Manufacture of Saliipro Particles

Abstract

The present invention relates to a method for preparing saposin lipoprotein particles comprising a saposin-like protein, a lipid, and optionally a hydrophobic agent, wherein the saposin-like protein or the hydrophobic agent is selectively bound to a support to allow self-assembly of the saposin lipoprotein particles. The method of the present invention comprises the steps of: a.) providing a hydrophobic agent and a lipid; b.1) / b.2) contacting the hydrophobic agent or the saposin-like protein with a support capable of selectively binding either of the two molecules to the support; c.1) / c.2) contacting the support-bound particle components with the remaining particle components, which are either the saposin-like protein or the hydrophobic agent, to allow self-assembly of the saposin lipoprotein particles on the support; and d) optionally eluting the support-bound saposin lipoprotein particles.

Description

Technical Field

[0001] The present invention relates to a method for preparing saposin-like lipoprotein particles comprising a saposin-like protein, a lipid, and optionally a hydrophobic agent.

Background Art

[0002] Membranes surround all living cells and form the envelopes of many viruses (e.g., HIV). In living cells, the cell membrane functions as a semipermeable barrier to preserve the cell's contents. The membrane components, particularly membrane proteins such as receptors and transporters, also determine and control how the cell interacts with its environment. The latter function, including biological processes such as signal transduction, molecular transport, energy metabolism, formation of cell-cell contacts, and cell homeostasis, is extremely important and depends on membrane proteins. Membrane proteins are encoded by approximately 30% of all ORFs (Wallin and von Heijne, Protein Science 1998 Apr;7(4):1029-38) and thus account for approximately one quarter of the cell's proteins. The membrane proteins of the viral envelope also help mediate interactions with the environment, such as the host cell membrane, and allow the viral capsid and viral genome to enter and infect the host cell.

[0003] Membrane proteins are of great interest for research in the life sciences, particularly for drug discovery, due to the extremely important functions described above. In fact, more than half of all drugs, i.e., over 60%, target membrane proteins (Overington et al., Nature Reviews Drug Discovery 5, 993 - 996 (December 2006)). For research purposes and clinical drug development, therefore, it is important that not only membrane proteins but also other hydrophobic agents (e.g., hydrophobic drugs) can be presented on state-of-the-art technology platforms without compromising the function of the hydrophobic agent or technology. This poses a paradox since most membrane proteins and other hydrophobic agents require a hydrophobic environment (ideally, being embedded in a structure that mimics their native membrane environment) to function properly. However, most biochemical assays commonly performed on state-of-the-art technology platforms are based on aqueous systems required for these assays and platforms to function properly. Accordingly, the object of the present invention is to provide an improved and more efficient method for formulating hydrophobic agents to render them soluble in an aqueous system while simultaneously preserving or mimicking the native membrane and lipid environment of the hydrophobic agent.

[0004] This will also enable the use of membrane proteins or other hydrophobic agents in high-throughput screening (HTS), which in its state-of-the-art setting also usually requires solubility in an aqueous system to function properly. Advances in molecular biology, computing, robotics, and detector technology have enabled HTS to progress to become the "core technology" in pharmaceutical, biotechnology, and life science research. Through the rapid and parallel conduct of biochemical tests, HTS has enabled the rapid identification of drugs, antibodies, molecular interactions, and proteins that modulate specific biomolecular pathways. The results obtained provide a starting point and important clues for further drug design and for understanding the role of specific biochemical processes in biological systems.

[0005] The desire to reduce the scale of HTS, combined with the progress of microfluidic technology, has led to the continued development of a biochemical lab-on-a-chip approach that can perform analyses more quickly and at lower cost in a small-scale setting. If membrane proteins and other hydrophobic agents could also be routinely used in such HTS or lab-on-a-chip applications, it would represent a major advance. For this purpose, it is often necessary to immobilize test agents on a surface such as a chip. Accordingly, a further object of the present invention is to provide an improved and more efficient method for immobilizing hydrophobic agents on a support, by rendering the hydrophobic agents compatible with an aqueous test system while simultaneously preserving or mimicking the native membrane and lipid environment of the hydrophobic agents.

[0006] Many state-of-the-art mechanisms for measuring biochemical interactions and reactions (e.g., the binding of a ligand to its corresponding receptor, the interaction of an antibody with its corresponding antigen, or the turnover of a substrate by an enzyme) rely on biosensors and analytes attached to the surface of a solid support. Well-known optical biosensor systems are based on surface plasmon resonance (SPR). Membrane proteins are attractive as analytes attached to the surface in biosensor systems because they play a central role in detecting various environmental stimuli. However, most of these biosensor systems are based on an aqueous environment, and thus membrane proteins and other hydrophobic agents cannot be easily used. Accordingly, a further object of the present invention is to provide an improved and more efficient method for immobilizing hydrophobic agents on a support, by rendering the hydrophobic agents compatible with an aqueous test system while simultaneously preserving or mimicking the native membrane and lipid environment of the hydrophobic agents.

[0007] In summary, as outlined above, the hydrophobic nature of membrane proteins and other hydrophobic agents is inconvenient for biochemical applications and research typically carried out in aqueous solutions. Since most biological systems, including human blood, exhibit a hydrophilic aqueous environment, the hydrophobic nature of membrane proteins and other hydrophobic agents is also inconvenient for their potential administration as therapeutic or diagnostic agents. Thus, hydrophobic agents (such as membrane proteins or hydrophobic compounds) present two major challenges for life science research and the pharmaceutical industry: (i) making membrane proteins and other hydrophobic agents soluble in aqueous solutions for assay and testing purposes without compromising their functions, and (ii) administering such hydrophobic agents as therapeutic and diagnostic agents without similarly compromising their desired and / or natural functions.

[0008] The second challenge, namely the administration and delivery of hydrophobic agents (e.g., hydrophobic compounds and / or proteins) as therapeutic or diagnostic agents, is mainly caused by their limited water solubility. In the case of drugs, this can lead to drug aggregation, high toxicity, undesirable immune responses, and / or highly concentrated local drug particles that can inactivate the drug (Allen and Cullis, SCIENCE, 303(5665):1818 - 1822, MAR 19, 2004).

[0009] An object of the present invention is to make hydrophobic agents such as membrane proteins or hydrophobic drugs more readily usable in state - of - the - art biochemical assay techniques such as HTS, lab - on - a - chip, and biosensor applications, and also to provide superior manufacturing and formulation methods for administering such hydrophobic agents in therapeutic or diagnostic applications. To achieve this object, an efficient, cost - and resource - saving method of incorporating hydrophobic agents into soluble particles while simultaneously preserving or mimicking the natural membrane and lipid environment of the hydrophobic agents is highly desirable.

[0010] Membrane proteins are most frequently purified in detergents that form micelles around the protein. However, detergents are very often harmful to the structure and activity of the protein. Furthermore, detergents can interfere with downstream analytical methods. Therefore, it is an object of the art to avoid the use of high concentrations of detergents when solubilizing hydrophobic agents. Prior art methods for addressing the problem of making insoluble hydrophobic agents soluble in an aqueous solution include, inter alia, nanodisc systems (Bayburt et al., Archives of biochemistry and biophysics, 450.2 (2006): 215-222 and Denisov et al., Journal of the American Chemical Society, 126.11 (2004): 3477-3487).

[0011] European Patent No. 1596828 describes discoidal bioactive agent delivery particles comprising an apolipoprotein that tightly surrounds a lipid bilayer in a double-belt shape. The interior of the particles is formed by the hydrophobic region of the lipid bilayer. This is in contrast to liposomes, which are closed spherical bilayer shells containing an aqueous interior. The discoidal bioactive agent delivery particles described in European Patent No. 1596828 have a Stokes diameter of about 10 nm and are proposed for use as delivery vehicles for hydrophobic pharmaceuticals such as amphotericin B or camptothecin.

[0012] European Patent No. 1345959 describes similar types of nanoscale particles having a diameter of about 10 nm and a height of about 5.5 nm. The particles are discoidal in shape and are composed of (i) an artificial membrane scaffold protein, (ii) a phospholipid bilayer, and (iii) at least one hydrophobic or partially hydrophobic membrane protein.

[0013] However, for example, in terms of the need to remove surfactants in a collection of particles, there are several drawbacks to this nanodisk technology. Furthermore, the size uniformity provided by the dense double-belt conformation of apolipoprotein-derived MSP seems to sacrifice constraints regarding a fixed minimum particle size and the maximum diameter obtainable by prior art methods.

[0014] Recently, a novel nanoparticle technology has been developed that includes the conserved saposin-like protein (SAPLIP) family of lipid-binding proteins (WO 2014 / 095576, WO 2015 / 036549, and WO 2018 / 033647).

[0015] The SAPLIP family is based on four saposin building members. These are saposins A - D, which are small (about 80 amino acids) proteins that bind and / or interact with lipids and function as essential cofactors for several lysosomal enzymes in sphingolipid catabolism (see Bruhn, Biochem J. (2005) 389, 249 - 257 and references cited therein). Saposins are described as preferring negatively charged lipids and low pH, showing significantly increased activity at acidic pH, and having an optimal pH at the lysosomal pH of 4.75. Saposins A, B, C, and D are proteolytically hydrolyzed from prosaposin, a single large precursor protein. The complete amino acid sequences of saposins A, B, C, and D, as well as the genomic and cDNA sequences of prosaposin, have been reported (O’Brien et al., (1988) Science 241, 1098 - 1101; Furst et al., (1992) Biochim Biophys Acta 1126:1 - 16).

[0016] Saposin C can induce the membrane fusion of phospholipid-containing vesicles in an acidic environment (Archives of Biochemistry and Biophysics 2003 Jul 1;415(1):43-53), which is a characteristic not shown by other saposins. Qi et al., (2009) Clin Cancer Res 15(18):5840-5851 reported on saposin C-conjugated dioleoylphosphatidylserine nanovesicles (SapC-DOPS) that contain an aqueous interior, have an average diameter of approximately 190 nm, and exhibit tumor-targeting activity in vivo. In SapC-DOPS, saposin C or a peptide derived therefrom acts as a homing peptide for the liposomes to which it is attached. Saposin C then targets the liposomes to cancer cells and exposes phosphatidylserine on the outer leaflet of the cell membrane. The authors believe that the unique acidic microenvironment surrounding cancer cells caused by the extracellular leakage of lysosomal enzymes targets tumor tissue as an optimal target for saposin C. According to Qi et al., SapC-DOPS liposomes are prepared by drying the solvent-dissolved purified phospholipids under N2(g), dispersing the dried phospholipids in an acidic buffer (pH 5) containing purified saposin C, diluting the mixture 50-fold in a physiological aqueous solution, and then promoting the aggregation of nanovesicles by subsequent sonication.

[0017] Popovic et al., PNAS, Vol.109, No.8 (2012) 2908-2912 reported on the structure of saposin A surfactant disks. Saposin A exists in soluble and lipid / surfactant-bound states. In the absence of lipids, saposin A adopts a closed monomeric apo conformation. In contrast, the saposin A surfactant disk structure reported by Popovic et al. is an open conformation that encloses 40 internally bound surfactant molecules organized in a highly ordered bilayer-like hydrophobic core, exposing two strands of saposin A.

[0018] In addition to the crystallization of the saposin A surfactant disk, Popovic et al. also describe the preparation of soluble lipid-saposin A complexes at pH 4.75 by a method that requires multiple steps. First, purified lipid dissolved in chloroform is dried under N2(g), the dried lipid is dispersed in acidic buffer (50 mM sodium acetate pH 4.8, 150 mM NaCl) by vortex mixing, the suspension is subjected to 10 cycles of freezing and thawing, mixed for 5 minutes with a vortex mixer, and the mixture is extruded through a 200 nm filter to prepare a homogeneous fraction of large unilamellar liposome vesicles. Mixing the large unilamellar artificial liposome vesicles thus prepared with purified saposin A in acidic buffer yielded soluble lipid-saposin A particles. The particles showed a narrow size distribution around an average hydrodynamic (Stokes) radius of 3.2 nm and contained approximately 5:1 lipid molecules per saposin A chain. The exact size of the particles was only mildly affected by the lipid-to-protein molar ratio and the composition of the liposomes. The authors observed similar 3.2 nm particles regardless of whether anionic phospholipids, cholesterol, or sphingoglycolipids were present in the liposome mixture. In all cases, a single peak was observed within the size range of a Stokes radius of 3.2 nm, indicating that the species distribution was relatively narrow. Thus, the technology of this publication is limited to a pH value of 4.75 and the above-described size of the particles and includes a cumbersome upstream liposome preparation step.

[0019] WO 2014 / 095576 pamphlet first showed that it is possible to incorporate purified, surfactant-solubilized hydrophobic cargo molecules or purified, surfactant-solubilized membrane proteins into sapocin lipoprotein particles using lipids solubilized and purified with surfactants. The method described in WO 2014 / 095576 pamphlet enables the self-assembly of sapocin lipoprotein particles by contacting sapocin-like proteins in a liquid environment with hydrophobic agents in the form of solubilized lipids and purified, surfactant-solubilized hydrophobic cargo molecules or purified, surfactant-solubilized membrane proteins. All individual components are present in free and soluble forms, and their self-assembly into sapocin lipoprotein particles is based on the free and random movement of all the participating particle components in the liquid environment.

[0020] WO 2015 / 036549 pamphlet extended the method described in WO 2014 / 095576 pamphlet to the incorporation of solubilized antigen molecules (shown for viral membrane proteins) derived from well-defined purified HIV-1 virus-like particles (VLPs). According to the examples in WO 2015 / 036549 pamphlet, pre-purified VLPs are lysed, HIV-1 membrane spike proteins are solubilized with surfactants, and then the free spike proteins are contacted with free sapocin A protein in a liquid environment. In this way, the sapocin-like protein provides lipids that mimic an environment in which the antigen molecules assemble. Again, all individual components are present in free and soluble forms, and their self-assembly into sapocin lipoprotein particles is based on the free and random movement of all the participating particle components in the liquid environment.

[0021] WO 2018 / 033647 describes a method for preparing a library of saposin lipoprotein particles from the membranes of cells or organelles. Instead of having to use solubilized and purified lipid and protein components, WO 2018 / 033647 enables the direct use of unpurified cell or organelle membranes as starting materials for assembling saposin lipoprotein particles. In this way, a library is obtained that contains a heterogeneous mixture of saposin lipoprotein particles having different membrane lipid and / or membrane protein compositions. Thus, the library of saposin lipoprotein particles obtained by the method of WO 2018 / 033647 presents a snapshot of the microdomains and components of the actual membrane from which the library was generated. The method of WO 2018 / 033647 enables the self-assembly of saposin lipoprotein particles by providing unpurified membranes or unpurified membrane vesicles that are contacted with saposin-like proteins in a liquid environment. Here too, during the assembly process, all individual components are present in a free (i.e., not bound to a support) form, and their self-assembly into saposin lipoprotein particles is based on the free and random movement of all the participating particle components present in the liquid environment.

[0022] WO 2014 / 095576, WO 2015 / 036549 and WO 2018 / 033647 present significant improvements by providing a new class of nanoparticles derived from saposin proteins that have many advantages over previous nanodisc technology, but further improvements in the process for preparing saposin lipoprotein particles are still needed.

[0023] For commercial use of saposin lipoprotein particles, it is necessary to provide them in large quantities at a reduced manufacturing cost. Thus, there is a need for an improved method for assembling saposin lipoprotein particles from the perspective of efficiency, cost and resource use.

[0024] In light of the increasing importance of the above-described technology platforms in pharmaceutical and life science research, it is important that hydrophobic agents incorporated into soluble nanoparticles be usable in these platforms. However, this often requires immobilizing the nanoparticles on a support, as is the case for biosensor applications.

[0025] Prior art methods for preparing saposin-lipoprotein particles can be further optimized for efficient, cost- and resource-saving production of the nanoparticles. In particular, prior art methods require, and assume, unrestricted mobility of the starting materials in a liquid environment between particle assemblies. Furthermore, the prior art does not offer an easy possibility to recycle surplus particle components for use in another particle assembly reaction. It is an object of the present invention to address one or more of these drawbacks. SUMMARY OF THE INVENTION

[0026] The underlying problem of the present invention is most generally seen in the provision of an improved, cost- and resource-saving method for generating saposin-lipoprotein particles.

[0027] This problem is solved by the method according to claim 1. Advantageous embodiments are described in the dependent claims and are described below in this specification.

[0028] The present invention is a method for generating saposin-lipoprotein particles, wherein the generated saposin-lipoprotein particles comprise: - a saposin-like protein, - a lipid, - optionally, a hydrophobic agent different from the lipid, and (I) the method comprises the following steps: a) providing the lipid and optionally the hydrophobic agent; b.1) contacting the support capable of selectively binding the saposin-like protein to a support in a liquid environment with the saposin-like protein; c.1) contacting the saposin-like protein bound to the support with the lipid and optionally the hydrophobic agent to enable self-assembly of the saposin lipoprotein particles on the support; d) optionally eluting the saposin lipoprotein particles bound to the support; comprising, or (II) alternatively, the method comprises the following steps: a) providing the hydrophobic agent and the lipid; b.2) contacting the hydrophobic agent with a support capable of selectively binding the hydrophobic agent to a support; c.2) contacting the hydrophobic agent bound to the support with the saposin-like protein to enable self-assembly of the saposin lipoprotein particles on the support; d) optionally eluting the saposin lipoprotein particles bound to the support; and provides a method.

[0029] In the method according to the invention, either the hydrophobic agent or the saposin-like protein is selectively bound to a support, and by contacting the hydrophobic agent or the saposin-like protein bound to the support with the remaining components of the saposin lipoprotein particles, particle aggregation occurs in a state bound to this support, thereby enabling self-assembly of the saposin lipoprotein particles on the support. This is different from the prior art methods in which all individual components are present in a free (i.e., not bound to a support) form and their self-assembly into saposin lipoprotein particles depends on an aggregation process based on the free and random movement of all the participating particle components in a liquid environment.

[0030] The method according to the invention has the advantage that a saposin-like protein or a hydrophobic agent is selectively bound to a support and that saposin lipoprotein particles bound to the support can be generated directly or later assembled particles can be eluted. This limits the amount of formed sariplo particles to the amount of pre-bound particle components, i.e., either the amount of bound saposin-like protein or the amount of bound hydrophobic agent. As a result, any unbound material can be recycled. Recycling can relate to saposin-like proteins or hydrophobic agents that are not bound to the support in steps b.2) and b.1), respectively. However, recycling can also relate to excess particle components that have been contacted with the material bound to the support to enable self-assembly of sariplo particles in steps c1) / c.2), but have not been incorporated into the particles. Such recycling of "unused" components enables particle assembly that saves costs and resources.

[0031] Direct assembly of saposin lipoprotein particles on a support is particularly advantageous in any case where it is necessary to use the particles in a bound state on the support. This applies to many chip-based analytical applications, such as biosensor applications, especially optical biosensor applications such as surface plasmon resonance. These will be explained in more detail below. Conventionally, free saposin lipoprotein particles had to be first prepared by prior art methods and then required a second coupling step to immobilize the free saposin lipoprotein particles on a support. In the method of the invention, it is possible to carry out the actual particle assembly on the support, saving additional costs and method steps.

[0032] It was surprising that salipro particles could be assembled even when one particle component, i.e., a saposin-like protein or a hydrophobic agent, was bound to the support. When one of the particle components binds to the support, the degree of freedom of contact with other particle components in the solution is restricted, and the degree of freedom of spatial interaction with the particle components is also restricted. As a result, a hindrance to particle assembly regarding efficiency was expected, which ultimately also affects the overall yield of the obtained particles.

[0033] It should be noted that the assembly of saposin lipoprotein particles must be assumed to involve a significant rearrangement of the structure of the saposin protein and the lipid composition (e.g., from the random movement of free lipids to a highly ordered nanomembrane bilayer structure). When switching from a lipid-free closed form to an open state with bound lipids, the saposin protein is likely to undergo a conformational change. The self-assembly of the particles is considered to include the process of the saposin-like protein capturing and encapsulating the lipids and hydrophobic agents incorporated into the particles. Although it was known that this self-assembly occurs in solution, it was completely unexpected to observe that the self-assembly functions equally well and rapidly when one of the main particle components (i.e., the saposin protein or the hydrophobic agent) is immobilized on a solid support.

[0034] It was unexpected to discover that high-quality, support-bound salipro particles can be obtained using the method according to the present invention, which provides an easy, rapid, and efficient method for producing support-bound salipro particles that maintain a uniform quality and composition over a long period. In principle, the present invention provides a more efficient and "direct" method for obtaining support-bound salipro particles by linking the assembly and coupling of salipro particles. In this way, the hydrophobic agent can be solubilized in stable particles that mimic its natural membrane environment in a single continuous method, and at the same time, it can be immobilized on a support for direct use in applications such as lab-on-a-chip or biosensor applications such as SPR.

