Process for the preparation of lipidated proteinaceous structures

Abstract

The present invention relates to a process for the preparation of conjugates comprising a biomolecule, an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety using enzymatic coupling.

Description

[Technical Field]

[0001] The present invention relates to a process for the preparation of peptide and protein-lipid conjugates that are soluble in neat aqueous dispersions yet exhibit strong interactions with hydrophobic surfaces and membranes. The conjugates obtained by such processes can be used in the surface engineering of hydrophobic polymer surfaces, drug delivery systems, and cells. The present invention relates to a process for the preparation of conjugates comprising a biomolecule, an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety using enzymatic coupling. The lipophilic moiety conjugated to the biomolecule is utilized to immobilize the biomolecule either on a hydrophobic polymer surface, on the surface of a lipid-based drug delivery system (DDS, such as a liposome), or on whole living cells.[1] [Background technology]

[0002] The immobilization of biomolecules onto drug delivery systems aims to achieve targeted drug delivery to cells, meaning that the drug encapsulated in the carrier is directed to the desired cell type via a ligand-target interaction. This interaction can be used to improve the pharmacokinetic and pharmacodynamic properties of the drug. This may include the delivery of toxic compounds to cancer cells in particular. Immobilization of biomolecules onto cells is particularly relevant for therapies involving adoptive cell transfer, where cells, such as stem cells or immune cells, derived 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 can benefit if the surface of the involved cells is modified in a supraphysiological manner, e.g., during immunotherapy by immobilization of cytokines [2], during regenerative therapy by immobilization of ligands that result in increased chemotaxis of mesenchymal stem cells [3, 4], or during the treatment of autoimmune diseases by immobilization of antigens and enzymes onto cells [5, 6].

[0003] Genetic engineering can be used to engineer cell membranes, thereby providing genetic inheritance to subsequent daughter cells, or to achieve simultaneous extracellular and intracellular modifications. However, genetic engineering suffers from several drawbacks. These include the use of viral vectors, which can cause immunogenic responses, hyperactivation (due to constitutive expression [7]), or de novo tumorigenesis [8, 9]. Genetic engineering is highly dependent on the efficiency of transduction or transfection, which is difficult to predict. Therefore, the therapeutic efficacy of modified cells can be inconsistent. Furthermore, modifications are not possible in all cell types (e.g., slowly dividing cells [1]). Ultimately, genetic engineering results in permanent, irreversible, specific modifications, which are not desirable in all therapeutic applications [1].

[0004] Non-genetic methods are important alternatives to genetic engineering of cell surfaces and can be divided into modifications based on covalent conjugation or hydrophobic insertion of structures into the cell membrane. Covalent conjugation utilizes reactive structures present on the cell surface to attach the desired structure [1]. The main drawback of these chemical methods is the resulting heterogeneity of conjugation products due to the large number of possible reactive sites, such as carboxylic acids, cysteines, lysines, or carbohydrate structures, involved in the binding of bioactive binders to the cell surface. Therefore, site-specific click chemistry [10, 11] has also been used to conjugate proteins to cell surfaces. These site-specific chemical reactions either require the (genetic) introduction of suitable accessor molecules on the cell surface, may require potentially toxic catalysts, or may still result in several by-products

[10] .

[0005] Hydrophobic insertion is an option to avoid direct modification of existing structures on the cell surface [1]. It involves lipidation of a bioactive binding agent as an isolated reaction, followed by the affinity-based spontaneous insertion of the lipidated structure into the membrane bilayer. Because liposomal drug delivery systems are also built on bilayer structures, their membranes can also be modified with lipidated biomolecules. This process is commonly referred to as "post-insertion"

[12] .

[0006] Immobilization of compounds into cell membranes via hydrophobic intercalation has been reported to be nontoxic. Furthermore, intercalated molecules can participate in dynamic membrane movement [1]. Compared to covalent attachment, hydrophobic intercalation avoids the dependency of the degree of cell modification on reaction efficacy. It also reduces physiological alterations, such as reduced membrane mobility or alterations of functional structures present on the cell surface [1]. Several reports have used hydrophobic intercalation to achieve cell membrane modification with polyethylene glycol [13, 14], glycans

[15] , oligonucleotides [6], and peptides or proteins [4, 16-19]. The above reports on protein lipidation and cell intercalation demonstrate promising processes and results for cell membrane engineering. However, several major disadvantages prevent its appropriate use (e.g., for cell membrane remodeling during human cell therapy). Martin et al. reported the lipidation of peptides with sterol-like lipid anchors

[17] . This step was performed on solid phase and required harsh reaction conditions, including extensive use of carboxylic acid protecting groups, because the reaction was based on a PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) / HOBT (hydroxybenzotriazole) activation step. It also required organic solvents such as dimethylformamide. Therefore, this procedure is not applicable to large proteins, which have several reactive sites and do not allow selective protection of amino acid side chains. Furthermore, a wide variety of side products can be expected from using this lipidation procedure on such large proteins.

[0007] To improve the treatment of myocardial infarction in a regenerative manner, Won et al. conjugated recombinant CXC chemokine receptor 4 (CXCR4) to a PEGylated lipid bearing two hydrophobic saturated myristyl chains to modify the surface of mesenchymal stem cells (MSCs) through hydrophobic intercalation [4]. This improved the migration of such modified MSCs in in vitro assays, potentially increasing their homing to ischemic sites in the myocardium [1]. Because CXCR4 has eight cysteines accessible for reaction with the maleimide-modified lipids utilized, various structural isomers of lipidated CXCR4 or multiple conjugated species can be expected after lipid modification [4]. These may have different biological activities or result in different interactions of the intercalated structures with the cell bilayer. This, in turn, may result in different steric accessibility of the receptor's binding region, ultimately leading to potentially reduced binding of the respective ligands. In another study, the same group conjugated dimyristylated PEGylated lipids to an antibody-drug conjugate similar to the commercially available product Kadcyla®

[20] . The lipids consist of a dimyristyl motif as a hydrophobic anchor and a polyethylene glycol spacer of unspecified length, the terminal carboxylic acid group of which was activated via carbodiimide-NHS chemistry. This structure preferentially binds to primary amines on lysine side chains of proteins, of which 88 to 70 have been described as reactive with Kadcyla® antibodies

[10] . The inevitable heterogeneity raises multiple concerns from therapeutic, safety, and even regulatory / analytical perspectives.

