Methods for the preparation of lipidated protein structures

Abstract

The present invention relates to a method for preparing a conjugate using enzymatic coupling, the conjugate comprising a biomolecule, an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety. A component comprising an enzyme label (e.g. a pentaglycine moiety), a hydrophilic spacer, a linker and a lipophilic moiety (e.g. a lipid) is enzymatically coupled to the biomolecule in an aqueous medium and then purified. Preferably there is a C-terminal motif (LPXTG) for enzymatic conjugation by a transpeptidase such as sortase A. It is intended for attaching biomolecules to, for example, liposomes, exosomes or for surface modification. Examples of components to be attached: DMA-PEG-G5.

Description

[0001] The present invention relates to a method for the preparation of peptide and protein-lipid conjugates, which exhibit strong interactions with hydrophobic surfaces and membranes while being soluble in pure aqueous dispersions. The conjugates obtained by this method can be used for hydrophobic polymer surfaces, drug delivery systems and surface artificial modification of cells.

[0002] The present invention relates to a method for preparing a conjugate using enzymatic coupling, wherein the conjugate comprises a biomolecule, an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety. The lipophilic moiety conjugated to the biomolecule is used to anchor the biomolecule on a hydrophobic polymer surface, the surface of a lipid-based drug delivery system (DDS, such as liposomes) or a whole living cell [1].

[0003] The anchoring of biomolecules on drug delivery systems has the purpose of achieving targeted drug delivery to cells, which means that the drug encapsulated by the carrier is directed to the desired cell type through ligand-target interactions. Using this interaction, the pharmacokinetic and pharmacodynamic properties of the drug can be improved. This may include, among other things, the delivery of toxic compounds to cancer cells.

[0004] The anchoring of biomolecules on cells is particularly relevant for therapies involving adoptive cell transfer. Here, cells such as stem cells or immune cells from the patient or another individual are cultured and modified ex vivo and then returned to the patient. Typical therapeutic applications include cancer immunotherapy, autoimmune diseases, regenerative medicine and tissue engineering. These therapeutic areas may benefit if the cell surfaces involved are modified in a supraphysiological manner, for example during immunotherapy by anchoring cytokines [2], during regenerative therapy by anchoring ligands leading to increased chemotaxis of mesenchymal stem cells [3,4] or during the treatment of autoimmune diseases by anchoring antigens and enzymes on cells [5, 6].

[0005] Genetic engineering can be used to engineer cell membranes, thereby providing genetic inheritance to subsequent daughter cells or the possibility of achieving both extracellular and intracellular modifications. However, genetic engineering has several disadvantages. These include the use of viral vectors, which may lead to immunogenic responses, overactivation (due to constitutive expression [7]), or de novo tumorigenesis [8, 9]. Genetic engineering is strongly dependent on transduction or transfection efficiency, which is difficult to predict. Therefore, the therapeutic efficacy of modified cells may show inconsistent therapeutic efficacy. Similarly, not all cell types are possible to modify, such as slowly dividing cells [1]. Finally, genetic engineering leads to permanent and irreversible intrinsic modifications, which may not be desired in every therapeutic case [1].

[0006] Non-genetic approaches are an important alternative to cell surface genetic engineering and can be divided into modifications based on covalent conjugation or hydrophobic insertion of structures into the cell membrane. Covalent conjugation exploits reactive structures present on the cell surface to attach the desired structure [1]. The main disadvantage of these chemical approaches is the heterogeneous conjugation products obtained, due to the plethora of possible reactive sites such as carboxylic acids, cysteines, lysines or carbohydrate structures involved in the connection of bioactive binders to the cell surface. Therefore, site-specific click chemistry [10, 11] has also been employed to conjugate proteins to the cell surface. These site-specific chemical reactions require the (genetic) introduction of appropriate auxiliary molecules on the cell surface, or may require potentially toxic catalysts, or may still result in the production of several side products

[10] .

[0007] Hydrophobic insertion is an alternative to directly modifying existing structures on the cell surface [1]. It involves lipidation of bioactive binders as a reaction to separation and subsequent affinity-based spontaneous insertion of the lipidated structure into the membrane bilayer. Since liposomal drug delivery systems are also built on bilayer structures, their membranes can also be modified with lipidated biomolecules. This process is often referred to as "post-insertion"

[12] .

[0008] Anchoring of compounds into cell membranes by hydrophobic insertion has been described as non-toxic. Also, the inserted molecules can participate in the dynamic movement of the cell membrane [1]. Compared to covalent attachment, hydrophobic insertion avoids the dependence of the degree of cell modification on the efficacy of the reaction. Also, physiological changes such as a decrease in cell membrane mobility or alteration of functional structures present on the cell surface are mitigated [1]. Hydrophobic insertion has been used in several reports to achieve cell membrane modification using polyethylene glycol [13, 14], polysaccharides

[15] , oligonucleotides [6] and peptides or proteins [4, 16-19]. The reports on protein lipidation and cell insertion listed above indicate promising approaches and results for cell membrane engineering. However, several major drawbacks have hampered their proper use, such as for cell membrane remodeling during cell therapy in humans. Martin et al. reported lipidation of peptides with sterol-like lipid anchors

[17] . The procedure is carried out on a solid phase and requires harsh reaction conditions, including the extensive use of carboxylic acid protecting groups, since the reaction is based on a PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) / HOBT (hydroxybenzotriazole) activation step. Also, an organic solvent like dimethylformamide is required. Therefore, the method is quite unsuitable for large proteins with several reactive sites and where selective protection of amino acid side chains is not possible. Moreover, a wide variety of by-products can be expected using this lipidation procedure for large proteins.

[0009] To improve the treatment of myocardial infarction with a regenerative approach, Won et al. conjugated recombinant CXC chemokine receptor 4 (CXCR4) to PEGylated lipids with two hydrophobic saturated myristyl chains and modified the surface of mesenchymal stem cells (MSCs) by hydrophobic insertion [4]. This improved the migration of the thus modified MSCs in in vitro assays, thus potentially increasing MSC homing to the site of myocardial ischemia [1]. Since CXCR4 has eight cysteines that facilitate reaction with the maleimide-modified lipids used, a wide variety of structural isomers of lipidated CXCR4 or multiple conjugated species can be expected after lipid modification [4]. These may have different biological activities or may result in different interactions of the inserted structure with the cell bilayer. This may in turn lead to different spatial accessibility of the receptor binding region and ultimately potentially lead to reduced binding of the corresponding ligand. In another study, the same team conjugated di-myristylated PEGylated lipids to an antibody drug conjugate similar to the marketed product Kadcyla®

[20] . The lipid consists of a dimyristyl motif as a hydrophobic anchor and a polyethylene glycol spacer of undescribed length, whose terminal carboxylic acid group is activated by carbodiimide-NHS chemistry. The structure preferentially couples to primary amines of lysine side chains in proteins, of which up to 70 out of 88 are described to be reactive in the Kadcyla® antibody

[10] . The inevitable heterogeneity leads to a variety of concerns from therapeutic, safety and also regulatory / analytical perspectives.

[0010] This heterogeneity can be mitigated with the help of appropriate site-selective conjugation strategies. Sortingase-mediated transpeptidation for lipidation of biomolecules is a promising approach for the above-mentioned problems due to the remarkable versatility of the reaction, the feasibility of conjugation under mild reaction conditions, and the inherent site specificity due to the conjugation between defined amino acid tags. The enzyme family of sortingases belongs to the transpeptidases, which were originally discovered as housekeeping enzymes in Gram-positive bacteria, where they mediate the anchoring of proteins to the peptidoglycan layer

[21] . Derived from Staphylococcus aureus ( Staphylococcus aureus ) recognizes the C-terminal LPxTG-motif (leucine-proline-any amino acid-threonine-glycine) in target proteins and forms a thioacyl intermediate with threonine and cysteine ​​in the catalytic center of the enzyme

[21] .

[0011] Reaction 1: General scheme of sortase A-mediated transpeptidation

[0012]

[0013] The amide bond towards the glycine is cleaved and subsequently replaced by a nucleophilic N-terminus of an incoming oligoglycine

[21] or other primary amine [22, 23] via a transpeptidation reaction.

[0014] An attempt to implement this sortase A-based conjugation strategy into hydrophobic insertion-based cell membrane engineering was conducted by Antos et al.