[0035] The supopsin lipoparticle obtained by the method of the present invention is only in a form directly bound to a support, and in principle, is the same as those known from the prior art. The supopsin lipoparticle is also referred to herein as a "saliPro particle". The supopsin lipoparticle is a nanoparticle containing a supopsin-like protein, a hydrophobic agent, and a lipid. These components and their advantageous embodiments are further defined below. The supopsin-like protein belongs to the well-known conserved SAPLIP family of lipid-interacting proteins, or is a derivative or truncated form thereof.

[0036] The actual experiments of the present inventors have revealed that the size of the saliPro particle self-adjusts according to the properties of the incorporated hydrophobic agent and lipid, for example, according to the size of the incorporated membrane protein. The size of the saliPro particle is surprisingly flexible. Also, in the method according to the present invention, it has been observed that the saliPro particle adjusts its size according to the properties of the incorporated hydrophobic agent. This is more advantageous than the size limitation of other prior art particles not derived from supopsin. This flexibility also allows the incorporation of very large hydrophobic agents such as (multimeric) membrane proteins. For these reasons, in particular, the method of the present invention has the advantage that the hydrophobic agent can be reconstituted in its natural environment, for example, in membrane lipids or other cellular components potentially required to associate with a membrane protein and maintain the structure and / or function of the protein.

[0037] Surprisingly, the sarpuro particles exhibit a certain degree of thermal stability and also seem to be suitable for lyophilization and rehydration when bound to a support, and no significant quality deterioration can be observed. This enables the sarpuro particles obtained by the method of the present invention to be used in applications where portability and storage stability are issues. Using the method of the present invention, it is possible to obtain sarpuro particles bound to a support that can be pre-made, stored according to specified specifications, and then shipped to the end user. It is also possible to use sarpuro particles bound to a support in applications over a longer period, for example, in lab-on-chip experiments where the same chip is reused multiple times.

[0038] The sarpuro particles bound to a support obtained by using the method of the present invention have been proven to be able to incorporate various lipids, membrane proteins, and hydrophobic compounds at physiological pH, are soluble in an aqueous environment, but are bound to the support and form stable nanoscale complexes. For the hydrophobic agents tested so far, especially many complex membrane proteins, incorporation into the sarpuro particles bound to the support did not adversely affect the biological function of the hydrophobic agent. On the contrary, in many of these cases, incorporation into the sarpuro particles efficiently mimics the natural membrane environment of the hydrophobic agent and thus has a positive effect on their biological function.

[0039] The present invention can indirectly immobilize a hydrophobic agent that cannot be directly immobilized on a support without impairment or loss of its biological function by incorporating it into sarpuro particles prepared according to method (I) of the present invention, i.e., the sarpuro particles also offer the advantage of binding to the support via the binding moiety in the saposin-like protein rather than via the hydrophobic agent. This enables the hydrophobic agent to remain in an unmodified state and / or not be in direct contact with the support while still being effectively bound to the support.

[0040] As described above, the provision of sarp proteins bound to a support that can optionally elute from the support is a very useful tool in chip-based applications such as lab-on-a-chip or biosensor (e.g., SPR) applications.

[0041] By selectively binding a hydrophobic agent or a saposin-like protein to a support and contacting them with their respective other essential particle components, the method of the present invention appears to provide a robust technique for assembling sarp particles, which are stable in aqueous solution over a wide pH range, particularly at physiological pH, and enable particles larger than the 3.2 nm saposin A-derived lipoprotein particles obtained from liposomes synthetically prepared according to the teachings of the prior art of Popovic et al.

[0042] As described in the introduction, due to the importance of membrane proteins in the development of therapeutic methods, there is a need to discover innovative methods for examining membrane proteins in a cell-free medium, preferably in an environment free of surfactants.

[0043] The sarp particles obtainable by the method of the present invention meet this requirement. Once obtained, these sarp particles are stable in a cell-free medium and an environment free of surfactants and can be directly bound to a support on which they can later be used.

[0044] In certain advantageous embodiments of the present invention, the hydrophobic agent and lipid to be incorporated into the saposin lipoprotein particles are provided in the form of a biological membrane comprising the hydrophobic agent and lipid to be incorporated into the saposin lipoprotein particles. In particular, membranes of viruses, archaea, eukaryotes or prokaryotes can be used directly in the method of the present invention as a source material for hydrophobic agents and lipids. The biological membrane can be provided in the form of cells, viruses or organelles, all of which can be untreated, treatable with an amphiphilic agent, or soluble. The amphiphilic agent can be selected from the group consisting of surfactants, amphiphilic peptides, amphiphilic polymers, other amphiphilic compounds and mixtures thereof. Examples of amphiphilic polymers that can be used according to the present invention are maleic acid copolymers, particularly styrene-maleic acid copolymers (SMA) or diisobutylene-maleic acid copolymers (DIBMA). Amphiphilic peptides and amphiphilic polymers can be regarded as special surfactants. Suitable SMAs are described, for example, in Postis, Vincent, et al.’’The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy.’’ Biochimica et Biophysica Acta (BBA)-Biomembranes 1848.2 (2015): 496-501. DIBMA is commercially available, for example, from anatrace as BMA101. Preferably, the amphiphilic agent is a surfactant as defined herein. Preferably, the cells are eukaryotic cells, particularly non-human animal or human cells. Surprisingly, the research leading to the present invention has shown that purification of the biological membrane is not required. Rather, when using the method of the present invention, the saposin lipoprotein particles can be prepared directly from untreated, amphiphilic agent-treated or lysed cells, viruses or organelles.

[0045] By using a biological membrane as a starting material in the method of the present invention, a hydrophobic agent can be incorporated into the saposin particles together with its natural membrane environment. Compared with the method of WO 2018 / 033647 pamphlet, the method alternative (II) of the present invention has the advantage that only saposin particles containing the hydrophobic agent of interest are prepared, and a complete library of saposin particles representing the entire membrane proteome / lipidome from which the particles containing the hydrophobic agent of interest need to be isolated again is not prepared. Furthermore, the method of the present invention has the advantage over the WO 2018 / 033647 pamphlet that it can obtain particles bound to a support in a direct, simplified and continuous "one-step method", which is cost-effective and particularly desirable when saposin particles should be used in a form bound to a support.

[0046] By using a biological membrane as a starting material in the method alternative (I) of the present invention, a library of saposin particles containing a heterogeneous mixture of saposin lipoprotein particles having different membrane lipid and membrane protein compositions can be obtained. Compared with the method of WO 2018 / 033647 pamphlet, this embodiment of the method of the present invention has the advantage that particles bound to a support can be obtained in a continuous "one-step method", which is cost-effective and particularly desirable when a library of saposin particles should be used in a form bound to a support.

[0047] The saposin particles or library of saposin particles obtainable by the method of the present invention can be for use in medicine, in diagnostic methods, in cosmetic treatments, or as a vaccine formulation. Furthermore, the particles obtainable by the method of the present invention can be used as tools for diagnosis, drug development, drug screening, drug discovery, antibody development, development of therapeutic biologics, membrane or membrane protein purification, membrane protein expression, research on membranes and / or membrane proteins, isolation, identification and / or research of membranes and / or membrane proteins, or creation of lipidome or membrane proteome databases.

Mode for Carrying Out the Invention

[0048] The present invention relates to a method for generating saposin-lipoprotein particles, wherein the saposin-lipoprotein particles comprise - a saposin-like protein, and - a lipid, and - optionally, a hydrophobic agent different from the lipid, and (I) The method comprises the following steps: a) providing the lipid and optionally the hydrophobic agent; and b.1) contacting a support capable of selectively binding the saposin-like protein in a liquid environment with the saposin-like protein; and c.1) contacting the saposin-like protein bound to the support with the lipid and optionally the hydrophobic agent to enable self-assembly of the saposin-lipoprotein particles on the support; and d) optionally eluting the saposin-lipoprotein particles bound to the support, or (II) Alternatively, the method comprises the following steps: a) providing the hydrophobic agent and the lipid; and b.2) contacting a support capable of selectively binding the hydrophobic agent with the hydrophobic agent; and c.2) contacting the hydrophobic agent bound to the support with the saposin-like protein to enable self-assembly of the saposin-lipoprotein particles on the support; and d) optionally eluting the saposin-lipoprotein particles bound to the support, and provides a method.

[0049] The method according to the invention enables, in particular, the provision of sarpol particles bound to a support, each sarpol particle comprising a saposin-like protein, a lipid and a hydrophobic agent. If a complex set of lipids and hydrophobic agents is provided in step a), for example in the form of a biological membrane, a library of sarpol particles is generated in alternative (I) of the method according to the invention. This library comprises sarpol particles comprising a saposin-like protein, a hydrophobic agent (e.g. a membrane protein) and a lipid, but may also comprise sarpol particles consisting only of a lipid and a saposin-like protein.

[0050] Saposin lipoprotein particles are also referred to herein as "sarpol particles". Saposin lipoprotein particles comprise a saposin-like protein and a lipid as essential components. Usually, a sarpol particle comprises one type of saposin-like protein. Typically, and according to a preferred embodiment, saposin lipoprotein particles prepared according to the invention comprise a saposin-like protein, a lipid and a hydrophobic agent.

[0051] The sarpol particles of the invention may also comprise a plurality of hydrophobic agents that may be the same as or different from each other. Examples of multimers of the same hydrophobic agent in a sarpol particle are multimeric membrane proteins or multiple hydrophobic compounds. Examples of different hydrophobic agents in a sarpol particle are sarpol particles comprising a membrane protein and a hydrophobic compound.

[0052] The sarpol particles of the invention comprise a plurality of lipids that may be the same as or different from each other.

[0053] The saposin-like proteins used in the method of the present invention are saposin-like proteins (SAPLIP) or derivatives or cleavage forms thereof. The term "saposin-like protein" (SAPLIP) is recognized in the art and includes all members of the conserved saposin-like protein (SAPLIP) family of lipid-interacting proteins. The abbreviation "SAPLIP" is used synonymously with the term "saposin-like protein". The SAPLIP family is characterized by a conserved alpha-helical three-dimensional structure stabilized by highly conserved intramolecular disulfide bonds, the saposin fold (Munford et al. (1995), Journal of Lipid Research, vol. 36, no. 8, 1653-1663 and Bruhn (2005), Biochem J 389(15):249-257). Examples of members of the saposin-like protein (SAPLIP) family according to the present invention are described in Munford et al. (1995), Journal of Lipid Research, vol. 36, no. 8, 1653-1663 and Bruhn (2005), Biochem J 389(15):249-257, both of which are hereby incorporated by reference in their entirety.

[0054] In the ligand-free (i.e., surfactant-free / lipid-free) "closed" state, SAPLIP adopts the saposin fold, a compact 4-helix bundle structure of monomers. This fold is exemplified by the structure of the closed apo form of human saposin A (Protein Data Bank (PDB) ID code: 2DOB, Ahn et al. (2006) Protein Sci. 15:1849-1857), or saposin C (PDB ID code: 1M12; de Alba et al. (2003), Biochemistry 42, 14729-14740), NK-lysin (PDB ID code: 1NKL; Liepinsh et al. (1997), Nat. Struct. Biol. 4, 793-795), amoebapore A (PDB ID code: 1OF9) and granulin (PDB ID code: 1L9L; Anderson et al. (2003) J. Mol. Biol. 325, 355-365), all of which are nearly identical and can be easily superimposed.

[0055] SAPLIP undergoes a conformational change upon binding to a ligand such as a lipid or surfactant molecule. In the "open" conformation bound to the ligand, SAPLIP adopts a V-shaped or boomerang-shaped conformation with an exposed hydrophobic surface that contacts the bound lipid. The open conformation is exemplified by the prior art saposin A surfactant discoidal structure (PDB ID code: 4DDJ; Popovic et al., PNAS, Vol. 109, No. 8 (2012) 2908-2912) and the structure of saposin C bound to an SDS surfactant micelle (PDB ID code: 1SN6; Hawkins et al. (2005) J. Mol. Biol. 346:1381-1392).

[0056] In sarpol particles, the saposin-like protein is preferably amphiphilic, with one part of its structure being more or less hydrophilic and facing an aqueous solvent, and the other part being more or less hydrophobic and facing the hydrophobic core of the lipid-containing particle. The saposin-like protein preferably has mainly more hydrophobic residues (such as A, C, F, G, I, L, M, V, W or Y) on one side of the helix and more polar or charged residues (such as D, E, N, Q, S, T, H, K or R) on the other side of the helix, and is characterized by an amphiphilic α-helix.

[0057] The abbreviations of amino acid residues used herein are as follows: A, Ala, alanine; V, Val, valine; L, Leu, leucine; I, Ile, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, Gly, glycine; S, Ser, serine; T, Thr, threonine; C, Cys, cysteine; Y, Tyr, tyrosine; N, Asn, asparagine; Q, Gln, glutamine; D, Asp, aspartic acid; E, Glu, glutamic acid; K, Lys, lysine; R, Arg, arginine; and H, His, histidine.

[0058] In contrast to prior art apolipoprotein-derived nanodiscs, the saposin-like proteins of the present invention do not surround lipids in a double-belt-like fashion. Rather, sarpol particles are integrated by a lipid-containing core surrounded by two or more substantially V-shaped or boomerang-shaped saposin-like proteins arranged in an orientation with substantially no direct protein-protein contact between individual saposin-like proteins within a given sarpol particle. Without wishing to be bound by this theory, this arrangement of saposin-like proteins and lipids in sarpol particles is thought to confer the size flexibility observed when bulky hydrophobic agents or increasing amounts of lipids are incorporated into the particles in the methods of the present invention.

[0059] The ability to interact with lipids as well as the amphiphilic nature and three-dimensional structure described above are highly conserved among SAPLIPs, but they are highly diverse at the amino acid sequence level, with sequence identity falling below the normal threshold zone of 25 - 30% identity to define homology (see sequence comparisons in Figures 4A and 4B of Bruhn (2005), Biochem J 389(1):249 - 257, reproduced in the attached Figures 13A and 13B).

[0060] In lipoprotein saproparticles, the saposin-like protein mainly functions as a structural protein, providing a scaffold for the structure of the lipoprotein saproparticle, such as a discoidal structure. For this reason, structural features, particularly the saposin fold characteristic of SAPLIPs, are more important for defining the saposin-like proteins of the present invention compared to mere sequence determinants.

[0061] Examples of SAPLIPs according to the invention are saposin A, B, C or D (e.g., from Homo sapiens [see SEQ ID NOs: 1-4], Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis); surfactant protein B (e.g., from Homo sapiens, Canis familiaris, Mus musculus, Oryctolagus cuniculus, Ovis aries or Rattus norvegicus); granulysin (e.g., from Homo sapiens; see SEQ ID NO: 5); NK-lysin (e.g., from Sus scrofa; see SEQ ID NO: 6); NK-lysin ortholog (e.g., from Equus caballus or Bos taurus); amoebapore (e.g., from Entamoeba histolytica); amoebapore ortholog (e.g., from Entamoeba dispar or Entamoeba invadens); amoebapore-like protein (e.g., from Fasciola hepatica); Negleria pore (e.g., from Naegleria fowleri); clonorine (e.g., from Clonorchis sinensis); prosaposin (e.g., from Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis) and MSAP (e.g., from Homo sapiens).

[0062] The sequences of the specific SAPLIPs used in accordance with the present invention are shown in FIGS. 4A and 4B of Bruhn (2005), Biochem J 389(15):249-257, reproduced in the attached FIGS. 13A and 13B, and the sequences are specifically incorporated herein by reference. The sequences of the specific SAPLIPs used in accordance with the present invention are shown in the following sequence listing.

[0063]

Table 1

[0064] The SAPLIPs used in accordance with the present invention can also be polypeptides that include a saposin fold as part of a multi-domain protein. This is the case, for example, for acid sphingomyelinase (from Homo sapiens, Caenorhabditis elegans, Ciona intestinalis, Anopheles, Drosophila, Mus musculus or Rattus norvegicus); GDSL (Gly-Asp-Ser-Leu) lipase, for example, acyl-oxy hydrolase (from Homo sapiens or Rattus norvegicus); coutin (from Dictyostelium discoideum); J3-crystallin (from Tripedalia cystophora) and plant aspartic protease (from Viridiplantae). A further SAPLIP used in accordance with the present invention can be bacteriocin AS-48. Bacteriocin AS-48 exhibits antibacterial activity, can also bind lipids, and has the same fold as the remaining SAPLIP family members but lacks disulfide bridges.

[0065] In the following, the present invention is described in more detail with respect to saposin A or its derivatives or cleavage forms as saposin-like proteins, which are preferred embodiments, but the present invention is not limited thereto. Rather, the present invention clearly extends to the entire family of saposin-like proteins (SAPLIPs) as saposin-like proteins of the present invention. Due to the high degree of structural and functional conservation among SAPLIPs, the features and advantages of certain embodiments using saposin A as a saposin-like protein have been shown to apply to other embodiments using other SAPLIPs or their derivatives or cleavage forms as saposin-like proteins.

[0066] According to a preferred embodiment, the SAPLIP is saposin A, saposin B, saposin C or saposin D. In one embodiment, the SAPLIP is saposin A, saposin B or saposin D. Saposin A, saposin B, saposin C or saposin D is preferably saposin A, saposin B, saposin C or saposin D derived from Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Ochotona cuniculus, Rattus norvegicus or Xenopus laevis. In one embodiment, the SAPLIP is of human origin (i.e., Homo sapiens SAPLIP).

[0067] In a preferred embodiment, the SAPLIP is saposin A, preferably saposin A derived from Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Ochotona cuniculus, Rattus norvegicus or Xenopus laevis, particularly preferably human saposin A, the amino acid sequence of which is given as SEQ ID NO: 1. The expression, purification and crystallization of saposin A as an LDAO-surfactant complex are described, for example, in PNAS, vol. 109, No. 8 (2012) 2908-2912 (Popovic et al.).

[0068] According to one embodiment, the saposin-like protein comprises the full-length sequence of SAPLIP. In another embodiment, the saposin-like protein is a derivative of SAPLIP, in particular a polypeptide comprising an amino acid sequence having at least 20, 25, 30, 40, 50 or 60%, preferably at least 75% identity to the full-length sequence of each SAPLIP. In particular, the saposin-like protein can comprise a sequence having at least 80%, 85%, 90% or 95% identity to the full-length sequence of SAPLIP.

[0069] Derivatives or truncated forms of SAPLIP can be used in the methods of the invention as long as they can self-assemble into saposin particles in the methods of the invention as further specifically defined in the claims and herein. This can be readily tested by one skilled in the art according to the examples described herein without undue burden.

[0070] In one embodiment, the saposin-like protein is saposin A, saposin B, saposin C, saposin D or a derivative or truncated form thereof that can form saposin lipoprotein particles in the method according to claim 1.

[0071] As used herein, the term "sequence identity" refers to the degree of identity between proteins that can be calculated by optimal alignment of sequences using a scoring matrix such as the Blosum62 matrix described in Henikoff S. and Henikoff JG., P.N.A.S. USA 1992, 89: 10915-10919. The percent identity and calculation of the optimal alignment of two sequences using the Blosum62 similarity matrix as well as the Needleman and Wunsch algorithms (J. Mol. Biol. 1970, 48: 443-453) can be performed using the GAP program of the Genetics Computer Group (GCG, Madison, WI, USA) with the program's default parameters. For comparison of amino acid alignments, the EMBL online tool "EMBOSS Stretcher" (http: / / www.ebi.ac.uk / Tools / psa / emboss_stretcher / ) is used with the program's default settings.

[0072] In another embodiment, a derivative of a SAPLIP is a polypeptide comprising a sequence having one or more amino acid deletions, additions, insertions and / or substitutions in the amino acid sequence of each SAPLIP. For example, a SAPLIP derivative can be a polypeptide comprising the sequence of a particular SAPLIP in which 1 to 40, preferably 1 to 30, particularly 1 to 20 or 1 to 15 amino acids have been deleted, added, inserted and / or substituted. As used herein, the term "deletion" refers to the removal of 1, 2, 3, 4, 5 or more amino acid residues from each starting sequence.

[0073] As used herein, the term "insertion" or "addition" refers to the insertion or addition of 1, 2, 3, 4, 5 or more amino acid residues to each starting sequence.

[0074] As used herein, the term "substitution" refers to the replacement of an amino acid residue located at a particular position with a different residue.

[0075] In preferred embodiments, the derivative or cleavage form of SAPLIP is i. a protein having at least 20% sequence identity to the full-length sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6; in particular, a protein having at least 20% sequence identity to the full-length sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6, which protein, when used in the method according to claim 1, can self-assemble with lipids into lipoprotein particles; and ii. a protein comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6, in which 1 to 40 amino acids are deleted, added, inserted and / or substituted; selected from.