[0008] Such heterogeneity can be mitigated by suitable site-selective conjugation strategies. Sortase-mediated transpeptidation for lipidation of biomolecules is a promising approach because the reaction is highly versatile, conjugation can be performed under mild reaction conditions, and it has inherent site specificity due to conjugation between defined amino acid tags. The sortase enzyme family belongs to the transpeptidase family and was originally discovered as a housekeeping enzyme in Gram-positive bacteria, mediating the anchoring of proteins on the peptidoglycan layer

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

[21] . Equation 1: General scheme of sortase A-mediated transpeptidation [ka]

[0009] The amide bond to the glycine is cleaved and subsequently replaced with an incoming nucleophilic N-terminus (e.g., of an oligoglycine

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

[0010] An attempt to implement such a sortase A-based conjugation strategy for hydrophobic insertion-based cell membrane engineering was performed by Antos et al.

[16] . Antos et al. used the transpeptidase sortase A to conjugate triglycine-modified alkyl chains ranging from 10 to 22 carbon atoms, as well as triglycine-modified cholesterol and adamantane derivatives, to LPETG-modified eGFP (enhanced green fluorescent protein (a model protein)). They demonstrated efficient association of lipidated eGFP with cells with alkyl chain lengths of 14 carbon atoms or longer, similar to cholesterol used as the lipid anchor. However, no association of adamantane-lipidated eGFP with cell bilayers was observed. Despite these promising results, the work demonstrated by Antos et al. has several disadvantages. First, only a single lipid chain was conjugated to the protein, whereas previous reports have stated the requirement of two lipid chains for reliable and stable anchoring of proteinaceous structures in membranes [24, 25]. Perhaps by utilizing a single lipid chain modification, Antos et al. circumvented the solubility issue, a major challenge during protein lipidation. These solubility issues of long-chain anchors were further alleviated by the use of detergent-quality n-dodecyl maltoside during lipidation of eGFP with a C22 alkyl chain, the most promising anchor for cell interaction. The use of detergents during protein lipidation is undesirable because both the detergent and lipid structure have similar polarity (which may, for example, prevent proper purification by chromatography). Similarly, lack of detergent from the lipidation product can lead to stability issues due to the hydrophobicity of the resulting product. Finally, detergents may be toxic and prevent the lipidation product from being utilized by cells (for example, during human autologous treatment), or detergents may affect the integrity of the lipidated protein through denaturation. Because eGFP lipidated with either cholesterol or a C22 alkyl chain anchor showed significant internalization in various cell lines after 5 h, it is questionable whether these lipid anchors would serve as suitable tools for stably reconstituting the outer membrane of the cell.Furthermore, because no spacer is used between the lipid anchor and the protein, it may be questionable whether potential binders immobilized on the cell surface have access to their target structures

[26] . Another drawback of the process demonstrated by Antos et al. is its affinity-based purification method. Because both eGFP and sortase A have His6 tags, which are cleaved from eGFP during the reaction with lipids, Ni-NTA resin containing 1 M NaCl and 40 mM imidazole was used to bind and subsequently remove all His6-tagged proteins. This strategy ignores the need to remove other compounds present in the reaction bulk, particularly unconjugated lipids. Therefore, the process presented by Antos et al. is not suitable for utilizing lipid adducts (e.g., in in vivo studies or cell therapy) in the disclosed form.

[0011] Nagamune et al. combined the hydrophobic insertion of triglycine lipids (containing a polyethylene glycol spacer between the hydrophobic moiety and the triglycine unit) into cells with subsequent conjugation of LPETG-modified proteins mediated by sortase A [19, 27]. Therefore, this process can 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, conjugation of the Fc fragment of immunoglobulin resulted in increased phagocytosis of cancer cells via co-incubation with dendritic cells. Although the combined strategy of hydrophobic insertion and covalent conjugation is a gentle approach, several significant disadvantages prevent its appropriate use (e.g., in in vivo treatments). First, sortase A is known to nonselectively conjugate LPETG-modified proteins to N-terminal glycines present on the surface of cell membranes

[28] or in the pericellular matrix / fluid. Therefore, various conjugates of either the model protein eGFP or the Fc fragment with cell surface proteins can be envisioned, thus losing the main advantage of sortase A: the generation of highly defined reaction products. Furthermore, determination of sortase A residues after cell washing was not demonstrated. Because sortase A is a protein that can adsorb to large surfaces such as cell membranes, thorough depletion from the cell bulk can be an extremely difficult task. Exposure of cells to sortase A can result in nonspecific adsorption

[29] , thus resulting in substantial residues on the cell surface even after purification. These can lead to reverse reactions, meaning cleavage of the conjugated structure from the cells, or, more dramatically, severe immune reactions upon administration of the cells.

[0012] Drug delivery systems are also modified by processes similar to hydrophobic insertion processes for cell membrane engineering. This so-called post-insertion process describes the insertion of lipidated ligands from either neat micelles or mixed micelles composed of lipidated ligands and PEGylated lipids [12, 30-32]. Derivatization of proteinaceous structures typically used as targeting ligands for drug delivery systems

[33] occurs primarily via nonspecific chemical reactions

[31] . To date, no site-selective strategies for post-insertion immunoliposome preparations have been disclosed, although several site-selective conjugation strategies for "in situ" (or "post-derivatization") conjugation of ligands to the liposome surface have been described

[31] . While these techniques offer distinct improvements over previously utilized non-selective conjugation methods, they still suffer from drawbacks such as the requirement for various catalysts, unknown toxicity of the linkers and / or reaction products used, or significant amounts of by-products

[10] . Enzymatic techniques can ameliorate this problem, and several reports have described the successful conjugation of model proteins [34-36] or targeting ligands [29, 37, 38] to the liposome surface. However, on-site conjugation with enzymes is challenging because enzymes can be difficult to remove from the reaction bulk, potentially affecting safety due to immunogenicity and stability of the drug delivery system (due to back reactions)

[29] .

