Process for preparation of lipidated protein structures

Abstract

The present invention relates to methods for the preparation of lipidated protein structures. The present invention relates to a method for preparing a conjugate comprising a biomolecule, an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety using enzymatic coupling. Components comprising an enzyme label (e.g., a pentaglycine moiety), a hydrophilic spacer, a linker, and a lipophilic moiety (e.g., a lipid) are enzymatically coupled with a 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. Planned for attachment of biomolecules to, for example, liposomes, exosomes or for surface modification. An example of the component to be linked is DMA-PEG-G5.

Description

[0001] This application is a divisional application of the invention patent application titled "Method for the Preparation of Lipidated Protein Structures" with the international application number PCT / EP2020 / 056339, an international filing date of March 10, 2020, and a Chinese application number of 202080020874.7. Technical Field

[0002] The present invention relates to methods for the preparation of peptide and protein-lipid conjugates that 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 the surface modification of hydrophobic polymer surfaces, drug delivery systems, and cells.

[0003] The present invention relates to a method for preparing conjugates using enzymatic coupling, the conjugates comprising 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 the surface of a hydrophobic polymer, the surface of a lipid-based drug delivery system (DDS, such as liposomes), or on the surface of an entire living cell [1]. Background Art

[0004] The anchoring of biomolecules on drug delivery systems aims to achieve targeted drug delivery to cells, which means that the drug encapsulated in the carrier is directed to the desired cell type through ligand-target interactions. Using such interactions, the pharmacokinetic and pharmacodynamic properties of the drug can be improved. This may particularly include the delivery of toxic compounds to cancer cells.

[0005] The anchoring of biomolecules on cells is particularly relevant to therapies including adoptive cell transfer. Here, cells such as stem cells or immune cells from a patient or another individual are cultured and modified ex vivo and then in 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 resulting in increased mesenchymal stem cell chemotaxis [3,4], or during the treatment of autoimmune diseases by anchoring antigens and enzymes on cells [5,6].

[0006] Genetic engineering can be used in cell membrane engineering, thus providing the possibility of genetic heritability of subsequent daughter cells or simultaneously achieving extracellular and intracellular modifications. However, genetic engineering has several drawbacks. These include the use of viral vectors, which may lead to immunogenic reactions, overactivation (due to constitutive expression [7]) or de novo tumorigenesis [8, 9]. Genetic engineering strongly depends 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 type modifications are possible, such as slowly dividing cells [1]. Finally, genetic engineering results in permanent and irreversible inherent modifications, which may not be desired in every therapeutic case [1].

[0007] Non-genetic methods are important alternative methods for cell surface genetic engineering and can be classified into modifications based on covalent conjugation or hydrophobic insertion with the cell membrane. Covalent conjugation utilizes reactive structures present on the cell surface to attach the desired structures [1]. The main drawback of these chemical methods is the resulting heterogeneous conjugation products, as excessive possible reaction sites such as carboxylic acids, cysteines, lysines or carbohydrate structures are involved in the linkage of bioactive adhesives and 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 suitable auxiliary molecules on the cell surface, or may require potentially toxic catalysts, or may still result in the production of several by-products

[10] .

[0008] Hydrophobic insertion is an option to avoid directly modifying the existing structures on the cell surface [1]. It involves the lipidation of bioactive adhesives as a separate reaction, and later the affinity-based spontaneous insertion of the lipidated structure into the membrane bilayer. Since liposome drug delivery systems are also based on bilayer structures, their membranes can also be modified with lipidated biomolecules. This process is usually referred to as "post-insertion"

[12] .

[0009] Anchoring of compounds into cell membranes by hydrophobic insertion has been described as non-toxic. Also, the inserted molecules can participate in the dynamic movements of cell membranes [1]. Compared to covalent attachment, hydrophobic insertion avoids the dependence of the reaction efficacy on the degree of cell modification. Also, physiological alterations such as a decrease in cell membrane mobility or a change in the 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], glycan

[15] , oligonucleotide [6], and peptide or protein [4,16-19]. The reports on protein lipidation and cell insertion listed above suggest promising methods and results for cell membrane engineering. However, several major drawbacks have hindered its proper use, such as cell membrane remodeling during cell therapy procedures in humans. Martin et al. reported the lipidation of peptides with sterol-like lipid anchors

[17] . The procedure was carried out on solid phase and required harsh reaction conditions, including extensive use of carboxylic acid protecting groups, since the reaction was based on the PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) / HOBT (hydroxybenzotriazole) activation step. Also, organic solvents such as dimethylformamide were required. Thus, the method is rather inapplicable to large proteins with several reaction sites and where selective protection of amino acid side chains is not possible. Additionally, a wide variety of by-products can be expected for the use of this lipidation procedure for large proteins.

[0010] To improve the treatment of myocardial infarction by a regenerative approach, Won et al. conjugated recombinant CXC chemokine receptor 4 (CXCR4) with a polyethylene glycolylated (PEGylated) lipid having two hydrophobic saturated myristoyl chains and modified the surface of mesenchymal stem cells (MSCs) by hydrophobic insertion [4]. This improved the migration of such modified MSCs in in vitro assays and thus potentially increased the homing of MSCs to the myocardial ischemic site [1]. Since CXCR4 has eight cysteines, which facilitated the reaction with the maleimide-modified lipid used, a wide variety of structural isomers or multiple conjugation species of lipidated CXCR4 could be expected after lipid modification [4]. These may have different biological activities or may lead to different interactions of the inserted structure with the cell bilayer. This may then result in different spatial accessibility of the receptor-binding region and ultimately potentially lead to a decreased binding of the corresponding ligand. In another study, the same team conjugated a di-myristylated polyethylene glycolylated lipid to an antibody-drug conjugate similar to a marketed product The lipid consisted of a di-myristoyl motif as a hydrophobic anchor and a polyethylene glycol spacer of an undescribed length, the terminal carboxylic acid group of which was activated by carbodiimide-NHS chemistry. The structure preferentially coupled to the primary amines of lysine side chains in the protein, up to 70 out of 88 of which were described as being in is reactive among antibodies

[10] . The inevitable heterogeneity leads to multiple concerns from the perspectives of therapy, safety, and also regulatory / analytical.

[0011] This heterogeneity can be alleviated by means of suitable site-selective conjugation strategies. Sortase-mediated transpeptidation for biomolecule lipidation is a promising method applicable to the above problems because the reaction has significant versatility, conjugation is feasible under mild reaction conditions, and it has inherent site specificity due to conjugation between defined amino acid tags. The enzyme family of sortases belongs to transpeptidases and was initially discovered as housekeeping enzymes in Gram-positive bacteria, where they mediate the anchoring of proteins to the peptidoglycan layer

[21] . The sortase A variant derived from Staphylococcus aureus recognizes the C-terminal LPxTG-motif (leucine-proline-any amino acid-threonine-glycine) in the target protein and forms a thioacyl intermediate with threonine and cysteine at the catalytic center of the enzyme

[21] .

[0012] Reaction Scheme 1: General scheme of sortase A-mediated transpeptidation The amide bond towards glycine is cleaved and then replaced by transpeptidation reaction with the incoming nucleophilic N-terminus of, for example, oligoglycine

[21] or other primary amines [22,23].