[0076] Embodiments where the sequence identity described herein, for example, a derivative of a saposin-like protein has at least 20% sequence identity to the full-length sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6, when used in the method according to claim 1, can be combined with the feature that the protein is amphiphilic, forms at least one alpha helix, and can self-assemble with lipids into lipoprotein particles.

[0077] According to a further embodiment, the sapocin-like protein is a derivative of sapocin A comprising one or more fragments of SEQ ID NO: 1. Preferred fragments correspond to helices a1, a2, a3 and a4 of sapocin A, where helix a1 is formed by the following continuous stretch of amino acids: "SLPCDICKDVVTAAGDMLK"; helix a2 is formed by the following continuous stretch of amino acids: "ATEEEILVYLEKTCDWL"; helix a3 is formed by the following continuous stretch of amino acids: "PNMSASCKEIVDSYLPVILDIIKGEMS"; helix a4 is formed by the following continuous stretch of amino acids: "PGEVCSAL". According to a particular embodiment, the derivative of sapocin A is a polypeptide comprising a sequence selected from helices a1, a2, a3, a4 of sapocin A and combinations thereof, in particular the polypeptide comprises the sequences of helices a1, a2 and a3 of sapocin A. Fragments of sapocin A, for example, helices a1, a2, a3, a4 of sapocin A, may have one or more amino acid deletions, additions, insertions and / or substitutions in the amino acid sequence.

[0078] According to another embodiment, the sapocin-like protein is a derivative of a sapocin-like protein comprising one or more of the fragments defined for sapocin A in the previous paragraph, the fragments being replaced by the corresponding sequences of the respective sapocin-like proteins.

[0079] According to one embodiment, when a derivative or cleaved form of a SAPLIP is used as the sapocin-like protein according to the present invention, the derivative or cleaved form should be amphiphilic and should form at least one alpha helix. In addition or alternatively, the derivative or cleaved form should be capable of self-assembling with lipids into lipoprotein particles when used in the method according to the present invention. As used herein, the term "amphiphilic" refers to a polypeptide or molecule having both a hydrophilic region and a hydrophobic region.

[0080] Preferably, when a derivative of SAPLIP is used, at least 3, 4, 5 or all of the 6 cysteine residues corresponding to the 6 cysteines in the saposin A of the SAPLIP building member should be present. In this regard, refer to the positions of cysteines in the sequence comparisons of Figures 4A and 4B of Bruhn (2005), Biochem J 389(15):249-257. The sequences and sequence alignments of Figures 4A and 4B of Bruhn et al. are reproduced in the attached Figures 13A and 13B and are specifically incorporated herein by reference.

[0081] The SAPLIP according to the present invention may also include one or more non-natural amino acids, amino acid analogs or peptidomimetic structures in which the peptide bond is replaced by a structure more resistant to metabolic degradation.

[0082] Step a) of the method of the present invention In step a) of the method of the present invention, the lipids and optionally hydrophobic agents to be incorporated into the sariplo particles are provided. Importantly, additional lipids and / or hydrophobic agents may also be provided in other steps of the method.

[0083] "A" and "the" are generally used herein to mean "at least one" or "at least one kind". For example, at least one hydrophobic agent is included in the sariplo particles of the present invention and is accordingly provided in step a).

[0084] The hydrophobic agent and / or lipid may be provided in a composition containing other components. Preferably, the hydrophobic agent and / or lipid is provided in a liquid composition. In one embodiment, the liquid composition contains an organic solvent. In another embodiment, the liquid composition is an aqueous composition. In another embodiment, the liquid composition is an aqueous composition containing a surfactant. In a further embodiment, the liquid composition is a dispersion in which the hydrophobic agent and / or lipid is dispersed in the aqueous phase.

[0085] The hydrophobic agent and the lipid can be provided together or in separate forms. When the hydrophobic agent and the lipid are provided together, they can be provided in the form of a biological membrane or a composition derived from a biological membrane. For example, the lipid and optionally the hydrophobic agent can be provided in the form of a cell membrane, an endosome, an exosome, a virus-like particle or a liposome. When different lipids and / or different hydrophobic agents are provided, these can likewise be provided together or in separate forms. The biological membrane, when used in the methods of the present invention, can be provided in the form of untreated, amphiphilic agent-treated or lysed cells, viruses or organelles.

[0086] The hydrophobic agent is different from the lipids otherwise contained in the particle. This means that when the hydrophobic agent itself is a lipid or a modified lipid, more than half of the lipids contained in the particle (i.e., more than 50 mol% based on the total amount of lipids present in the particle) should be different from the lipids forming the hydrophobic agent. In one embodiment, the hydrophobic agent is not a lipid, and in another embodiment, the hydrophobic agent is neither a lipid nor a surfactant.

[0087] As used herein, "hydrophobic agent" means any molecule that is substantially hydrophobic. "Hydrophobic" is a technical term that refers to the property of being immiscible with water or having a strong lack of affinity / solubility in water. The hydrophobic agent can be a hydrophobic organic compound and / or a hydrophobic biomolecule. The hydrophobic agent can be a therapeutically or biologically active hydrophobic agent, or a hydrophobic agent that simply stabilizes the discoidal shape of the salipro particle. The hydrophobic agent is an agent, i.e., a compound and / or a biomolecule, that does not completely penetrate water or remains insoluble in water and / or tends to aggregate and / or partition into a hydrophobic environment when present in an aqueous phase. By being contained in the salipro particle, the hydrophobic agent is effectively solubilized in the hydrophobic interior of the particle. Thereby, the hydrophobic agent can maintain its native functionality, such as catalytic activity or ligand binding.

[0088] The hydrophobic agent contained in the salipro particles generally includes at least one hydrophobic (e.g., lipophilic) region that can associate with or integrate into the hydrophobic portion of the lipid bilayer. Thus, the hydrophobic agent can also be a chimeric molecule in which a hydrophobic (e.g., lipophilic) moiety, module or compound that can associate with or integrate into the hydrophobic portion of the lipid bilayer is bound to another molecule. For example, a compound to which a lipid or fatty acid is bound, particularly a drug to which a lipid or fatty acid is bound, can be used as a hydrophobic agent according to the present invention. In these cases, the compound or drug itself does not necessarily have to be hydrophobic. In some embodiments, at least a portion of the hydrophobic agent is inserted between or penetrates into the hydrophobic portions (e.g., fatty acid acyl chains) of the lipid molecules inside the particle.

[0089] In one embodiment, the hydrophobic organic compound and / or hydrophobic biomolecule can be, for example, a biologically active agent, a drug, an active ingredient of a drug, an active ingredient of a cosmetic, an active ingredient of a plant protection product, a food and / or dietary supplement, a diagnostic probe, a contrast agent, a label and / or an indicator.

[0090] The hydrophobic drug contained in the salipro particles and administrable to a patient in need thereof can be any drug having low solubility in an aqueous environment. The low solubility in an aqueous environment may only be apparent under certain conditions, for example, within a certain pH or temperature range, or when the concentration of the hydrophobic agent exceeds a certain threshold value.

[0091] For example, the drugs contained in the salipro particles and administrable to a patient in need thereof are, inter alia, for the treatment of cancer, inflammatory or infectious conditions, cardiovascular diseases, neurological disorders and rheumatism. The hydrophobic agent can be an antioxidant, a vitamin, an anti-proliferative agent, a hormone, a steroid or an enzyme. The hydrophobic agent can be a herbicide or fungicide compound.

[0092] Some specific examples of hydrophobic drugs that can be incorporated into the silypro particles include curcumin, sulfonamides such as sulfonamide, sulfamethoxazole and sulfacetamide; trimethoprim, especially in combination with sulfamethoxazole; quinolines such as norfloxacin and ciprofloxacin; penicillins such as penicillin G, penicillin V, ampicillin, amoxicillin and piperacillin, cephalosporins such as cephalosporin C, cephalothin, cefoxitin and ceftazidime, other beta-lactam antibiotics such as imipenem and aztreonam including beta-lactam compounds; beta-lactamase inhibitors such as clavulanic acid; aminoglycosides such as gentamicin, amikacin, tobramycin, neomycin, kanamycin and netilmicin; tetracyclines such as chlortetracycline and doxycycline; chloramphenicol; macrolides such as erythromycin; or other antibiotics such as clindamycin, polymyxin and bacitracin for antibacterial use and in some cases for antifungal infections; polyene antibiotics such as amphotericin B, nystatin and hamycin; flucytosine; imidazole or triazole such as ketoconazole, miconazole, itraconazole and fluconazole; griseofulvin for antifungal diseases such as aspergillosis, candidiasis, histoplasmosis; zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, interferon (e.g., interferon alpha-2a or interferon alpha-2b) and ribavirin for antiviral diseases; aspirin, phenylbutazone, phenacetin, acetaminophen, ibuprofen, indomethacin, sulindac, piroxicam, diclofenac; gold and steroid anti-inflammatory agents for inflammatory diseases such as arthritis; ACE inhibitors such as captopril, enalapril and lisinopril; organic nitrates such as amyl nitrite, nitroglycerin and isosorbide dinitrate; calcium channel blockers such as diltiazem, nifedipine and verapamil; beta-adrenergic antagonists such as propranolol for cardiovascular diseases;Diuretics, for example, thiazides; for example, benzothiadiazines or loop diuretics, for example, furosemide; sympatholytics, for example, methyldopa, clonidine, guanabenz, guanethidine and reserpine; vasodilators, for example, hydralazine and minoxidil; calcium channel blockers, for example, verapamil; ACE inhibitors, for example, captopril for the treatment of hypertension; quinidine, procainamide, lidocaine, encainide, propranolol, esmolol, bretylium and diltiazem for the treatment of cardiac arrhythmias; lovastatin, lipitor, clofibrate, cholestyramine, probucol and nicotinic acid for the treatment of hypolipoproteinemia; anthracyclines, for example, doxorubicin, daunorubicin and idarubicin; covalent DNA-binding compounds, covalent DNA-binding compounds, as well as platinum compounds, for example, cisplatin and carboplatin; folic acid antagonists, for example, methotrexate and trimethoprim; metabolic antagonists and pyrimidine antagonists, for example, fluorouracil, 5-fluorouracil and fluorodeoxyuridine; metabolic antagonists as well as purine antagonists, for example, mercaptopurine, 6-mercaptopurine and thioguanine; metabolic antagonists as well as sugar-modified analogs, for example, cytarabine and fludarabine; metabolic antagonists and ribonucleotide reductase inhibitors, for example, hydroxyurea; covalent DNA-binding compounds as well as nitrogen mustard compounds, for example, cyclophosphamide and ifosfamide; covalent DNA-binding compounds and alkanesulfonates, for example, busulfan; nitrosoureas, for example, carmustine; covalent DNA-binding compounds and methylating agents, for example, procarbazine; covalent DNA-binding compounds and aziridines, for example, mitomycin; non-covalent DNA-binding compounds; non-covalent DNA-binding compounds, for example, mitoxantrone and bleomycin; inhibitors of chromatin function and topoisomerase inhibitors, for example, etoposide, teniposide, camptothecin and topotecan; inhibitors of chromatin function as well as microtubule inhibitors, for example, vincristine, vinblastine, vindesine and paclitaxel, taxotere or other taxanes including vinca alkaloids;Compounds that affect endocrine function, such as antibodies like prednisone, prednisolone, tamoxifen, leuprolide, ethinyl estradiol, herceptin; genes, such as the p-53 gene, p16 gene, MIT gene and E-cadherin gene; cytokines, such as interferons, especially IL-I, IL-2, IL-4, IL-6, IL-8 and IL-12, tumor necrosis factors, such as tumor necrosis factor-alpha and tumor necrosis factor-beta, colony stimulating factors, such as granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF), interferons, such as interferon-alpha, interferon-beta1, interferon-beta2 and interferon-gamma; all-trans retinoic acid or another retinoid for the treatment of cancer; immunosuppressive agents, such as cyclosporine, immunoglobulins; Sulfasalazine, methoxsalen, Thalidomide; insulin and glucagon for diabetes; calcitonin and sodium alendronate for the treatment of osteoporosis, hypercalcemia and Paget's disease; morphine and related opioids; meperidine or analogs; methadone or analogs; opioid antagonists, such as nalorphine; centrally acting antitussives, such as dextromethorphan; tetrahydrocannabinol or marinol for pain management, lidocaine and Bupivacaine; chlorpromazine, prochlorperazine; cannabinoids, such as tetrahydrocannabinol, butyrophenones, such as droperidol; benzamides, such as metoclopramide for the treatment of nausea and vomiting; heparin, coumarin, streptokinase, tissue plasminogen activator (t-PA) as anticoagulants, antithrombotic agents or antiplatelet agents; heparin, sulfasalazine, nicotine and steroids and tumor necrosis factor-alpha for the treatment of inflammatory bowel disease; nicotine for the treatment of smoking addiction; growth hormone, luteinizing hormone, corticotropin and somatotropin for hormone therapy; and adrenaline for general anaphylaxis are included.

[0093] One skilled in the art can readily determine experimentally whether a particular compound is well incorporated into the salipro particles by the methods and examples described in this specification and in the examples of WO 2014 / 095576 pamphlet, for example Example 9. For compounds that cannot be detected by spectroscopy, one skilled in the art can rely on, for example, LC-MS or thin layer chromatography to determine whether a particular compound is well incorporated into the salipro particles.

[0094] The advantage of including a hydrophobic agent in the salipro particles is that the hydrophobic agent is effectively solubilized in the stable structure of the particles, thereby functioning as a storage and / or delivery vehicle for the hydrophobic agent in, for example, the aqueous environment present in most of the body fluids and tissues. Compared to classical solubilization means via surfactants or organic solvents, salipro particles have the advantage that the hydrophobic agent is effectively solubilized inside the hydrophobicity of the particles while being hydrophilic from the outside, whereby the hydrophobic agent can maintain its natural functionality such as catalytic activity or ligand binding. Furthermore, in contrast to most surfactants and organic solvents, salipro particles appear to be biocompatible.

[0095] In addition to hydrophobic organic compounds, it has also been demonstrated that lipoprotein salipro particles can stably incorporate hydrophobic biomolecules, such as proteins containing hydrophobic moieties. According to a preferred embodiment, the hydrophobic agent is a hydrophobic protein, particularly a membrane protein. Membrane proteins can be selected from integral membrane-spanning proteins, integral monotopic membrane proteins, surface membrane proteins, amphitropic proteins in a lipid-bound state, lipid-anchored proteins, and chimeric proteins having fused hydrophobic and / or membrane-spanning domains. The term "membrane protein" as used herein does not include saposin-like proteins.

[0096] An integral membrane protein is a membrane protein that is constantly bound to the lipid bilayer and usually requires a surfactant or nonpolar solvent to be removed from the membrane. A transmembrane protein is an integral membrane protein that penetrates the membrane at least once. Examples of transmembrane proteins that can be incorporated into sariplo particles are G protein-coupled receptors (GPCRs), transporters, such as uniporters, cotransporters or antiporters, channels, such as ion channels or enzymes.

[0097] An integral monotopic membrane protein is constantly attached to the membrane only from one side and does not penetrate the membrane. This class includes membrane proteins that are tethered to the membrane via an alpha-helical transmembrane anchor. Examples include cytochrome P450 oxidase and glycophorin A.

[0098] A peripheral membrane protein only associates transiently or indirectly with the lipid bilayer or an integral membrane protein incorporated therein. Peripheral membrane proteins usually dissociate from the membrane after treatment with polar reagents having an elevated pH or high salt concentration. Examples of peripheral membrane proteins include phospholipase A2 or C, lipoxygenase and cytochrome c.

[0099] A lipid-anchored protein binds to the lipid bilayer via a lipid-added, especially prenylated or GPI-anchored amino acid residue. Examples include bacterial lipoproteins, G proteins and certain kinases.

[0100] An amphitropic protein is a protein that exists in at least two conformational states, a lipid-free aqueous state and a lipid-bound state. When associating with lipids, amphitropic proteins undergo a conformational change and can bind to the membrane reversibly or irreversibly. Examples of amphitropic proteins are membrane pore-forming toxins and antimicrobial peptides.

[0101] The hydrophobic agent for use in the method of the present invention can be obtained by various means. The hydrophobic agent for use in the method of the present invention can be of synthetic or natural origin. The hydrophobic agent for use in the method of the present invention can be provided in purified form or still in unpurified form, for example in the form of an unpurified membrane or an unpurified reaction mixture, in step a). This applies in particular when alternative (II) of the method of the present invention is used, which allows the use of step b.2) as an additional purification step for the hydrophobic agent. This can be achieved, for example, by carrying out a washing step between steps b.2) and c.2). The washing step used here can be limited to the removal of a part of the unbound composition provided in step a) and / or contacted with the support in step b.2).

[0102] If the hydrophobic agent is a hydrophobic biomolecule, the hydrophobic agent can be obtained by purification from natural sources. Purification strategies are known to those skilled in the art, and those skilled in the art can select an appropriate purification method according to the purification goal to be achieved. The properties of the hydrophobic biomolecule determine the purification method, which can include separation based on charge or hydrophobicity, such as in size exclusion chromatography, hydrophobic interaction chromatography or ion exchange chromatography, and / or affinity chromatography.

[0103] As used herein, the term "purified" is recognized in the art and does not represent a specific purity. "Purified" means that a biological molecule has been subjected to a purification method. Removal of any amount of unwanted molecules is considered purification.

[0104] Next to the saposin-like protein and at least one hydrophobic agent, the sariplo particles obtained in the method of the present invention also contain lipids as a third essential component. At least a part of the lipids to be incorporated into the sariplo particles is provided in step a) of the method of the present invention.

[0105] As used herein, the term "lipid" is recognized in the art and refers to natural or synthetic substances of biological origin, which are soluble or partially soluble in organic solvents or partition into hydrophobic environments when present in an aqueous phase. The term "lipid" as used herein does not mean a single type of lipid molecule in the saposin lipoprotein particles obtained by the methods of the present invention.

[0106] Salipro particles typically contain a mixture of lipids. In one embodiment, the lipids of the Salipro particles are a mixture that naturally occurs in the archaea, virus, prokaryote, eukaryotic cell or organelle membrane of eukaryotic cells from which the lipids are obtained. For example, the lipids can be brain lipids, lipids from heart extracts, lipids from liver extracts, lipids from yeast extracts or lipids from E. coli extracts. In another embodiment, the lipids can be of synthetic origin. In a further embodiment, the lipids can be of semi-synthetic origin, i.e., a mixture of synthetic and natural lipids is used and / or, for example, natural lipids are chemically modified to obtain fluorescently labeled lipids or lipids with modified heads, such as glycosylated lipids, functionalized lipids, pH-sensitive lipids or adhesive lipids. In one embodiment, the Salipro particles contain at least 3, 5, 10 or 20 different lipids.

[0107] In a preferred embodiment, the lipids comprise or consist of amphiphilic lipids. These can be selected from the group consisting of phospholipids, glycolipids, sterols and mixtures thereof.

[0108] Phospholipids have a polar part (phosphate "head") that dissolves in water and a hydrophobic non-polar part ("lipid tail") that does not dissolve in water. These parts are linked by a glycerol moiety. In water, phospholipids can form clusters where the heads face the water and the tails are away from the water. The fatty acid chains in phospholipids and glycolipids usually contain an even number of carbon atoms, typically 16 to 20 carbon atoms. Fatty acids with 16 and 18 carbon atoms are the most common. Fatty acids can be saturated or unsaturated. The configuration of the double bond is typically the so-called cis configuration. Cis and trans isomers are terms used in organic chemistry to represent the stereoisomers that occur in the relative orientation of functional groups within a molecule according to Cahn-Ingold-Prelog (CIP; Cahn, R.S. & Ingold, C.K.; Prelog, V., ''Specification of Molecular Chirality''. Angewandte Chemie International Edition, 5(4), p. 385-415, 1966). Typical fatty acids present in cell membranes are also described in Alberts et al., ''The Cell'', 4 th edition, Macmillian Magazines Ltd, 2002 on pages 61 and 62. Further examples of membrane lipids are phospholipids such as phosphatidylcholine such as POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), phosphatidylethanolamine and phosphatidylserine such as POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) phosphatidylinositol and sphingomyelin.

[0109] Glycolipids are lipids to which carbohydrates are attached by glycosidic bonds. Carbohydrates are typically found on the outer surface of eukaryotic cell membranes. The carbohydrates extend from the phospholipid bilayer into the extracellular aqueous environment. Examples of glycolipids are glyceroglycolipids, galactolipids, sulfolipids, sphingoglycolipids, glucocerebrosides, sulfatides, gangliosides, globosides, phosphosphingoglycolipids and glycosylphosphatidylinositol.

[0110] Sterols are a subgroup of steroids. Sterols as part of membranes are typically present in the membranes of eukaryotes such as plants, animals and fungi. Examples of sterols are cholesterol, campesterol, sitosterol, stigmasterol and ergosterol.