[0013] The above-described processes known in the art for lipidation of proteinaceous structures and subsequent derivatization of drug delivery systems or cell membranes have several disadvantages and / or result in either chemical or cellular products with insufficient properties in terms of homogeneity or purity. The object of the present invention is to provide a process for the production of suitable protein-lipid conjugates that overcomes such disadvantages, meaning that it should produce a site-selectively lipidated product that is free of residues from the reaction process, a product that dissolves without the need for additives such as surfactants, and that provides unaltered biological function and activity when present on complex surfaces such as cell membranes. Summary of the Invention

[0014] Thus, one object of the present invention is directed to a process for preparing a conjugate, wherein the conjugate comprises a biomolecule, an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety, the process comprising enzymatic coupling of the components comprising the enzyme tag, the hydrophilic spacer, the linker, and the lipophilic moiety with the biomolecule in an aqueous medium, and purification of the conjugate. Advantageously, the process can be carried out without the use of detergents. 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. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the chemical structure. [Figure 2] Figure 2 shows the chemical reaction formula. [Figure 3] FIG. 3 shows the experimental data. [Figure 4] FIG. 4 shows the experimental data. [Figure 5] FIG. 5 shows the experimental data. [Figure 6] FIG. 6 shows the experimental data. [Figure 7] FIG. 7 shows the experimental data.

[0016] [Figure 8] FIG. 8 shows the experimental data. [Figure 9] FIG. 9 shows the experimental data. [Figure 10] FIG. 10 shows the experimental data. [Figure 11] FIG. 11 shows the experimental data. [Figure 12] FIG. 12 shows a schematic diagram. [Figure 13] FIG. 13 shows the experimental data. [Figure 14] FIG. 14 shows the experimental data.

[0017] As used herein, the term "component comprising an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety" is also referred to as "component A." According to a preferred embodiment, the enzyme tag, hydrophilic spacer, linker, and lipophilic moiety that constitute component A that is bound to the biomolecule are linked in the order in which they are mentioned (i.e., enzyme tag-hydrophilic spacer-linker-lipophilic moiety).

[0018] As used herein, "a" or "an" shall mean one or more. As used herein, when used in connection with the word "comprising," the words "a" or "an" shall mean one or more. As used herein, "another" means at least a second or more. Furthermore, unless the context requires otherwise, singular terms include the plural and plural terms include the singular.

[0019] As used herein, the term "biomolecule" refers primarily to natural or synthetic molecules having a molecular weight greater than approximately 300, preferably polysaccharides or oligosaccharides, oligopeptides or polypeptides, proteins, peptides, polynucleotides or oligonucleotides, and glycosylated lipid derivatives thereof. Most typically, the biomolecule is an immunotherapeutic agent, an antibody or fragment thereof, or a functional derivative of any of these antibodies or fragments, including fusion proteins.

[0020] As used herein, the term "enzyme tag" refers to a moiety that can be recognized by an enzyme and identifies a molecule linked to such a tag as a substrate for a reaction catalyzed by such an enzyme. When reacting with an enzyme, the enzyme tag can be partially or completely removed. In the present invention, the enzyme tag is located at the end of a component comprising such an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety, and is directly linked to the hydrophilic spacer. The enzyme tag allows such a component to be bound to a biomolecule via an enzymatic reaction. After coupling the component to a biomolecule, all or part of the enzyme tag moiety remains in the conjugate resulting from such coupling.

[0021] As used herein, the term "hydrophilic spacer" refers to a moiety present between certain elements of a conjugate or component A that 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. As used herein, the term "linker" refers to a chemical moiety containing a covalent bond or chain of atoms that covalently attaches a first molecule to a second molecule through a chemical bond. In the present invention, a linker is used to connect a hydrophilic spacer with a lipophilic moiety.

[0022] As used herein, the term "lipophilic moiety" refers to any hydrophobic group that is soluble or miscible in fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene. As used herein, the term "purification" refers to a process step that reduces the amount of extraneous elements, such as side reaction products (e.g., fragments of the enzyme substrate that inevitably appear during conjugation, the enzyme itself or a cofactor required for the reaction, or unreacted substrate), that are not conjugates and may be present in the medium after carrying out an enzymatic reaction. Purification can involve various methods known in the art, such as chromatographic methods, and can include one or more steps, such as a chromatographic step.

[0023] The biomolecule present in the conjugate is a polypeptide. Consequently, the present invention is directed to processes in which the biomolecule is a polypeptide. As used herein, the term "polypeptide" refers to a polymer of amino acids, typically L-amino acids, joined to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids, typically by amide linkages, where the number of amino acid residues can range from about 5 to about 1,000,000. Preferably, a polypeptide has about 10 to about 2,000 amino acid residues, and even more preferably about 20 to about 500 amino acid residues. Thus, as used herein, polypeptide includes what are often referred to in the art as oligopeptides (5 to 10 amino acid residues), polypeptides (11 to 100 amino acid residues), and proteins (more than 100 amino acid residues).

[0024] Suitable polypeptides that may be present as biomolecules include antigens, cell adhesion proteins including integrins and cadherins, peptide hormones, particularly growth factors, cytokines, particularly interleukins, receptors for any of these molecules, enzymes, and natural or artificial antibodies and fragments thereof. Thus, the present invention is further directed to processes in which the biomolecule conjugated to "component A" is a cell adhesion protein such as an antigen, integrin, or cadherin, a peptide hormone such as a growth factor, a cytokine such as an interleukin, a receptor for any of these molecules, an enzyme, or a natural or artificial antibody or fragment thereof.