[0013] Antos et al. made an attempt to implement this sortase A-based conjugation strategy into hydrophobic insertion-based cell membrane engineering

[16] . Antos et al. used the transpeptidase sortase A to attach tri-glycine-modified alkyl chains ranging from 10 to 22 carbons, as well as tri-glycine-modified cholesterol and adamantane derivatives, to LPETG-modified eGFP (enhanced green fluorescent protein, a model protein). They demonstrated the efficient association of lipidated eGFP with cells for alkyl chains equal to or longer than 14 carbon atoms and for cholesterol used as a lipid anchor. However, no association of adamantane-lipidated eGFP with the cell bilayer was found. Despite these promising results, the study shown by Antos et al. had several drawbacks. First, only a single lipid chain was attached to the protein, although previous reports indicated that two lipid chains were required for reliable and stable anchoring of protein structures in the membrane [24, 25]. Most likely, Antos et al. circumvented the solubility problem by using single-lipid chain modification - a major challenge during protein lipidation. These solubility problems of long-chain anchors were further alleviated by using the detergent n-dodecyl maltoside 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 polarities, which may impede proper purification, e.g., by chromatography. Similarly, due to the hydrophobicity of the obtained product, the exclusion of the detergent from the lipidated product may lead to stability problems. Finally, the detergent may be toxic, thus precluding the use of the lipidated product on cells, e.g., during autotherapy in humans, or the detergent may affect the integrity of the lipidated protein by denaturation. Since eGFP lipidated with cholesterol or a C22 alkyl chain anchor showed significant internalization in different cell lines after 5 hours, it is also questionable whether these lipid anchors serve as suitable tools to stably reshape the outer side of the cell membrane. Similarly, since no spacer was used between the lipid anchor and the protein, it may be questionable whether the potential adhesives immobilized on the cell surface are accessible to their target structures

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

[0014] Nagamune et al. combined the hydrophobic insertion of a triglycine lipid (which contains a polyethylene glycol spacer between the hydrophobic moiety and the triglycine unit) into cells with the subsequent conjugation of a sortase A-mediated LPETG-modified protein [19, 27]. The method can thus 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 the triglycine-modified membranes of cancer cell lines. In addition, the conjugation of the Fc fragment of an immunoglobulin led to increased cancer cell phagocytosis by co-incubation with dendritic cells. Although the combined strategy of hydrophobic insertion and covalent conjugation is a mild method, several important drawbacks prevent its proper use, such as during in vivo therapy. First, sortase A is known to conjugate LPETG-modified proteins non-selectively to the N-terminal glycine present on the cell membrane surface

[28] or in the extracellular matrix / fluid surrounding the cell. Thus, the major advantage of sortase A - the generation of highly defined reaction products - is lost, as multiple conjugates of the model proteins eGFP or Fc fragments with cell surface proteins can be assumed. In addition, the determination of the residues of sortase A after cell washing was not shown. Since sortase A is a protein that can adsorb to large surfaces such as cell membranes, its complete exclusion from the whole cell may be a very challenging task. Exposure of cells to sortase A may lead to non-specific adsorption

[29] , and thus a considerable amount of residue on the cell surface even after purification. These may lead to reverse reactions, meaning the cleavage of the conjugated structure from the cell, or more significantly, a severe immune response after the administration of the cells.

[0015] Drug delivery systems are also modified using methods similar to the hydrophobic insertion methods used for cell membrane engineering. The so-called post-insertion method describes the insertion of lipidated ligands from pure micelles or from mixed micelles composed of lipidated ligands and pegylated lipids [12, 30 - 32]. Derivatization of protein structures, which are generally used as targeting ligands on drug delivery systems

[33] , mainly occurs through non-specific chemical reactions

[31] . Until now, no site-selective strategy for using post-inserted immunoliposome formulations has been disclosed, although several site-selective conjugation strategies for conjugating ligands "in situ" (or "post-derivatization") to the liposome surface have been revealed

[31] . Although these techniques provide significant improvements over previously used non-selective conjugation methods, they still have drawbacks such as the need for various catalysts, the unknown toxicity of the linkers and / or reaction products used, or a significant amount of by-products

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

[29] .

[0016] The above-mentioned methods known in the art for the lipidation of protein structures and subsequent derivatization of drug delivery systems or cell membranes have several drawbacks and / or lead to chemical or cellular products with inadequate properties regarding homogeneity or purity. The object of the present invention is to provide a method for producing suitable protein-lipid conjugates that overcomes such drawbacks, meaning that it should produce site-selectively lipidated products free of residues from the reaction process, products that are soluble without the need for additives such as detergents, and that should provide unaltered biological functions and activities when present on complex surfaces such as cell membranes. Summary of the Invention

[0017] Accordingly, 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 the enzymatic coupling in an aqueous medium of a component comprising the enzyme label, the hydrophilic spacer, the linker, and the lipophilic moiety with the biomolecule and the purification of the conjugate. Advantageously, this method can be run without using any detergent.

[0018] A further object of the present invention is to insert these molecules into a hydrophobic environment, such as the surface of a hydrophobic polymer, a lipid-based drug delivery system, or the membrane of a living cell.

[0019] 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".

[0020] According to a preferred embodiment, the enzyme label, hydrophilic spacer, linker, and lipophilic moiety that make up Component A to be conjugated to a biomolecule are interconnected in the order in which they are mentioned (i.e., enzyme label - hydrophilic spacer - linker - lipophilic moiety).

[0021] As used herein, "a" or "an" shall mean one or more. As used herein, when used in conjunction with the word "comprising", the word "a" or "an" means one or more than one. As used herein, "another" means at least a second or more. Further, unless the context otherwise requires, singular terms include the plural and plural terms include the singular.

[0022] As used herein, the term "biomolecule" refers to natural or synthetic molecules that generally have a molecular weight greater than about 300, and are preferably polysaccharides or oligosaccharides, oligopeptides or polypeptides, proteins, peptides, polynucleotides or oligonucleotides, and their glycosylated lipid derivatives. Most generally, biomolecules are immunotherapeutic agents, antibodies or fragments thereof, functional derivatives of any of these antibodies or fragments including fusion proteins.

[0023] As used herein, the term "enzyme label" refers to a moiety that can be recognized by an enzyme and identifies the molecule linked to such a label as a substrate for a reaction catalyzed by such an enzyme. When reacted 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 the component comprising such an enzyme label, a hydrophilic spacer, a linker, and a lipophilic moiety, and is directly linked to the hydrophilic spacer. The enzymatic label allows such a component to be conjugated to a biomolecule by an enzymatic reaction. After the component is conjugated to the biomolecule, all or part of the moiety of the enzyme label remains in the conjugate produced by such conjugation.

[0024] As used herein, the term "hydrophilic spacer" refers to a moiety present between specific elements of a conjugate or Component A that is hydrophilic and thus provides some hydrophilicity to such a conjugate or Component A. In the present invention, the hydrophilic spacer is present between the enzyme label and the linker. The hydrophilic spacer used in the present invention is not limited in any way as long as it increases the water solubility of the component or conjugate.

[0025] As used herein, the term "linker" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches a first molecule to a second molecule. In the present invention, a linker is used to link the hydrophilic spacer to the lipophilic moiety.

[0026] As used herein, the term "lipophilic moiety" refers to any hydrophobic group that is soluble in or miscible with fats, oils, lipids, and lipophilic nonpolar solvents such as hexane or toluene.