[0111] According to a preferred embodiment, the lipid is a lipid forming a lipid bilayer and / or a biocompatible lipid. As used herein, the term "biocompatible" means being biologically compatible by not causing toxicity, harmfulness, or an immunological response in living tissue. As used herein, "bilayer-forming lipid" refers to a lipid capable of forming a lipid bilayer having a hydrophobic interior and a hydrophilic exterior. Any bilayer-forming lipid that can associate with SAPLIP or its derivatives or cleavage forms and aggregate into a particulate structure can be used according to the present invention. Bilayer-forming lipids include, but are not limited to, phospholipids, sphingolipids, glycolipids, alkylphospholipids, ether lipids and plasmalogens. One type of bilayer-forming lipid or a mixture of two or more types can be used.

[0112] Lipids can include lipids that are not bilayer-forming lipids. Such lipids include, but are not limited to, cholesterol, cardiolipin, phosphatidylethanolamine (which can form bilayers under certain circumstances), oxysterols, phytosterols, ergosterol, sitosterol, cationic lipids, cerebrosides, sphingosine, ceramides, diacylglycerol, monoacylglycerol, triacylglycerol, gangliosides, ether lipids, alkyl phospholipids, plasmalogens, prostaglandins, and lysolipids.

[0113] Lipids can be, but are not limited to, mixtures of the lipids listed above. Typically, the lipids used in the methods of the present invention include at least phospholipids, glycolipids, cholesterol, and mixtures thereof. According to a preferred embodiment, the lipids are eukaryotic lipids and / or prokaryotic lipids such as are typically present in any one of the membranes present in eukaryotic or prokaryotic cells. Preferred lipids are, for example, phospholipids, sphingoglycolipids, sterols, phosphatidylcholine, phosphatidylserine (PS), 2- Ray oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 2- Ray oleoyl-1-palmitoyl (pamlitoyl)-sn-glycero-3-glycerol (POPG), 2- Ray oleoyl-1-palmitoyl (pamlitoyl)-sn-glycero-3-phosphoethanolamine (POPE), diacylglycerol, cholesterol, sphingomyelin, galactosylceramide, ganglioside, phosphatidylinositol, and sulfogalactosylceramide or combinations thereof.

[0114] In another embodiment, the lipid comprises a phospholipid. Examples of suitable phospholipids include, but are not limited to, DMPC, DMPG, POPC, dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine (DPPS), cardiolipin, dipalmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), egg yolk phosphatidylcholine (egg PC), soy phosphatidylcholine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and cationic lipids.

[0115] The lipids used in the methods of the invention are typically heterogeneous mixtures of lipids such as are found in cell or organelle membranes. However, it is also possible to further include synthetic or non-conventional lipids which are modified lipids containing one or more attached functional moieties such as targeting or bioactive moieties.

[0116] The lipids used in the methods of the invention can comprise, or consist of, naturally occurring lipids, synthetic lipids, modified lipids, fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and prenol lipids, or combinations thereof.

[0117] In a further embodiment, one or more of the hydrophobic agent, lipid, and / or saposin-like protein is in a solubilized state with a surfactant. In one embodiment, the hydrophobic agent is in a solubilized state with a surfactant. In another embodiment, the hydrophobic agent and lipid are in a solubilized state with a surfactant. Being in a solubilized state with a surfactant means that the surfactant renders each constituent soluble in, and / or maintains it in, an aqueous solution.

[0118] As used herein, the term "surfactant" is recognized in the art and is not included in the definition of "lipid" as used herein. While many surfactants have a similar amphiphilic general structure as compared to lipids, i.e., a polar hydrophilic head and a nonpolar hydrophobic tail, surfactants differ from lipids in the shape of the monomer, the type of aggregates formed in solution, and the concentration range required for aggregation. The structure of lipids is generally substantially cylindrical, with the volume occupied by the hydrophobic tail being similar to the volume occupied by the polar head. Surfactant monomers are generally more conical in shape, with the volume occupied by the hydrophobic tail being smaller than the volume occupied by the polar head. Surfactants tend to aggregate into spherical or ellipsoidal micelles that are water-soluble and do not form bilayer structures in the absence of lipids (see the handbook "Detergents and their uses in membrane protein science" by Anatrace, www.anatrace.com).

[0119] Surfactants that can be used can be anionic, cationic, nonionic, zwitterionic, and mixtures thereof.

[0120] A typical anionic surfactant is alkylbenzene sulfonate. The alkylbenzene moiety of these anions is lipophilic and the sulfonate is hydrophilic. Anionic surfactants can include, for example, branched or straight-chain alkyl groups. Examples of suitable anionic surfactants are bile acids such as deoxycholic acid (DOC), alkylbenzene sulfonates such as sodium branched dodecylbenzene sulfonate, sodium linear dodecylbenzene sulfonate, and mixtures thereof.

[0121] Cationic surfactants are similar to anionic surfactants having a hydrophobic component, but instead of an anionic sulfonate group, cationic surfactants have a quaternary ammonium as the polar terminus. The ammonium center is positively charged.

[0122] Nonionic surfactants are characterized by their uncharged hydrophilic heads. Typical nonionic surfactants are based on, for example, polyoxyethylene or glycosides. Common examples of the former include the Tween, Triton, Brij series, etc. These substances are also known as ethoxylates or PEGylates and their metabolite nonylphenol. Glycosides have sugar as their uncharged hydrophilic head. Examples include octylthioglucoside and maltoside. The glycoside head can also be of high molecular weight such as saponin. Saponin consists of a sugar moiety linked to a triterpene or steroid aglycone. Depending on the non-sugar part of the saponin molecule, saponin can be divided into triterpene glycosides, steroid glycosides and steroid alkaloid glycosides. HEGA and MEGA series surfactants have sugar alcohol as their head. Examples of suitable nonionic surfactants that can be used in the method according to the present invention are saponins such as escin, bacopaside, bacoside, chaconin, charantin, digitonin, glycyrrhizin, ginsenoside, holothurin, protodioscin and solanine and mixtures thereof. Synthetic surfactants structurally similar to saponins such as glycol-diosgenin are also suitable for use in the method according to the present invention. Further examples of suitable nonionic surfactants that can be used in the method according to the present invention are alkyl glycosides such as short-chain, medium-chain or long-chain alkyl maltosides such as n-dodecyl-β-maltoside (DDM), decanoyl-N-hydroxyethyl glucamide (HEGA), n-decanoyl-N-methyl-D-glucamide (MEGA), and mixtures thereof.

[0123] Particularly preferred surfactants according to the present invention are selected from the class of saponins, particularly digitonin. Actual experiments with digitonin have shown that when digitonin is used as a surfactant, the method of the present invention is more efficient with respect to the production of sariplo particles compared to the absence of a surfactant or the use of other types of surfactants.

[0124] Amphoteric surfactants have a net zero charge resulting from the presence of equal numbers of oppositely charged chemical groups, i.e., the total number of negative and positive charges is equal and the overall charge is net zero. Examples include Fos-choline, CHAPS / CHAPSO, lauryl-dimethylamine-oxide (LDAO), and mixtures thereof.

[0125] In one embodiment of the present invention, the surfactant is selected from the group consisting of alkylbenzene sulfonates or bile acids, cationic surfactants, and nonionic or amphoteric surfactants such as lauryl-dimethylamine oxide (LDAO), Fos-choline, CHAPS / CHAPSO, saponins such as digitonin and structurally related synthetic surfactants such as glycol-diosgenin, alkyl glycosides such as short-chain, medium-chain or long-chain alkyl maltosides, particularly n-dodecyl β-D-maltoside, glucosides, maltose-neopentyl glycol (MNG) amphiphilic substances, amphiphilic polymers (amphipols), styrene maleic acid copolymers (SMA), macrocyclic molecules or cyclic oligomers (calixarenes) based on hydroxyalkylated products of phenols and aldehydes, and mixtures thereof.

[0126] When the hydrophobic agent used in the method of the present invention is solubilized in a surfactant, it is advantageous for the surfactant to have a short-chain to medium-chain hydrophobic tail. This is particularly true when membrane proteins are incorporated as hydrophobic agents. As used herein, "short-chain hydrophobic tail" means C2-C9, such as in n-nonyl-β-maltoside (NM), and "medium-chain hydrophobic tail" as used herein means C10-C15, such as in n-decyl-β-maltoside (DM) or n-dodecyl-β-maltoside (DDM). In one embodiment, the surfactant has 2 to 12 carbon atoms, preferably 2 to 10, most preferably 2 to 9 carbon atoms in its hydrophobic tail.

[0127] Actual experiments have shown that generally, the saposin-like proteins do not require surfactants or other solvents during purification, storage or handling. However, optionally, the saposin-like proteins may also be in a solubilized state with a surfactant.

[0128] When the hydrophobic agent is in the form of a hydrophobic biomolecule solubilized with a surfactant, the hydrophobic agent may further form a complex with a lipid, particularly a cyclic lipid. The cyclic lipid, also referred to herein as "shell lipid" or "boundary lipid", represents a selected group of lipids that preferentially bind or adhere to the surface of the hydrophobic biomolecule during purification from the membrane. The cyclic lipid constitutes a layer or ring or shell of highly immobilized lipids due to the presence of strong lipid-protein binding interactions.

[0129] In one embodiment, the surfactant used to solubilize the hydrophobic agent, lipid and / or saposin-like protein is not carried over in substantial amounts into the salipro particles formed in the method of the present invention. In particular, the amount of surfactant in the particles obtainable by the method according to the present invention can be low to undetectable. In one embodiment, the obtained salipro particles do not contain a substantial amount of surfactant, particularly less than 0.1% by weight, preferably less than 0.01% by weight, particularly preferably less than 0.001% by weight of surfactant, based on the weight of the particles. The amount of surfactant (or other constituent) present in the particles can be determined, for example, by mass spectrometry.

[0130] In another embodiment, the hydrophobic agent and lipid are provided in step a) in the form of a biological membrane comprising the hydrophobic agent and lipid to be incorporated into the saposin lipoprotein particles. The biological membrane can be a membrane from a virus, archaea, eukaryote or prokaryote, i.e., a membrane derived from a cell or organelle of a virus, archaea, eukaryote or prokaryote. The biological membrane can be used in the method of the present invention in the form of an untreated, amphiphilic agent-treated or lysed cell, virus or organelle. In particular, the biological membrane can be provided in the form of a cell, virus or organelle contacted with a surfactant. The membrane can contain hydrophobic agents and lipids that are to be naturally incorporated into the saposin lipoprotein particles. However, the membrane can also be derived from a virus, archaea, eukaryote or prokaryote cell or organelle engineered to express or contain a hydrophobic agent and / or lipid in its membrane. For example, if the hydrophobic agent is a hydrophobic protein, the membrane can be obtained from a cell expressing the hydrophobic protein as a transgene. The hydrophobic protein can be expressed, in particular, in a genetically modified (e.g., tagged) form. If the hydrophobic agent is a hydrophobic drug, the membrane can be derived from a cell exposed to the hydrophobic drug. If the drug is sufficiently lipophilic, this results in incorporation of the drug into the membrane, which can be readily tested by extracting the membrane fraction and testing for the presence of the drug.

[0131] The membrane that can be used in the method of the present invention is preferably selected from the membranes of cells or organelles of viruses, archaea, eukaryotes or prokaryotes. The term "membrane" refers to any membrane containing a lipid layer. Preferably, the membrane is a lipid bilayer. The term "membrane" may be used interchangeably herein with "cell membrane" and / or "organelle membrane".

[0132] When the membranes are provided in the form of cells, these can be selected from prokaryotic cells, eukaryotic cells and archaebacterial cells. Preferably, the cells are eukaryotic cells, in particular non-human animal or human cells. Said cells can be obtained from cell cultures, but can also be obtained from natural sources such as tissue samples, biopsy samples and other biological materials.

[0133] The term "cell membrane" refers to the biological membrane that separates the interior of the cell from the external environment. The complex structures and multiple components contained in the cell membrane, such as membrane lipids and membrane proteins, are also described in detail in Alberts et al., ’’The Cell’’, 4 th edition, Macmillian Magazines Ltd, 2002, pp. 583 - 614 and Campbell et al., ’’Biologie’’, 6 th edition, Spektrum Verlag, 2003, pp. 163 - 177.

[0134] In one embodiment, the saposin-like particles obtained by the method of the present invention consist essentially of a saposin-like protein and a membrane component obtained from the membrane of a virus, cell or organelle.

[0135] Hydrophobic agents and lipids can be provided in step a) in the form of an unpurified membrane comprising the hydrophobic agents and lipids to be incorporated into the saposin lipoprotein particles. As used herein, an "unpurified membrane" refers to a membrane that is no longer completely untreated but still contains an essentially native membrane composition, particularly with respect to membrane lipids and membrane proteins. The unpurified membrane can be an unpurified cell membrane, an unpurified organelle membrane, or a part thereof, each derived from an archaeal cell, a prokaryotic cell, or a eukaryotic cell. The unpurified membrane can also be an unpurified viral envelope membrane derived from an enveloped virus. For example, an unpurified membrane fraction obtained after cell disruption or lysis of a cell or organelle (of archaeal, eukaryotic, or prokaryotic origin) is an "unpurified cell or organelle membrane". After disruption of the viral envelope, an unpurified viral membrane fraction can be obtained. An "unpurified viral, cell, or organelle membrane" necessarily contains the native membrane components present in the viral envelope, cells, and organelles. In particular, an "unpurified viral, cell, or organelle membrane" contains both membrane lipids and membrane proteins. Cell disruption or lysis can occur not only by mechanical means but also by contacting the cells with an amphiphilic agent, particularly a surfactant.

[0136] The unpurified cell membrane, unpurified organelle membrane, or unpurified viral membrane is no longer a completely untreated cell membrane, organelle membrane, or viral envelope. They spontaneously form unpurified membrane vesicles due to hydrophobic interactions between two given membrane rupture sites.

[0137] Hydrophobic agents and lipids can be provided in step a) in the form of a membrane still contained in untreated, amphiphilic agent-treated, or lysed cells, viruses, or organelles. Thus, cells, viruses, or organelles can be used directly in step a). They can be untreated, pretreated with an amphiphilic agent, or lysed. In particular, hydrophobic agents and lipids can be provided in step a) in the form of cells, viruses, or organelles that have been contacted with an amphiphilic agent, particularly a surfactant.

[0138] Membranes particularly suitable for use in the method of the present invention can be obtained directly from any natural cell, virus or organelle by simply treating them with an amphiphilic agent, in particular a surfactant. Preferably, the cells contacted with the amphiphilic agent are eukaryotic cells, in particular non-human animal or human cells. Eukaryotic cells treated with an amphiphilic agent can be, for example, neoplastic cells or cancer / tumor cells. Cells contacted with an amphiphilic agent in the method of the present invention can also be organized, for example, as tissues or solid tumors.

[0139] Treatment of cells, viruses or organelles with an amphiphilic agent is typically carried out in a liquid environment. Treatment with an amphiphilic agent, particularly when the amphiphilic agent is a surfactant, appears to make the membrane structure of cells, viruses or organelles more fluid and has a dissociating effect. Without wishing to be bound by this scientific theory, amphiphilic agents, particularly surfactants, also appear to activate saposin proteins, perhaps by inducing an open conformation. In a preferred embodiment, no further treatment of the cells, viruses or organelles is carried out in addition to treatment with the amphiphilic agent, in particular no further treatment is carried out to achieve disruption of the cells, viruses or organelles, such as by chemical and / or mechanical treatment. Suitable chemical and / or mechanical treatments for achieving disruption of cells, viruses or organelles are known to those skilled in the art.

[0140] In a preferred embodiment, treatment with a surfactant in a liquid environment involves contacting a cell, virus or organelle with a surfactant at a concentration that is from 0.01 to 2 million times the CMC of the surfactant, preferably from 0.1 to 20,000 times the CMC of the surfactant, and most preferably from 1 to 2000 times the CMC of the surfactant. In many embodiments, the best results are achieved when the surfactant concentration in the liquid environment exceeds the CMC of the surfactant. Apparently, surfactant concentrations below the CMC also already have a positive effect, perhaps by the surfactant actually activating saposin without solubilizing the membrane. The critical micelle concentration (CMC) is defined as the concentration of surfactant at which micelles form and all additional surfactant molecules added to the system are incorporated into the micelles. Typically, a cell, virus or organelle is incubated with the amphiphilic agent for 0.5 minutes to 180 minutes, preferably 30 minutes to 90 minutes, especially when the amphiphilic agent is a surfactant. Further, the incubation with the surfactant is typically carried out at a temperature of 1 to 37 °C, preferably 2 to 6 °C.

[0141] Step b.1) / b.2) of the method of the present invention In step b.1) / b.2) of the method of the present invention, either a saposin-like protein (step b.1) or a hydrophobic agent (step b.2) is contacted with a support that can selectively bind the hydrophobic agent or the saposin-like protein. In step b.2) of the method of the present invention, the hydrophobic agent is contacted with a support that can selectively bind the hydrophobic agent to the support. Alternatively, in step b.1) of the method of the present invention, the saposin-like protein is contacted with a support that can selectively bind to the saposin-like protein.

[0142] The hydrophobic agent in step b.2) or the saposin-like protein in step b.1) is preferably brought into contact with the support in a liquid environment, i.e., in a contacting state. Preferably, the liquid environment is an aqueous liquid environment. In certain embodiments, the aqueous liquid environment is a buffer solution having a pH of 2.0 to 10.0, 5.0 to 10.0, or 5.0 to 8.5, particularly 6.0 to 8.0, and most preferably 7.0 to 8.0.

[0143] As used herein, the term "support" refers to any support material capable of selectively binding a target molecule of interest. In the case of the support used in step b.1) of the method (Alternative I) of the present invention, the target molecule is a saposin-like protein. In the case of the support used in step b.2) of the method (Alternative II) of the present invention, the target molecule is a hydrophobic agent. The molecular structure in the support that binds the target molecule is referred to herein as the "capture moiety". The molecular structure in the target molecule bound by the support is referred to herein as the "binding moiety". Thus, the support comprises, or is functionalized to comprise, a capture moiety that binds the corresponding binding moiety present in the target molecule of interest, i.e., the saposin-like protein (step b.1) or the hydrophobic agent (step b.2).

[0144] The support used herein can in particular be a "carrier", "carrier material" or "stationary phase" comprising such a capture moiety. In a preferred embodiment, the support is a solid support.

[0145] The support can be in the form of beads, beds, membranes or solid supports having planar, curved, kinked, twisted and / or angled surfaces.

[0146] In one embodiment, the support is in the form of beads. Particularly good results are achieved by beads used as chromatographic supports in the prior art, for example in bioaffinity chromatography. These beads are most commonly made of materials based on polysaccharides such as agarose, cellulose, lignocellulose and dextran, or other polymers such as polyethylene oxide, which can be crosslinked. For use in the method of the present invention, the bead material comprises or is functionalized to comprise a capture moiety that binds to the corresponding binding moiety present in the target molecule of interest, i.e., the saposin-like protein (step b.1) or the hydrophobic agent (step b.2). The beads used in the method of the present invention, such as affinity beads, can have an average diameter in the range of 1 to 900 μm or 10 to 500 μm. In one embodiment, the diameter is less than 500 μm, less than 250 μm, or less than 100 μm. Preferably, the beads used in the method of the present invention have an accessible pore structure. The support can also form a gel due to the hydration of the solid. These porous materials or gels are preferred because they have a large amount of binding sites available per volume of material. A large number of binding sites provide greater binding, reactivity and / or separation capabilities. In another embodiment, the beads are magnetic, allowing for facile attachment to other surfaces of the support. Magnetic beads are typically smaller than standard affinity beads. Thus, the beads according to the present invention can also have an average diameter in the range of 600 nm to 10 μm, 400 nm to 7 μm or 200 nm to 5 μm. In another embodiment, the diameter of the magnetic beads is less than 7 μm, less than 4 μm, or less than 1 μm.

[0147] In one embodiment, the support is in the form of a bed. The bed can be formed by beads or fibers. However, the bed may be a continuous structure such that the bed comprises a single piece of material with pores traversing it. Examples of continuous beds are beads or fibers crosslinked by covalent bonds. The bed can be a chromatography bed well-known to those skilled in the field of protein and lipid chromatography. The bed can be based on materials made of polysaccharides such as agarose, cellulose, lignocellulose and dextran, or other polymers such as polyethylene oxide, which can be crosslinked. For use in the method of the present invention, the bed material comprises, or is functionalized to comprise, a capture moiety that binds to a corresponding binding moiety present in the target molecule of interest, i.e., the saposin-like protein (step b.1) or the hydrophobic agent (step b.2).