[0025] 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 organisms, particularly animals, more particularly mammals, including humans. The term "antigen" includes regions known as antigenic determinants or epitopes, which refer to portions of an antigen that contact or play an important role in supporting contact residues of the antigen that are responsible for its antigenicity or antigenic determinant. As used herein, the term "cell adhesion protein" refers to a large family of cell adhesion proteins that have extracellular regions in their structure as cell recognition sites and are involved in mediating cell-cell interactions. The term "cell adhesion protein" includes biologically active equivalents of the native sequences of adhesion proteins, including proteins from natural sources or recombinant cell culture, and synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof.

[0026] As used herein, the term "integrin" refers to a cell adhesion protein that allows cells to both bind to and respond to the extracellular matrix and is involved in various cellular functions, such as wound healing, cell differentiation, tumor cell homing, and apoptosis. Functional integrins consist of two non-covalently bound transmembrane glycoprotein subunits, termed alpha and beta. All alpha subunits, as well as the beta subunits, share some homology with each other. Receptors always contain one alpha chain and one beta chain. Examples include alpha6beta1, alpha3beta1, alpha7beta1, LFA-1, etc. As used herein, the term "integrin" refers to a cell adhesion protein that allows cells to bind to and respond to the extracellular matrix and is involved in various cellular functions, such as wound healing, cell differentiation, tumor cell homing, and apoptosis. Functional integrins consist of two non-covalently bound transmembrane glycoprotein subunits, termed alpha and beta. All alpha subunits share some homology with each other, as do the beta subunits. Receptors always contain one alpha chain and one beta chain. Examples include alpha6beta1, alpha3beta1, alpha7beta1, LFA-1, etc.

[0027] 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 desmosomal protein subfamily (specific examples of which include desmogleins 1, 2, and 3, and desmocollin 3). Natural sources of cadherins can be found in vertebrates, including humans, livestock, sport animals, primates, rodents, and pets, such as chickens, pigs, sheep, horses, cows, rabbits, mice, and rats.

[0028] As used herein, the term "peptide hormone" refers to proteins with endocrine function, e.g., 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).

[0029] As used herein, the term growth factor refers to proteins or polypeptides that can stimulate cell growth. These include, but are not limited to, epidermal growth factor (EGF), human growth hormone (HGF), nerve growth factor (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 (Eph), erythropoietin (EPO), glial cell-stimulating factor (GSF); colony-stimulating factors (CSFs) 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 hematopoietic stem cell 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.

[0030] As used herein, the term "cytokine" refers to a protein released by a cell population that acts on another cell as an intercellular mediator. Examples of such cytokines are lymphokines and monokines. Cytokines include interleukins; prolactin; placental lactogen; mouse gonadotropin-related peptide; inhibin; activin; thrombopoietin (TPO); interferons such as IFNα, IFNβ, and IFNγ; and TNFα or TNFβ.

[0031] 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. 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.

[0032] The term "antibody" or "immunoglobulin" herein is used in the broadest sense and specifically covers 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 heteroantibodies, which are composed of two or more antibodies or fragments thereof of different binding specificities linked together.

[0033] Depending on the amino acid sequence of their constant regions, intact antibodies can be assigned to various "antibody (immunoglobulin) classes." There are five major classes of intact antibodies (IgA, IgD, IgE, IgG, and IgM), and some of these can be further divided into "subclasses" (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2). The heavy chain constant domains corresponding to the various classes of antibodies are called α, δ, ε, γ, and μ, respectively. The preferred major class of antibody to be used as a biomolecule is IgG, more particularly IgG1 and IgG2.

[0034] Antibodies are typically glycoproteins with a molecular weight of approximately 150,000 and consist 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, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several 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 aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the variable domain of the light chain and the variable domain of the heavy chain. The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0035] An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. 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 antibody fragment(s). An "intact" antibody is one 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.

[0036] As used herein, the term "artificial" refers to compositions and systems that are designed or prepared by humans and do not occur in nature. For example, an artificial polypeptide (e.g., an antibody or antibody fragment) is one that contains a non-native sequence (e.g., a polypeptide that does not have 100 percent identity to a naturally occurring protein or fragment thereof). As used herein, consistent with the definition of "artificial" above, the term "artificial antibody" refers to an antibody that has an amino acid sequence or chemical makeup distinct from that found in naturally occurring antibodies. An artificial antibody is not a subsequence of a naturally occurring protein, either the wild-type (i.e., most abundant) or a mutant version 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.).

[0037] According to a preferred embodiment of the present invention, the antibody used as the biomolecule is a monoclonal antibody or a fragment thereof (such as a single-chain variable fragment (scFv), a variable fragment (Fv), or a fragment antigen-binding (Fab, Fab', or F(ab')2), a heavy chain-only antibody or a fragment thereof (such as a VHH or vNAR) from camelids or cartilaginous fish), or wherein the artificial polypeptide is a DARPin, adnectin, anticalin, or affibody.

[0038] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible 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, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Methods for making 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, Volume 13, Elsevier Science Publishers, Amsterdam), or can be made by well-known recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:58, 1-597 (1991).

[0039] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing a single antigen-binding site and the CL and CH1 regions, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The "Fc" region of an antibody generally comprises CH2, CH3, and the hinge region of the major classes of IgG1 or IgG2 antibodies. The hinge region is a group of approximately 15 amino acid residues that combines the CH1 region with the CH2-CH3 regions.

[0040] Pepsin treatment yields an "F(ab')2" fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Further treatment with a suitable reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethylamine, or dithiothreitol, yields Fab fragments. "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, noncovalent association. In this configuration, 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 the three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with 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 carboxy 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 which have hinge cysteines between them.

[0041] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of 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 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, Vol. 113, Rosenburg and Moore eds., 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 Plückthun (1988, Science 240, 1038).

[0042] 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 normally found in antibodies. Camelids (such as camels, llamas, or alpacas) or cartilaginous fish (such as sharks, rays, or skates) are known to produce HCAbs. Camelid and cartilaginous fish antibodies contain heavy chains but lack light chains. 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.