[0027] As used herein, the term "purification" refers to a method step of reducing the amount of foreign components, such as side reaction products that are not conjugates and may be present in the medium after an enzymatic reaction (e.g., fragments of enzyme substrates, the enzyme itself, or cofactors required for the reaction or unreacted substrates that inevitably occur during conjugation). Purification can include different methods known in the art, such as chromatography methods, and can include one or more steps, such as chromatography steps.

[0028] The biomolecule present in the conjugate is a polypeptide. Accordingly, the present invention relates to a method in which the biomolecule is a polypeptide.

[0029] As used herein, the term "polypeptide" refers to a polymer of amino acids (generally L - amino acids) that are generally linked to one another by peptide bonds of amide bonds between the α - amino and carboxyl groups of adjacent amino acids, where the number of amino acid residues can range from about 5 to about one million. Preferably, the polypeptide has from about 10 to about 2000 amino acid residues, and even more preferably from about 20 to about 500 amino acid residues. Thus, as used herein, polypeptides include those that are often referred to in the art as oligopeptides (5 - 10 amino acid residues), polypeptides (11 - 100 amino acid residues), and proteins (more than 100 amino acid residues).

[0030] Suitable polypeptides that can 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 their fragments. Accordingly, the present invention further relates to methods in which the biomolecule conjugated to "Component A" is an antigen, a cell adhesion protein such as integrin or cadherin, a peptide hormone such as a growth factor, a cytokine such as interleukin, a receptor associated with any of these molecules, an enzyme, or a natural or artificial antibody or a fragment thereof.

[0031] As used herein, the term "antigen" refers to an entity or a fragment thereof that can bind an antibody. An antigen can induce an immune response in an organism, especially an animal, more especially a mammal including a human. The term "antigen" includes regions called antigenic determinants or epitopes, which refer to a part of the antigen (which is contacted or plays an important role among the contacting residues in the antigen responsible for antigenicity or the antigenic determinant).

[0032] 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 cultures and bioactive equivalents of the natural sequences of adhesion proteins, including synthetically produced small molecule entities and their pharmaceutically acceptable derivatives and salts.

[0033] As used herein, the term "integrin" refers to a cell adhesion protein that allows cells to bind to the extracellular matrix and respond to the extracellular matrix and is associated with a variety of cell functions, such as wound healing, cell differentiation, tumor cell homing, and apoptosis. Functional integrins are composed of two transmembrane glycoprotein subunits called α and β, which are non-covalently bound. The α subunits, like the β subunits, share some homology with each other. The receptor always contains one α chain and one β chain. Examples include α6β1, α3β1, α7β1, LFA-1, etc. As used herein, the term "integrin".

[0034] 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 desmoglein subfamily, specific examples of which include desmoglein 1, 2, and 3, and desmocollin 3. Natural sources of cadherins can be found in vertebrates, including humans, farm animals, sports animals, primates, rodents, and pets, such as chickens, pigs, sheep, horses, cows, rabbits, mice, and rats.

[0035] As used herein, the term "peptide hormone" refers to proteins with endocrine functions, 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).

[0036] 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 factor (HGF), nerve growth factors such as NGFβ, N-methionyl human growth hormone, bovine growth hormone, hepatic 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 steel factor); stromal cell-derived factor (SDF), its active 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 their active fragments.

[0037] As used herein, the term "cytokine" refers to a protein released by a cell population that acts as an intercellular mediator on another cell. Examples of such cytokines are lymphokines and monokines. Included in cytokines are interleukins; prolactin; placental lactogen; murine gonadotropin-associated peptide; inhibin; activin; thrombopoietin (TPO); interferons, such as IFNα, IFNβ and IFNγ; and TNFα or TNFβ.

[0038] As used herein, the term "interleukin" refers to any of a variety of cytokines secreted by immune cells that regulate a series of immune system functions. Those skilled in the art will understand 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.

[0039] The term "antibody" or "immunoglobulin" is used herein in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two intact antibodies, and antibody fragments, provided that they exhibit the desired biological activity. The term generally includes heterologous antibodies composed of two or more antibodies or fragments thereof with different binding specificities linked together.

[0040] Depending on the amino acid sequence of their constant regions, intact antibodies can be assigned to different "antibody (immunoglobulin) classes". There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of antibodies are designated α, δ, ε, γ, and μ, respectively. The preferred main class of antibodies for use as biomolecules is IgG, and more particularly IgG1 and IgG2.

[0041] Antibodies are generally glycoproteins having a molecular weight of approximately 150,000 and are composed 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 varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable domain (VH) 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 heavy-chain variable domain. Specific amino acid residues are thought to form an interface between the light-chain and heavy-chain variable domains. Based on the amino acid sequence of their constant domains, the "light chains" of antibodies from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ).

[0042] "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. An "intact" antibody is an antibody that comprises an antigen-binding variable region as well as the light-chain constant domain (CL) and the heavy-chain constant domains CH1, CH2, and CH3. Preferably, the intact antibody has one or more effector functions.

[0043] As used herein, the term "artificial" refers to compositions and systems that are designed or prepared by a human and are not naturally occurring. For example, an artificial polypeptide (e.g., an antibody or antibody fragment) is a polypeptide that comprises a non-natural sequence (e.g., a polypeptide that does not have 100% identity with a naturally occurring protein or a fragment thereof).

[0044] 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 that is 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.).

[0045] According to a preferred embodiment of the present invention, the antibody used as a biomolecule is a monoclonal antibody or a fragment thereof, such as a single-chain variable fragment (scFv), variable fragment (Fv), or antigen-binding fragment (Fab, Fab' or F(ab')2), a heavy-chain only antibody or a fragment thereof from camelid or chondrichthyes origin, such as VHH or vNAR, or wherein the artificial polypeptide is a DARPin, adnectine, anticalin or affibody.

[0046] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they can be synthesized without contamination by other antibodies. Methods for preparing monoclonal antibodies include the hybridoma method described by Kohler and Milstein (1975, Nature 256, 495) and in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" (1985, Burdon et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevier Science Publishers, Amsterdam), or they 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 by Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:58,1-597 (1991).

[0047] Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each containing a single antigen-binding site and the CL and CH1 regions, as well as the residual "Fc" fragment, the name of which reflects its ability to crystallize readily.

[0048] The "Fc" region of an antibody typically contains the CH2, CH3, and hinge regions of the major classes of IgG1 or IgG2 antibodies. The hinge region is a group of approximately 15 amino acid residues that links the CH1 region to the CH2-CH3 region.

[0049] Pepsin treatment produces "F(ab’)2" fragments that have two antigen-binding sites and are still able to cross-link antigens. Further treatment with a suitable reducing agent such as tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethylamine, or dithiothreitol provides Fab’ fragments. "Fv" is the minimal antibody fragment that contains the complete antigen recognition and antigen-binding site. This region consists of a dimer of one heavy-chain and one light-chain variable domain that are tightly non-covalently associated. 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. Together, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv that contains only three antigen-specific hypervariable regions) has the ability to recognize and bind antigen, although 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. The "Fab’" fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy-chain CH1 domain, including one or more cysteines from the antibody hinge region. The F(ab’)2 antibody fragment was originally produced as pairs of Fab’ fragments that had hinge cysteines between them.