[0148] In one embodiment, the support is in the form of a membrane. The membrane is a non-biological membrane that does not consist of typical biological membrane components such as lipids and / or membrane proteins. Preferably, a chromatography membrane well-known to those skilled in the field of protein and lipid membrane chromatography is used. The membrane can be based on materials made of polysaccharides such as agarose, cellulose, lignocellulose and dextran, or other polymers such as polyethylene oxide, which can be crosslinked. For use in the method of the present invention, the membrane comprises, or is functionalized to comprise, a capture moiety that binds to a corresponding binding moiety present in the target molecule of interest, i.e., the saposin-like protein (step b.1) or the hydrophobic agent (step b.2).

[0149] In one embodiment, the support is a solid support having a plane. As used herein, "plane" means "substantially planar" in the sense that the surface is macroscopically substantially planar. This means that the microstructure can include non-planar structures and that the macrostructure can include regions that are not perfectly planar. In one embodiment, bent or inclined structures are also considered to be substantially planar in the sense of the present invention if they have a large surface area. The term "solid support having a plane" serves to distinguish solid substantially planar supports such as chips or surface planes from the bead embodiments described above. The term "solid" as used herein does not mean including gels. In one embodiment, a solid is substantially rigid and has no flexibility. In another embodiment, the solid support having a plane includes a non-porous surface.

[0150] Examples of solid supports having a plane that can be used in the method of the present invention are chips or biosensor surfaces. In one embodiment, the plane of the solid support is made of metal. In particular, the metal can be selected from the group consisting of gold, silver, copper, aluminum, and mixtures thereof. In another embodiment, the surface is made of glass and / or synthetic resin, i.e., a polymeric material. For use in the method of the present invention, the plane of the solid support includes or is functionalized to include a capture moiety that binds to a corresponding binding moiety present in the target molecule of interest, i.e., the saposin-like protein (step b.1) or the hydrophobic agent (step b.2). In one embodiment, the dimensions of the solid support having a plane are in the range of 1 to 50 mm 2 and particularly 5 to 25 mm 2 in range.

[0151] The support includes a capture moiety that enables selective binding of the target molecule of interest via a binding moiety in the target molecule. In the case of the support used in step b.1) of the method of the present invention (Alternative I), the target molecule is a saposin-like protein. In the case of the support used in step b.2) of the method of the present invention (Alternative II), the target molecule is a hydrophobic agent.

[0152] The terms "capture moiety" or "binding moiety" are used herein to mean "at least one capture moiety" and "at least one binding moiety". The support used in the method of the present invention typically comprises a plurality of target moieties which may be the same or different. Typically, the support comprises a plurality of capture moieties of the same kind. The target molecule can comprise one or more binding moieties. For example, where the binding is based on an affinity interaction, the target molecule may comprise one or more copies of an affinity tag. Where the binding is based on a hydrophobic interaction, the target molecule may comprise one or more hydrophobic binding sites or the like.

[0153] The target molecule comprises a binding moiety that is selectively bound by the capture moiety of the support. The term "binding moiety" refers to any molecular structure in the target molecule that enables selective binding to the corresponding capture moiety of the support, i.e., a hydrophobic agent or a saposin-like protein according to the method of the present invention. The binding moiety of the target molecule and the capture moiety of the support form a complementary interaction pair. The terms "capture moiety" and "binding moiety" are collectively referred to herein as "recognition moiety".

[0154] Thus, in alternative (I) of the method of the present invention, the support comprises a capture moiety, the saposin-like protein comprises a binding moiety, and the capture moiety can selectively bind the binding moiety in the saposin-like protein. In alternative (II) of the method of the present invention, the support comprises a capture moiety, the hydrophobic agent comprises a binding moiety, and the capture moiety can selectively bind the binding moiety in the hydrophobic agent. With respect to binding to the support, "hydrophobic agent" and "saposin-like protein" are collectively referred to herein as "target molecule".

[0155] As used herein, the term "bind" can mean "immobilize", "capture", "fix", "bind" or "retain" a target molecule on a support. The term "selectively" indicates that the support preferentially, mainly and / or almost exclusively binds to the target molecule, i.e., a hydrophobic agent or a saposin-like protein. Selective binding can be achieved by electrostatic interactions, hydrophobic interactions, affinity binding and / or covalent bond formation between the capture moiety of the support and the binding moiety of the target molecule. Usually, the binding is reversible. However, in one embodiment, the binding to the support is substantially irreversible. In another embodiment, the binding to the support is by a covalent bond.

[0156] As a general note, whenever a suitable pair of capture moiety / binding moiety is referred to herein, the reverse pair is also considered to be described. In other words, in many cases, the capture moiety on the support and the binding moiety in the target molecule can be used interchangeably.

[0157] The binding can include a linker or spacer that connects the binding moiety and the capture moiety. The linker or spacer can be a bifunctional molecule that reacts with or binds to both the binding moiety and the capture moiety, or the linker or spacer can be included within the binding moiety or the capture moiety.

[0158] The capture moiety of the support can enable selective binding of the target molecule by various modes that will be described in more detail below. For example: (i) capturing the target molecule by chemically bonding the binding moiety of the target molecule to the capture moiety of the support by a chemical bond; (ii) capturing the target molecule via an interaction based on the affinity between the capture moiety of the support and the natural or engineered binding moiety in the target molecule; (iii) indirectly binding the target molecule by engineering the capture moiety in the support to bind to a crosslinking agent (e.g., a peptide epitope, a substrate analog, or a ligand) that is selectively bound by the binding moiety in the target molecule; (iv) capturing the target molecule via a hydrophobic interaction between the capture moiety of the support and the natural or engineered binding moiety in the target molecule; and / or (v) capturing the target molecule via a charge-based interaction between the capture moiety of the support and the natural or engineered binding moiety in the target molecule.

[0159] In one embodiment, the selective binding of the target molecule to the support in steps b.1) / b.2) is achieved by chemically bonding the binding moiety of the target molecule to the capture moiety of the support by a chemical bond. This can be achieved by utilizing functional groups in the target molecule such as thiol groups, amino groups, hydroxyl groups, aldehyde groups or carboxyl groups. Suitable capture moieties of the support for reacting with such binding moieties of the target molecule are known to those skilled in the art. Those skilled in the art can select an appropriate pair of capture moiety / binding moiety according to the type of bond to be achieved. The thiol group in the target molecule can be reacted with, for example, maleimide, disulfide or iodoacetamide used as a capture moiety on the support, or vice versa. The primary amine in the target molecule can be reacted with, for example, a succinimide ester (such as N-hydroxysuccinimide, sulfosuccinimide or other succinimidyl esters), imido ester or isothiocyanate (such as phenyl isothiocyanate) used as a capture moiety on the support, or vice versa. The carboxyl group of the target molecule can be reacted with, for example, a carbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) used as a capture moiety on the support, or vice versa.

[0160] Depending on the chemistry selected, the binding of the target molecule to the support via the formation of a chemical bond can be substantially irreversible, which can be beneficial for certain applications that require a particularly tight binding of the formed sariplo particles to the support. On the other hand, capture by affinity-based recognition moieties, hydrophobic interactions or electrostatic interactions is usually reversible and has the advantages of easy elution in step d) and the possibility of regeneration and reuse of the support.

[0161] In another embodiment, the selective binding of the target molecule to the support in steps b.1) / b.2) is achieved by an interaction based on the affinity between the capture moiety of the support and the natural or engineered binding moiety in the target molecule. In this way, a target molecule having a natural or engineered binding moiety is selectively bound by the capture moiety of the support using the affinity-based interaction.

[0162] Examples of pairs based on the affinity of natural capture / binding moieties are lectins that bind to specific sugar moieties in the target molecule (e.g., when the target molecule is a glycosylated membrane protein), or antibodies that recognize the natural epitope of the target molecule (or vice versa). Post-translational modifications such as glycosylation, sulfation, palmitoylation, myristoylation, ubiquitination or SUMOylation can also serve as binding moieties according to the present invention. Various other affinity-based interactions between the capture moiety and the binding moiety are known to those skilled in the art.

[0163] A particular important example of an engineered affinity-based capture / binding moiety pair useful in the method of the present invention is derived from well-known regions of affinity tags. Thus, in one embodiment, the capture moiety or the binding moiety is an affinity tag, and the corresponding other recognition moiety is a moiety having a high affinity for the affinity tag. For example, protein A-Sepharose resin can be used as the support together with a hydrophobic agent-antibody fusion (in this case, the Fc portion of the antibody is the binding moiety in the target molecule) (in this case, protein A is the affinity tag on the support). When a hydrophobic agent-antibody complex is used, the antibody acts as a linker as described above. Preferably, the binding moiety in the target molecule is an affinity tag. For example, together with a target molecule with a His tag added (in this case, the His tag is the affinity tag and the binding moiety in the target molecule), Ni 2+ -NTA resin can be used as the support (in this case, Ni 2+ -NTA is the capture moiety).

[0164] Affinity tags have the advantage of being easily introduced, for example, by standard cloning or genetic engineering, and binding to capture moiety-containing supports that are well-known and often commercially available. The saposin-like protein used in step b.1) or the hydrophobic agent (when the hydrophobic agent is a hydrophobic protein) used in step b.2) can be in a form with an added affinity tag. This can be easily obtained by introducing an affinity tag in the form of a peptide or polypeptide into the target molecule by cloning or genetic engineering. Thus, in this embodiment, the hydrophobic agent (when the hydrophobic agent is a hydrophobic protein) and / or the saposin-like protein are each used as a fusion protein with an added affinity tag in steps b.2) and b.1), respectively. The term "fusion protein" is recognized in the art and refers to a protein fused to another peptide or polypeptide by recombinant DNA technology. The term "fusion protein" may be used interchangeably herein with "chimeric protein".

[0165] The interaction between the affinity tag and the corresponding capture moiety was originally developed to assist in the purification and immobilization of proteins. Protein target molecules can be modified at the genetic level by a specific peptide sequence known as an affinity tag that binds to a known capture moiety. Affinity tags used herein are generally classified into three categories: a) peptide sequences that bind to small molecules; b) proteins that bind to small molecules; c) peptides or proteins that bind to antibodies. An affinity tag can also be a small molecule compound (e.g., a ligand) having an appropriate binding partner. An affinity tag can be covalently attached to the target molecule used in the method of the present invention. For example, nitrilotriacetic acid forms a complex (Ni 2+ -NTA) with Ni 2+ and is a common capture moiety that binds to proteins modified with a run of histidines that define an affinity tag that can be used as a binding moiety in the target molecule.

[0166] The term "affinity tag" is given its ordinary meaning in the art. An affinity tag is any biological or chemical material that can be readily attached to a target biological or chemical molecule. An affinity tag can be attached to a target biological or chemical molecule by any suitable method. For example, in some embodiments, an affinity tag can be attached to a target molecule using genetic methods. For example, a nucleic acid sequence encoding an affinity tag can be placed at any location within a nucleic acid that enables the affinity tag to be expressed with a biological molecule, such as an internal, adjacent, or nearby location. In other embodiments, an affinity tag can be attached to a target biological or chemical molecule even after the target biological or chemical molecule has been produced (e.g., expressed or synthesized). As an example, to facilitate binding to a target streptavidin, an affinity tag such as biotin can be chemically bound to a target protein or peptide, e.g., by a covalent bond.

[0167] Examples of affinity tags include metal-binding tags such as a histidine tag, GST (in glutathione / GST binding), streptavidin (in biotin / streptavidin binding), or maltose (which binds to MBP, i.e., maltose-binding protein). Other affinity tags include Myc or Max in the Myc / Max pair, or polyamino acids such as polyhistidine. Specific affinity tags are described at various places herein in connection with binding interactions. Generally, the molecular structure in a support with which an affinity tag, which is its known biological or chemical binding partner, interacts (e.g., selectively binds) is the "capture moiety" as used herein.

[0168] Pairs of affinity tags or antigens (used as binding moieties) / capture moieties useful in steps b.1) / b.2) of the method of the present invention are, for example, polyhistidine / NTA / Ni2+, glutathione S-transferase / glutathione, maltose-binding protein / maltose, streptavidin / biotin, biotin / streptavidin, antigen (or antigen fragment) / antibody (or antibody fragment), and the like.

[0169] Further pairs of affinity tags or antigens (used as binding moieties) / capture moieties useful in steps b.1) / b.2) of the method of the present invention are, for example, antibody / peptide interaction, antibody / antigen interaction, antibody fragment / antigen interaction, nucleic acid / nucleic acid interaction, protein / nucleic acid interaction, peptide / peptide interaction, protein / protein interaction, small molecule / protein interaction, glutathione / GST interaction, maltose / maltose-binding protein interaction, carbohydrate / protein interaction, carbohydrate derivative protein interaction, peptide tag / metal ion-metal chelate interaction, peptide / NTA-Ni interaction, epitope tag (e.g., V5 tag, Myc tag, FLAG tag or HA tag) / antibody interaction, protein A / antibody interaction, protein G / antibody interaction, protein L / antibody interaction, fluorescent protein (e.g., GFP) / antibody interaction Fc receptor / antibody interaction, biotin / avidin interaction, biotin / streptavidin interaction, zinc finger / nucleic acid interaction, small molecule / peptide interaction, small molecule / target interaction, and metal ion / chelating agent / polyamino acid interaction.

[0170] Any of the above affinity tags can be present on a hydrophobic agent or saposin-like protein that selectively binds to the support in steps b.1) / b.2) of the method of the present invention. The reverse can also be true, i.e., the affinity tag can be present on the support and on the respective capture moiety of the target molecule or optionally on an intermediate linker. Any of the above interaction pairs based on affinity can be applied to the method of the present invention.

[0171] In another embodiment, the affinity tag of the target molecule is a fluorescent tag. Such fluorescent tags are useful for detecting and tracking specific hydrophobic agents or specific types (species) of saposin particles during the generation of saposin particles in the method of the present invention. GFP and its variants are examples of commonly used fluorescent tags.

[0172] The inventors' research has revealed that in the method of the present invention, it is also possible to use specifically tagged membrane proteins present in the crude membrane as hydrophobic agents. This is possible by adding tags to the membrane proteins of interest in the underlying cells or viruses from which the crude membrane is derived. The crude membrane containing the tagged hydrophobic protein can then be used in step a) of the method of the present invention to provide the hydrophobic protein and lipid. Tagging is preferably done at the genetic level, for example, by genetic manipulation or by inserting a vector with a tagged transgene into the virus, cell, or organelle from which the crude membrane vesicles are obtained.

[0173] In another embodiment, the selective binding of the target molecule to the support in steps b.1) / b.2) is achieved by indirect binding of the target molecule to the capture portion of the support via the binding portion of the target molecule. This indirect binding of the target molecule can be achieved by engineering the capture portion of the support to bind to a cross-linking agent (e.g., a peptide epitope, substrate analog, or ligand) that is selectively bound by the binding portion in the target molecule. Thus, secondary interactions between the captured cross-linking agents can be used to selectively bind the saposin-like protein or hydrophobic agent to the support. Examples of cross-linking agents are enzyme cofactors, substrate analogs or inhibitors, ligands, or peptide epitopes. These cross-linking agents can bind to the capture portion of the support, where the target molecule can be selectively bound. For example, when an enzyme cofactor, substrate analog, or inhibitor is used as a cross-linking agent, the respective enzyme is the target molecule, and each binding pocket in the enzyme constitutes the binding portion.

[0174] In a further embodiment, the selective binding of the target molecule to the support in step b.1) / b.2) captures the target molecule by means of hydrophobic interactions between the capture moiety of the support and the natural or engineered binding moiety in the target molecule, and / or by means of charge-based interactions between the capture moiety of the support and the natural or engineered binding moiety in the target molecule. Capture moieties suitable for mediating such interactions are known to those skilled in the art. When hydrophobic interactions are used to selectively bind the target molecule to the support, the support contains, for example, as a capture moiety, an aromatic group, C8-C 24 alkyl, C8-C 24 alkenyl or C8-C 24 hydrophobic groups such as fatty acids. When electrostatic interactions are used to selectively bind the target molecule to the support, suitable anion exchange moieties and cation exchange moieties can be used as capture moieties on the support. In particular, commercially available anion or cation exchange resins can be used as the support. Functional groups commonly used in anion exchangers include, for example, quaternary ammonium, diethylaminopropyl or diethylaminoethyl. Functional groups commonly used in cation exchangers include, for example, sulfonic acid, methylsulfonate or carboxymethyl.

[0175] Step c.1) / c.2) of the method of the present invention In step c.1) / c.2) of the method of the present invention, the self-assembly of the saposin lipoprotein particles occurs by contacting the target molecule (hydrophobic agent or saposin-like protein) bound to the support with the remaining components of the saposin lipoprotein particles.

[0176] In the case of alternative (I) of the method of the present invention, step c.1) requires contacting the supposin-like protein bound to the support with a hydrophobic agent in order to enable self-assembly of the supposin lipoprotein particles. Without wishing to be bound by this theory, it is highly likely that adjacent captured supposin-like proteins contribute to the formation of individual sarpol particles. Further, the support can be present in the form of a very dynamic and dense 3D structure such that the interaction of adjacent captured supposin-like proteins is promoted. In a preferred embodiment, an additional supposin-like protein is added before, during or after step c.1) in order to further assist in the self-assembly into supposin lipoprotein particles.

[0177] In the case of alternative (II) of the method of the present invention, step c.2) requires contacting the hydrophobic agent bound to the support with a supposin-like protein in order to enable self-assembly of the supposin lipoprotein particles.

[0178] Preferably, the self-assembly of the particles in step c.1) and / or c.2) is carried out at a pH of 2.0 to 10.0, particularly 6.0 to 10.0, preferably 6.0 to 9.0, particularly preferably 7.0 to 9.0, and most preferably 7.0 to 8.0. Preferably, the contact in step c.1) / c.2) is carried out in a liquid environment. Preferably, the liquid environment is an aqueous liquid environment. In certain embodiments, the aqueous liquid environment is a buffer solution having a pH of 2.0 to 10.0, 5.0 to 10.0 or 5.0 to 8.5, particularly 6.0 to 8.0, and most preferably 7.0 to 8.0.

[0179] Any material that remains unbound after contacting the particle components bound to the support in step c.1) / c.2) can be recycled from the liquid environment, stored and reused for another use.

[0180] It is a remarkable and surprising property of the saposin-like protein that it can self-assemble hydrophobic agents into sariplo particles simply by being incubated with lipids and hydrophobic agents. The research underlying the present invention has surprisingly revealed that this self-assembly functions extremely efficiently when either one of the primary components, namely, the saposin-like protein (method alternative I) or the hydrophobic agent itself (method alternative II), is bound and immobilized to a support. The self-assembly then occurs under more difficult conditions at the interface between the support and the liquid environment containing the remaining components.

[0181] The inventors' research has shown that the particles obtained by the method of the present invention are disc-shaped like prior art sariplo particles formed purely in a liquid environment based on the free and random movement of all the participating particle components in the liquid environment. In a preferred embodiment, the assembled sariplo particles are disc-shaped. In another preferred embodiment, the sariplo particles do not contain an aqueous or hydrophilic core. In yet another embodiment, the sariplo particles are disc-shaped and do not contain an aqueous or hydrophilic core.

[0182] In a preferred embodiment, the salipro particles are generally considered to be disc-shaped. In particular, the salipro particles can have a Stokes radius (hydrodynamic radius) RS in the range of 2 nm to 500 nm, particularly 2 nm to 200 nm or 3 nm to 150 nm, preferably 3 nm to 100 nm. The method for determining the Stokes radius is known to those skilled in the art. This is preferably done by eluting the particles from a support and subjecting the particles to analytical gel filtration (size exclusion chromatography) in comparison with a standard of known Stokes radius. In particular, the particles can be subjected to a gel filtration step on, for example, a Superdex 200 HR10 30 gel filtration column and eluted with a suitable buffer at room temperature at pH 7.5 and 0.5 ml / min. For proteins, the absorbance is monitored at 280 nm. The column is calibrated using a mixture of protein standards of known Stokes radius such as, for example, thyroglobulin 669 kDa (RS = 8.5 nm), ferritin 440 kDa (RS = 6.1 nm), catalase 232 kDa (RS = 4.6 nm), lactate dehydrogenase 140 kDa (RS = 4.1 nm), bovine serum albumin 66 kDa (RS = 3.55 nm) and horse heart cytochrome c 12.4 kDa (RS = 1.8 nm). The standard proteins should span RS values above and below the Rs value of the particle of interest. The calibration curve is created by plotting the elution positions of the standard proteins against RS. This generally gives a more or less linear plot, but if not, it is sufficient to draw a line between the points and read the Rs of the protein of interest from its elution position on this standard curve.

[0183] In some embodiments, for example, when bulky hydrophobic agents such as membrane proteins or a larger amount of lipids are present in the particles, the Stokes radius is greater than 3.2 nm, particularly at least 3.5 nm, at least 5.0 nm or at least 10.0 nm.

[0184] SALipro particles can be examined by transmission electron microscopy either in a state bound to a support or in a "free" state. If the particles are large enough, they can be analyzed by negative stain electron microscopy and single particle analysis.