[0043] As used herein, the term "VHH" refers to the variable region of the heavy chain of a camelid antibody. Thus, VHH regions from such camelid antibodies represent the minimum structural elements required for specific binding to the 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).

[0044] As used herein, the term "vNAR" refers to a single variable new antigen receptor (NAR) domain antibody fragment. A vNAR fragment is a single domain antibody fragment derived from a heavy chain antibody, such as a shark immunoglobulin new antigen receptor antibody (IgNAR).

[0045] The term "designed ankyrin repeat protein" or "DARPin" refers to an artificial polypeptide prepared through 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 two or at least three ankyrin repeat motifs are repeated, for example, three, four, or five ankyrin repeat motifs. For example, DARPins containing three, four, or five ankyrin repeat motifs can have molecular weights of approximately 10 kDa, approximately 14 kDa, and approximately 18 kDa, respectively. DARPins contain a core portion that performs a structural function and a target-binding portion outside the core that binds to the target. The core portion contains a conserved amino acid sequence, while the target-binding portion contains a different amino acid sequence depending on the target.

[0046] As used herein, the term "Adnectin" refers to a monobody, an artificial antibody constructed using a fibronectin type III domain (FN3). As used herein, the term "anticalin" refers to an artificial antibody derived from lipocalin that specifically binds to GPD1 and inhibits GPD1 activity. Anticalins share a barrel structure with naturally occurring lipocalins, formed by eight antiparallel beta strands connected in pairs by loops and connected α-helices.

[0047] As used herein, the term "affibody" refers to a recombinant protein composed of a single polypeptide chain containing a domain responsible for selective interaction with an antigen (e.g., a specific tumor marker such as HER2) (see, e.g., C. Steffen, M. Wikman, V. Tolmachev, GP Adams, FY Nilsson, S. Stahl, J. Carlsson: "In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics," CancerBiother. Radiopharm., 20 (2005), pp. 239-248). It can be conjugated to another polypeptide, ensuring better antigen-binding properties, greater stability, and additional functions (e.g., the possibility of controllably binding to another substance or solid surface). Its mass is usually in the range of several to several tens of kilodaltons.

[0048] According to a preferred embodiment, the biomolecule present in the component used in the process of the invention is a single domain antibody derived from the variable domain of a camelid heavy chain only antibody (VHH). Enzymatic coupling of the biomolecule to component A can be achieved by the use of a transpeptidase such as sortase. A preferred transpeptidase for use in the process of the present invention is sortase A. To enable coupling of the biomolecule to component A, the biomolecule has a C-terminal motif as a recognition signal for a transpeptidase. Thus, in a suitable embodiment of the process of the present invention, the biomolecule has a C-terminal motif for enzymatic conjugation by a transpeptidase, preferably sortase A, prior to its coupling to a component comprising an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic component portion (component A).

[0049] According to a preferred embodiment, the biomolecule has as a C-terminal motif the amino acid sequence consisting of "Leucine-Proline-X-Threonine-Glycine" (LPXTG). In such a C-terminal motif, "X" can be any proteinogenic amino acid except cysteine ​​and tryptophan. Thus, the present invention is also directed to a process in which the biomolecule, prior to coupling to the building block, comprises a C-terminal motif consisting of the amino acid sequence "Leucine-Proline-X-Threonine-Glycine" (LPXTG), where "X" can be any proteinogenic amino acid.

[0050] As used herein, the term "proteinogenic amino acid" refers to one of the 21 amino acids directly encoded for protein synthesis by the eukaryotic genetic code, excluding cysteine ​​and tryptophan. Consequently, the proteinogenic amino acid X present in the C-terminal motif is one of the 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 is further directed to a process in which 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.

[0051] The enzyme tag comprises an N-terminal amino functionality. According to a suitable embodiment, such an N-terminal amino functionality is provided by a polypeptide sequence comprising an aliphatic amine, a single glycine, or one or more N-terminal glycines, preferably pentaglycines, linked at the C-terminus to a hydrophilic spacer. Thus, the present invention is also directed to a process in which the enzyme tag is a polypeptide sequence comprising an aliphatic amine, a single glycine, or one or more N-terminal glycines, preferably pentaglycines, linked at the C-terminus to a hydrophilic spacer.

[0052] The "N-terminus" with respect to 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 a polypeptide chain. Similarly, the "C-terminus" with respect to an amino acid or polypeptide chain refers to the free carboxy group on the amino acid or the free carboxy group on the final amino acid residue of a polypeptide chain.

[0053] As explained above, hydrophilic spacers increase the water solubility of the building blocks or conjugates. Suitable hydrophilic spacers that may be present in building block A used in the processes of the present invention include hydrophilic polymeric radicals (which have increased affinity for aqueous solutions), i.e., polymers containing repeating structural units containing one or more hydrophilic (or polar) groups in their alkylene backbone. Examples of usable hydrophilic polymeric radicals include polyoxy(C2-C3) alkylenes (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)), polysaccharides (e.g., dextran, pullulan, chitosan, hyaluronic acid), and polyethyleneimine, with polyethylene glycol being preferred. Accordingly, suitable embodiments of the present invention are further directed to processes in which the building blocks containing the enzyme tag, hydrophilic spacer, linker, and lipophilic moiety bearing the biomolecule used in such processes are polyoxy(C2-C3) alkylenes (e.g., polyethylene glycol or polypropylene glycol), polysaccharides (e.g., dextran, pullulan, chitosan, hyaluronic acid), polysialic acid, polyethyleneimine, and preferably polyethylene glycol.

[0054] A preferred hydrophilic spacer is "PEG" or "polyethylene glycol," which includes any water-soluble poly(ethylene oxide). Typically, "PEG" refers to a polymer containing a majority (e.g., >50%) of subunits that are -CH2CHO-. Different forms of PEG can differ in molecular weight, structure, or shape (e.g., branched, linear, forked PEG, multifunctional, etc.). PEG that may be present in component A used in the process of the present invention is "-OCH2CHO(CH2CHO) m -" but also includes a form in which one terminal "-O-" group is replaced with a "-NH-" group, which is represented by the formula "-NHCH2CH2O(CH2CH2O) m The 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.