[0050] The "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Preferably, the scFv polypeptide further contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. Single-chain Fv antibodies are known, for example, from Plückthun (The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 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).

[0051] As used herein, the term "heavy-chain only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain that is typically found in an antibody. It is known that camelids (such as camels, llamas or alpacas) or cartilaginous fish (such as sharks, skates or rays) produce HCAbs. Camelid and cartilaginous fish antibodies contain a heavy chain but lack a light chain. HCAbs derived from camelids or cartilaginous fish are referred to as "camelid-derived heavy-chain only antibodies" and "cartilaginous fish-derived heavy-chain only antibodies", respectively.

[0052] As used herein, the term "VHH" refers to the variable region of the heavy chain of a camelid antibody. Thus, the VHH region from such camelid antibodies represents the minimal structural element required to specifically bind an antigen of interest in these species. It has been found that camelid VHH domains 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).

[0053] As used herein, the term "vNAR" refers to a single variable new antigen receptor (NAR) domain antibody fragment. The vNAR fragment is a single-domain antibody fragment derived from a heavy-chain only antibody, such as the shark immunoglobulin new antigen receptor antibody (IgNARs).

[0054] 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. DARPins are derived from natural ankyrin proteins and have 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 containing 3, 4, or 5 ankyrin repeat motifs can have molecular weights of approximately 10 kDa, approximately 14 kDa, and approximately 18 kDa, respectively. A DARPin includes a core portion that performs a structural function and a target-binding portion outside the core that binds to the target. The core portion includes a conserved amino acid sequence, and the target-binding portion includes different amino acid sequences depending on the target.

[0055] As used herein, the term "adnectine" refers to a monobody, which is an artificial antibody constructed using the fibronectin type III domain (FN3).

[0056] As used herein, the term "anticalin" relates to an artificial antibody derived from lipocalin that specifically binds to GPD1 and inhibits the activity of GPD1. An anticalin has a barrel-shaped structure formed by eight anti-parallel β-strands connected in pairs by loops and an attached α-helix and shared with the naturally occurring lipocalin.

[0057] As used herein, the term "affibody" refers to a recombinant protein consisting of a single polypeptide chain that contains 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, G. P. Adams, F. Y. Nilsson, S. J. Carlsson: In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics, Cancer Biother. Radiopharm., 20 (2005), pp. 239-248), which can bind to another polypeptide, thereby ensuring better properties for antigen binding, higher stability, and additional functions, such as the possibility of controllable binding to another substance or a solid surface. Its mass generally falls within the range of a few thousand daltons to over twelve thousand daltons.

[0058] According to a preferred embodiment, the biomolecule present in the components used in the method of the present invention is a single-domain antibody derived from the variable domain (VHH) of the heavy-chain only antibody of camelids.

[0059] The enzymatic coupling of a biomolecule with component A can be accomplished by using a transpeptidase such as sortase. A preferred transpeptidase used in the method of the present invention is sortase A.

[0060] In order for the biomolecule to be coupled with component A, the biomolecule carries a C-terminal motif as a recognition signal for the transpeptidase. Thus, in a suitable embodiment of the method of the present invention, the biomolecule carries a C-terminal motif for enzymatic conjugation by a transpeptidase, preferably sortase A, prior to its coupling with a component (component A) comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety.

[0061] According to a suitable embodiment, the biomolecule carries an amino acid sequence consisting of "leucine-proline-X-threonine-glycine" (LPXTG) as the C-terminal motif. In such a C-terminal motif, "X" can be any proteinogenic amino acid other than cysteine and tryptophan. Thus, the present invention also relates to a method wherein the biomolecule comprises a C-terminal motif prior to its coupling with a component, said C-terminal motif consisting of the amino acid sequence "leucine-proline-X-threonine-glycine" (LPXTG), wherein "X" can be any proteinogenic amino acid.

[0062] As used herein, the term "proteinogenic amino acid" refers to one of the 21 amino acids directly encoded by the genetic code of eukaryotes for protein synthesis other than 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, with glutamic acid being preferred. Thus, 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.

[0063] The enzyme label comprises an N-terminal amino functionality. According to a suitable embodiment, such an N-terminal amino functionality is provided by an aliphatic amine, monoglycine or a polypeptide sequence comprising one or more N-terminal glycines, preferably pentaglycine, which is C-terminally linked to a hydrophilic spacer. Thus, the present invention also relates to a method wherein the enzyme label is an aliphatic amine, monoglycine or a polypeptide sequence comprising one or more N-terminal glycines, preferably pentaglycine, which is C-terminally linked to a hydrophilic spacer.

[0064] The "N-terminus" together 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 of the polypeptide chain. Similarly, the "C-terminus" together 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 of the polypeptide chain.

[0065] As explained above, hydrophilic spacers increase the water solubility of the component or conjugate. Suitable hydrophilic spacers that can be present in component A used in the methods of the present invention include hydrophilic polymeric moieties (which have an increased affinity for aqueous solutions), i.e., polymers that contain repeating structural units in their alkylene backbone that contain one or more hydrophilic (or polar) groups. Examples of available hydrophilic polymeric moieties include polyoxy(C2-C3)alkylene (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)), polysaccharides (e.g., dextran, amylopectin, chitosan, hyaluronic acid), and polyethyleneimine, with polyethylene glycol being preferred. Accordingly, a suitable embodiment of the present invention further relates to a method wherein the component for use with a biomolecule in such method that comprises an enzyme label, a hydrophilic spacer, a linker, and a lipophilic moiety is polyoxy(C2-C3)alkylene (e.g., polyethylene glycol or polypropylene glycol), polysaccharide (e.g., dextran, amylopectin, chitosan, hyaluronic acid), polysialic acid, polyethyleneimine, preferably polyethylene glycol.

[0066] A preferred hydrophilic spacer is "PEG" or "polyethylene glycol", which includes any water-soluble poly(ethylene oxide). Generally, "PEG" means a polymer that contains 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, dendritic PEGs, multifunctional, etc.). The PEGs that may be present in component A used in the methods of the present invention can be "-OCH2CH2O(CH2CH2O) m -", but also include forms in which one terminal "-O-" group is replaced by an "-NH-" group, resulting in the formula "-NHCH2CH2O(CH2CH2O) m -, where m is from 10 to 300, preferably from 15 to 100, more preferably from 20 to 70, even more preferably from 25 to 50, particularly preferably from 30 to 40, and most preferably 35.

[0067] The linker connects the hydrophilic spacer and the lipophilic moiety via covalent bonds such as C-C, C-O, C-N, and C-S. Linkers useful for coupling to hydrophilic spacers and lipophilic moieties, as well as the coupling of linkers to hydrophilic spacers and lipophilic moieties, are known in the art and described, for example, in EP 2825156B1. In principle, bifunctional reagents (i.e., reagents having two functional (terminal) groups), preferably heterobifunctional reagents (i.e., reagents having two different functional (terminal) groups), react and thereby couple to a component comprising a hydrophilic spacer and a lipophilic moiety. Typical functional groups include, but are not limited to, groups such as succinimidyl esters, maleimides, and pyridyldisulfides. In some embodiments, the bifunctional reagent is selected from, but not limited to, for example, carbodiimides, N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), dimethyl pimelimidate dihydrochloride (DMP), dimethyl suberimidate (DMS), 3,3'-dithiobispropionimidate (DTBP), N-succinimidyl 3-[2-pyridyldithio]-propionamide (SPDP), succinimidyl α-methylbutyrate, biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (SMCC), succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol] ester (NH SPEO12), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl S-acetylthioacetate (SATA), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and N-γ-maleimidobutyryloxy-succinimide ester (GMBS), succinimidyl dicarbonyl pentane or disuccinimidyl suberate.