[0185] Structural analysis has often shown that in SALipro particles, the membrane lipids aggregate into disc-shaped bilayer-like structures of distinct sizes inside the particles. The saposin-like protein components generally delineate the boundaries of the disc-shaped bilayer and confer structure and stability to the particles. In most embodiments, the interior of the particles contains a hydrophobic region (e.g., composed of lipid aliphatic acyl chains). In contrast to liposomes, SALipro particles usually do not contain a hydrophilic core or an aqueous core. The particles are preferably disc-shaped and have a flat disc-shaped nearly circular lipid bilayer circumscribed by an amphipathic α-helix provided by two or more saposin-like proteins that associate with the hydrophobic surface of the bilayer at the periphery of the disc.

[0186] In some embodiments, depending on the size of the hydrophobic agent incorporated into the SALipro particles, the main disc shape of the (empty) SALipro particles can be approximated by a square, a triangle, or a cylinder. For example, SALipro particles approximated by a cylinder can have a ratio of maximum height to maximum diameter (major axis length) of at least 1.0:1.1, particularly 1.0:1.5, 1.0:2.0, 1.0:4.00, 1.0:8.00, or 1.0:9.00. The maximum height of the disc-shaped particles is generally at least 3.5 nm, particularly at least 5 nm, as determined by transmission electron microscopy of the eluted free particles or, if the particles are large enough, by negative stain electron microscopy and single particle analysis. Preferably, the SALipro particles have a top, a bottom, and a circumferential side surface, and the maximum diameter (major axis length) of the top and bottom surfaces is greater than the height of the circumferential side surface. In some embodiments of the SALipro particles, the saposin-like protein is at least partially located so as to surround the circumferential side surface of the particle.

[0187] In some embodiments, the average maximum diameter (major axis length) of the discoidal sarpop particles, as determined by transmission electron microscopy or, if the particles are sufficiently large, by negative stain electron microscopy and single particle analysis, is from 2 nm to 200 nm, particularly from 3 nm to 150 nm, preferably from 3 nm to 100 nm. In another embodiment, the average maximum diameter (major axis length) of the discoidal particles is from 3 nm to 80 nm, particularly from 3 nm to 60 nm. Actual experiments have shown that particles having an average maximum diameter (major axis length) of from 3 nm to 20 nm are particularly readily obtained by the method of the present invention.

[0188] In another embodiment, the particles are defined by a substantially monodisperse population of discoidal structures, as evaluated, for example, by the gel filtration elution profile of the free particles eluted on a HiLoad Superdex™ 200 16 / 60 GL column.

[0189] Generally, the main interaction between the saposin-like protein and the lipid bilayer in the sarpop particles is due to hydrophobic interactions between the residues on the hydrophobic surface of the amphipathic α-helix of the saposin-like protein molecule and the hydrophobic surface of the lipid at the edge of the bilayer at the periphery of the bioactive agent delivery particle, such as the hydrophobic acyl chains of phospholipids. The amphipathic α-helix of the saposin-like protein includes both a hydrophobic surface that contacts the hydrophobic surface of the lipid bilayer at the periphery of the particle and a hydrophilic surface that faces the outside of the particle and contacts the aqueous environment when the particle is suspended in an aqueous medium.

[0190] In another embodiment, the saposin lipoprotein particles are stable in aqueous solution and can be lyophilized for long-term storage and then reconstituted in aqueous solution. As used herein, "stability" or "stable" means that particle fragmentation during particle preparation, transport, and storage is at a low to undetectable level and aggregation or quality degradation is at a low to undetectable level.

[0191] In a preferred embodiment, the particles and particle libraries according to the method of the present invention are stable in an aqueous solution at a pH of 2.0 to 10.0, particularly 6.0 to 10.0, preferably 6.0 to 9.0, particularly preferably 7.0 to 9.0, and most preferably 7.0 to 8.0. In another embodiment, the libraries and particles according to the method of the present invention are, for example, determined by visual inspection (a clear solution containing no precipitate) or analytical gel filtration (fragmentation of less than 50%, particularly 1 to 40% of the particles), stable in an aqueous solution at a temperature of -210°C to 80°C, particularly -210°C to 40°C, -210°C to 30°C or -210°C to 4°C for at least 1 day, at least 2 days, at least 7 days, at least 2 weeks, at least 1 month, at least 6 months or at least 12 months. Actual experiments have shown that the sariplo particles are also stable in an aqueous solution at a pH of 5.0 to 8.0 and a temperature of 4°C to 40°C for at least 1 day, at least 2 days, at least 7 days, at least 2 weeks, at least 1 month or at least 3 months, when determined by, for example, visual inspection (a clear solution containing no precipitate) or analytical gel filtration (fragmentation of less than 50%, particularly 1 to 40% of the particles). It has also been demonstrated that the sariplo particles are stable for at least 10 minutes in an aqueous solution at a pH of 5.0 to 8.0 and a temperature of 40°C to 75°C, when determined by, for example, visual inspection (a clear solution containing no precipitate) or analytical gel filtration (fragmentation of less than 50%, particularly 1 to 40% of the particles). In some embodiments, the particles can be reconstituted in an aqueous solution after being lyophilized for long-term storage. In some embodiments, the sariplo particles are stable in a lyophilized form at -210°C to 80°C, particularly -210°C to 40°C, -210°C to 30°C or -210°C to 4°C for at least 1 day, at least 2 days, at least 7 days, at least 2 weeks, at least 1 month, at least 6 months or at least 12 months, when determined by, for example, analytical gel filtration after reconstitution in a suitable buffer at pH 7.5 (fragmentation of less than 50%, particularly less than 40%, or 1 to 40% of the particles).As used herein, "fragmentation" means that in the gel filtration elution profile, the size of the peak corresponding to the saposin particles (i.e., peak height) is reduced at the expense of the peak sizes of free non-lipid-bound SAPLIP and / or free lipids and / or aggregates, as compared to the peak size of newly prepared saposin particles. Thus, for example, 40% fragmentation means that the peak size (i.e., the height of the peak in the gel filtration elution profile) is reduced by 40% compared to the peak size before storage (100%).

[0192] Actual experiments have shown that saposin particles are particularly stable even in an aqueous solution substantially free of surfactant. Preferably, the aqueous solution contains a surfactant concentration lower than the critical micelle concentration (CMC) of the surfactant used. CMC is defined as the concentration of surfactant above which micelles are formed and all additional surfactant molecules added to the system are incorporated into the micelles.

[0193] At least a portion of the lipids to be incorporated into the saposin particles is provided in step a) of the method of the invention. Additional lipids can be provided during any further step of the method, in particular between one or more of steps b.1) / b.2) and / or c.1) / c.2) of the method of the invention. In one embodiment, the lipids are selected from the group consisting of lipids of archaea, prokaryotes, eukaryotes or viruses and mixtures thereof.

[0194] In a specific embodiment specific to alternative (I) of the method of the invention, in step c.1), the saposin-like protein bound to the support is contacted with the membrane of archaea, prokaryotes, eukaryotes or viruses provided in step a). The membrane contains a hydrophobic agent and at least a portion of the lipids to be incorporated into the saposin particles. This enables the formation of a library of saposin particles containing a heterogeneous mixture of saposin lipoprotein particles having different membrane lipid and optionally membrane protein compositions.

[0195] The supposin-like protein bound to the support does not seem to distinguish the membrane components it contacts. Thus, in step c.1) of the method of the present invention, when the supposin-like protein bound to the support is brought into contact with a complex biological membrane, a library of suppro particles bound to the support is obtained, which reflects the complexity and presents a snapshot of the membrane lipid and protein composition of the biological membrane used. Preparing a library of the lipidome / proteome of the biological membrane bound to the support in this way has the advantage that distinct units of the original membrane structure appear to be preserved in the suppro particles. Such a library bound to the support is advantageous for special applications such as high-throughput screening and biosensor use.

[0196] The term "library" according to the present invention means a set (a plurality of complexes) of different suppro particles. In particular, the differences can exist in the size and composition of the particles, especially in the membrane components contained therein, i.e., the membrane lipids, and optionally the composition of the membrane proteins. Typically, the library is a mixture of suppro particles containing different types of membrane proteins as opposed to "lipid-only particles". This is represented by the term "different membrane lipid and optionally membrane protein composition" used herein. The particles in the library can also differ in the content and composition of different membrane lipids. Preferably, some of the particles in the library differ in the fact whether they contain membrane proteins and which membrane proteins they contain.

[0197] In step c.1), a library of supposin-like particles having different membrane lipid and optionally membrane protein compositions is obtained by contacting the supposin-like protein bound to the support with the membrane of archaea, prokaryotes, eukaryotes or viruses provided in step a).

[0198] The library of saposin particles that can be obtained by the method of the present invention can include saposin particles lacking membrane proteins, i.e., saposin particles ( "empty" saposin particles) consisting essentially of only saposin-like proteins and membrane lipids from unpurified membranes. However, the library can also include saposin particles ( "filled" saposin particles) containing one or more membrane proteins.

[0199] A eukaryote is any cell or organism whose cells contain a nucleus and may optionally contain additional membrane-bound organelles. In one embodiment, the membrane used in the method according to the present invention is a membrane derived from a eukaryote, such as a cell membrane and / or a membrane derived from an organelle. Examples of organelles are the Golgi apparatus, mitochondria, peroxisomes, endoplasmic reticulum, chloroplasts, nucleus, etc.

[0200] Examples of eukaryotes are plants, animals, and fungi such as yeast and mold. Preferred eukaryotic cells that can be used in the method according to the present invention are selected from the group consisting of mammalian cells, particularly animal and human cells, insect cells, avian cells, fungal cells such as yeast cells, plant cells, and mixtures thereof. The term mammalian cells particularly includes animal and human cells maintained in a culture medium.

[0201] Prokaryotes are single-celled organisms that lack a nucleus. Prokaryotic cells are simpler and smaller than eukaryotic cells and lack membrane-bound organelles. An example of a prokaryote is a bacterium. Exemplary bacterial phyla are Acidobacteria, Actinobacteria, Aquificae, Armationadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctobacteria, Proteobacteria, Spirochaetae, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia. Preferred prokaryotic cells that can be used in the method according to the present invention are bacteria, particularly pathogenic bacteria, and mixtures thereof.

[0202] Archaea are only distantly related to prokaryotes and eukaryotes. A detailed overview can be found, for example, in De Rosa et al., ’’Structure, Biosynthesis, and Physicochemical Properties of Archaebacterial Lipids’’, MICROBIOLOGICAL REVIEWS, p. 70 - 80 Vol. 50, No. 1, 1986 or Albers et al., ’’The archaeal cell envelope’’, Nature Reviews Microbiology, 9, p. 414 - 426, 2011. De Rosa et al. reported that archaeal membranes contain molecules that are very different from those of prokaryotic and eukaryotic membranes. Prokaryotes and eukaryotes mainly contain membranes with glycerol - ester lipids, while archaea contain membranes with glycerol - ether lipids. The ether bond is more chemically resistant than the ester bond. This stability may help archaea survive in extreme temperatures and extremely acidic or alkaline environments. Prokaryotes and eukaryotes may contain ether lipids, but in contrast to archaea, these lipids are either minor components of the membrane or not membrane components at all.

[0203] Furthermore, archaeal lipids are based on isoprenoid side chains. Isoprenoid side chains are long chains containing 20, 25, or up to 40 carbon atoms that may optionally have multiple side branches. Isoprenoid side chains may also contain cyclopropane or cyclohexane rings. This is in contrast to the fatty acids found in the membranes of the other organisms mentioned above. Isoprenoids play important roles in the biochemistry of many organisms, but only archaea use isoprenoids to make phospholipids. In some archaea, the lipid bilayer can be replaced by a monolayer.

[0204] Examples of archaea are methanogenic archaea, halophilic bacteria, and thermoacidophilic archaea. Preferred archaea that can be used in the method according to the present invention are extremophilic archaea and mixtures of different extremophilic archaea.

[0205] The structure of the virus and the components of the viral membrane, where applicable, differ from the membranes of eukaryotes, prokaryotes, and archaea. This is reported in more detail, for example, in Lorizate et al., ’’Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines’’, Cellular Microbiology, 15(2), p.292-304, 2013 and Brugger et al., ’’The HIV lipidome: A raft with an unusual composition’’, PNAS, vol.103, No.8, p.2641-2646, 2006.

[0206] Lorizate et al. reported that various studies have shown that the HIV-1 membrane is different from the cell membrane of the producing cells, suggesting virus budding from existing subdomains or virus-mediated induction of a special budding membrane. Lipid analysis of cell membranes and HIV-1 purified from two different cell lines revealed a significantly different lipid composition of the viral membrane compared to the host cell membrane, regardless of the cell type investigated. Viral particles were significantly enriched in phosphatidylserine, sphingomyelin, hexosylceramide, and saturated phosphatidylcholine species compared to the host cell membrane of the producing cells. Viral particles showed reduced levels of unsaturated phosphatidylcholine species, phosphatidylethanolamine, and phosphatidylinositol. For plasmalogen-phosphatidylethanolamine and phosphatidylglycerol, cell type-specific differences were observed in the lipid composition of HIV-1 and donor cell membranes, and plasmalogen-phosphatidylethanolamine and phosphatidylglycerol were enriched in intensity only in HIV-1 derived from MT-4 cells. HIV-1 derived from MT-4 cells also contained dihydrosphingomyelin. In summary, these data reported by Lorizate et al. support the fact that HIV-1 selects a specific lipid environment for its morphogenesis and is not identical or similar to the cell membrane of its host. Usually, the particles and libraries obtained by the method of the present invention do not contain viral proteins and / or viral membranes.

[0207] Enveloped viruses include orthomyxoviruses, flaviviruses, and retroviruses. Examples of enveloped viruses are influenza virus A, influenza virus B, influenza virus C, influenza virus D, Batken virus, Bourbon virus, Dhori virus, hepatitis C virus, dengue virus, Japanese encephalitis virus, Ebola virus, yellow fever virus, Zika virus, HTLV1, and HIV.

[0208] Step d) of the method of the present invention In step d) of the method of the present invention, the sarpuro particles bound to the support can be eluted from the support. In certain applications (e.g., biosensors or lab-on-a-chip), this step is optionally performed since the sarpuro particles bound to the support are the product of interest. Thus, in these cases, it is not necessary to elute the particles from the support.

[0209] The covalent attachment of a hydrophobic agent or saposin-like protein to the support via the formation of chemical bond (i) is often substantially irreversible. For elution, the chemical bond has to be cleaved, for example, by performing a reverse bonding reaction or a different cleavage reaction. For example, a linker with a cleavable bond can be used. This can be a linker containing a protease cleavage site that allows elution, for example, by incubation with a protease (e.g., TEV protease). In contrast, the binding of sarpuro particles via affinity interaction (ii), indirect interaction via a crosslinker (iii), hydrophobic interaction (iv) or electrostatic interaction (v) is usually reversible and allows for relatively easy elution of the particles, as well as often regeneration of the support and its reuse.

[0210] Elution of the assembled sarpuro particles from the support can be achieved by various techniques depending on the type of interaction between the support and the target molecule, in particular between the capture moiety and the binding moiety as described above. Suitable elution methods are known to those skilled in the art. Those skilled in the art can select an appropriate elution strategy depending on the type of interaction by which the target molecule is selectively bound to the support.

[0211] As used herein, the term "elution" refers to the removal or replacement of sarpuro particles bound to the support.

[0212] For example, sarpuro particles bound by an affinity interaction (ii) between one of its components and the support are eluted by providing a specific molecule in the liquid environment that disrupts and / or replaces the affinity interaction. For example, if the target molecule is tagged with a His tag and the support is Ni2+ When having an -NTA capture moiety, elution is performed by contacting the support-bound salipro particles with imidazole. Similarly, when the target molecule is tagged with a GST tag and the support contains glutathione as a capture moiety, elution can be performed by contacting the support-bound salipro particles with glutathione. Elution of the salipro particles bound by a cross-linking agent that mediates the indirect interaction (iii) between the target molecule and the support can be carried out by cleaving the cross-linking agent or linker, or by disrupting the interaction between the cross-linking agent and the capture and / or binding moieties. In the case of a receptor / ligand-like interaction between the capture moiety and the binding moiety, such as in the case of lectin attached to the support that binds to the sugar moiety on the target molecule, elution can be performed by using a competitive binding substance such as a sugar. Elution of the salipro particles bound to the support by hydrophobic interaction (iv) can be achieved by reducing the salt concentration in the liquid environment in order to promote solvation and thus elution of the bound material. When the salipro particles are bound to the support by electrostatic interaction (v), elution can be performed by changing the pH or salt concentration in the liquid environment surrounding the support-bound salipro particles.

[0213] According to certain embodiments, the above-described steps, particularly steps a), b.1) / b.2), c.1) / c.2) and d), are carried out at a temperature of 1°C to 85°C, particularly 1°C to 40°C, particularly preferably 1°C to 30°C. Temperatures of 1°C to 25°C, particularly 1°C to 20°C, and most particularly 3°C to 15°C are sufficient for most applications. However, by the methods taught herein, one of ordinary skill in the art can determine the optimal incubation temperature with respect to the temperature stability of the membranes, compounds, lipids and proteins used.

[0214] As used herein, the term "essentially" means that in the method of the present invention, trace amounts of additional components used, for example, additional components of the crude membrane used to provide a hydrophobic agent and / or lipid, or agents used in the method such as surfactants, may also be present in the suripro particles. However, the fact that the suripro particles consist essentially of at least one saposin-like protein and membrane components obtained from the membranes of cells or organelles is specifically meant to mean that no additional lipids and / or proteins have been added.

[0215] Suripro particles or libraries of suripro particles obtainable according to the method of the present invention can be used in medicine, particularly for use in the prevention, treatment or reduction of the severity of diseases, or for use in diagnostic methods, cosmetic treatments, or for use as a vaccination preparation.

[0216] For example, the suripro particles can be included in a pharmaceutical composition for delivering one or more membrane proteins and / or lipids to an individual in need of one or more membrane proteins and / or lipids, the composition comprising the above-mentioned suripro particles.

[0217] In addition to the suripro particles, the pharmaceutical composition can optionally contain a (further) pharmaceutically acceptable vehicle, carrier or adjuvant.

[0218] When suripro particles are used in a pharmaceutical composition, the individual components of the particles and the pharmaceutical composition should be pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, without excessive toxicity, irritation, allergic reaction, etc., and refers to a component, compound or agent that meets a reasonable benefit / risk ratio.

[0219] Pharmaceutical compositions containing salipro particles can be administered orally, rectally, parenterally, intravaginally, intraperitoneally, topically (such as by powders, ointments or drops), buccally, by aerosol, oral or nasal spray, etc. to humans and other animals, depending on the severity of the disease or condition being treated. In particular, the pharmaceutical compositions can be formulated for enteral, parenteral and / or topical administration. The pharmaceutical compositions can be administered as capsules, infusions or injections, as brushable or drinkable compositions, or as aerosols. Also described herein are pharmaceutical compositions in which the salipro particles are present as a dispersion or in solid form in solution.

[0220] The salipro particles are also useful for diagnostic and / or cosmetic applications. For example, salipro particles containing a detectable antigen or tag as described above can be used as diagnostic agents and applied for diagnostic purposes. In the latter case, it can also be very useful to use salipro particles in a form bound to a support obtainable according to the invention when elution step d) is omitted. Examples of diagnostic and life science research tools according to the invention include incorporated membrane proteins with tags, saposin-like proteins with tags, lipids with tags, particles having incorporated fluorophores or contrast agents (for example for magnetic resonance imaging). The tag can be, for example, a fluorescent tag.

[0221] In another aspect, the sarpuro particles are useful as a vaccination preparation, as its carrier, or as a drug delivery vehicle. Many pathogenic antigens that can be particularly effective in vaccination are exposed on the surface of eukaryotic or prokaryotic pathogens or diseased cells (e.g., cancer cells) in a patient and / or are contained in the outer cell membrane. These antigens can be derived from, for example, pathogenic lipids, other hydrophobic biomolecules, or membrane proteins. In sarpuro particles, such antigens can then be effectively incorporated into particles that can be used as antigen-presenting delivery vehicles in a vaccination preparation. Similarly, sarpuro particles are also useful for serving as antigen-presenting delivery vehicles for generating antibodies against lipids or membrane proteins in a suitable host animal, preferably in mammals such as rabbits, goats, llamas, mice, and primates.

[0222] The use of sarpuro particles as tools for drug development, drug screening, and drug discovery is also described herein.

[0223] For example, specific membrane protein drug targets such as cell surface receptors or ion channels can be incorporated into sarpuro particles, thereby solubilizing them in their native state. Such particles can then be utilized in assays for studying the activity of the drug target membrane protein in its native lipid bilayer environment or can be used in drug screening for identifying new drugs. According to the method of the present invention, sarpuro particles are assembled while being selectively bound to a support with one particle component. The sarpuro particles bound to the support thus obtained can be directly used in chip-based applications such as lab-on-a-chip, surface plasmon resonance (SPR), grating-coupled interferometry (GCI), or other biosensor applications.