[0055] The linker connects the hydrophilic spacer to the lipophilic moiety through a covalent bond such as CC, CO, CN, and CS. Linkers that can be used for coupling between a hydrophilic spacer and a lipophilic moiety, and between a linker and a hydrophilic spacer and a lipophilic moiety, are known in the art and are described, for example, in EP 2825156 B1. In principle, bifunctional agents (i.e., agents having two functional (terminal) groups), preferably heterobifunctional agents (i.e., agents having two different functional (terminal) groups), react and thereby bond with components containing a hydrophilic spacer and a lipophilic moiety. 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 the group consisting of, for example, carbodiimide, N-hydroxysuccinimidyl-4-azidosalicylate (NHS-ASA), dimethyl pimelimidate dihydrochloride (DMP), dimethyl suberimidate (DMS), 3,3′-dithiobispropionimidate (DTBP), N-succinimidyl 3-[2-pyridyldithio]-propionamide (SPDP), succinimidyl α-methylbutanoate, biotinamide hexanoyl-6-amino-hexanoic acid N-hydroxy-succinimide ester (SMCC), succinimidyl [(N-maleimidopropionamido)-dodecaethyleneglycol] ester (NHS-ASA), and succinimidyl α-methylbutanoate. SPEO12), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl S-acetylthioacetate (SATA), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and Nh-maleimidobutyryloxy-succinimide ester (GMBS), succinimidyl dicarbonylpentane, or disuccinimidyl suberate, but are not limited to these.

[0056] The components comprising an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety used in the process of the present invention can be prepared with any of such linkers. According to a preferred embodiment, the linker used to prepare such components is an isoglutamine on the δ-position amide linked to 3-amino-1,2-propanediol and on the α-standing amine function linked to a hydrophilic spacer. Within the components comprising an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety, the linker moiety has the following formula (I): [ka]

[0057] In this equation, the vertical dotted line [ka] indicates an atom bond to a lipophilic moiety, and a vertical dashed line [ka] indicates attachment to a hydrophilic spacer.

[0058] According to a suitable embodiment of the present invention, component A used in the process comprises, as lipophilic moieties, one or more, independently of one another, saturated or unsaturated, linear or branched hydrocarbon chains (such as fatty alcohols or fatty acids having a chain length of 6 to 30 carbon atoms), or sterols (such as cholesterol). Thus, the present invention is also directed to a process in which the lipophilic moieties present in component A are, independently of one another, one or more saturated or unsaturated, linear or branched hydrocarbon chains, 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 moieties are saturated linear hydrocarbon chains.

[0059] 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, with R representing a linear alkyl group, optionally substituted with one or more hydroxyl groups, containing 6 to 30, even better 10 to 30, or even 10 to 22, and even better 14 to 18 carbon atoms. 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-octadecanol), arachidyl alcohol (1-eicosanol), behenyl alcohol (1-docosanol), lignoceryl alcohol (1-tetracosanol), seryl alcohol (1-hexacosanol), montanyl alcohol (1-octacosanol), and myricyl alcohol (1-triacontanol), alone or in mixtures. In compound A, the fatty alcohol and linker are preferably ether-linked R 1 -OR 2 are connected to each other by R 1 is the linker and R 2 is the alkyl group of the fatty alcohol.

[0060] As used herein, the term "fatty acid" refers to a long-chain aliphatic acid containing at least six carbon atoms and one or two, preferably one, carboxylic acid group. Preferably, the fatty acid has the structure R-COOH with R representing a linear alkyl group containing 6 to 30, even better 10 to 30, or even 10 to 22, and even better 14 to 18 carbon atoms. Suitable fatty acids include, for example, n-decanoate (C10, caprate), n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), cis-6 9-octadecanoate (C18, oleate), all cis-6 5,8,11,14-eicosatetraenoate (C20, arachidonate), etc. In Compound A, the fatty acid is preferably connected to a linker having a hydroxyl group, wherein the carboxylic acid group of the fatty acid is preferably linked to the hydroxyl group of the linker via an ester linkage.

[0061] 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 gonane 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 linker are preferably ether-linked R 1 -OR 2’ are connected to each other by, where R 1 is the linker and R 2’ is a sterol ring system.

[0062] According to a particularly preferred embodiment of the present invention, component A used in the process comprises two myristyl alcohols each ether-linked to the diol group of the linker 3-amino-1,2-propanediol. Thus, the present invention is also directed to a process in which the lipophilic component portion comprises two myristyl alcohols each ether-linked to the diol group of 3-amino-1,2-propanediol.

[0063] According to a suitable embodiment of the present invention, the process comprises the following steps: (a) preparing an aqueous dispersion of components comprising an enzyme tag, 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; and (d) purifying the conjugate obtained in step (c). Thus, the present invention is also directed to a process comprising the following steps: (a) preparing an aqueous dispersion of components including an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety; (b) adding enzymes and biomolecules; (c) incubating the mixture obtained in step (b) to produce a conjugate; (d) purifying the conjugate obtained in step (c).

[0064] According to a suitable embodiment of the present invention, the process uses a ligase as the enzyme. As used herein, the term "ligase" refers to an enzyme that can catalyze the joining of two large molecules by forming a new chemical bond, usually involving the hydrolysis of a small pendant chemical group on one of the larger molecules, or an enzyme that catalyzes the joining of two compounds together (e.g., enzymes that catalyze the joining of CO, CS, CN, etc.). Generally, ligases catalyze the following reaction: Ab + C → A-C + b. In the present invention, ligases are used to catalyze the joining of hydrophilic spacers with biomolecules.

[0065] Ligases that can be used in the present invention include sortase, butelase, trypsiligase, subtiligase, peptiligase, and omniligase, with sortase A being preferred. Thus, the present invention is further directed to a process wherein the enzyme in step (b) is a ligase, including sortase, butelase, trypsiligase, subtiligase, peptiligase, and omniligase, with sortase A being preferred.