[16] . Antos et al. used the transpeptidase sortase A to attach triglycine-modified alkyl chains ranging from 10 to 22 carbons as well as triglycine-modified cholesterol and adamantane derivatives to LPETG-modified eGFP (enhanced green fluorescent protein, a model protein). They showed efficient association of lipidated eGFP with cells for alkyl chains equal to or longer than 14 carbon atoms and for cholesterol used as lipid anchor. However, no association of adamantane-lipidated eGFP with cell bilayers was found. Despite these promising results, the study shown by Antos et al. has several drawbacks. First, only a single lipid chain was attached to the protein, although previous reports have indicated that two lipid chains are required for reliable and stable anchoring of protein structures in membranes [24, 25]. Most likely, Antos et al. circumvented the solubility issue—a major challenge in protein lipidation—by using a single lipid chain modification. These solubility issues of long-chain anchors were further alleviated by using the detergent n-dodecylmaltoside during the lipidation of eGFP with a C22 alkyl chain, the most promising anchor for cell interaction. The use of detergents during protein lipidation is disadvantageous because both the detergent and the lipid structure have similar polarity, which may prevent proper purification, e.g. by chromatography. Similarly, the exclusion of detergents from the lipidated product may lead to stability issues due to the hydrophobicity of the obtained product. Finally, detergents may be toxic, thereby preventing the use of the lipidated product on cells, e.g. during autologous therapy in humans, or detergents may affect the integrity of the lipidated protein by denaturation. Since eGFP lipidated with cholesterol or C22 alkyl chain anchors showed significant internalization in different cell lines after 5 h, it is also questionable whether these lipid anchors serve as suitable tools to stably remodel the outer side of the cell membrane. Similarly, since no spacer between the lipid anchor and the protein was used, it may be questionable whether potential binders immobilized on the cell surface would be accessible to their target structures

[26] . Another disadvantage of the method shown by Antos et al. is the affinity-based purification method. Since both eGFP and sortase A carry a His6-tag, and the His6-tag is cleaved from eGFP during the reaction with lipids, Ni-NTA resin containing 1 M NaCl and 40 mM imidazole is used to bind and then remove all His6-tagged proteins. The strategy ignores that other compounds present in the bulk of the reaction must also be removed, especially non-conjugated lipids. Therefore, the method proposed by Antos et al. is not suitable for the use of lipidated products in a published form during, for example, in vivo studies or cell therapy.

[0015] Nagamune et al. combined hydrophobic insertion of triglycine lipids (containing a hydrophobic moiety and a polyethylene glycol spacer between triglycine units) into cells with subsequent sortase A-mediated conjugation of LPETG-modified proteins [19, 27]. The approach can therefore be considered a combination of hydrophobic insertion and covalent conjugation strategies for cell membrane engineering. The authors reported that eGFP could be successfully conjugated to triglycine-modified membranes of cancer cell lines. Furthermore, conjugation of the Fc fragment of an immunoglobulin resulted in increased phagocytosis of cancer cells by co-incubation with dendritic cells. Although the combined strategy of hydrophobic insertion and covalent conjugation is a gentle approach, several important drawbacks prevent its proper use, for example during in vivo therapy. First, sortase A is known to non-selectively conjugate LPETG-modified proteins to N-terminal glycines present on the cell membrane surface

[28] or in the pericellular matrix / fluid. Therefore, the main advantage of sortase A - the production of highly defined reaction products - is lost, as a variety of conjugates of the model protein eGFP or the Fc fragment to cell surface proteins can be assumed. Furthermore, the determination of the residues of sortase A after washing the cells is not shown. Since sortase A is a protein that can adsorb to large surfaces like the cell membrane, its complete exclusion from the cell mass may be a very challenging task. Exposure of cells to sortase A may result in nonspecific adsorption

[29] and thus considerable residues on the cell surface even after purification. These may lead to reverse reactions, implying cleavage of the conjugated construct from the cells or, more significantly, severe immune reactions after administration to the cells.

[0016] Drug delivery systems have also been modified using methods similar to the hydrophobic insertion methods used for cell membrane engineering. The so-called post-insertion methods describe the insertion of lipidated ligands from pure micelles or from mixed micelles composed of lipidated ligands and PEGylated lipids [12, 30-32]. The derivatization of protein structures that are generally used as targeting ligands in drug delivery systems

[33] occurs mainly through non-specific chemical reactions

[31] . Until now, no site-selective strategies have been published for the use of post-insertion immunoliposome formulations, although several site-selective conjugation strategies have been revealed for the “in situ” conjugation (or “post-derivatization”) of ligands onto the liposome surface

[31] . Although these techniques offer a clear improvement over previously used non-selective conjugation methods, they still have disadvantages such as the need for various catalysts, unknown toxicity of the linkers and / or reaction products used, or significant amounts of side products

[10] . Enzymatic techniques may improve these issues, and several reports describe the successful conjugation of model proteins [34–36] or targeting ligands [29, 37, 38] onto the liposome surface. However, in situ conjugation with enzymes is challenging because the enzymes can be difficult to remove from the reaction complex, potentially affecting safety due to immunogenicity and also stability (due to reverse reactions) of the drug delivery system

[29] .

[0017] The above-mentioned methods known in the art for lipidation of protein structures and subsequent derivatization of drug delivery systems or cell membranes have several disadvantages and / or result in chemical or cell products with insufficient properties regarding homogeneity or purity. The object of the present invention is to provide a method for producing suitable protein-lipid conjugates which overcomes such disadvantages, meaning that it should produce a site-selectively lipidated product free of residues originating from the reaction process, a product that is soluble without the need for additives such as detergents, and that it should provide unchanged biological functions and activities when present on complex surfaces such as cell membranes.

[0018] Thus, one object of the present invention relates to a method for preparing a conjugate, wherein the conjugate comprises a biomolecule, an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety, the method comprising enzymatically coupling a component comprising the enzyme label, the hydrophilic spacer, the linker and the lipophilic moiety with the biomolecule and purification of the conjugate in an aqueous medium. Advantageously, the method can be run without the use of any detergent.

[0019] A further object of the present invention is to insert these molecules into hydrophobic environments, such as hydrophobic polymer surfaces, lipid-based drug delivery systems or membranes of living cells.

[0020] As used herein, the term "component comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety" is also referred to as "component A".

[0021] According to a preferred embodiment, the enzyme label, hydrophilic spacer, linker and lipophilic moiety constituting component A to be coupled to the biomolecule are interconnected in the order as they are mentioned (ie enzyme label - hydrophilic spacer - linker - lipophilic moiety).

[0022] As used herein, "a" or "an" shall mean one or more. As used herein, when used with the word "including", the word "a" or "an" shall mean one or more than one. As used herein, "another" shall mean at least a second or more. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0023] As used herein, the term "biomolecule" refers to a natural or synthetic molecule generally having a molecular weight greater than about 300, and is preferably a polysaccharide or oligosaccharide, an oligopeptide or polypeptide, a protein, a peptide, a polynucleotide or oligonucleotide, and glycosylated lipid derivatives thereof. Most generally, the biomolecule is an immunotherapeutic agent, an antibody or fragment thereof, a functional derivative of any of these antibodies or fragments including a fusion protein.

[0024] As used herein, the term "enzyme label" refers to a portion that can be recognized by an enzyme and that identifies the molecule connected to the label as a substrate for a reaction catalyzed by the enzyme. When reacting with an enzyme, the enzyme label can be partially or completely removed. In the present invention, the enzyme label is located at the end of a component comprising the enzyme label, a hydrophilic spacer, a linker, and a lipophilic portion, and is directly connected to the hydrophilic spacer. The enzymatic label allows the component to be coupled to a biomolecule by an enzymatic reaction. After the component is coupled to the biomolecule, all or part of the enzyme-labeled portion is retained in the conjugate produced by the coupling.

[0025] As used herein, the term "hydrophilic spacer" refers to a portion present between specific elements of a conjugate or component A, which is hydrophilic and thereby provides some hydrophilicity to such conjugate or component A. In the present invention, the hydrophilic spacer is present between the enzyme tag and the linker. The hydrophilic spacer used in the present invention is not subject to any limitation as long as it increases the water solubility of the component or conjugate.

[0026] As used herein, the term "linker" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches a first molecule to a second molecule via a chemical bond. In the present invention, a linker is used to connect a hydrophilic spacer to a lipophilic moiety.

[0027] As used herein, the term "lipophilic moiety" refers to any hydrophobic group that is soluble or miscible with fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene.

[0028] As used herein, the term "purification" refers to a process step to reduce the amount of extraneous elements, such as side reaction products that are not conjugates and that may be present in the medium after the enzymatic reaction has been carried out (e.g. fragments of enzyme substrates that inevitably appear during conjugation, the enzyme itself or cofactors required for the reaction or unreacted substrate). Purification may include different methods known in the art, such as chromatography methods, and may include one or more steps, such as chromatography steps.

[0029] The biomolecule present in the conjugate is a polypeptide. Thus, the present invention relates to a method, wherein the biomolecule is a polypeptide.

[0030] The term "polypeptide" as used herein refers to a polymer of amino acids (generally L-amino acids) connected to one another generally by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids through amide bonds, wherein the number of amino acid residues can be from about 5 up to about one million. Preferably, the polypeptide has about 10 to about 2000 amino acid residues, and even more preferably about 20 to about 500 amino acid residues. Therefore, as used herein, polypeptides include those often referred to in the art as oligopeptides (5-10 amino acid residues), polypeptides (11-100 amino acid residues) and proteins (greater than 100 amino acid residues).

[0031] Suitable polypeptides that may be present as biomolecules include antigens, cell adhesion proteins including integrins and cadherins, peptide hormones, especially growth factors, cytokines, especially interleukins, receptors associated with any of these molecules, enzymes and natural or artificial antibodies and fragments thereof. Therefore, the present invention further relates to a method, wherein the biomolecule conjugated to "component A" is an antigen, a cell adhesion protein such as an integrin or a cadherin, a peptide hormone such as a growth factor, a cytokine such as an interleukin, a receptor associated with any of these molecules, an enzyme or a natural or artificial antibody or a fragment thereof.

