID NO:29), SdyTag-SdyCatcher (SEQ ID NO:18-SEQ ID NO:19), SdyTag-SpyCatcher (SEQ ID NO:18-SEQ ID NO:11), SdyTag-SpyCatcher002 (SEQ ID NO:18-SEQ ID NO:15), SdyTag-SpyCatcher003 (SEQ ID NO:18-SEQ ID NO:17), KTag- SpyTag (SEQ ID NO:38-SEQ ID NQ:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SnoopTag2-SnoopCatcher (SEQ ID NO:7-SEQ ID NO:13), DogTag2- SnoopCatcher (SEQ ID NO:8-SEQ ID NO:13), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), MoonCake-RumTrunkTagD9N (SEQ ID NO:34-SEQ ID NQ:40), MoonCake- RumTag (SEQ ID NO:34-SEQ ID NO:42), MoonCake-SpyTag (SEQ ID NO:34-SEQ ID NQ:10), MoonCake-SdyTag (SEQ ID NO:34-SEQ ID NO:18), Katl-SpyTag (SEQ ID NO:35-SEQ ID NQ:10), Katl-SdyTag (SEQ ID NO:35-SEQ ID NO:18), Katl- RumTrunkTagD9N (SEQ ID NO:35-SEQ ID NQ:40), Katl-RumTag (SEQ ID NO:35-SEQ ID NO:42), and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to itscoupling moiety, respectively, and exhibits at least 70 % identity thereto, such as a variant selected from the group consisting of SEQ ID NO:10-19, 22-25, 34 and 38-40 and variants exhibits at least 70 % identity thereto.

25. The method according to any one of claims 39-40, wherein the coupling system is a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first and second moieties are capable of being isopeptide bonded by means of the third moiety.

26. The method according to claim 25, wherein said tripartite system comprises a first and second moiety selected from the group consisting of KTag-SpyTag (SEQ ID NO:38-SEQ ID NO:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SpyTag- BDTag (SEQ ID NQ:10-SEQ ID NO:39), DogTag2-SnoopTagJr (SEQ ID NO:8-SEQ ID NO:24), and wherein the tripartite system further comprises a third moiety which is a catalytic moiety, such as wherein said catalytic moiety is a ligase.

45. The method according to any one of claims 1-37, wherein the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as selected from SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and variants thereof exhibiting at least 70 % identity thereto, wherein a variant of a coupling moiety exhibiting at least 70 % identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70 % identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as wherein said coupling system is SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ IDNO:11).

27. The method according to any one of the preceding claims, wherein said at least one target polypeptide moiety is selected from the group consisting of aggregation- prone polypeptides and proteins; insoluble polypeptides and proteins; disordered proteins; misfolded proteins; hydrophobic polypeptides and proteins; and selfassociating polypeptides and proteins; such as the group consisting of aggregation- prone polypeptides and proteins; and insoluble polypeptides and proteins.

28. The method according to any one of the preceding claims, wherein said at least one target polypeptide moiety is selected from the group consisting of aggregation- prone polypeptides and proteins and insoluble polypeptides and proteins.

29. The method according to claim 28, wherein said aggregation-prone polypeptides and proteins are amyloid-forming polypeptides and proteins.

30. The method according to any one of claims 1-26, wherein said at least one target polypeptide moiety is a polypeptide comprising multiple tandem-fused neoantigen derived epitopes.

31. The method according to any one of the preceding claims, wherein said at least one POI further comprises at least one solubility-enhancing moiety.

32. The method according to claim 31, wherein said at least one solubility-enhancing moiety is linked directly or indirectly to said at least one target polypeptide moiety.