[0068] The component comprising an enzyme label, a hydrophilic spacer, a linker, and a lipophilic moiety 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 component is isoglutamine, at the amide in the δ-position bonded to 3-amino-1,2-propanediol and at the amine functionality in the α-position bonded to the hydrophilic spacer. In the component comprising an enzyme label, a hydrophilic spacer, a linker, and a lipophilic moiety, the linker moiety has the following formula (I): In such a formula, the vertical dashed line represents the atomic bond to the lipophilic moiety, and the vertical dashed line represents the bond to the hydrophilic spacer.

[0069] According to a suitable embodiment of the present invention, component A used in the method contains one or more saturated or unsaturated, straight-chain or branched hydrocarbon chains independent of each other as the lipophilic moiety, such as fatty alcohols or fatty acids having a chain length of 6 to 30 carbon atoms, or sterols such as cholesterol. Accordingly, the present invention also relates to a method in which the lipophilic moiety present in component A is one or more saturated or unsaturated, straight-chain or branched hydrocarbon chains independent of each other, such as fatty alcohols or fatty acids having a chain length of 6 to 30 carbon atoms, or sterols such as cholesterol. Preferably, the lipophilic moiety is a saturated straight-chain hydrocarbon chain.

[0070] As used herein, the term "fatty alcohol" refers to a long-chain aliphatic alcohol containing 6 to 30 carbon atoms and containing at least one hydroxyl group OH. Preferably, the fatty alcohol has the structure R-OH, where R represents a linear alkyl group, optionally substituted by one or more hydroxyl groups, including 6 to 30, more preferably 10 to 30, or even 10 to 22, and even more preferably 14 to 18 carbon atoms. The fatty alcohols that can be used can be selected individually or as a mixture from lauryl alcohol (1-dodecanol), myristyl alcohol (1-tetradecanol), cetyl alcohol (1-hexadecanol), stearyl alcohol (1-octadecanol), arachidyl alcohol (1-eicosanol), behenyl alcohol (1-docosanol), lignoceryl alcohol (1-tetracosanol), cerotinyl alcohol (1-hexacosanol), montanyl alcohol (1-octacosanol), and melissyl alcohol (1-triacontanol). In compound A, the fatty alcohol and the linker are preferably connected by an ether bond R 1 -O-R 2 to each other, where R 1 is the linker and R 2 is the alkyl group of the fatty alcohol.

[0071] As used herein, the term "fatty acid" refers to a long-chain aliphatic acid containing at least 6 carbon atoms and containing one or two, preferably one carboxylic acid group. Preferably, the fatty acid has the structure R-COOH, where R represents a linear alkyl group containing 6 to 30, more preferably 10 to 30, or even 10 to 22, and even more preferably 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-69-octadecenoic acid (C18, oleic acid), all-cis-65,8,11,14-eicosatetraenoic acid (C20, arachidonic acid), etc. In compound A, the fatty acid is preferably connected to a linker having a hydroxyl group, whereby the carboxyl group of the fatty acid is preferably connected to the hydroxyl group of the linker by an ester bond.

[0072] 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]heptacos-7-en-5-ol). In Compound A, the sterol and the linker are preferably connected via an ether bond R 1 -O-R 2’ to each other, whereby R 1 is the linker and R 2’ is the cyclic system of the sterol.

[0073] According to a particularly preferred embodiment of the present invention, Component A used in the method comprises two myristyl alcohols each ether-linked to the diol group of 3-amino-1,2-propanediol of the linker. Accordingly, the present invention also relates to a method in which the lipophilic component part comprises two myristyl alcohols each ether-linked to the diol group of 3-amino-1,2-propanediol.

[0074] According to a suitable embodiment of the present invention, the method comprises the following steps: (a) preparing an aqueous dispersion of a component comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic part; (b) adding an enzyme and a biomolecule; (c) incubating the mixture obtained in step (b) to produce a conjugate; (d) purifying the conjugate obtained in step (c). Accordingly, the present invention also relates to a method comprising the following steps (a) preparing an aqueous dispersion of a component comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic part; (b) adding an enzyme and a biomolecule; (c) incubating the mixture obtained in step (b) to produce a conjugate; (d) purifying the conjugate obtained in step (c).

[0075] According to a suitable embodiment of the present invention, the method uses a ligase as the enzyme. As used herein, the term "ligase" refers to an enzyme that can catalyze the ligation 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 joining together of two compounds, e.g., an enzyme that catalyzes C-O, C-S, C-N, etc. linkages. Generally, a ligase catalyzes the following reaction: Ab + C → A-C + b. In the present invention, the ligase is used to catalyze the hydrophilic spacer and the biomolecule.

[0076] Ligases that can be used in the present invention include sortase, butelase, trypsiligase, subtiligase, peptiligase, and omniligase, with sortase A being preferred. Accordingly, the present invention further relates to a method in which the enzyme in step (b) is a ligase, including sortase, butelase, trypsiligase, subtiligase, peptiligase, and omniligase, preferably sortase A.

[0077] 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, sortase is 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; butelase 1 is described by GKT Nguyen et al.: Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 2014, 10:732-738; Trypsiligase is described by S Liebscher et al.: N-Terminal Protein Modification by Substrate-Activated Reverse Proteolysis, Angewandte Chemie International Edition, 53-11, 1433-7851; Subtiligase is 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.

[0078] According to a preferred embodiment of the present invention, component A used in the method has the formula II wherein m is any integer from 15 to 60, preferably from 25 to 45, more preferably from 30 to 40, and most preferably 36; n is any integer from 3 to 27, preferably from 7 to 19, more preferably from 11 to 15, and most preferably 11; p is any integer from 0 to 9, preferably from 2 to 7, more preferably from 3 to 5, and most preferably 4.

[0079] Accordingly, the present invention also relates to a method in which a component comprising an enzyme label, a hydrophilic spacer, a linker, and a lipophilic moiety has the formula II, where m represents from 15 to 60; n represents from 2 to 27; p represents from 0 to 9.

[0080] In a particularly preferred embodiment, the method of the present invention uses a compound according to formula I as compound A, where n is 11, m is 36, and p is 4.

[0081] 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. Accordingly, the present invention also relates to the use of the conjugate obtained by the said method for modifying lipid-based drug delivery systems such as solid-lipid nanoparticles, nanoemulsions, micelles, or liposomes, preferably liposomes.

[0082] As used herein, the term "nanoparticle" refers to particles having an average size of less than 1 μm. Nanoparticles preferably have a regular shape, such as spherical, but may also have an irregular shape.

[0083] The term "nanoemulsion" refers to a colloidal dispersion, generally a water-in-oil two-phase system. The colloidal dispersion comprises droplets having an average size of from 10 to 500 nm, preferably from 20 to 200 nm. As used herein, the term "average size" or "mean size" refers to the average diameter of the droplets.

[0084] The average size of these systems can be measured by standard methods known to those skilled in the art, such as dynamic light scattering.