[0032] As used herein, the term "antigen" refers to an entity or fragment thereof that can bind to an antibody. Antigens can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term "antigen" includes a region known as an antigenic determinant or epitope, which refers to a portion of an antigen that is contacted or plays an important role in supporting the contact residues in the antigen that are responsible for antigenicity or an antigenic determinant.

[0033] As used herein, the term "cell adhesion protein" refers to a large family of cell adhesion proteins that have an extracellular region within the structure as a cell recognition site and are involved in the mediation of cell-to-cell interactions. The term "cell adhesion protein" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequences of adhesion proteins, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof.

[0034] As used herein, the term "integrin" refers to cell adhesion proteins that allow cells to bind and respond to the extracellular matrix and are involved in a variety of cellular functions, such as wound healing, cell differentiation, tumor cell homing and apoptosis. Functional integrins consist of two transmembrane glycoprotein subunits called α and β, which are non-covalently bound. Like the β subunits, the α subunits all share some homology with each other. Receptors always contain one α chain and one β chain. Examples include α6β1, α3β1, α7β1, LFA-1, etc. As used herein, the term "integrin".

[0035] As used herein, the term "cadherin" refers to a cell adhesion protein that is a member of the cadherin superfamily of proteins. The cadherin superfamily includes, for example, the classical cadherin subfamily, specific examples of which include E-cadherin, N-cadherin, and P-cadherin, and the desmoplakin subfamily, specific examples of which include desmoglein 1, 2, and 3, and desmocollin 3. Natural sources of cadherin can be found in vertebrates, including humans, farm animals, sports animals, primates, rodents, and pets, such as chickens, pigs, sheep, horses, cattle, rabbits, mice, and rats.

[0036] As used herein, the term "peptide hormone" refers to proteins with endocrine function, such as insulin, proinsulin, parathyroid hormone, relaxin, prorelaxin, insulin, glucagon, calcitonin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH).

[0037] As used herein, the term growth factor refers to a protein or polypeptide capable of stimulating cell growth. They include, but are not limited to, epidermal growth factor (EGF), human growth hormone (HGF), nerve growth factors such as NGFβ, N-methionyl human growth hormone, bovine growth hormone, liver growth factor, platelet growth factor; transforming growth factors (TGFs), such as TGFα and TGFβ; fibroblast growth factor ephrin (Eph), erythropoietin (EPO), glial cell stimulating factor (GSF); colony stimulating factors (CSF) including macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF); stem cell growth factor (SCGF) (also known as cyan factor); stromal cell-derived factor (SDF), its effective fragments and combinations thereof; and vascular endothelial growth factor (VEGF). Other growth factors may include hepatocyte growth factor (HGF), angiopoietin-1, angiopoietin-2, b-FGF and FLT-3 ligand, and effective fragments thereof.

[0038] As used herein, the term "cytokine" refers to a protein released by one cell population that acts as an intercellular mediator on another cell. Examples of such cytokines are lymphokines and monokines. Included among the cytokines are interleukins; prolactin; placental lactogen; mouse gonadotropin-related peptide; inhibin; activin; thrombopoietin (TPO); interferons, such as IFNα, IFNβ, and IFNγ; and TNFα or TNFβ.

[0039] As used herein, the term "interleukin" refers to any of a variety of cytokines secreted by immune cells that regulate a range of immune system functions. One skilled in the art will appreciate the presence or level of one or more interleukins, including, but not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24.

[0040] The term "antibody" or "immunoglobulin" is used in the broadest sense herein and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The term generally includes heterogeneous antibodies composed of two or more antibodies or fragments thereof with different binding specificities linked together.

[0041] Depending on the amino acid sequence of their constant region, intact antibodies can be assigned to different "antibody (immunoglobulin) classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into "subclasses" (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The preferred major class of antibodies used as biomolecules is IgG, and in more detail IgG1 and IgG2.

[0042] Antibodies are usually glycoproteins with a molecular weight of about 150,000, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds in the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. Based on the amino acid sequence of its constant domain, the "light chain" of an antibody from any vertebrate species can be assigned to one of two clearly distinct types called kappa (κ) and lambda (λ).

[0043] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv and Fc fragments, diabodies, linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from one or more antibody fragments. A "complete" antibody is an antibody that comprises an antigen binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. Preferably, the intact antibody has one or more effector functions.

[0044] As used herein, the term "artificial" refers to compositions and systems that are designed or prepared by humans and do not occur naturally. For example, an artificial polypeptide (e.g., an antibody or antibody fragment) is a polypeptide comprising a non-natural sequence (e.g., a polypeptide that is not 100% identical to a naturally occurring protein or fragment thereof).

[0045] As used herein, consistent with the above definition of "artificial", the term "artificial antibody" refers to an antibody having an amino acid sequence or chemical composition different from those found in natural antibodies. An artificial antibody is not a subsequence of a naturally occurring protein, either wild-type (i.e., the most abundant) or a mutant form thereof. As used herein, an "artificial antibody" can be produced or synthesized by any suitable method (e.g., recombinant expression, chemical synthesis, enzymatic synthesis, purification from whole animals, etc.).

[0046] According to a preferred embodiment of the present invention, the antibody used as biomolecule is a monoclonal antibody or a fragment thereof, such as a single chain variable fragment (scFv), a variable fragment (Fv), or an antigen binding fragment (Fab, Fab' or F(ab')2), a heavy chain-only antibody or a fragment thereof of camelid or cartilaginous fish origin, such as VHH or vNAR, or wherein the artificial polypeptide is a DARPin, an adnectine, anticalin or affibody.

[0047] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for the naturally occurring mutation that may exist in a small amount, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to the polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of an antigen. In addition to their specificity, monoclonal antibodies are advantageous because they can be synthesized without being contaminated by other antibodies. The method for preparing monoclonal antibodies includes Kohler and Milstein (1975, Nature 256, 495) and the hybridoma method described in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" (1985, Burdon et al., ed., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevier Science Publishers, Amsterdam), or can be prepared by well-known recombinant DNA methods (see, e.g., US 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:58, 1-597 (1991).

[0048] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each containing a single antigen-binding site and CL and CH1 regions, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily.

[0049] The "Fc" region of an antibody typically includes the CH2, CH3 and hinge regions of the IgG1 or IgG2 antibody major classes. The hinge region is a group of about 15 amino acid residues that connects the CH1 region to the CH2-CH3 region.

[0050] Pepsin treatment produces a "F(ab')2" fragment that has two antigen binding sites and is still able to cross-link antigen. Further treatment with a suitable reducing agent such as tri(2-carboxyethyl)phosphine (TCEP), β-mercapto-ethylamine or dithiothreitol provides a Fab' fragment. "Fv" is the minimum antibody fragment that contains a complete antigen recognition and antigen binding site. The region consists of a dimer of one heavy chain and one light chain variable domain in tight non-covalent association. It is in this configuration that the three hypervariable regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. "Fab'" fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them.

[0051] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired structure for antigen binding. Single-chain Fv antibodies are known, for example, from Plückthun (The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)), WO93 / 16185; US 5,571,894; US 5,587,458; Huston et al. (1988, Proc. Natl. Acad. Sci. 85, 5879) or Skerra and Plueckthun (1988, Science 240, 1038).

[0052] As used herein, the term "heavy chain antibody only" or "HCAb" refers to a functional antibody that includes a heavy chain but lacks the light chain typically found in an antibody. Camelids (e.g., camels, llamas, or alpacas) or cartilaginous fish (e.g., sharks, mullets, or rays) are known to produce HCAbs. Camelid and cartilaginous fish antibodies include a heavy chain but lack a light chain. HCAbs derived from camelids or cartilaginous fish are referred to as "heavy chain antibodies derived from camelids" and "heavy chain antibodies derived from cartilaginous fish," respectively.

[0053] The term "VHH" as used herein refers to the variable region of the heavy chain of a camelid antibody. Thus, the VHH region from such camelid antibodies represents the minimum structural elements required to specifically bind an antigen of interest in these species. Camelid VHH domains have been found to bind antigens with high affinity (Desmyter et al. (2001), J. Biol. Chem. 276:26285-90) and have high stability in solution (Ewert et al. (2002), Biochemistry 41:3628-36).

[0054] As used herein, the term "vNAR" refers to a single variable neoantigen receptor (NAR) domain antibody fragment. vNAR fragments are single domain antibody fragments derived from heavy chain antibodies, such as shark immunoglobulin neoantigen receptor antibodies (IgNARs).

[0055] The term "designed ankyrin repeat protein" or "DARPin" refers to an artificial polypeptide prepared by genetic engineering that has high specificity and high binding affinity for a target protein. DARPin originates from natural ankyrin protein and has a structure in which at least 2 or at least 3 ankyrin repeat motifs, such as 3, 4 or 5 ankyrin repeat motifs, are repeated. For example, DARPins comprising 3, 4 or 5 ankyrin repeat motifs may have molecular weights of about 10 kDa, about 14 kDa and about 18 kDa, respectively. DARPin includes a core portion that performs structural functions and a target binding portion outside the core that binds to a target. The core portion includes a conserved amino acid sequence, and the target binding portion includes a different amino acid sequence depending on the target.