[0224] Label-free optical SPR biosensors are the gold standard for measuring the binding affinity and kinetics of molecular interactions. There are mainly four SPR biosensor platforms: Biacore T100 from GE Healthcare, ProteOn XPR36 from Bio-Rad, Octet RED384 from ForteBio, and IBIS MX96 from Wasatch Microfluidics.

[0225] Instead of SPR biosensors, waveguide interferometers based on GCI can be used to characterize molecular interactions and / or to determine the kinetic rates, affinity constants, and concentrations of the interacting molecules.

[0226] SPR- and GCI-based optical measurements detect changes in the refractive index within the evanescent field near the sensor surface due to mass changes caused by the formation of complexes of the interacting molecules, while other biosensor systems apply different detection strategies. Calorimetric biosensors record the heat absorbed or released by the reaction. Potentiometric biosensors detect the distribution of charges that give rise to a potential. Alternatively, amperometric biosensors can represent the movement of electrons generated in redox reactions. Furthermore, biosensors can also detect the difference in light output during the course of the reaction or the absorbance between reactants and products (optical biosensors). Additionally, piezoelectric biosensors utilize the effect due to the mass of reactants or products bound to the biosensor surface (piezoelectric biosensors). All of these biosensor applications have in common that they benefit from binding one of the molecules of interest to a support. The method of the present invention provides an easy and direct "one-step" process for directly incorporating a hydrophobic agent of interest into the sariplo particles bound to the support. This eliminates the need for an additional binding step and is thus advantageous in terms of process efficiency and cost.

[0227] The method of the present invention can also be used as a tool for membrane protein purification, for membrane and / or membrane protein research for membrane protein expression, particularly for lipidomics and proteomics, preferably for the isolation, identification and / or research of membranes and / or membrane proteins or for the creation of lipidome or proteome libraries or databases.

[0228] SALipro particles are generally useful for solubilizing originally insoluble membrane proteins and membrane domains or components in an aqueous solution in their native membrane bilayer microenvironment. The SALipro particles obtained by using the method of the present invention can be used in a variety of new applications in membrane protein research. For example, the SALipro particles enable the study of membrane proteins incorporated into the SALipro particles by methods such as nuclear magnetic resonance (NMR), X-ray crystallography, electron microscopy (EM), mass spectrometry, isothermal titration calorimetry (ITC), differential light scattering, small-angle X-ray scattering (SAXS), enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), biochemical assays, high-throughput screening (HTS), and the like.

[0229] A library of particles obtained by certain embodiments of the method of the present invention is particularly useful for research in the field of systems biology, particularly in the areas of lipidomics and membrane proteomics. The library of the present invention may be suitable for capturing and solubilizing the lipidome and / or proteome of viruses, cells or organelles. Typical analytical techniques in lipidomics and proteomics are techniques such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, and computational methods. By applying these methods or techniques to the sariplo particles, for example, it becomes possible to further elucidate the roles of natural lipids and membrane proteins in many metabolic diseases such as cancer, autoimmune diseases, obesity, atherosclerosis, stroke, hypertension and diabetes. When the library is in a state of being bound to a support, particularly when it is bound to a solid support having a substantially flat surface, spatial two-dimensional separation and immobilization of the sariplo particles are achieved, which is useful for the analysis of such complex mixtures.

[0230] To identify new molecular drug targets such as membrane proteins or lipids present in the membrane of target cells or organelles, the entire library of sariplo particles obtained from a specific cell or organelle that may be a disease target can be used for drug screening purposes.

Brief Description of the Drawings

[0231] Hereinafter, the present invention will be described with reference to the drawings illustrating specific embodiments of the present invention. However, the present invention is defined in the claims and as generally described herein. The present invention should not be limited to the embodiments shown for illustrative purposes in the following figures.

[0232]

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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Figure 12

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Figure 13

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Figure 14

[0246] Figure 1 illustrates prior art apolipoprotein A-1 (see the above-mentioned European Patent No. 1596828 specification) containing nanodisc particles (10) comprising a lipid (3) and apolipoprotein A-1 as a lipid-binding polypeptide (11). In contrast to prior art apolipoprotein-derived nanodiscs, the lipid-binding polypeptide of the present invention, i.e., the protein for saposin, does not surround the lipid in a double-belt-like manner (see Figures 2, 3 and 4). Rather, the particles of the present invention are integrated by a core containing membrane lipids surrounded by two or more substantially V-shaped or boomerang-shaped lipid-binding polypeptides arranged in a head-to-tail orientation with substantially no direct protein-protein contact between the individual saposin-like proteins within a given sariplo particle obtained by the method of the present invention (see Figures 2, 3 and 4).

[0247] Figures 2a to 2c are schematic diagrams of sialipro particles obtained according to a specific embodiment. The particles of Figures 2a, 2b, and 2c include a saposin-like protein (2), a plurality of different lipids (3), a membrane protein (4a) / oligomeric membrane protein (4b), and optionally a hydrophobic compound (4c). The membrane proteins (4a, 4b) in Figures 2a to 2c include a binding portion. The binding portion in the membrane protein can be a native or engineered binding portion. The binding portion of the membrane protein can be present at the N-terminus, C-terminus of the amino acid sequence of the membrane protein or within the amino acid sequence of the membrane protein. When the membrane protein contains an engineered binding portion, the engineered binding portion can be attached to the membrane protein during or after protein synthesis. In another embodiment not shown, the membrane proteins (4a, 4b) can have a plurality of binding portions of the same or different types. The lipids (3) of the sialipro particles illustrated in Figures 2a to 2c are different from each other, meaning that the lipid composition of the sialipro particles is not uniform or homogeneous. Depending on how the membrane proteins (4a, 4b) in step a) are provided, the lipid composition changes. In a further embodiment not shown, the sialipro particles can also include additional components typically present in the membranes of viruses, archaea, eukaryotes, and / or prokaryotes. The lipids (3) and the membrane proteins (4a, 4b) can be derived from the same viral, archaeal, eukaryotic or prokaryotic membrane source or from different sources.

[0248] The particles in FIGS. 2a - 2c are not drawn to scale. Depending on the size of the membrane proteins (4a, 4b) incorporated into the particles, the particles can be substantially different in size compared to other particles. The size of the salipro particles obtained by the method of the present invention is flexible. For example, the particles in FIG. 2b that contain oligomeric membrane proteins are larger and contain more saposin subunits (2) compared to the particles in FIG. 2a that contain monomeric membrane proteins. Depending on the size of the salipro particles, the particles contain two or more saposin - like molecules arranged in a head - to - tail fashion per particle. The particles illustrated in FIGS. 2a and 2c contain two saposin - like molecules, while the particles illustrated in FIG. 2b contain three saposin - like molecules.

[0249] FIGS. 2a, 2b, and 2c illustrate salipro particles containing saposin - like proteins (2), lipids (3) from a viral, archaeal, eukaryotic, and / or prokaryotic membrane source, membrane proteins (4a, 4b), and optionally a hydrophobic compound, in a simplified form as side views and top views. The membrane protein (4a) can be an integral membrane - spanning protein in monomeric form. However, the membrane protein can also be an integral membrane - spanning protein in oligomeric form as illustrated in FIG. 2b, or a surface membrane protein, a lipid - bound amphitropic protein, a lipid - anchored protein, or a chimeric protein having fused hydrophobic and / or membrane - spanning domains, all of which can be in monomeric or oligomeric states.

[0250] The particles illustrated in FIG. 2c differ from 2a and 2b in that they further contain a hydrophobic compound (4c) of natural or synthetic origin. The number of hydrophobic compounds within one salipro particle can vary. The hydrophobic compound can form tight interactions with lipids (3) and / or any type of membrane protein. The membrane protein can be, for example, a monomeric membrane - spanning protein (4a) as illustrated in FIG. 2c.

[0251] Figures 3a - 3c illustrate, in a similarly simplified schematic form (left side view and right top view), the sarpol particles obtained according to a particular embodiment, not to scale. The particles illustrated in Figures 3a, 3b, and 3c include a saposin-like protein (2), a lipid (3), and optionally transmembrane proteins (4a, 4b) in monomeric or oligomeric form, and the saposin-like protein (2) has a binding moiety (5). In another embodiment not illustrated, the saposin-like protein (2) can have a plurality of binding moieties of the same or different types. As described for the particles of Figure 2, the particles illustrated in Figure 3 can vary with respect to their lipid (3) composition and their size in order to incorporate any type of hydrophobic protein by simply incorporating more than two saposin-like proteins (2) to form the particles.

[0252] Figures 4a and 4b show schematic diagrams, not to scale, of the sarpol particles of a particular embodiment of the present invention, from the side (left) and top (right). The particles illustrated in Figures 4a and 4b include a saposin-like protein (2), a lipid (3), a hydrophobic compound (4c), and optionally a membrane protein (4a). The hydrophobic compound presents a binding moiety that can be of natural or engineered origin. The natural binding moiety can form an internal part of the hydrophobic compound (4c), while the engineered binding moiety is usually attached to a suitable terminal reactive group after synthesis of the hydrophobic compound or after purification of a natural hydrophobic compound. In a further embodiment not illustrated, the sarpol particles can include different types of hydrophobic compounds having the same natural or engineered binding moiety. The particles of Figures 4a and 4b can differ in the number and type of lipid (3), hydrophobic compound (4c), membrane protein (4a), and saposin-like protein (2) incorporated into the particles.

[0253] Figs. 5a and 5b show saposin-like particles of a particular embodiment in schematic form (left side view and right top view), not to scale. The particles illustrated in Figs. 5a and 5b differ from the particles illustrated in Figs. 4a and 4b in that they have a binding moiety for saposin-like protein (2) rather than hydrophobic compound (4c). The same considerations as described for Figs. 4a and 4b apply to the different compositions of the particles illustrated in Figs. 5a and 5b, which are not shown.

[0254] Figures 6a to 6f show specific embodiments of providing a hydrophobic agent in the form of a hydrophobic biomolecule (i.e., for example, a transmembrane protein in monomeric form (4a) or oligomeric form (4b)) or a hydrophobic compound in step a) of the method of the present invention. In Figure 6a, the membrane proteins (4a, 4b) are provided as unpurified membrane vesicles (7, 7'). The membrane vesicles (7, 7') can be of viral, archaeal, eukaryotic or prokaryotic origin. The membrane vesicles (7, 7') contain a plurality of lipids (3), and in the case of vesicle (7), it contains a plurality of membrane proteins exemplified in purified form only by two membrane proteins (4a, 4b) in this figure, and the membrane protein (4a) has a binding moiety (5). Vesicle (7') is a particle of "empty" lipids only. The unpurified membrane vesicles exemplified as vesicles (7, 7') can be obtained directly, for example, by lysing cells or organelles of archaeal, prokaryotic or eukaryotic origin. The unpurified membrane vesicles can also be obtained by rupturing the viral envelope. Vesicles such as (7) and (7') are usually formed naturally upon lysis or membrane rupture. The unpurified membrane vesicles can contain surfactant molecules (not shown here) or can associate with surfactant molecules. Figure 6b shows a membrane protein (4a) having a binding moiety (5), which is not present in a natural membrane or unpurified membrane vesicles but is in a solubilized state with an artificial surfactant (see the association of membrane protein (4a) with lipids (3) and surfactant molecules (6) shown in Figure 6b). Figure 6c shows a hydrophobic compound having a binding moiety (5) in a solubilized state with a surfactant, meaning that the hydrophobic compound is embedded within a micelle containing lipids (3) and surfactant molecules (6). Optionally, the membrane protein (4a) shown in Figure 6b or the hydrophobic compound shown in Figure 6c may be solubilized by surfactant molecules only. The manner for delivering the hydrophobic agent illustrated in Figures 6d to 6f is different from Figures 6a to 6c in that the hydrophobic biomolecule exemplified as a transmembrane protein (4a, 4b) or a hydrophobic compound (4c) does not contain a binding moiety.

[0255] Figures 7a and 7b illustrate specific embodiments for providing a saposin-like protein in the method of the present invention. Figure 7a differs from Figure 7b in that the saposin-like protein has a binding moiety of natural or engineered origin. The natural binding moiety forms part of the natural amino acid sequence of the saposin-like protein, while the binding moiety of engineered origin can be attached during or after synthesis of the saposin-like protein. The engineered binding moiety is optimized to bind with high affinity to their corresponding binding partners that are capture moieties in the method of the present invention. In a further embodiment, the saposin-like protein can associate with surfactant molecules (not shown here).

[0256] Figures 8a to 8d show non-to-scale views of a saposin-like protein (Figure 8a) and hydrophobic agents (Figures 8b to 8d) bound to a support. The selective binding of the binding moiety in the saposin-like protein (see Figure 8a) or hydrophobic agent (see Figures 8b to 8d) to the capture moiety (13) of the support (12) is illustrated for a single capture moiety on the support. In practice, the support typically comprises a plurality of capture moieties of the same or different types. The interaction of the binding moiety / capture moiety recognition pair can be based on chemical bond formation, affinity-based interactions (including mediation by cross-linking agents), hydrophobic interactions and / or electrostatic interactions. The saposin-like protein (2) bound to the support can be in a solubilized state with a surfactant (not shown here). Also, the crude membrane vesicles (7) can contain surfactant molecules (not shown here) or can associate with surfactant molecules. The membrane proteins (4a, 4b) in Figure 8d are embedded in their native lipid environment in the form of crude membrane vesicles (7), while the hydrophobic compound (4c) and membrane protein (4a) are in a solubilized state with an artificial surfactant, whereby micelles around the hydrophobic agent can be formed by lipids and surfactants. In a further embodiment, the surfactants used to solubilize the saposin-like protein (2) and / or hydrophobic agent can be of the same or different chemical nature.

[0257] Figures 9a - 9c show in a simplified schematic form specific embodiments of method steps b.2) and c.2). The hydrophobic agent bound to the support, in the form of a solubilized membrane protein (4a) (Figure 9a), a solubilized hydrophobic compound (4c) (Figure 9b), and a membrane protein (4c) embedded in unpurified membrane vesicles (7) (Figure 9c), is contacted in step c.2) with a saposin - like protein (2), which may optionally be in a solubilized state with a surfactant. Usually, the self - assembly of the particles occurs directly on the support when the hydrophobic agent bound to the support is contacted with the saposin - like protein (2). Optionally, additional solubilized lipids of viral, archaeal, eukaryotic, and / or prokaryotic origin may be added to the particle assembly reaction, although this is not illustrated in Figures 9a - 9c. The solubilized membrane protein (4a) and solubilized hydrophobic compound (4c) of Figure 9a are provided in a purified form for selective binding to the support in step b.2). The assembled particles of Figures 9a and 9b are substantially composed of lipids (3) associated with the solubilized membrane protein (4a) or hydrophobic compound (4c). Optionally, additional lipids may be added to the liquid environment of the support. In Figure 9c, unpurified membrane vesicles (7) are bound to the support (12). As shown in Figure 9c, only the membrane protein (4a) bound to the capture portion (13) of the support (12) by its binding portion (5) remains bound to the solid support when contacted with the saposin - like protein (2), enabling the self - assembly of the sariplo particles on the support. Initially, in step b.2), a portion of the unpurified membrane vesicles bound to the support is formed, and other membrane proteins, exemplified by the multimeric membrane protein (4b) in the unpurified membrane vesicles (7), which do not present a complementary binding portion to the capture portion of the support, are not incorporated into the sariplo particles bound to the support. Thus, the embodiment illustrated in Figure 9c enables the production of sariplo particles containing a single type of membrane protein (4c) from a plurality of unpurified membrane vesicles bound to the support in step b.2). The assembled particles are substantially free of surfactant. The particles bound to the support in Figures 9a - 9c can be eluted.The dissolution strategy depends on the type of interaction between the capture moiety (13) of the support (12) and the binding moiety (5) in the hydrophobic agent.

[0258] Figures 10a - 10d show in a simplified schematic form specific embodiments of method steps b.1) and c.1). A saposin - like protein bound to a support is contacted with a detergent - solubilized membrane protein (exemplified as monomeric transmembrane protein (4a), see Figure 10a), unpurified membrane vesicles (exemplified as vesicles (7, 7’), see Figure 10b), a detergent - solubilized hydrophobic compound (4c) (see Figure 10c), or both, i.e., a detergent - solubilized hydrophobic compound (4c) and a membrane protein (4a) (see Figure 10d) to enable self - assembly of respective sariPro particles on the support in step c.1). Adjacent captured saposin - like proteins or additionally added free saposin - like proteins contribute to forming individual sariPro particles (not shown). To enable self - assembly of saposin - lipoprotein particles (not shown), for example, additional saposin - like protein is added during or after step c.1). The assembled particles in Figure 10a are substantially composed of lipids (3) associated with the solubilized membrane protein (4a). Solubilized proteins are usually obtained by protein purification methods and, in these circumstances, are often embedded in micelles containing detergent molecules and lipids (3). Lipids associated with the solubilized membrane protein are often the “shell lipids” of the membrane from which the membrane protein (4a) was purified. The membrane protein can be purified from its “native” membrane, but it doesn't have to be. For example, a eukaryotic membrane protein can be overexpressed from a transgene in a prokaryotic cell such as a bacterium or a virus from which the membrane protein is purified. Thus, the lipids (3) remaining associated with the detergent - solubilized eukaryotic membrane protein may not form part of the “native” lipid environment of this purified protein. The shell lipids adhere tightly to the hydrophobic surface of the membrane protein (4a). Optionally, additional lipids may be added to the liquid environment for incorporation into the sariPro particles.

[0259] In step c.1), by contacting unpurified membrane vesicles (exemplified as vesicles (7, 7') in Fig. 10b) with a saposin-like protein bound to a support (see Fig. 10b), it becomes possible to generate a library of sarylpro particles bound to the support, i.e., the sarylpro particles bound to the support differ in their size and in the composition containing the saposin-like protein (2), lipid (3) and / or membrane protein (4a, 4b). It is even possible to produce "empty" particles that contain no hydrophobic agent at all. In the library particles, each membrane protein (4a, 4b) is embedded in the membrane environment from which it was obtained, i.e., the membrane protein remains embedded in its "native" lipid environment. Thus, the lipid (3) associated with the membrane protein (4a, 4b) in the assembled sarylpro particles is preferably carried over from the native lipid environment of the membrane protein present in the unpurified membrane vesicles (7). Membranes of viruses, archaea, eukaryotes or prokaryotes can serve as the source membrane. The assembled particles in Fig. 10c contain three hydrophobic compounds per particle. Of course, the number of hydrophobic compounds incorporated into a single particle can be adjusted by adjusting the molar ratio of the hydrophobic compound to the lipid used in the self-assembly reaction of step c.1). The hydrophobic compounds in Fig. 10c are added to the self-assembly reaction in a solubilized state. Solubilization of the hydrophobic compound is usually achieved by surfactant molecules, similar to the solubilization of hydrophobic membrane proteins. As shown in Fig. 10c, the lipid can form part of the solubilized state of the hydrophobic compound. Optionally, additional lipid can be added. As shown in Fig. 11d, hydrophobic agents in the form of solubilized hydrophobic compounds (4c) and membrane proteins (4a) can be applied together in step c.1). Any desired molecular ratio of the solubilized hydrophobic compound to the membrane protein can be selected, which affects the composition of the resulting particles. The assembled particles shown in Figs. 10a - 10d usually contain substantially no surfactant. The particles bound to the support in Figs. 10a - 10d can be eluted. The elution strategy depends on the type of interaction between the capture moiety (13) / binding moiety (5) pair.

Example

[0260] The following examples serve to explain the present invention in more detail with reference to specific embodiments and drawings, but the specific embodiments and drawings are not intended to limit the present disclosure.