33. The method according to any one of claims 30-31, wherein said at least one solubility enhancing moiety is selected from the group consisting of -, PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NO:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA (SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA(SEQ ID NO:74), RpoS (SEQ ID NO:75), TF (SEQ ID NO:196), SUMO (SEQ ID NO:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), Fh8 (SEQ ID NO:197), H tag (SEQ ID NO:198), PpiB (SEQ ID NO:199), Bfr (SEQ ID NQ:200), YjdC (SEQ ID NQ:201), FolA (SEQ ID NQ:202), CheZ (SEQ ID NQ:203), GshB (SEQ ID NQ:204), ArsC (SEQ ID NQ:205), N-ePGK (SEQ ID NQ:206), KDPG aldolase (SEQ ID NQ:207), Tsf (SEQ ID NQ:208), MsyB (SEQ ID NQ:209), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), Z-tag (SEQ ID NQ:210), ZZ-tag (SEQ ID NO:211), GB1 (SEQ ID NO:212), DsbA (SEQ ID NO:213), DsbAmut (SEQ ID NO:214), IF2 domain I, CaBP (SEQ ID NO:215), FTN-H (SEQ ID NO:216), Skp (SEQ ID NO:217), T7PK (SEQ ID NO:218), Ecotin (SEQ ID NO:219), Halo Tag7 (SEQ ID NO:87), NusA (SEQ ID NO:88), s / GFP (SEQ ID NQ:220), SNUT (SEQ ID NO:222), EspA (SEQ ID NO:221), BLA, / n / Bl-21 (SEQ ID NO:223), SmbP (SEQ ID NO:224), Ffu, TDX (SEQ ID NO:225), HE-MBP(Pyr), N-terminal domain of rpoD (SEQ ID NO:226), yjgD (SEQ ID NO:227), (Arg)i-3, (Arg)4(SEQ ID NO:58), (Arg)5(SEQ ID NO:59), (His)s (SEQ ID NQ:108), (Arg)io (SEQ ID NQ:109), (Arg-Gly-Gly)3-Gly (SEQ ID NO:117), Poly(Arg), (Gly-Arg)3-(Arg)3(SEQ ID NO:118), (Gly-Arg)4(SEQ ID NO:158), Gly-(Arg)5(SEQ ID NO:159), Gly(Arg-Gly-Gly)3(SEQ ID NQ:160), Gly(Lys-Gly)6(SEQ ID NO:161), (Gly)2-(Arg)2-Gly-Arg (SEQ ID NO:162), Gly-Lys-Gly-(Lys)2(SEQ ID NO:163), (Gly)2-(Lys)4(SEQ ID NO:164), (Lys)i-3, (Lys)4(SEQ ID NO:165), (Lys)5(SEQ ID NO:166), (Lys)6(SEQ ID NO:167), (Lys)io (SEQ ID NO:168), (Asp)5(SEQ ID NO:169), (Glu)5(SEQ ID NQ:170), [Gly]-(Asp)3]3(SEQ ID NO:171), (Asn)5(SEQ ID NO:172), (Gln)5(SEQ ID NO:173), and (Ser)s (SEQ ID NO:174), such as selected from the group consisting of, PotD from E. coli (SEQ ID NO:71), PotD from 5. suis (SEQ ID NQ:70), Crr (SEQ ID NO:72), TrxA (SEQ ID NO:77), GST (SEQ ID NO:78), NusA(SEQ ID NO:88), SlyD (SEQ ID NO:73), RpoA (SEQ ID NO:74), RpoS (SEQ ID NO:75), SUMO (SEQ ID NQ:80), UB (SEQ ID NO:79), SUMO1 (SEQ ID NO:81), SUMO2 (SEQ ID NO:82), SUMO3 (SEQ ID NO:83), NEXT tag (SEQ ID NO:84), NEXT33 Tag (SEQ ID NO:85), NEXT16 Tag (SEQ ID NO:86), HALO Tag7 (SEQ ID NO:87), and NusA (SEQ ID NO:88).

34. The method according to claim 33, wherein said PotD is derived fromStreptococcus suis.

35. The method according to any one of claims 33-34, wherein said PotD is selected from group consisting of PotD comprising an amino acid sequence as defined in SEQ ID NO:70 and any polypeptide exhibiting at least 80 %, such as at least 81 %, such as at least 82 %, such as at least 83 %, such as at least 84 %, such as at least 85 %, such as at least 86 %, such as at least 87 %, such as at least 88 %, such as at least 89 %, 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 %, thereto, such as wherein said PotD is as defined in SEQ ID NO:70.

36. The method according to any one of the preceding claims, wherein said at least one POI further comprises at least one moiety promoting translocation of POI into microsomes, such as wherein said at least one moiety promoting translocation of POI into the microsomes is a signaling sequence.

37. The method according to any one of the preceding claims, wherein said at least one POI is provided as a nucleic acid encoding said POI in vector comprising at least one moiety promoting translocation of POI into the microsomes, such as wherein said at least one moiety promoting translocation of said POI into the microsomes is a signaling sequence.

38. The method according to any one of claims 36-37, wherein said signaling sequence selected from the group consisting of honeybee melittin signal sequence, such as wherein said signaling sequence is SEQ ID NO:229.

39. The method according to any one of the preceding claims, wherein the method further comprises the step of recovering the at least one vesicle coupled to said at least one polypeptide of interest.