[0056] The term "adnectine" as used herein refers to a monobody, which is an artificial antibody, which is constructed using the fibronectin type III domain (FN3).

[0057] The term "anticalin" as used herein relates to an artificial antibody derived from lipocalin that specifically binds to GPD1 and inhibits GPD1 activity. Anticalin has a barrel-like structure formed by eight antiparallel β-strands connected in pairs by loops and attached α-helices and shared with naturally occurring lipocalin.

[0058] The term "affibody" as used herein refers to a recombinant protein consisting of one polypeptide chain, which comprises a domain responsible for selective interaction with an antigen, such as a specific tumor marker like HER2 (e.g. C. Steffen, M. Wikman, V. Tolmachev, GP Adams, FY Nilsson, S. Ståhl, J. Carlsson: Invitro characterization of a bivalent anti-HER-2 affibody with potential forradionuclide-based diagnostics, Cancer Biother. Radiopharm., 20 (2005), pp. 239-248), which can be combined with another polypeptide to ensure better properties of antigen binding, higher stability and additional functions, such as the possibility of controlled binding to another substance or a solid surface. Its mass usually falls within the range of several thousand daltons to more than twelve thousand daltons.

[0059] According to a preferred embodiment, the biomolecule present in the composition used in the method of the invention is a single domain antibody derived from the variable domain (VHH) of a heavy chain only antibody of a camelid.

[0060] Enzymatic coupling of the biomolecule to component A can be accomplished by using a transpeptidase such as sortase. A preferred transpeptidase for use in the method of the invention is sortase A.

[0061] In order to enable the biomolecule to be coupled to component A, the biomolecule carries a C-terminal motif as a recognition signal for a transpeptidase. Thus, in a suitable embodiment of the method of the invention, the biomolecule carries a C-terminal motif for enzymatic conjugation by a transpeptidase, preferably sortase A, before it is coupled to a component (component A) comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic component portion.

[0062] According to a suitable embodiment, the biomolecule carries as a C-terminal motif an amino acid sequence consisting of "Leucine-Proline-X-Threonine-Glycine" (LPXTG). In this C-terminal motif, "X" can be any proteinogenic amino acid except cysteine ​​and tryptophan. Therefore, the present invention also relates to a method, wherein the biomolecule comprises a C-terminal motif prior to its coupling to a component, the C-terminal motif consisting of the amino acid sequence "Leucine-Proline-X-Threonine-Glycine" (LPXTG), wherein "X" can be any proteinogenic amino acid.

[0063] As used herein, the term "proteinogenic amino acid" refers to one of the 21 amino acids directly encoded by the genetic code of eukaryotic organisms for protein synthesis, except cysteine ​​and tryptophan. Thus, the proteinogenic amino acid X present in the C-terminal motif is one of the following amino acids: glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine, whereby glutamic acid is preferred. Therefore, the present invention further relates to a method, wherein the proteinogenic amino acid present in the LPXTG motif is glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine, preferably glutamic acid.

[0064] The enzyme label comprises an N-terminal amino functionality. According to a suitable embodiment, this N-terminal amino functionality is provided by an aliphatic amine, a monoglycine or a polypeptide sequence comprising one or more N-terminal glycines, preferably penta-glycines, whose C-terminus is linked to a hydrophilic spacer. Therefore, the present invention also relates to a method, wherein the enzyme label is an aliphatic amine, a monoglycine or a polypeptide sequence comprising one or more N-terminal glycines, preferably penta-glycines, whose C-terminus is linked to a hydrophilic spacer.

[0065] The "N-terminus" in conjunction with an amino acid or polypeptide chain refers to the free amine group on the amino acid, or the free amine group on the first amino acid residue in the polypeptide chain. Similarly, the "C-terminus" in conjunction with an amino acid or polypeptide chain refers to the free carboxyl group on the amino acid, or the free carboxyl group on the last amino acid residue in the polypeptide chain.

[0066] As explained above, the hydrophilic spacer increases the water solubility of the component or conjugate. Suitable hydrophilic spacers that may be present in component A used in the method of the present invention include hydrophilic polymeric radicals (having increased affinity for aqueous solutions), i.e., polymers containing repeating structural units containing one or more hydrophilic (or polar) groups in their alkylene backbones. Examples of available hydrophilic polymeric radicals include polyoxy (C2-C3) alkylenes (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)), polysaccharides (e.g., dextran, amylopectin, chitosan, hyaluronic acid) and polyethylene imine, whereby polyethylene glycol is preferred. Therefore, suitable embodiments of the present invention further relate to a method, wherein the components comprising enzyme labels, hydrophilic spacers, linkers and lipophilic moieties used in this method with biomolecules are polyoxy (C2-C3) alkylenes (e.g., polyethylene glycol or polypropylene glycol), polysaccharides (e.g., dextran, amylopectin, chitosan, hyaluronic acid), polysialic acid, polyethylene imine, preferably polyethylene glycol.

[0067] The preferred hydrophilic spacer is "PEG" or "polyethylene glycol", which includes any water-soluble poly(ethylene oxide). In general, "PEG" means a polymer containing a majority, e.g., >50%, of subunits that are -CH2CH2O-. Different forms of PEG may differ in molecular weight, structure, or geometry (e.g., branched, linear, forked PEGs, multifunctional, etc.). The PEGs that may be present in component A used in the methods of the present invention may be "-OCH2CH2O(CH2CH2O) m -", but also includes a form in which one of the terminal "-O-" groups is replaced by a "-NH-" group, resulting in the formula "-NHCH2CH2O(CH2CH2O) m wherein m is 10 to 300, preferably 15 to 100, more preferably 20 to 70, even more preferably 25 to 50, particularly preferably 30 to 40, and most preferably 35.

[0068] The linker connects the hydrophilic spacer to the lipophilic part via a covalent bond such as CC, CO, CN and CS. Linkers that can be used to couple with the hydrophilic spacer and the lipophilic part and the coupling of the linker to the hydrophilic spacer and the lipophilic part are known in the art and are described, for example, in EP 2825156 B1. In principle, a bifunctional reagent (i.e., a reagent having two functional (terminal) groups), preferably a heterobifunctional reagent (i.e., a reagent having two different functional (terminal) groups) reacts and thereby couples with a component comprising a hydrophilic spacer and a lipophilic part. Typical functional groups include, but are not limited to, groups such as succinimidyl esters, maleimides and pyridyl disulfides. In some embodiments, the bifunctional agent is selected from, but not limited to, for example, carbodiimide, N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), dimethyl pimelimidate dihydrochloride (DMP), dimethyl suberimidate (DMS), 3,3'-dithiobispropionic acid (DTBP), N-succinimidyl 3-[2-pyridyldithio]-propionamido (SPDP), α-methylbutyric acid succinimidyl ester, biotinamidohexanoyl-6-amino-hexanoic acid N-hydroxy-succinimidyl ester (SMCC), succinimidyl-[(N-maleimidopropionamido)-dodecanol] ester (NHSPEO12), (4-iodoacetyl) aminobenzoic acid N-succinimidyl ester (SIAB), S-acetylthioacetic acid N-succinimidyl ester (SATA), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and Nh-maleimidobutyryloxy-succinimide ester (GMBS), succinimidyl dicarbonylpentane or disuccinimidyl suberate.

[0069] The composition comprising an enzyme tag, a hydrophilic spacer, a linker and a lipophilic part used in the method of the present invention can be prepared with any such linker. According to a preferred embodiment, the linker used to prepare such a composition is isoglutamine, on the δ-position amide connected to 3-amino-1,2-propanediol, and on the α-position amine function connected to the hydrophilic spacer. In the composition comprising an enzyme tag, a hydrophilic spacer, a linker and a lipophilic part, the linker part has the following formula (I):

[0070]

[0071] In this formula, the vertical dashed line indicates bonding to the atoms of the lipophilic part, and the vertical dashed line Indicates bonding to a hydrophilic spacer.

[0072] According to a suitable embodiment of the invention, the component A used in the method contains one or more saturated or unsaturated, linear or branched hydrocarbon chains independently of one another, such as fatty alcohols or fatty acids with a chain length of 6 to 30 carbon atoms, or sterols such as cholesterol. Therefore, the present invention also relates to a method, wherein the lipophilic moiety present in component A is one or more saturated or unsaturated, linear or branched hydrocarbon chains independently of one another, such as fatty alcohols or fatty acids with a chain length of 6 to 30 carbon atoms, or sterols such as cholesterol. Preferably, the lipophilic moiety is a saturated linear hydrocarbon chain.