[0085] As used herein, the term "micelle" refers to an aggregate of amphiphilic molecules, such as lipids, that are assembled so as to form particles having a hydrophobic interior and a hydrophilic exterior. Micelles are generally spherical assemblies having a diameter below 100 nm, although a range of micelle diameters and various micelle shapes, such as discoidal micelles, are known in the art.

[0086] As used herein, the term "liposome" refers to a vesicle composed of one or more lipids, phospholipids, and / or surfactants, which can be used to deliver drugs (such as chemotherapeutic agents) to mammals. The components of liposomes are arranged in a bilayer structure, similar to the lipid arrangement of biological membranes.

[0087] The conjugate obtained by the method of the present 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 said method for modifying the membrane of living cells, preferably T cells.

[0088] As used herein, the term "T cell" refers to a class of lymphocytes that mature 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 the 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 (Tregs), and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic reactions.

[0089] The conjugate obtained by the method of the present invention is further very suitable for modifying a surface having an affinity for hydrophobic substances such as hydrophobic polystyrene. Therefore, the present invention further relates to the use of the conjugate obtained by the said method for modifying a surface having an affinity for hydrophobic substances such as hydrophobic polystyrene.

[0090] As used herein, the term "hydrophobic" refers to the degree of affinity of a substance for water. Hydrophobic substances substantially lack an affinity for water, thus tending to repel rather than absorb water and tending to be insoluble in water or not mix with or be wetted by water.

[0091] Similarly, the conjugate obtained by the method of the present invention is further very suitable for modifying the membrane of exosomes. Therefore, the present invention also relates to the use of the conjugate obtained by the said method for modifying the membrane of exosomes.

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[35] J.R. Silvius, R. Leventis, A Novel “Prebinding” Strategy Dramatically Enhances Sortase-Mediated Coupling of Proteins to Liposomes, Bioconjugate Chemistry 28(4) (2017) 1271-1282.

[36] A. Tabata, N. Anyoji, Y. Ohkubo, T. Tomoyasu, H. Nagamune, Investigation on the Reaction Conditions of Staphylococcus aureus Sortase A for Creating Surface-modified Liposomes as a Drug-delivery System Tool, Anticancer Research 34(8) (2014) 4521-4527.

[37] A. Tabata, Y. Ohkubo, N. Anyoji, K. Hojo, T. Tomoyasu, Y. Tatematsu, K. Ohkura, H. Nagamune, Development of a Sortase A-mediated Peptide-labeled Liposome Applicable to Drug-delivery Systems, Anticancer Research 35(8) (2015) 4411-4417.

[38] S. S. Schiller, C. Bachran, L.K. Swee, R. Scherliefβ, Pentaglycine lipid derivates-rp-HPLC-analytics for bioorthogonal anchor molecules in targeted, multiple-composite liposomal drug delivery systems, International Journal of Pharmaceutics (2018). Description of the Drawings

[0093] Figure 1 Displays the formula of DMA-PEG-G5.

[0094] Figure 2 Illustrates schematically the preparation of the conjugate as described in Example 1.

[0095] Figure 3 Shows the effective lipidation in Example 1 via analysis by reverse-phase HPLC.

[0096] Figure 4 Mass spectrometry confirming the expected molecular weight of the lipidated single-domain antibody (VHH ENH), as described in Example 7.

[0097] Figure 5 Mass spectrometry confirming the expected molecular weight of another lipidated single-domain antibody (VHH DC13), as described in Example 7.

[0098] Figure 6 Shows the purity of the lipidated VHH analyzed by reverse-phase FIPLC together with UV detection at 214 nm, as described in Example 7.

[0099] Figure 7 Shows the fluorescence intensity at 485 nm excitation and 535 nm emission of three different batches of lipidated single-domain antibodies incubated with eGFP compared to native VHH ENH, as described in Example 7.

[0100] Figure 8 Shows flow cytometry analysis data of cell binding or association of FITC-labeled liposomal drug delivery systems after incubation with VHH ENH and VHH DC13, as described in Example 7.

[0101] Figure 9 Shows the binding of liposomes incubated with different ratios of lipidated ligand to phospholipid concentration, as described in Example 7.

[0102] Figure 10 Shows flow cytometry confirmation of the presence of VHH on the cell surface of certain cells after staining with FITC-anti-lama antibody, as described in Example 7.

[0103] Figure 11 Shows flow cytometry data revealing selective capture of eGFP by cells treated with lipidated VHH ENH, as described in Example 7.

[0104] Figure 12 Schematically shows a method for assessing cell interactions between MDSC and T cells after incubation of T cells with lipidated VHHs, as described in Example 7.

[0105] Figure 13 Shows the results of flow cytometry as described in Example 7, which indicate that VHH DC13 promotes cell interactions between MDSCs and T cells.

[0106] Figure 14 Shows that lipidated VHHs incubated in the presence of eGFP in a 96-well plate after different washing steps coat and retain eGFP on hydrophobic polystyrene, as described in Example 7. 96-well plate in the presence of eGFP coat and retain eGFP on hydrophobic polystyrene, as described in Example 7. Detailed Description

[0107] The examples illustrate the invention by way of example and not limitation.

[0108] Methods For protein expression, the method described by Bachran et al. (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) was used.

[0109] For this purpose, VHHs and recombinant SrtA28 were expressed in Escherichia coli (E. coli) strains WK6 (VHHs) and BL21(DE3) (SrtA expression). In the presence of the appropriate antibiotic, expression cultures 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) were grown at 37 °C to an OD600nm 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 h. 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) per 400 mL culture. The resuspended samples were 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 agarose (Protino Ni-NTA, Macherey-Nagel) column. All expressed proteins contained a 6×His-tag, allowing purification of the proteins by metal chelate chromatography. The column was then washed with PBS, PBS + 20 mM imidazole, and PBS + 50 mM imidazole. Proteins were eluted with PBS + 250 mM imidazole. The eluted proteins were concentrated in an Amicon centrifugal filtration device (Millipore) with a 3 kDa cutoff (for VHHs) and a 10 kDa cutoff (for SrtA). The concentrated proteins were dialyzed against PBS overnight. The concentration of the dialyzed proteins was determined by absorbance measurement at 280 nm. The purity of the purified proteins was analyzed by reducing SDS-PAGE (12% gel) and Coomassie staining. The final material was determined to be >90% pure.

[0110] The extinction coefficient was calculated based on the primary protein sequence from the online software ExPASy ProtParam, SIB, Lausanne, Switzerland.

[0111] Sequences of the proteins used (single letter code according to Nomenclature and symbolism for aminoacids andpeptides (Recommendations 1983), Pure andApplied Chemistry, 1984, p. 595]) Sortase A (SrtA7m) [SEQ ID NO 1] eGFP (enhanced green fluorescent protein) [SEQ ID_NO 4] DMA-PEG-G5 DMA-PEG-G5 is a Figure 1 The substance of the formula shown.

[0112] Example 1 Further referred to herein as DMA-PEG-G5 Figure 1 The stock solution of structure 2 (25mg / mL in chloroform) was equally divided in HPLC vials. Chloroform was evaporated under a gentle nitrogen stream to produce a thin lipid film, which was hydrated to micellar aqueous lipid dispersion (2mM) with DPBS (Dulbecco's phosphate buffered saline, Sigma Aldrich, #D1408) pH 7.4. On the other hand, DMA-PEG-G5 was dissolved in DPBS pH 7.4 (5-10mg / 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 reversed-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 binary gradient mode (Table).