40. A vesicle coupled to at least one polypeptide of interest (POI), wherein said vesicle on its outer surface displays at least one complementary moiety of a coupling system and at least one POI; wherein said at least one POI comprises at least one target polypeptide moiety, at least one solubility enhancing moiety and at least one coupling moiety of said coupling system; and wherein said vesicle is coupled to said at least one POI via an isopeptide bond formed between the respective moieties of the coupling system.

41. The vesicle coupled to at least one POI according to claim 40, wherein said vesicle further displays on its outer surface at least one autotransporter (AT) fusion protein and wherein said at least one AT fusion protein comprises an AT protein and at least one complementary moiety of said coupling system; and wherein said at least one AT fusion protein is coupled to said at least one POI via an isopeptide bond formed between the moieties of the coupling system on said at least one AT fusion protein and said at least one POI.

42. The vesicle coupled to at least one POI according to claim 40 or 41, wherein said vesicle is as defined in any one of claims 12-13.

43. The vesicle coupled to at least one POI according to any one of claims 40-42, wherein said autotransporter (AT) protein is as defined in any one of the claims 14- 21.

44. The vesicle coupled to at least one POI according to any one of claims 40-43, wherein said coupling system is as defined in any one of claims 22-26.

45. The vesicle coupled to at least one POI according to any one of claims 40-44, wherein said at least one target polypeptide moiety is as defined in any one ofclaims 27-30.

46. The vesicle coupled to at least one POI according to claim 40-45, wherein said at least one solubility-enhancing moiety is selected from the group as defined in any one of claims 31-35.

47. The vesicle coupled to at least one POI obtainable by the method according to any one of claims 1-39.

48. A vesicle coupled to at least one POI according to any one of claims 40-47, for use as a medicament.

49. A vesicle coupled to at least one POI for use according to 48, wherein said at least one POI comprises at least one target polypeptide moiety having therapeutical activity.

50. The vesicle coupled to at least one POI to claim 48 or 49, wherein said medicament is a vaccine.

51. A vesicle coupled to at least one POI according to any one of claims 40-47 for use in the treatment of a disease selected from the group consisting of cancer, autoimmune disease, inflammatory disease, infectious disease and transplant rejection.

52. The vesicle coupled to at least one POI according to any one of claims for use according to claim 51, wherein said disease is selected from the group consisting of infectious disease and cancer.

53. A pharmaceutical composition comprising a vesicle coupled to at least one POI according to any one of claims 40-47 or a vesicle coupled to at least one POI for useaccording to any one of times 48-52 and at least one pharmaceutically acceptable excipient, wherein said at least one POI comprises at least one target polypeptide moiety having therapeutical activity.

54. A vaccine composition comprising a vesicle coupled to at least one POI as defined in any one of claims 40-47 or a vesicle coupled to at least one POI for use according to any one of times 48-52, and at least one pharmaceutically acceptable excipient, wherein said at least one POI comprises at least one target polypeptide moiety which is an antigen or an immunogenic fragment thereof and wherein said composition does not comprise any additional agent with adjuvant effect.

55. Use of a vesicle coupled to at least one POI as defined in any one of claims 40-47 as a display platform for delivery of an antigen or an immunogenic fragment thereof.

56. Drug delivery platform comprising a vesicle coupled to at least one POI as defined in any one of claims 40-47 wherein said at least one POI comprises at least one target polypeptide moiety having therapeutic activity.

57. A method of diagnosis in vitro, comprising the steps:- providing a vesicle coupled to at least one polypeptide of interest (POI) according to any one of claims 40-47, wherein said at least one POI is capable of binding to a target of interest (TOI);- contacting a sample suspected to contain said TOI with the vesicle coupled to said at least one POI, thereby allowing said at least one POI to bind to said TOI;- detecting the binding of said at least one POI to indicate the presence of said TOI in the sample; and- using the information obtained to establish a diagnosis.

58. A method of prognosis in vitro, comprising the steps:- providing a vesicle coupled to at least one polypeptide of interest (POI) according toany one of claims 40-47, wherein said at least one POI is capable of binding to a target of interest (TOI);- contacting a sample suspected to contain said TOI with the vesicle coupled to said at least one POI, thereby allowing said at least one POI to bind to said TOI; - detecting the binding of said at least one POI to indicate the presence of said TOI in the sample; and- using the information obtained to establish a prognosis.

59. A method of detection of a target of interest (TOI) in a sample, comprising the steps:- providing a vesicle coupled to at least one polypeptide of interest (POI) according to any one of claims 40-47, wherein said at least one POI is capable of binding to said TOI;- contacting a sample suspected to contain said TOI with the vesicle coupled to said at least one POI, thereby allowing said at least one POI to bind to said TOI;- detecting the binding of said at least one POI to indicate the presence of said TOI in the sample.

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