[0073] The term "fatty alcohol" as used herein refers to a long-chain aliphatic alcohol comprising 6 to 30 carbon atoms and comprising at least one hydroxyl group OH. Preferably, the fatty alcohol has the structure R-OH, wherein R represents a linear alkyl group, optionally substituted with one or more hydroxyl groups, comprising 6 to 30, better 10 to 30, or even 10 to 22, and even better 14 to 18 carbon atoms. The fatty alcohols that can be used can be selected from lauryl alcohol (1-dodecanol), myristyl alcohol (1-tetradecanol), cetyl alcohol (1-hexadecanol), stearyl alcohol (1-octadecyl alcohol), arachidyl alcohol (1-eicosyl alcohol), behenyl alcohol (1-docosanol), wood wax alcohol (1-tetracosyl alcohol), wax alcohol (1-dodecanol), montanol (1-octacosyl alcohol) and myricol (1-triacontol) alone or as a mixture. In compound A, the fatty alcohol and the linker are preferably connected by an ether bond R 1 -OR 2 connected to each other, where R 1 is a connector and R 2 It is the alkyl group of a fatty alcohol.

[0074] The term "fatty acid" as used herein refers to a long-chain aliphatic acid comprising at least 6 carbon atoms and comprising one or two, preferably one, carboxylic acid group. Preferably, the fatty acid has the structure R-COOH, wherein R represents a linear alkyl group comprising 6 to 30, better 10 to 30, or even 10 to 22 and even better 14 to 18 carbon atoms. Suitable fatty acids include, for example, n-decanoic acid (C10, decanoic acid), n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosanoic acid (C22, behenic acid), cis-6 9-octadecanoic acid (C18, oleic acid), all-cis-6 5,8,11,14-eicosatetraenoic acid (C20, arachidonic acid), etc. In compound A, the fatty acid is preferably linked to a linker having a hydroxyl group, whereby the carboxyl group of the fatty acid is preferably linked to the hydroxyl group of the linker via an ester bond.

[0075] As used herein, the term "sterol" refers to a steroid containing at least one hydroxyl group. Steroids are characterized by the presence of a fused tetracyclic sterane ring system. Sterols include, but are not limited to, cholesterol (i.e., 2,15-dimethyl-14-(1,5-dimethylhexyl)tetracyclo[8.7.0.02,7.011,15]heptacosa-7-ene-5-ol). In compound A, the sterol and the linker are preferably connected by an ether bond R 1 -OR 2’ connected to each other, so R 1 is a connector and R 2’ It is the ring system of sterols.

[0076] According to a particularly preferred embodiment of the present invention, component A used in the process comprises two myristyl alcohols each ether-linked to a diol group of 3-amino-1,2-propanediol of the linker. Therefore, the present invention also relates to a process in which the lipophilic component part comprises two myristyl alcohols each ether-linked to a diol group of 3-amino-1,2-propanediol.

[0077] According to a suitable embodiment of the present invention, the method comprises the following steps: (a) preparing an aqueous dispersion of components comprising an enzyme tag, a hydrophilic spacer, a linker and a lipophilic part; (b) adding the enzyme and the biomolecule; (c) incubating the mixture obtained in step (b) to produce a conjugate; (d) purifying the conjugate obtained in step (c). Therefore, the present invention also relates to a method comprising the following steps:

[0078] (a) preparing an aqueous dispersion of components comprising an enzyme tag, a hydrophilic spacer, a linker and a lipophilic moiety;

[0079] (b) adding enzymes and biomolecules;

[0080] (c) incubating the mixture obtained in step (b) to produce a conjugate;

[0081] (d) Purification of the conjugate obtained in step (c).

[0082] According to a suitable embodiment of the present invention, the method uses a ligase as an enzyme. As used herein, the term "ligase" refers to an enzyme that can catalyze the connection of two macromolecules by forming a new chemical bond, usually accompanied by the hydrolysis of a small chemical side group on one of the larger molecules, or an enzyme that catalyzes the connection of two compounds together, for example, an enzyme that catalyzes the connection of CO, CS, CN, etc. Typically, a ligase catalyzes the following reaction: Ab + C → AC + b. In the present invention, a ligase is used to catalyze the connection of a hydrophilic spacer with a biomolecule.

[0083] Ligase that can be used in the present invention includes sortase, butelase, trypsiligase, subtiligase, peptiligase and omniligase, whereby sortase A is preferred. Therefore, the present invention further relates to a method, wherein the enzyme in step (b) is a ligase, including sortase, butelase, trypsiligase, subtiligase, peptiligase and omniligase, preferably sortase A.

[0084] For example, M Schmidt et al.: Enzyme-mediated ligation technologies for peptides and proteins, Current Opinion in Chemical Biology (2017) 38, pp. 1-7) describes the enzymes used in the present invention.More specifically, sortases are described, for example, by TT Hung et al.: Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif, PNAS, 1999, 96 (22) 12424-12429; butterfly pea mucins are described by GKT Nguyen et al.: Butelase1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 2014, 10:732-738; trypsiligases are described by S Liebscher et al.: N-Terminal Protein Modification by Substrate-Activated Reverse Proteolysis, Angewandte Chemie International Edition, 53-11, 1433-7851; subtiligases are described by AC Braisted et al.: Synthesis of proteins by subtiligase. Methods Enzymol 1997, 289:298-313; Omniligase and Peptiligase are described by T Nuijens T et al.: Engineering of a diverse ligase toolbox for peptide segment condensation. Adv Synth Catal 2016, 358:4041-4048 and by T Nuijens et al.: Omniligase and selective Peptiligases, efficient biocatalysts for assembling linear and cyclic peptides and protein conjugates, Chem. Today 2016, 34:16-19.

[0085] According to a preferred embodiment of the present invention, component A used in the process has the formula II

[0086]

[0087] in

[0088] m is any integer from 15 to 60, preferably from 25 to 45, more preferably from 30 to 40, and most preferably 36;

[0089] n is any integer from 3 to 27, preferably from 7 to 19, more preferably from 11 to 15, and most preferably 11;

[0090] p is any integer from 0 to 9, preferably from 2 to 7, more preferably from 3 to 5, and most preferably 4.

[0091] Therefore, the present invention also relates to a method wherein the composition comprising the enzyme tag, the hydrophilic spacer, the linker and the lipophilic part has the formula II,

[0092] in

[0093] m means 15 to 60;

[0094] n represents 2 to 27;

[0095] p represents 0 to 9.

[0096] In a particularly preferred embodiment, the process according to the invention uses as compound A a compound according to formula I in which n is 11, m is 36 and p is 4.

[0097] The conjugate obtained by the method of the present invention is very suitable for modifying lipid-based drug delivery systems, such as solid-lipid nanoparticles, nanoemulsions, micelles or liposomes, preferably liposomes. Therefore, the present invention also relates to the use of the conjugate obtained by the method for modifying lipid-based drug delivery systems such as solid-lipid nanoparticles, nanoemulsions, micelles or liposomes, preferably liposomes.

[0098] As used herein, the term "nanoparticle" refers to particles having an average size of less than 1 μm. The nanoparticles preferably have a regular shape, such as a sphere, but may also have an irregular shape.

[0099] The term "nanoemulsion" refers to a colloidal dispersion, generally a two-phase system of oil in water. The colloidal dispersion comprises droplets having an average size of 10 to 500 nm, preferably 20 to 200 nm. As used herein, the term "average size" or "mean size" refers to the average diameter of the droplets.

[0100] The average size of these systems can be measured by standard methods known to those skilled in the art, such as dynamic light scattering.

[0101] As used herein, the term "micelle" refers to aggregates of amphiphilic molecules, such as lipids, that are assembled to form particles having a hydrophobic interior and a hydrophilic exterior. Micelles are typically spherical assemblies with a diameter of less than 100 nm, although a range of micelle diameters and distinct micelle shapes, such as disc-shaped micelles, are known in the art.

[0102] As used herein, the term "liposome" refers to a vesicle composed of one or more lipids, phospholipids and / or surfactants that can be used to deliver drugs (e.g., chemotherapeutic agents) to mammals. The components of the liposome are in a bilayer structure, similar to the lipid arrangement of biological membranes.

[0103] The conjugate obtained by the method of the invention is further very suitable for modifying the membrane of living cells, preferably T cells. Therefore, the present invention further relates to the use of the conjugate obtained by the method for modifying the membrane of living cells, preferably T cells.

[0104] The term "T cell" as used herein refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes (such as B cells) by the presence of T cell receptors on the cell surface. T cells can be isolated (from spleen of animal origin or human blood donation) or obtained from commercially available sources. "T cells" include all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T-regulatory cells (Treg) and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells and neutrophils, which are capable of mediating cytotoxic reactions.

[0105] The conjugates obtained by the process of the invention are further very suitable for modifying surfaces having an affinity for hydrophobic substances such as hydrophobic polystyrene. Therefore, the present invention furthermore relates to the use of the conjugates obtained by the process for modifying surfaces having an affinity for hydrophobic substances such as hydrophobic polystyrene.

[0106] As used herein, the term "hydrophobic" refers to the degree of affinity a substance has for water. Hydrophobic substances substantially lack affinity for water, tending to repel rather than absorb water and tending not to dissolve in, mix with, or be wetted by water.

[0107] Likewise, the conjugates obtained by the method of the invention are furthermore very suitable for modifying the membrane of exosomes, and the present invention therefore also relates to the use of the conjugates obtained by said method for modifying the membrane of exosomes.