[0261] I. Abbreviations The following abbreviations are used. TIFF0007695198000004.tif244170TIFF0007695198000005.tif68170

[0262] II. Purification of Saposin A The purified saposin A used in the following experiments was prepared as follows. The coding region of human saposin A (SEQ ID NO: 1) was inserted into the pNIC-Bsa4 plasmid and transformed into the Escherichia coli Rosetta gami-2(DE3) (Novagen) strain. Using the expressed vector, the expression of saposin A protein was carried out. Cells were grown at 37 °C in TB medium supplemented with tetracycline, chloramphenicol, and kanamycin and induced with 0.7 mM IPTG. Three hours after induction, cells were collected by centrifugation at 12,000 × g for 15 minutes. The supernatant was discarded, and the cell pellet was resuspended in lysis buffer LB1 (20 mM HEPES pH 7.5, 150 mM NaCl, 20 mM imidazole) and disrupted by sonication. The solubilized solution was subjected to centrifugation at 26,000 × g for 30 minutes, and the supernatant was heated to 85 °C for 10 minutes and then subjected to an additional centrifugation step at 26,000 × g for 30 minutes. Batch adsorption of the supernatant by end-over-end rotation for 60 minutes using Ni Sepharose™ 6 Fast Flow medium was used for preparative IMAC purification. After binding of saposin A to the IMAC resin, the chromatography medium was packed into a 10 mm (inner diameter) open gravity flow column and unbound proteins were removed by washing with 15 bed volumes of lysis buffer LB1. The resin was washed with 15 bed volumes of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 40 mM imidazole). Saposin A was eluted by the addition of 5 bed volumes of elution buffer EB1 (20 mM HEPES pH 7.5, 150 mM NaCl, 400 mM imidazole). The eluate was dialyzed overnight against gel filtration buffer GF pH 7.5 (20 mM HEPES pH 7.5, 150 mM NaCl) supplemented with recombinant TEV protease. TEV protease containing non-cleavable His tags was removed from the eluate by passing the eluate through 2 ml of IMAC resin. The cleaved target protein was concentrated to a volume of 5 ml using a centrifugal filter unit and loaded onto a HiLoad Superdex™ 200 16 / 60 GL column (both from GE Healthcare) using an AKTAexplorer™ 10 chromatography system.The peak fractions were pooled and concentrated to 1.2 mg / ml protein. The protein sample was snap-frozen in liquid nitrogen and stored at -80 °C.

[0263] III. Generation of saposin particles on a support Example 1: In this example, a large transmembrane transporter (SLC) is used as a hydrophobic agent in alternative (II) of the method according to the invention. The lipid and the hydrophobic agent are provided in the form of an unpurified membrane fraction obtained from HEK293F cells overexpressing SLC. The SLC transporter contains a Strep II-tag as a binding moiety. Anti-Strep-II affinity purification beads were used as the support according to the invention. The anti-Strep-II affinity purification beads contain an anti-Strep-II capture moiety that can bind to the Strep II binding moiety contained in the SLC transporter protein. When saposin A is added to the solubilized membrane containing the SLC transporter bound to the support, it becomes possible to form saposin lipid particles containing the SLC transporter that remain attached to the support via the Strep-II tag contained in the SLC transporter protein. Thus, the assembly of saposin lipid particles occurred entirely on the support, originating from the cell membrane and occurring with endogenous lipids that still form a complex with the SLC transporter protein bound to the support.

[0264] 1.a. Overexpression of membrane proteins The coding sequence of the human SLC transporter was introduced into an expression vector encoding an N-terminal Strep-tag II, followed by daGFP and a PreScission protease cleavage site. Before transfection, HEK293F cells (ATCC cell line, mycoplasma test negative) were grown to a density of 2.5×10 -1 cells / ml in Excell293 medium (Sigma) supplemented with 4 mM L-glutamine (Sigma) and 5 μg / ml 6 of phenol red (Sigma-Aldrich). Using polyethyleneimine (PEI) (Polysciences), 2.5×10 -1 cells / ml 6 cells / ml -1At a density of, cells were transiently transfected with the expression vector in Freestyle293 medium (Invitrogen), diluted with an equal volume of Excell293 6 hours after transfection, and treated with 2.2 mM valproic acid (Sigma) 12 hours after dilution of the culture. Subsequently, the transfected cells overexpressed the fusion protein Strep II - daGFP - SLC. All cells were collected approximately 48 hours after transfection.

[0265] 1.b. Preparation of crude cell membranes Essentially as described in a. above, large - scale expression of the fusion protein Strep II - daGFP - SLC was carried out in 5 l of culture. Cells were collected in lysis buffer (LB2) containing 50 mM HEPES / Tris base, pH 7.4, 50 mM NaCl supplemented with 1 mM L - Asp, 1 mM EDTA, 1 mM PMSF, 1 mM TCEP and a 1:200 (v / v) dilution of mammalian protease inhibitor cocktail (Sigma), and disrupted in a cell homogenizer (EmulsiFlex - C5, Avestin) by three runs at approximately 103,000 kPa. The resulting homogenate was clarified by centrifugation (4,500 g for 0.5 h), and crude membranes were recovered by ultra - centrifugation (186,000 g for 1.5 h). The membranes were washed once with LB2 buffer and finally homogenized with a douncer in a buffer containing 50 mM HEPES / Tris base, pH 7.4, 200 mM NaCl, 1 mM L - Asp, 1 mM EDTA, 1 mM TCEP, and 10% glycerol, snap - frozen in liquid N2, and stored at - 80 °C at 0.5 g membrane / ml -1 and stored at - 80 °C.

[0266] 1.c. Binding to and elution from the affinity support The following buffers were used: - Solubilization buffer (SB): 50 mM HEPES pH 7.5, 2% DDM, 0.4% CHS, 200 mM NaCl, 1 mM L - Asp, 1 mM EDTA, 1 mM TCEP and 5% glycerol. - Working buffer (WB): 50 mM HEPES pH 7.5, 200 mM NaCl, 1 mM L-Asp, 1 mM TCEP, and 5% glycerol. - Elution buffer EB2: WB supplemented with 2.5 mM desthiobiotin (dBiotin). - HNG buffer: 50 mM HEPES pH 7.5, 200 mM NaCl, and 5% glycerol.

[0267] 800 μl of crude membrane containing overexpressed SLC transporter (0.5 g membrane / ml -1 ) was solubilized with 4.2 ml of SB and incubated for 90 min at 4 °C using a rotating wheel. Membrane debris was removed by centrifugation at 30,000 g for 30 min, followed by the addition of 900 μl of equilibrated anti-Strep-II affinity purification beads (StrepTactin Sepharose beads, GE Healthcare), and the total volume was adjusted to 5 ml using WB. The sample was then incubated at 4 °C for 1 h to bind the Strep-II-tagged SLC transporter to the affinity beads. The sample was then split into five separate columns to allow removal of unbound material by gravity flow-through. The affinity beads were not washed at this stage and contained the affinity-bound SCL transporter in an environment that partially contained native cell membrane lipids, surfactant micelles, and WB components (the "dead volume" of the beads).

[0268] Different amounts (0 - 4 ml) of 3.6 mg / ml saposin were added to the corresponding columns. The mixture was then transferred to five new tubes and the total sample volume was adjusted to 5 ml using WB as follows: - Sample 1: 0 ml of saposin A + 4 ml of WB - Sample 2: 0.5 ml of saposin A + 3.5 ml of WB - Sample 3: 1 ml of saposin A + 3 ml of WB - Sample 4: 2 ml of saposin A + 2 ml of WB - Sample 5: 4 ml of saposin A

[0269] Subsequently, samples 1-5 were incubated at 4 °C for 1 hour using a rotating wheel and then returned to the column. After removing the unbound material from the column using gravity flow-through, it was washed with 6 CV by WB, and an elution step was performed with 4 ml of elution buffer EB2. SEC was performed using a Superose 6 Increase, 5 / 150 GL column run in detergent-free WB as the SEC buffer (protein detection at 280 nm), and 50 μl of each eluted sample was analyzed.

[0270] The results are illustrated in Figure 11. The results demonstrate that sapropin particles can be obtained even when the membrane protein forming a complex with the lipid is bound to the affinity support. As the amount of saposin A increases (0.5 mL, 1 mL, 2 mL, and 4 mL of saposin A at a concentration of 3.6 mg / ml), the formation of sapro particles also increases. As a negative control under these conditions, when saposin A was excluded from the liquid environment, sapro particles could not be obtained (Figure 11, sample 1).

[0271] The results illustrated in Figure 11 also reinforce that the sapro particles bound to the support are stable during thorough washing steps of the column before elution with detergent-free buffer.

[0272] 1.d. Analysis of the obtained sapro particles The eluate obtained after incubating the affinity beads with 4 ml of SapA (see sample 5 in the previous section c) was concentrated using an Amicon Ultra-2 centrifugal filter with a molecular size cut-off of 100 kDa. The concentrated sample of 40 μl was further analyzed by SEC using a Superose 6 Increase 5 / 150 GL column in detergent-free HNG buffer supplemented with 1 mM L-Asp.

[0273] Analysis of the SEC fractions by SDS-PAGE shows that mainly fractions 13, 14, and 15 contain purified sapro particles containing the SLC transporter and saposin (Figure 12a).

[0274] To further verify the uniformity of the reconstituted saposin particles, 20 μl from fraction 14 was further analyzed by SEC using a Superose 6 Increase 5 / 150 GL column in surfactant-free HNG buffer supplemented with 1 mM L-Asp. The corresponding SEC profile (Figure 12b) further demonstrates the stability and uniformity of the saposin particles when reconstituted on the affinity beads.

[0275] The data presented in this example clearly demonstrate that it is possible to reconstitute a hydrophobic agent into saposin particles while one of the particle components is bound to an affinity support. The data also show that unpurified membranes can be used in the method of the present invention.

[0276] IV. Generation of Saposin Particles from Whole Cells Example 2: In this example, a membrane protein is used as a hydrophobic agent in alternative (II) of the method according to the invention. The lipid and the hydrophobic agent are provided in the form of untreated cells that overexpress the membrane protein, namely human embryonic kidney (HEK) cells. The HEK cells are only contacted with a surfactant without performing a mechanical cell lysis step. The eukaryotic membrane protein contains a FLAG tag as a binding moiety. Anti-FLAG affinity purification beads are used as the support according to the invention. The anti-FLAG affinity purification beads contain an anti-FLAG capture moiety that can bind to the FLAG binding moiety contained in the eukaryotic membrane protein. By adding saposin A to the eukaryotic membrane protein bound to the support, which is contained in the surfactant-treated membrane, the formation of saposin lipid particles containing the eukaryotic membrane protein becomes possible. Thus, in this example, the assembly of saposin lipid particles occurs entirely on the support and occurs with the endogenous lipids provided in the form of surfactant-treated whole cells that express the eukaryotic membrane protein to be included of interest.

[0277] 2.a. Overexpression of Membrane Protein Introduce the coding sequence of the eukaryotic membrane protein into an expression vector encoding an N-terminal FLAG tag. Before transfection, grow HEK293F cells in 293Freestyle culture medium and transfect using the PEI-Max reagent using the protocol provided by the manufacturer (ThermoFisher). The transfected cells then overexpress the membrane protein. All cells are collected approximately 48 hours after transfection.

[0278] 2.b. Preparation of solubilized membranes Collect the cells overexpressing the eukaryotic membrane protein with an added FLAG tag into a cell pellet. Then dissolve the cell pellet in HNG buffer II further containing a 25-fold protein inhibitor cocktail at a final concentration of 2-fold. Subsequently, add a solution containing 10% GDN (w / v) in water to the resuspended cells to a final concentration of 1% GDN (w / v). Then incubate the sample on a rotating wheel in a cold cabinet for 5 minutes. Thereafter, centrifuge the sample at 5000 g for 5 minutes at 4 °C. Collect the supernatant containing the solubilized material including the surfactant-treated membrane and incubate it on a rotating wheel in a cold cabinet for an additional 50 minutes. After this incubation step, centrifuge the supernatant at 30000 g and 4 °C for 30 minutes to remove membrane debris and then use it in the next step 2.c for binding to the affinity support.

[0279] 2.c. Binding and elution to the affinity support The following buffers are used. - HNG buffer II: 50 mM HEPES pH 7.5, 200 mM NaCl and 10% glycerol - EB3: 50 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol, 250 μg / mL FLAG-peptide

[0280] Four columns are prepared that allow removal of unbound material by gravity flow-through by loading 100 μl of equilibrated M2 anti-FLAG affinity purification beads (Sigma Aldrich) onto each column. Subsequently, 500 μl of the solubilized membrane obtained in step 2.b is added to each column. Then, to allow efficient binding of the eukaryotic membrane protein tagged with FLAG to the affinity beads, the flow-through is passed through the column three more times. The affinity beads loaded with the eukaryotic membrane protein tagged with FLAG are not washed at this stage and contain the affinity-bound eukaryotic membrane protein in an environment that partially contains native cell membrane lipids and surfactant micelles and HNG buffer II components (the "dead volume" of the beads).

[0281] Add different amounts (0 - 6 ml) of 1 mg / ml saposin A to the corresponding columns. - Sample 1: 1 ml of HNG buffer II - Sample 2: 1 ml of saposin A - Sample 3: 3 ml of saposin A - Sample 4: 6 ml of saposin A

[0282] The mixture is then transferred to four new tubes, incubated at 4 °C for 25 minutes using a rotating wheel, and then returned to the columns. After removing unbound material from the columns using gravity flow-through, wash with 10 CV of HNG buffer II and perform an elution step with 500 μl of elution buffer EB3.

[0283] 2.d. Analysis of sarpoparticles The eluates obtained after incubating the affinity beads with different amounts of SapA (see samples 1 - 4 in section 2.c above) are concentrated at 13000 g and 4 °C using an Amicon Ultra-2 centrifugal filter (10 kDa NMWL). To detect the formed sarpoparticles, the concentrated samples are further analyzed by SEC using a Superose 6 Increase 5 / 150 GL column in surfactant-free HNG buffer II.

[0284] Using the above experimental workflow, it is expected that sapropin lipoprotein particles can be obtained from untreated cells as starting material while the eukaryotic membrane protein of interest is bound to the affinity support and has not been subjected to mechanical cell lysis. As a negative control under these conditions, excluding saposin A from the liquid environment should prevent the formation of sariplo particles.

[0285] V. Generation of sariplo particles on a support Example 3: In this example, the reconstitution of sariplo particles was carried out according to alternative (I) of the method of the invention, i.e., saposin was immobilized on an affinity support. For this purpose, saposin was biotinylated and bound to an avidin affinity bead matrix. The formation of sariplo particles according to the invention was enabled by contacting the saposin bound to the support with saposin without additional tags, lipids, and optionally hydrophobic agents. Thus, an assembly of saposin lipid particles occurred on the support.

[0286] 3.a. Preparation of biotinylated saposin A Saposin A was biotinylated using the EZ-Link® NHS-biotin reagent (Thermo Fisher, reference number 21343) according to the manufacturer's protocol. Subsequently, quantification of the number of biotins per saposin A was performed using the Quant*Tag Biotin Kit (Vector laboratory, BDK-2000), and it was shown that there was 1.1 biotins per saposin A molecule.

[0287] 3.b. Binding and elution to the affinity support A monomer avidin matrix (Thermo Fisher 20228) was prepared and washed according to the manufacturer's protocol. Biotinylated saposin A was bound to the prepared avidin affinity matrix. For each sample, 100 μl of biotinylated saposin A (1.2 mg / ml) was bound to 25 μl of avidin affinity matrix by passing the biotinylated saposin A through the matrix contained in a column (BioRad, Polyprep Chromatography column, product number 7311550) three times. Subsequently, the affinity matrix was extensively washed with HNG buffer II to ensure removal of unbound saposin A.

[0288] Two different particle assembly conditions were evaluated using an avidin affinity matrix loaded with saposin A. In sample 1, saposin A without added brain lipids and tags was added to an affinity resin with pre-immobilized saposin A. In sample 2, saposin A without added brain lipids, membrane protein (bacterial ion channel membrane protein T2), and tags was added to an affinity resin with pre-immobilized saposin A.

[0289] The brain lipid solution was prepared by dissolving 5 mg / ml brain lipids (Sigma-Aldrich) in 0.5% DDM and pre-incubating at 37 °C for 5 minutes.

[0290] The bacterial ion channel membrane protein T2 was purified as previously described in F Guettou et al., Nature structural & Molecular Biology, 21;728-731, 2014.

[0291] The particle assembly conditions for samples 1-2 were as follows. - Sample 1: 16 μl of the brain lipid solution was added to an affinity resin with pre-immobilized saposin A and incubated at room temperature for 5 minutes, followed by the addition of 100 μl of saposin A without added tags (1.2 mg / ml). -Sample 2: 16 μl of the brain lipid solution was mixed with 8 μl of T2 (10 mg / ml) and incubated at 37 °C for 5 minutes. After that, the mixture was added to an affinity resin having pre-immobilized saposin A. Then, after incubating the sample at room temperature for 5 minutes, 100 μl of untagged saposin A (1.2 mg / ml) was added.

[0292] Next, the two samples were incubated simultaneously on a rotary wheel at room temperature for 25 minutes. Thereafter, the sample column was treated using the following buffers: -HNG buffer II: 50 mM HEPES pH 7.5, 150 mM NaCl and 10% glycerol -EB4: HNG buffer supplemented with 2 mM biotin (Thermo Fisher 29129)

[0293] The affinity beads were extensively washed with surfactant-free HNG buffer II (3 times using 10 CV), and the immobilized sample was eluted using elution buffer EB4.

[0294] 3.c. Analysis of the obtained sariplo particles The eluted sample was subjected to analytical SEC using a Superdex™ 200 5 / 150 GL analytical gel filtration column run in HNG buffer II.

[0295] The results are shown in Figure 13. For Samples 1 and 2, sariplo particles were detected in the elution profiles (see the SEC peak at 6.4 minutes for Sample 1 and the SEC peak at 4.5 minutes for Sample 2 in Figure 13). Thus, immobilized saposin A enabled the occurrence of sariplo particle aggregation on the affinity support.

[0296] Collectively, the data presented in this example clearly demonstrate that it is possible to reconstitute sariplo particles using different starting materials while one of the particle components is bound to the affinity support.

Claims

1. A method for generating saposin lipoparticle proteins, wherein the generated saposin lipoparticle proteins are - a saposin-like protein belonging to the SAPLIP family of lipid-interacting proteins, - a lipid, - a hydrophobic agent that is a membrane protein, and where the saposin-like protein is characterized by a saposin fold having a conserved alpha-helical three-dimensional structure stabilized by highly conserved intramolecular disulfide bonds, the lipid is a mixture of lipids that are naturally present in archaea, viruses, prokaryotes, eukaryotic cells or eukaryotic cell organelle membranes from which the lipid is obtained, (I) The method comprises the following steps: a) providing the lipid and the hydrophobic agent; b.1) contacting the saposin-like protein with a support capable of selectively binding the saposin-like protein in a liquid environment; c.1) contacting the saposin-like protein bound to the support with the lipid and the hydrophobic agent to enable self-assembly of the saposin lipoparticle proteins on the support; d) eluting the saposin lipoparticle proteins bound to the support; and (II) The method comprises the following steps: a) providing the hydrophobic agent and the lipid; b.2) contacting the hydrophobic agent and the lipid with a support capable of selectively binding the hydrophobic agent and the lipid; c.2) contacting the hydrophobic agent and the lipid bound to the support with the saposin-like protein to enable self-assembly of the saposin lipoparticle proteins on the support; d) a step of eluting the suppressor lipoprotein particles bound to the support; A method comprising.

2. The method according to option (I) of claim 1, wherein the support comprises a capture portion, the suppressor-like protein comprises a binding portion, and the capture portion can selectively bind the binding portion in the suppressor-like protein.

3. The method according to option (II) of claim 1, wherein the support comprises a capture portion, the hydrophobic agent comprises a binding portion, and the capture portion can selectively bind the binding portion in the hydrophobic agent.

4. The support is i. beads, ii. a bed, and iii. a membrane, and / or iv. a solid support The method according to any one of claims 1 to 3, which is in the form of.

5. In step a), the hydrophobic agent and the lipid are provided in the form of a membrane of a virus, archaea, eukaryote or prokaryote containing the hydrophobic agent and the lipid to be incorporated into the suppressor lipoprotein particles. The method according to any one of claims 1 to 4.

6. In step c.1), in order to enable the formation of a library of suppressor-like particles, the suppressor-like protein bound to the support is contacted with the membrane of the virus, archaea, eukaryote or prokaryote provided in step a), and the library comprises a heterogeneous mixture of suppressor lipoprotein particles having different membrane lipid and membrane protein compositions. The method according to any one of claims 1 to 5.

7. The method according to any one of claims 1 to 6, wherein the suppressor-like protein is suppressor A, suppressor B, suppressor C, or suppressor D.

8. The hydrophobic agent, the lipid and / or the saposin-like protein are in a solubilized state with a surfactant, and the surfactant is selected from the group consisting of alkylbenzene sulfonate or bile acid, cationic surfactants and nonionic or zwitterionic surfactants, fos-choline, CHAPS / CHAPSO, saponin, glycol-diosgenin, alkyl glycoside, glucoside, maltose-neopentyl glycol (MNG) amphiphilic substances, amphiphilic polymers (amphipol), styrene maleic acid copolymers (SMA), macrocyclic molecules or cyclic oligomers (calixarenes) based on hydroxyalkylated products of phenols and aldehydes, and mixtures thereof, the method according to any one of claims 1 to 7. **Claim 9** i. the particles obtained in step c.2) and / or c.1) are disc-shaped; ii. the particles of step c.2) and / or step c.1) have an average maximum diameter of 2 nm to 200 nm; iii. the self-assembly of the particles in step c.2) and / or step c.1) is carried out at a pH of 2.0 to 10.0, The method according to any one of claims 1 to 8.

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