[0113] 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) Eluent A is water with 0.1% TFA, and eluent B is acetonitrile with 0.05% TFA. The analysis was carried out on an Agilent 1110 HPLC system equipped with a degassing device, a binary pump, a temperature-controlled autosampler, a column oven, a diode array detector (DAD), and an analytical fraction collector (AFC), and 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 analytical or separation injection volume was 5 μL or 25 - 100 μL, respectively. The flow rate was 0.5 mL / min. Data were recorded using DAD at 214 and 280 nm.

[0114] The conjugate peaks were collected manually or using an automatic fraction collector. The pooled single injections were stored on ice until the eluent mixture was removed using a vacuum centrifuge (RVC 2 - 33IR, Martin Christ, Osterode am Harz, Germany; speed: 1500 rpm; temperature: 40 °C; 100 millibar (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 the 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. The purity was determined by rp-HPLC analysis at 214 nm with automatic peak detection for 2 - 15 minutes and the threshold level obtained from the background noise of the water blank.

[0115] To verify the reaction product, the method was transferred to a similar HPLC system equipped with an electrospray ionization mass spectrometer (ESI-MS, amaZon SL, Bruker Corporation, Billerica, Massachusetts, USA). The ion source type was adjusted to ESI with positive polarity. The capillary exit was 140 V, and the trap drive 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 mass was calculated using deconvolution of the original spectra.

[0116] Example 2 The separated VHH ENH conjugate or native VHH ENH (100 nM) from Example 1 was incorporated into 100 nM eGFP. VHH ENH is well known for its ability to increase the intrinsic fluorescence of eGFP upon binding. Using a Spark Plate Reader (Tecan Group, Switzerland) with excitation and emission wavelengths tuned to 485 nm and 535 nm respectively, the increase in fluorescence intensity was measured in a black 96-well plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) compared to eGFP alone.

[0117] Example 3 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, mole fraction) 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. The lipid concentration was determined by rp-HPLC method with evaporative light scattering detection as described elsewhere [5]. To prepare immunoliposomes, lipidated VHH ENH or VHH DC13 was 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.

[0118] Mouse bone marrow-derived suppressive CD11b+Gr-1+ cells (MDSCs) are derived from bone marrow-derived NUP-ancestor 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) at 4 °C for 4 hours. Cells were washed with FACS buffer (1× PBS + 2% heat-inactivated fetal bovine serum) and antibody staining of the cells was performed in the presence of Fc receptor blockade (TruStain FcX, BioLegend, #422302) in FACS buffer. SytoxBlue (ThermoFisher Scientific, S34857) was used to exclude dead cells. Liposomes were detected by encapsulated FITC-dextran.

[0119] Example 4 T cells were isolated from the spleens of C57BL / 6j mice maintained in a specific pathogen-free condition in the animal facility of the University of Heidelberg and euthanized under the guidance of the registered protocol T47 / 16. The 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 column, #130-042-401, Miltenyi Biotec) according to the manufacturer's instructions after erythrocyte lysis (ACK lysis buffer, #A1049201, Thermo Fisher Scientific). The purified CD8+ T cells were stained with 1 nM Cell Tracer Far Red (#C34564, Thermo Fisher Scientific) for 5 minutes at 35 °C and washed with FACS buffer. The stained T cells (1.65×108 cells / mL) were incubated with 650 nM native or lipidated VHH DC13 and VHH ENH at 4 °C for 1 hour. The binding of lipidated VHH to T cells was detected by FITC-anti-camelid antibody.

[0120] Mouse bone marrow-derived suppressive CD11b+Gr-1+ cells (MDSCs) were derived from bone marrow-derived NUP-ancestor 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 of native or lipidated VHH ENH or VHH DC13 at 4 °C for 30 minutes. 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 blockade (TruStain FcX, BioLegend, #422302) in FACS (fluorescence-activated cell sorting) buffer. SytoxBlue (Thermo Fisher Scientific, S34857) was used to exclude dead cells. Lipidated VHH inserted into the cell membrane was 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).

[0121] Example 5 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 FACSAria II (Beckton, Dickinson and Company, Franklin Lakes, NJ, USA) and the results were analyzed by FlowJo (Tree Star, V.10.0.8).

[0122] Example 6 For the cell-cell interaction experiment, T cells were isolated from the spleens of C57BL / 6j mice that were maintained in the animal facility of the University of Heidelberg under specific pathogen-free conditions and euthanized under the guidance of the registered protocol T47 / 16. The spleens were mashed and after erythrocyte lysis (ACK lysis buffer, #A1049201, Thermo Fisher Scientific), mouse CD8a + T cell isolation kit (#130-104-075, Miltenyi Biotec, Bergisch-Gladbach, Germany) and magnetic cell separation (LS column, #130-042-401, Miltenyi Biotec) were used to isolate CD8 + cells. Purified CD8 +T cells were stained with 1 nM Cell Tracer FarRed (#C34564, Thermo Fisher Scientific) at 35 °C for 5 minutes and washed with FACS buffer. The stained T cells (1.65×10 8 cells / mL) were incubated with 650 nM native or lipidated VHH DC13 and VHH ENH at 4 °C for 1 hour. The VHH-labeled T cells were washed twice with FACS buffer and 3×10 7 T cells were incubated with 1.1×10 7 MDSCs (obtained as described in Example 4) at 4 °C for 1 hour. The T cells and MDSCs were loaded onto an LS column for magnetic bead separation of CD8 + T cells and co-purification of MDSCs bound to T cells. The eluted cells were stained with anti-CD11b-BV605 and anti-Gr-1-FITC (#101237 and #108405, Biolegend) and analyzed by flow cytometry as described above.

[0123] Example 7 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. The 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 at 37 °C for 1.5 hours in an orbital shaker at 60 rpm. Later, the plate was centrifuged at 300 g for 1 minute to collect all the liquid at the bottom of the wells. Then the plate was measured using a TecanReader Spark (ThermoFischer) with excitation at 485 nm and emission at 525 nm. Later, the liquid in the wells was exchanged 5 times. Fluorescence was measured after each 1st, 2nd, 3rd, and 5th exchange step.

[0124] Surprisingly, it has been found that a compound consisting of an enzyme label, a hydrophilic spacer, a linker, and a lipophilic moiety ( Figure 1 Structure 2 in) can be dissolved in an aqueous buffer up to 10 mg / mL (4 mM) (Example 1).

[0125] After preparation of a mixture of 1 mM of Structure 2, 25 μM of the transpeptidase sortase A, and 50 μM of the LPETG-modified single-domain antibody (Example 1) (Structure 1), efficient lipidation of Structure 1 was observed 4 h after analysis by reverse-phase HPLC ( Figure 3 , exemplary chromatogram of the VHH ENH of Structure 1).

[0126] Furthermore, no aggregation or precipitation of the lipidated product was observed during the reaction, as analyzed by visual inspection (Example 1).

[0127] Mass spectrometry confirmed the expected molecular weights for two different single-domain antibodies (VHH ENH ( Figure 4 ), VHHDC13 ( Figure 5 ), (Example 1).

[0128] The reaction bulk was purified by collecting the column effluent of the reverse-phase HPLC method for 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).