[0108] The term "exosome" as used herein refers to a cell-derived small (20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane surrounding an internal space, and which is produced by the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides and optionally contain a payload (e.g., a therapeutic agent), a recipient (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a simple sugar, a polysaccharide or a polysaccharide) or other molecules. Exosomes can be derived from production cells and isolated from production cells based on their size, density, biochemical parameters or a combination thereof. Exosomes are an extracellular vesicle.

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] The examples illustrate the present invention without limiting it thereto.

[0115] method

[0116] For protein expression, the method described by Bachran et al. was used (Bachran, M. et al.: The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent, Scientific Reports 7(1) (2017) 17916).

[0117] To this end, VHHs and recombinant SrtA28 were expressed in E. coli strains WK6 (VHHs) and BL21 (DE3) (SrtA expression). Expression cultures were grown in terrific broth (12 g / L tryptone, 24 g / L yeast extract, 5 g / L glycerol, 2.3 g / L KH2PO4, 12.5 g / L K2HPO4) at 37°C in the presence of appropriate antibiotics to an OD600 nm of 0.8. Isopropyl β-D-thiogalactopyranoside (BioChemica, #A1008,0025) was added to a final concentration of 1 mM and the cultures were further incubated at 37°C for 3 hours. The cells were harvested by centrifugation (15 min, 4000 × g, 4 ° C) and the pellet was resuspended in 20 mL PBS (150 mM NaCl, 8.3 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.4) / 400 mL culture. The resuspended sample was lysed by sonication (2 × 1 min sonication, 100% intensity, 50% duty cycle, Sonicator HD2070, Sonotrode KE76, Bandelin), centrifuged (30 min, 38000 × g, 4 ° C) and the supernatant was applied to a nickel-nitrilotriacetic acid agar (Protino Ni-NTA, Macherey-Nagel) column. All expressed proteins contained a 6×His-tag, allowing protein purification by metal chelate chromatography. The column was then washed with PBS, PBS + 20 mM imidazole and PBS + 50 mM imidazole. The protein was eluted with PBS + 250 mM imidazole. The eluted protein was concentrated in an Amicon centrifugal filter device (Millipore) with a 3 kDa cutoff (for VHHs) and a 10 kDa cutoff (for SrtA). The concentrated protein was dialyzed overnight against PBS. The concentration of the dialyzed protein was determined by measuring the absorbance at 280 nm. The purity of the purified protein was analyzed by reducing SDS-PAGE (12% gel) and Coomassie staining. The final material was determined to be >90% pure.

[0118] The absorption coefficients were based on the primary protein sequences and calculated from the online software ExPASy ProtParam, SIB, Lausanne, Switzerland.

[0119] The sequence of the protein used (the single letter code is based on Nomenclature and symbolism for aminoacids and peptides (Recommendations 1983), Pure and Applied Chemistry, 1984, p. 595])

[0120] Sortase A (SrtA7m) [SEQ ID NO 1]

[0121]

[0122] eGFP (enhanced green fluorescent protein) [SEQ ID_NO 4]

[0123]

[0124] DMA-PEG-G5

[0125] DMA-PEG-G5 is a Figure 1 The substance of the formula shown.

[0126] Example 1

[0127] Further referred to herein as DMA-PEG-G5 Figure 1 A stock solution of structure 2 (25 mg / mL in chloroform) was aliquoted in HPLC vials. Chloroform was evaporated under a gentle stream of nitrogen to produce a thin lipid film, which was hydrated with DPBS (Dulbecco's phosphate buffered saline, Sigma Aldrich, #D1408) pH 7.4 to form a micellar aqueous lipid dispersion (2 mM). On the other hand, DMA-PEG-G5 was dissolved in DPBS pH 7.4 (5-10 mg / mL) using ultrasound. 400 μL of 50 μM VHH, 25 μM Sortase-A, and 1 mM DMA-PEG-G5 (which corresponds to a target product mass of 320 μg) were incubated at 4°C for 4 hours until the conjugate was separated using reverse phase HPLC (rp-HPLC). The reaction bulk and products were separated using an Aeris Widepore C4 column (3.6 μm particle size, 100 mm length, 2.1 mm diameter, Waters Corporation, Milford, Massachusetts, USA) and a binary gradient mode (Table).

[0128] Table 2: rp-HPLC gradient mode used to separate the reaction mass and products (A: water with 0.1% trifluoroacetic acid (TFA v / v); B: acetonitrile with 0.05% TFA v / v)

[0129]

[0130] Eluent A was water with 0.1% TFA and eluent B was acetonitrile with 0.05% TFA. The analysis was performed on an Agilent 1110 HPLC system equipped with a degasser, binary pump, temperature-controlled autosampler, column oven, diode array detector (DAD) and analytical fraction collector (AFC), controlled by EZChrom Elite software (Agilent Technologies, Santa Clara, CA, USA). The column temperature was adjusted to 30°C and the autosampler temperature was 4°C. The standard analysis or separation injection volume was 5 μL or 25-100 μL, respectively. The flow rate was 0.5 mL / min. Data were recorded at 214 and 280 nm using DAD.

[0131] The conjugate peak was collected manually or using an automatic fraction collector. The pool of single injections was stored on ice until the eluent mixture was removed using a vacuum centrifuge (RVC 2-33 IR, Martin Christ, Osterodeam Harz, Germany; speed: 1500 rpm; temperature: 40°C; 100 mbar for 10 minutes, followed by 20 mbar for 20 minutes and further evaporation at 2 mbar). The resulting precipitate was hydrated with water, and the protein concentration was determined by UV spectroscopy (NP80, Implen, Westlake Village, CA, USA) using an extinction coefficient calculated by the ExPASy ProtParam web application (https: / / web.expasy.org / protparam, SIB, Lausanne, Switzerland). The yield was calculated based on the mass and concentration of the recovered protein solution and the mass of the target product. Purity was determined by rp-HPLC analysis at 214 nm with automatic peak detection for 2-15 minutes and a threshold level obtained from the background noise of the water blank.

[0132] To verify the reaction products, the method was transferred to a similar HPLC system equipped with an electrospray ionization mass spectrometer (ESI-MS, amaZonSL, Bruker Corporation, Billerica, Massachusetts, USA). The ion source type was adjusted to ESI with positive polarity. The capillary exit was 140 V and the trapdrive was adjusted to "94". The mass range mode was adjusted to enhanced resolution, and the scan range was 100-2200 m / z. Five spectra were averaged for each run. The masses were calculated using deconvolution of the raw spectra.

[0133] Example 2

[0134] Isolated VHH ENH conjugates or native VHH ENH from Example 1 (100 nM) were spiked into 100 nM eGFP. VHH ENHs are well known for their ability to increase the intrinsic fluorescence of eGFP upon binding. The increase in fluorescence intensity compared to eGFP alone was measured in a black 96-well plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) using a Spark Plate Reader (Tecan Group, Männedorf, Switzerland) with excitation and emission adjusted to 485 nm and 535 nm, respectively.

[0135] Example 3

[0136] FITC-dextran (fluorescein isothiocyanate) labeled liposomes were prepared as described elsewhere [4]. Briefly, a mixture of DPPC (1,2-dipalmitoylphosphatidylcholine) cholesterol, DPPG (1,2-dipalmitoyl-phosphatidylglycerol) and DMA-PEG-G5 (59.4:34.6:5.0:1.0, molar fractions) was dissolved in methanol to 32 mM and injected into a 10 mg / mL FITC (fluorescein isothiocyanate) dextran solution in DPBS pH 7.4 using a custom-made T-piece with a 27G needle via a computer-controlled binary pump system. The dispersion was purified and concentrated by tangential flow filtration. Lipid concentrations were determined by rp-HPLC with evaporative light scattering detection as described elsewhere [5]. To prepare immunoliposomes, lipidated VHH ENH or VHH DC13 were added to the liposome dispersion to 0.25–2 nM VHH per μM phospholipid (PL). The mixture was vortexed thoroughly and incubated at 50°C for 30 minutes.

[0137] Murine myeloid-derived suppressor CD11b+Gr-1+ cells (MDSCs) are derived from bone marrow-derived NUP- progenitor cells [6]. MDSCs were differentiated for four days in complete RPMI (RPMI 1640 medium, Life Technologies, #21875-034, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin (Life Technologies, #15140122), 100 μg / mL streptomycin (Life Technologies, #15140122), 1 mM sodium pyruvate (Life Technologies, #11360070), 50 μM 2-mercaptoethanol (Life Technologies, #31350-010), and 1× non-essential amino acids (Life Technologies, #11140-035) supplemented with 20 ng / mL interleukin-6 and 20 ng / mL granulocyte-macrophage colony-stimulating factor (Biolegend, #576304, San Diego, USA). To investigate the binding of VHH-modified liposomes, MDSCs were incubated with 500 μM liposomes (based on total lipid content) for 4 h at 4°C. Cells were washed with FACS buffer (1× PBS + 2% heat-inactivated fetal bovine serum) and antibody staining of cells was performed in the presence of Fc receptor blocking (TruStain FcX, BioLegend, #422302) in FACS buffer. SytoxBlue (Thermo Fisher Scientific, S34857) was used to exclude dead cells. Liposomes were detected by encapsulated FITC-dextran.