[0066] The enzymes used in the present invention are described, for example, by M Schmidt et al: Enzyme-mediated ligation technologies for peptides and proteins, Current Opinion in Chemical Biology (2017) 38, pp 1-7.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; butelases 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; and subtiligases are described by AC Braisted et al.: Synthesis of proteins by subtiligase. Methods Enzymol 1997, 289:298-313; omniligases and peptiligases 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 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.

[0067] According to a preferred embodiment of the present invention, component A used in the process has formula II: [ka] where: m is an integer of 15 to 60, preferably 25 to 45, more preferably 30 to 40, and most preferably 36; n is an integer of 3 to 27, preferably 7 to 19, more preferably 11 to 15, and most preferably 11; p is an integer of 0 to 9, preferably 2 to 7, more preferably 3 to 5, and most preferably 4.

[0068] Thus, the present invention is also directed to a process wherein the components comprising the enzyme tag, the hydrophilic spacer, the linker, and the lipophilic moiety have formula II: where m indicates 15–60; n indicates 2 to 27; p represents 0 to 9.

[0069] In a particularly preferred embodiment, the process of the present invention utilizes as compound A a compound according to formula I, wherein n is 11, m is 36 and p is 4. The conjugates obtained by the process of the present invention are well suited for the modification of lipid-based drug delivery systems, such as solid lipid nanoparticles, nanoemulsions, micelles, or liposomes, preferably liposomes. Accordingly, the present invention is also directed to the use of the conjugates obtained by the process for the modification of lipid-based drug delivery systems, such as solid lipid nanoparticles, nanoemulsions, micelles, or liposomes, preferably liposomes. 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 a sphere, but can also have an irregular shape.

[0070] The term "nanoemulsion" refers to a colloidal dispersion, typically 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. The term "average size" or "mean size," as used herein, relates to the average diameter of the droplets. The average size of these systems can be measured by standard processes known to those skilled in the art, such as dynamic light scattering.

[0071] As used herein, the term "micelle" refers to an aggregate of amphiphilic molecules, such as lipids, assembled to form a particle with a hydrophobic interior and a hydrophilic exterior. Micelles are generally spherical aggregates with diameters less than 100 nm, although a range of micelle diameters and various micelle shapes (such as discoidal micelles) are known in the art. As used herein, the term "liposome" refers to a vesicle composed of one or more lipids, phospholipids, and / or surfactants, which is useful for delivery of drugs (such as chemotherapeutic agents) to a mammal. The components of a liposome form a bilayer similar to the lipid arrangement of biological membranes.

[0072] The conjugates obtained by the process of the invention are further well suited for the modification of the membrane of living cells, preferably T cells. Thus, the invention is further directed to the use of the conjugates obtained by the process for the modification of the membrane of living cells, preferably T cells. As used herein, the term "T cell" 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 a T cell receptor on the cell surface. T cells can be isolated (from animal spleens or human blood donations) or obtained from commercial sources. "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and gamma delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses.

[0073] The conjugates obtained by the process of the invention are further well suited for the modification of surfaces with an affinity for hydrophobic substances, such as hydrophobic polystyrene. Consequently, the invention is further directed to the use of the conjugates obtained by the process for the modification of surfaces with an affinity for hydrophobic substances, such as hydrophobic polystyrene. As used herein, the term "hydrophobicity" refers to the degree of affinity a material has for water. Hydrophobic materials substantially lack affinity for water, tend to repel and not absorb water, and tend not to dissolve in, mix with, or become wetted by water.

[0074] Likewise, the conjugates obtained by the process of the invention are moreover well suited for the modification of exosomal membranes, and consequently the invention is also directed to the use of the conjugates obtained by the process for the modification of exosomal membranes. As used herein, the term "exosome" refers to a small (between 20 and 300 nm in diameter, more preferably between 40 and 200 nm in diameter) cell-derived vesicle that encloses an internal space and includes a membrane generated from the cell by direct plasma membrane budding or by fusion of a late endosome with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a simple sugar, polysaccharide, or glycan), or other molecules. Exosomes can be derived from producer cells or isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle.

[0075] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0076] The examples illustrate the invention without limiting it. method Protein expression was performed using 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).

[0077] For this VHH, recombinant SrtA28 was expressed in E. coli strains WK6 (VHH) and BL21(DE3) (SrtA expression). 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) in the presence of appropriate antibiotics 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 incubated at 37°C for an additional 3 hours. Cells were harvested by centrifugation (15 min, 4000 × g, 4 °C), and the pellet was resuspended in 20 mL of PBS (150 mM NaCl, 8.3 mM NaHPO, 1.7 mM KHPO, pH 7.4) per 400 mL of 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 agarose (Protino Ni-NTA, Macherey-Nagel) column. All expressed proteins contained a 6xHis tag, allowing for 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 with Amicon centrifugal filter devices (Millipore) with a 3 kDa cutoff (for VHH) 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 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.

[0078] Absorption coefficients were based on the primary protein sequence and calculated from the online software ExPASy ProtParam, SIB, Lausanne, Switzerland. The sequence of the protein used (one-letter code according to Nomenclature and symbolism for amino acids and peptides (Recommendations 1983), Pure and Applied Chemistry, 1984, p. 595).

[0079] Sortase A (SrtA7m) [SEQ ID NO: 1] MGHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENASLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVK VHH DC13 [SEQ ID NO: 2] QVQLQESGGGLVQAGGSHNLSCTASGITFSSLAMGWFRQTPGKEREFVANIMRSGSSVFYADSVRGRFTISRDNAKNTAHLQMNSLKPEDTAVYFCAATRGAWPAEYWGQGTQVTVSSGGLPETGGHHHHHH

[0080] VHH ENH [SEQ ID NO: 3] QVQLQESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGGLPETGGHHHHHH eGFP (enhanced green fluorescent protein) [SEQ ID NO: 4] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH DMA-PEG-G5 DMA-PEG-G5 is a material having the formula shown in FIG.