[0129] The purity of the lipidated single-domain antibody thus obtained was analyzed by reverse-phase FIPLC combined with UV detection at 214 nm. It revealed a purity of >95% (based on UV area) for the following batches ( Figure 6 , Table 1) (Example 1).

[0130] Table 1 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 the VHH ENH batch #3 prepared without cooling the column effluent (Example 1).

[0131] The biological activity of the conjugate purified by reverse-phase HPLC was analyzed by the ability of lipidated VHH ENH to increase the fluorescence of eGFP upon binding (Example 2) [1].

[0132] Three different batches were incubated with eGFP, and the fluorescence intensity excited at 485 nm and emitted at 535 nm was compared with that of native VHH ENH. The data revealed no loss of binding in batches #1 and #2 ( Figure 7 ). Batch #3 was prepared without cooling the column effluent and showed a slightly reduced fluorescence enhancement of eGFP (Example 2).

[0133] The lipidated and separated single-domain antibodies VHH ENH and VHH DC13 were incubated with the FITC-labeled liposomal drug delivery system (Example 3) at different VHH to phospholipid ratios. The 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 significant cell association with CD11b+Gr-1+ cells during flow cytometry analysis ( Figure 8 , showing data of the VHH DC13 surface density on liposomes of 2 nM / μM phospholipid, and the percentage in each dot plot represents the number of positive cells in the gated population. FSC: forward scatter) (Example 3).

[0134] When liposomes were incubated with different ratios of lipidated VHH DC13 to phospholipid concentration, optimal binding to the cells was observed for 0.5 nM VHH DC13 / μM phospholipid ( Figure 8 ) (Example 3).

[0135] 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 VHH on the cell surface was confirmed by flow cytometry after staining the cells with FITC-anti-camelid antibody (Example 4, Figure 10 ). Lipidated VHHs significantly increased the fluorescence signal obtained from the cells in flow cytometry (Example 4). If VHH DC13 was incubated with CD11b+ cells, no difference between the lipidated and non-lipidated forms was detected because the direct binding of VHH DC13 to CD11b overrides the hydrophobic insertion effect (Example 4).

[0136] To evaluate whether the 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 washing the cells, the cells were incubated with the antigen eGFP corresponding to VHH ENH. Flow cytometry revealed that cells treated with lipidated VHH ENH selectively captured eGFP (Example 5, Figure 11 ).

[0137] To assess whether lipidated VHHs present on the cell surface are also able to promote cell-cell interactions (Example 6), isolated CD8+ T cells were incubated with lipidated or native VHH ENH or VHH DC13. Subsequently, the thus modified cells 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, the CD8 + retentate was stained for CD11b and Gr-1 and analyzed by flow cytometry( Figure 14 )(Example 6). The cell population pre-incubated with lipidated VHH DC13 resulted in a higher signal of MDSCs in the CD8-positive column retentate, indicating cell interactions between VHH-promoted MDSCs and T cells( Figure 13 )(Example 6).

[0138] To assess whether lipidated VHHs can be immobilized on a hydrophobic surface such as hydrophobic polystyrene, lipidated or native VHH ENH was incubated in a 96-well plate 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 were observed for 25 nM and 50 nM coated in the wells( Figure 14 ). The data indicate the availability of lipidated VHHs for antigen or cell capture or immobilization on cells (Example 7). It can also be used for protein purification.

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 moiety, the method comprising the enzymatic coupling in an aqueous medium of a component comprising the enzyme label, the hydrophilic spacer, the linker and the lipophilic moiety with the biomolecule and the purification of the conjugate.

2. The method according to claim 1, wherein the biomolecule is a polypeptide.

3. The method according to claim 2, wherein the biomolecule is an antigen, a cell adhesion protein such as integrin or cadherin, a peptide hormone such as a growth factor, a cytokine such as interleukin, or a receptor associated with any of these molecules, an enzyme or a natural or artificial antibody or a fragment thereof.

4. The method according to claim 3, wherein the antibody 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 from camelids or chondrichthyans, such as VHH or vNAR, or wherein the artificial antibody is a DARPin, an adnectine, an anticalin or an affibody.

5. The method according to claim 3 or 4, wherein the biomolecule is a single-domain antibody derived from the variable domain (VHH) of a camelid heavy-chain only antibody.

6. The method according to any one of claims 1-5, wherein the biomolecule carries a C-terminal motif for enzymatic conjugation by a transpeptidase, preferably sortase A, prior to its conjugation with the component comprising the enzyme label, the hydrophilic spacer, the linker and the lipophilic moiety.

7. The method according to claim 6, wherein the C-terminal motif consists of the amino acid sequence "leucine-proline-X-threonine-glycine" (LPXTG), wherein "X" can be any proteogenic amino acid.

8. The method according to claim 7, wherein the proteogenic 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.

9. The method according to one or more of claims 1-8, wherein the enzyme label is an aliphatic amine, monoglycine, a peptide sequence having one or more N-terminal glycines, preferably pentaglycine, the C-terminus of which is linked to the hydrophilic spacer.

10. The method according to one or more of claims 1-9, wherein the hydrophilic spacer is polyoxy(C2-C3)alkylene (such as polyethylene glycol or polypropylene glycol), a polysaccharide (such as dextran, amylopectin, chitosan, hyaluronic acid), polysialic acid, polyethylenimine, preferably polyethylene glycol.

11. The method according to one or more of claims 1-10, wherein the linker is isoglutamine, amide on the δ-position linked to 3-amino-1,2-propanediol and amine-functional on the α-position linked to the hydrophilic spacer.

12. A method according to one or more of claims 1 - 11, wherein the lipophilic moiety is one or more saturated or unsaturated, straight-chain or branched hydrocarbon chains independent of each other, such as fatty alcohols or fatty acids having a chain length of 6 - 30 carbon atoms, or sterols such as cholesterol.

13. A method according to claim 12, wherein the lipophilic moiety comprises two myristyl alcohols each ether-linked to the diol group of 3-amino-1,2-propanediol.

14. A method according to one or more of claims 1 - 13, wherein the component (component A) comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety is DMA-PEG-G5.

15. A method according to one or more of claims 1 - 14, which comprises the steps (a) preparing an aqueous dispersion of a component comprising an enzyme label, a hydrophilic spacer, a linker and a lipophilic moiety; (b) adding an enzyme and a biomolecule; (c) incubating the mixture obtained in step (b) to produce a conjugate; (d) purifying the conjugate obtained in step (c).

16. A method according to claim 15, wherein the enzyme in step (b) is a ligase, including sortase, Clavibacter michiganensis subsp. michiganensis adhesin, trypsiligase, subtiligase, peptiligase and omniligase, preferably sortase A.

17. Use of a conjugate obtained by a method according to one or more of claims 1 - 16 for modifying a lipid-based drug delivery system such as solid-lipid nanoparticles, nanoemulsions, micelles or liposomes, preferably liposomes.

18. Use of a conjugate obtained by a method according to one or more of claims 1 - 16 for modifying the membrane of a living cell, preferably a T cell.

19. Use of a conjugate obtained by a method according to one or more of claims 1 - 16 for modifying a surface having an affinity for a hydrophobic substance such as hydrophobic polystyrene.

20. Use of a conjugate obtained by a method according to one or more of claims 1 - 15 for modifying the membrane of exosomes.

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