[0138] Example 4

[0139] T cells were isolated from spleens of C57BL / 6j mice maintained under specific pathogen-free conditions at the animal facility of the University of Heidelberg and euthanized under the guidance of registered protocol T47 / 16. Spleens were mashed and CD8+ cells were isolated using a mouse CD8a+ T cell isolation kit (#130-104-075, Miltenyi Biotec, Bergisch-Gladbach, Germany) and magnetic cell separation (LS columns, #130-042-401, Miltenyi Biotec) used according to the manufacturer's instructions after erythrocyte lysis (ACK lysis buffer, #A1049201, Thermo Fisher Scientific). Purified CD8+ T cells were stained with 1 nM Cell Tracer Far Red (#C34564, ThermoFisher Scientific) for 5 minutes at 35°C and washed with FACS buffer. Stained T cells (1.65 × 108 cells / mL) were incubated with 650 nM native or lipidated VHH DC13 and VHH ENH for 1 h at 4°C. Binding of lipidated VHH to T cells was detected by FITC-anti-llama antibody.

[0140] Murine myeloid-derived suppressor CD11b+Gr-1+ cells (MDSCs) are derived from bone marrow-derived NUP- progenitor cells [6]. MDSCs were differentiated for four days in complete RPMI (RPMI 1640 medium, Life Technologies, #21875-034, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin (Life Technologies, #15140122), 100 μg / mL streptomycin (Life Technologies, #15140122), 1 mM sodium pyruvate (Life Technologies, #11360070), 50 μM 2-mercaptoethanol (Life Technologies, #31350-010), and 1× non-essential amino acids (Life Technologies, #11140-035) supplemented with 20 ng / mL interleukin-6 and 20 ng / mL granulocyte-macrophage colony-stimulating factor (Biolegend, #576304, San Diego, USA). To investigate the membrane insertion of lipidated VHHs, MDSCs (10 7Cells / mL) were incubated with 500 nM native or lipidated VHH ENH or VHH DC13 for 30 min at 4°C. Cells were washed with FACS buffer (1× PBS + 2% heat-inactivated fetal bovine serum) and antibody staining of cells was performed in the presence of Fc receptor blocking (TruStain FcX, BioLegend, #422302) in FACS (fluorescence-assisted cell sorting) buffer. SytoxBlue (Thermo Fisher Scientific, S34857) was used to exclude dead cells. Lipidated VHHs inserted into the cell membrane were detected by FITC-anti-llama antibody (Invitrogen, #A16061). All cell analyses were performed by flow cytometry. Flow cytometry was performed on a FACSAria II (Beckton, Dickinson and Company, Franklin Lakes, NJ, USA), and the results were analyzed by FlowJo (Tree Star, V.10.0.8).

[0141] Example 5

[0142] CD11b+Gr-1+ cells from Example 4 were incubated with 100 μg / mL eGFP at 4°C for 30 minutes to detect the binding of eGFP to lipidated VHH ENH inserted into the cell membrane. For the fluorescence of eGFP, flow cytometry was performed on a FACSAriaII (Beckton, Dickinson and Company, Franklin Lakes, NJ, USA), and the results were analyzed by FlowJo (Tree Star, V.10.0.8).

[0143] Example 6

[0144] For cell-cell interaction experiments, T cells were isolated from spleens of C57BL / 6j mice maintained under specific pathogen-free conditions at the animal facility of the University of Heidelberg and euthanized under the guidance of registered protocol T47 / 16. Spleens were mashed and mouse CD8a was used according to the manufacturer's instructions after erythrocyte lysis (ACK lysis buffer, #A1049201, ThermoFisher Scientific). + CD8 T cell isolation kit (#130-104-075, Miltenyi Biotec, Bergisch-Gladbach, Germany) and magnetic cell separation (LS columns, #130-042-401, Miltenyi Biotec) were used to isolate CD8+ Cells. Purified CD8 + T cells were stained with 1 nM CellTracer Far Red (#C34564, Thermo Fisher Scientific) at 35°C for 5 minutes and washed with FACS buffer. 8 10 cells / mL) were incubated with 650 nM native or lipidated VHH DC13 and VHH ENH at 4°C for 1 h. VHH-labeled T cells were washed twice with FACS buffer and 3 × 10 7 T cells and 1.1×10 7 MDSCs (obtained as described in Example 4) were incubated at 4°C for 1 hour. T cells and MDSCs were loaded onto LS columns for CD8 + Magnetic bead separation of T cells and co-purification of MDSCs associated with T cells. Eluted cells were stained with anti-CD11b-Brilliant Violet 605 and anti-Gr-1-FITC (#101237 and #108405, Biolegend) and analyzed by flow cytometry as described above.

[0145] Example 7

[0146] In a ThermoFisher Polysorp 96-well plate (# Nunc 475094), native or lipidated VHH ENH (Batch #3 of Example 1) was mixed with eGFP in DPBS pH 7.4 (1×, Sigma, #D1408) in different variations. Variations included: PBS, PBS with eGFP (50 nM), PBS with eGFP (50 nM) + native VHH ENH (50 nM), PBS with eGFP (50 nM) + lipidated VHH ENH (5-50 nM). The plate was incubated in an orbital shaker at 60 rpm, 37°C for 1.5 hours. Afterwards, the plate was centrifuged @ 300g for 1 minute to collect all the liquid at the bottom of the well. The plate was then measured using a TecanReader Spark (ThermoFischer) with excitation at 485 nm and emission at 525 nm. Afterwards, the liquid in the well was exchanged 5 times. After each 1., 2., 3. and 5. exchange step, the fluorescence was measured.

[0147] Surprisingly, compounds consisting of an enzyme tag, a hydrophilic spacer, a linker and a lipophilic moiety have been found Figure 1 Structure 2) can be dissolved in aqueous buffer up to 10 mg / mL (4 mM) (Example 1).

[0148] After preparing a mixture of 1 mM structure 2, 25 µM transpeptidase sortase A, and 50 µM LPETG-modified single domain antibody (Example 1) (structure 1), efficient lipidation of structure 1 was observed after 4 hours after analysis by reverse phase HPLC ( Figure 3 , an exemplary chromatogram of VHH ENH used as structure 1).

[0149] Furthermore, no aggregation or precipitation of the lipidated product was observed during the reaction as analyzed by visual inspection (Example 1).

[0150] Mass spectrometry confirmed the expected molecular weights for two different single domain antibodies (VHH ENH ( Figure 4 ), VHHDC13 ( Figure 5 ), (Example 1).

[0151] The reaction mass was purified by collecting the column effluent of the reverse phase HPLC method of the "lipidated VHH" peak in a glass vial. The eluent mixture consisting of water, acetonitrile and trifluoroacetic acid was removed by vacuum centrifugation. The precipitate thus obtained could be dissolved in water to a protein content of ~1 mg / mL (Example 1).

[0152] The purity of the lipidated single domain antibodies thus obtained was analyzed by reverse phase FIPLC combined with UV detection at 214 nm. It revealed that for the following batches ( Figure 6 , Table 1) with a purity greater than >95% (based on UV area) (Example 1).

[0153] Table 1

[0154]

[0155] The yield was calculated based on the mass and concentration of the recovered protein solution and the target product mass (320 μg protein per batch). Good yields of >50% were obtained, except for VHH ENH batch #3, which was prepared without cooling the column effluent (Example 1).

[0156] The biological activity of the conjugate purified by reverse phase HPLC was analyzed by the ability of the lipidated VHH ENH to increase the fluorescence of eGFP upon binding (Example 2) [1].

[0157] Three different batches were incubated with eGFP and the fluorescence intensity at 485 nm excitation and 535 nm emission was compared to that of native VHH ENH. The data revealed no loss of binding in batch #1 and batch #2 ( Figure 7) (Example 2). Batch #3 was prepared without cooling the column effluent and showed a slightly reduced fluorescence enhancement of eGFP (Example 2).

[0158] Lipidated and isolated single domain antibodies VHH ENH and VHH DC13 were incubated with FITC-labeled liposomal drug delivery system (Example 3) at different VHH to phospholipid ratios. Liposomes modified by this post-insertion method were then incubated with cultured murine CD11b+Gr-1+ cells. VHH DC13 binds to the cell surface receptor CD11b, and VHH DC13-modified liposomes showed clear cell association with CD11b+Gr-1+ cells during flow cytometry analysis ( Figure 8 , shows the data of VHH DC13 surface density on liposomes at 2 nM / µM phospholipids, the percentage in each dot plot represents the number of positive cells in the marked gate. FSC: forward scatter) (Example 3).

[0159] When liposomes were incubated with different ratios of lipidated VHH DC13 to phospholipid concentrations, optimal binding on cells was observed for 0.5 nM VHH DC13 / µM phospholipid ( Figure 8 ) (Example 3).

[0160] To evaluate whether lipidated VHHs can be used for cell membrane remodeling, CD11b+Gr-1+ cells or T cells were incubated with lipidated or native VHH ENH or VHH DC13 (Example 4). After washing the cells, the presence of VHHs on the cell surface was confirmed by flow cytometry after staining the cells with FITC-anti-llama antibody (Example 4, Fig.10 ). Lipidated VHHs clearly increased the fluorescent signal obtained from cells in flow cytometry (Example 4). If VHH DC13 was incubated with CD11b+ cells, no difference between lipidated and non-lipidated forms was detected, since direct binding of VHH DC13 to CD11b overrides the hydrophobic insertion effect (Example 4).

[0161] To assess whether lipidated VHHs present on the cell surface are also accessible to soluble antigens, CD11b+Gr-1+ cells were incubated with lipidated or native VHH ENH or VHH DC13 (Example 5). After cell washing, the cells were incubated with the antigen corresponding to the VHH ENH, eGFP. Flow cytometry revealed that cells treated with lipidated VHH ENH selectively captured eGFP (Example 5, Fig.11 ).

[0162] In order to evaluate whether lipidated VHHs present on the cell surface can also promote cell-cell interactions (Example 6), isolated CD8+ T cells were incubated with lipidated or native VHH ENH or VHH DC13. Subsequently, the cells thus modified were incubated with CD11b+Gr-1+ cells. The cell mixture was first separated by magnetic bead-assisted cell sorting specific for the antigen CD8. Subsequently, CD8 + The retentate was stained for CD11b and Gr-1 and analyzed by flow cytometry ( Fig.14 ) (Example 6). Cell populations pre-incubated with lipidated VHH DC13 resulted in a higher signal of MDSCs in the CD8-positive column retentate, indicating a VHH-promoted cellular interaction between MDSCs and T cells ( Fig.13 ) (Example 6).

[0163] To assess whether lipidated VHHs can be immobilized on hydrophobic surfaces such as hydrophobic polystyrene, lipidated or native VHH ENH were incubated in Polysorp® 96-well plates in the presence of 50 nM eGFP (the corresponding antigen of VHH ENH) (Example 7). After several washing steps, significant coating and retention of eGFP ( Fig.14 ). The data demonstrate the usefulness of lipidated VHHs for antigen or cell capture or immobilization on cells (Example 7). They can also be used for protein purification. Sequence Listing <110> Wöll Steffen Schiller Stefan Geißler Simon Scherließ Regina <120> Methods for the preparation of lipidated protein structures <130> I <160> 4 <170> BiSSAP 1.3.6 <210> 1 <211> 186 <212> PRT <213> Homo sapiens <400> 1 Met Gly His His His His His Ser Ser Gly Leu Val Pro Arg Gly 1 5 10 15 Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met 20 25 30 Asp Ser Pro Asp Leu Gly Thr Gln Ala Lys Pro Gln Ile Pro Lys Asp 35 40 45 Lys Ser Lys Val Ala Gly Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys 50 55 60 Glu Pro Val Tyr Pro Gly Pro Ala Thr Arg Glu Gln Leu Asn Arg Gly 65 70 75 80 Val Ser Phe Ala Lys Glu Asn Ala Ser Leu Asp Asp Gln Asn Ile Ser 85 90 95 Ile Ala Gly His Thr Phe Ile Asp Arg Pro Asn Tyr Gln Phe Thr Asn 100 105 110 Leu Lys Ala Ala Lys Lys Gly Ser Met Val Tyr Phe Lys Val Gly Asn 115 120 125 Glu Thr Arg Lys Tyr Lys Met Thr Ser Ile Arg Asn Val Lys Pro Thr 130 135 140 Ala Val Glu Val Leu Asp Glu Gln Lys Gly Lys Asp Lys Gln Leu Thr 145 150 155 160 Leu Ile Thr Cys Asp Asp Tyr Asn Glu Glu Thr Gly Val Trp Glu Thr 165 170 175 Arg Lys Ile Phe Val Ala Thr Glu Val Lys 180 185 <210> 2 <211> 132 <212> PRT <213> Homo sapiens <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser His Asn Leu Ser Cys Thr Ala Ser Gly Ile Thr Phe Ser Ser Leu 20 25 30 Ala Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Asn Ile Met Arg Ser Gly Ser Ser Val Phe Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala His 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ala Thr Arg Gly Ala Trp Pro Ala Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser Gly Gly Leu Pro Glu Thr Gly Gly His His 115 120 125 His His His His 130 <210> 3 <211> 129 <212> PRT <213> Homo sapiens <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr 20 25 30 Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Gly Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Val Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Gly Gly Leu Pro Glu Thr Gly Gly His His His His His 115 120 125 His <210> 4 <211> 245 <212> PRT <213> Aequorea victoria <400> 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys His 225 230 235 240 His His His His His 245

Claims

1. A method for preparing a conjugate, wherein the conjugate comprises a biomolecule, an enzyme label, a hydrophilic spacer, a linker and a lipophilic portion; wherein the method comprises enzymatically coupling a component comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic portion with a biomolecule in an aqueous medium, and wherein the method comprises the steps of: a) preparing an aqueous dispersion of components having the formula (II): Where m is any integer from 15 to 60; n is any integer from 3 to 27; p is any integer from 0 to 9; b) adding a transpeptidase and a biomolecule which is a polypeptide and is selected from a cell adhesion protein, a peptide hormone, a cytokine or a receptor associated with any of these molecules, an enzyme or a natural or artificial antibody or a fragment thereof, c) incubating the mixture obtained in step (b) to produce a conjugate with said biomolecule, and d) purifying the conjugate obtained in step (c), wherein said purification is a process step to reduce the amount of extraneous elements.

2. The method according to claim 1, wherein the foreign element is a side reaction product which is not a conjugate and which may be present in the medium after the enzymatic reaction is performed.

3. The method according to claim 1, wherein the foreign element is a fragment of an enzyme substrate which inevitably occurs during conjugation, the enzyme itself or a cofactor required for the reaction or an unreacted substrate.

4. The method according to claim 1, wherein the component of formula (II) is 5. The method according to claim 1, wherein the biomolecule is an integrin, a cadherin, a growth factor or an interleukin.

6. The method according to claim 1, wherein the antibody is a monoclonal antibody or a fragment thereof; a heavy chain-only antibody or a fragment thereof of camelid or cartilaginous fish origin, or wherein the artificial antibody is a DARPin, an adnectine, anticalin or affibody.

7. The method according to claim 6, wherein the monoclonal antibody or fragment thereof is a single chain variable fragment, a variable fragment, or an antigen binding fragment.

8. The method according to claim 7, wherein the antigen binding fragment is Fab, Fab' or F(ab')2.

9. The method according to claim 6, wherein the heavy chain-only antibody or fragment thereof of camelid or cartilaginous fish origin is a VHH or a vNAR.

10. The method according to claim 1, wherein the biomolecule is a single domain antibody derived from the variable domain (VHH) of a camelid heavy chain only antibody.

11. The method according to claim 1, wherein the biomolecule carries a C-terminal motif for enzymatic conjugation by a transpeptidase before its coupling with the component according to formula (II).

12. The method according to claim 11, wherein the transpeptidase is sortase A.

13. The method according to claim 11, wherein the C-terminal motif consists of the amino acid sequence "Leucine-Proline-X-Threonine-Glycine" (LPXTG), wherein "X" can be any proteinogenic amino acid.

14. The method according to claim 13, wherein the proteinogenic amino acid present in the LPXTG motif is glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine.

15. The method according to claim 14, wherein the proteinogenic amino acid present in the LPXTG motif is glutamic acid.

16. The method according to claim 1, wherein the transpeptidase in step (b) is a sortase.

17. The method according to claim 16, wherein the sortase is sortase A.

18. The method according to claim 1, wherein: m is any integer from 25 to 45; n is any integer from 7 to 19; p is any integer from 2 to 7.

19. The method according to claim 1, wherein: m is any integer from 30 to 40; n is any integer from 11 to 15; p is any integer from 3 to 5.

20. The method according to claim 1, wherein: m is 36; n is 11; p is 4.

21. The method according to claim 1, wherein said purification comprises one or more chromatography steps.

22. The method according to claim 21, wherein said purification comprises reverse phase HPLC.

23. The method according to claim 4, wherein said purification comprises one or more chromatography steps.

24. The method according to claim 11, wherein said purification comprises one or more chromatography steps.

25. The method according to claim 4, wherein the transpeptidase in step (b) is sortase A, and wherein the purification comprises one or more chromatography steps.

26. Use of a conjugate obtained by a method according to any one of claims 1 to 25 for modifying a lipid-based drug delivery system.

27. Use according to claim 26, wherein the lipid-based drug delivery system is a solid-lipid nanoparticle, a nanoemulsion, a micelle or a liposome.

28. Use according to claim 26, wherein the lipid-based drug delivery system is a liposome.

29. Use of a conjugate obtained by a method according to any one of claims 1 to 25 for modifying the membrane of a living cell.

30. The use according to claim 29, wherein the living cells are T cells.

31. Use of a conjugate obtained by a method according to any one of claims 1 to 25 for modifying a surface having an affinity for a hydrophobic substance.

32. The use according to claim 31, wherein the hydrophobic substance is hydrophobic polystyrene.

33. Use of a conjugate obtained by a method according to any one of claims 1 to 25 for modifying the membrane of exosomes.

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