[0081] Example 1 A stock of DMA-PEG-G5 (25 mg / mL in chloroform), further referred to herein as Structure 2 in Figure 1, was dispensed into an HPLC vial. The chloroform was evaporated under a gentle nitrogen stream to produce a thin lipid film, which was then hydrated in DPBS (Dulbecco's Phosphate-Buffered Saline, Sigma-Aldrich, #D1408) pH 7.4 as a micellar aqueous lipid dispersion (2 mM). Alternatively, DMA-PEG-G5 was dissolved in DPBS (pH 7.4) (5–10 mg / mL) using sonication. 400 μL of 50 μM VHH, 25 μM Sortase A, and 1 mM DMA-PEG-G5 (corresponding to a target product mass of 320 μg) was incubated at 4 °C for 4 h until the conjugate was isolated using reverse-phase HPLC (rp-HPLC). An Aeris Widepore C4 column (particle size 3.6 μm, length 100 mm, diameter 2.1 mm, Waters Corporation, Milford, Massachusetts, USA) and a binary gradient pattern were used to separate the reaction bulk and products (Table).

[0082] Table 2: rp-HPLC gradient pattern for separation of reaction bulk and products (A: water with 0.1% trifluoroacetic acid (TFA v / v); B: acetonitrile with 0.05% TFA v / v) [Table 2]

[0083] 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), and was controlled by EZChrom Elite Software (Agilent Technologies, Santa Clara, CA, USA). The column temperature was set at 30 °C, and the autosampler temperature was set at 4 °C. The standard analytical or isolated injection volume was 5 μL or 25–100 μL, respectively. The flow rate was 0.5 mL / min. Data were recorded using the DAD at 214 and 280 nm.

[0084] The conjugate peak was collected manually or using an automated fraction collector. The collection of a single injection was stored on ice until the eluent mixture was removed using a vacuum centrifuge (RVC 2-33 IR, Martin Christ, Osterode am Harz, Germany; speed: 1500 rpm; temperature: 40 °C; 10 min at 100 mbar, followed by 20 min at 20 mbar, and evaporation at 2 mbar). The resulting pellet 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. Purity was determined by RP-HPLC analysis at 214 nm using automated peak detection between 2 and 15 min and a threshold level derived from the background noise of a water blank.

[0085] To verify the reaction products, 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 set to ESI with positive polarity. The capillary outlet voltage was 140 V, and the trap drive was set to 94. The mass range mode was set to enhanced resolution with a scan range of 100–2200 m / z. Five spectra were averaged per run. Masses were calculated using deconvolution of the raw spectra.

[0086] Example 2 Isolated VHH ENH conjugates from Example 1 or native VHH ENH (100 nM) were spiked into 100 nM eGFP. VHH ENH is known for its 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, Maennedorf, Switzerland) with excitation and emission set to 485 nm and 535 nm, respectively.

[0087] 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-dipalmitoylphosphatidylglycerol), and DMA-PEG-G5 (59.4:34.6:5.0:1.0 molar 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 customized 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 was added to the liposome dispersion to give 0.25–2 nM VHH per 1 μM phospholipid (PL). The mixture was vortexed thoroughly and incubated at 50 °C for 30 min.

[0088] Murine myeloid-derived suppressor CD11b+Gr-1+ cells (MDSCs) were derived from bone marrow-derived NUP progenitor cells [6]. MDSCs were differentiated for 4 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), and supplemented with 20 ng / mL interleukin-6 and 20 ng / mL granulocyte-macrophage colony-stimulating factor (Biolegend, #576304, San Diego, USA). To examine VHH-modified liposome binding, MDSCs were incubated with 500 μM liposomes (based on total lipid content) for 4 h at 4°C. Cells were washed with FACS buffer (1x PBS + 2% heat-inactivated fetal bovine serum) and subjected to antibody staining in the presence of Fc receptor blocker (TruStain FcX, BioLegend, #422302) in FACS buffer. SytoxBlue (Thermo Fisher Scientific, S34857) was used for exclusion of dead cells. Liposomes were detected via encapsulated FITC-dextran.

[0089] Example 4 T cells were isolated from the spleens of C57BL / 6j mice maintained under specific pathogen-free conditions in the animal facility at the University of Heidelberg and euthanized under registered protocol T47 / 16. Spleens were crushed, and red blood cells were lysed (ACK lysing buffer, #A1049201, Thermo Fisher Scientific) using a mouse CD8a+ T cell isolation kit (#130-104-075, Miltenyi Biotec, Bergisch-Gladbach, Germany). CD8+ cells were isolated using magnetic cell isolation (LS columns, #130-042-401, Miltenyi Biotec) according to the manufacturer's instructions. Purified CD8+ T cells were stained with 1 nM Cell Tracer Far Red (#C34564, Thermo Fisher Scientific) for 5 min at 35°C and washed with FACS buffer. Stained T cells (1.65 x 108 cells / mL) were incubated with 650 nM of native or lipidated VHH DC13 and VHH ENH for 1 hour at 4°C. Lipidated VHH binding to T cells was detected by FITC-anti-llama antibody.

[0090] Murine myeloid-derived suppressor CD11b+Gr-1+ cells (MDSCs) were derived from bone marrow-derived NUP progenitor cells [6]. MDSCs were differentiated for 4 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), and supplemented with 20 ng / mL interleukin-6 and 20 ng / mL granulocyte-macrophage colony-stimulating factor (Biolegend, #576304, San Diego, USA). To examine membrane insertion of lipidated VHHs, MDSCs (10 7 Cells (1000 cells / 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 (1x PBS + 2% heat-inactivated fetal bovine serum) and subjected to antibody staining in the presence of Fc receptor blocker (TruStain FcX, BioLegend, #422302) in FACS (fluorescence-assisted cell sorting) buffer. SytoxBlue (Thermo Fisher Scientific, S34857) was used to exclude dead cells. Lipidated VHH inserted into the cell membrane was detected with 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 results were analyzed using FlowJo (Tree Star, V.10.0.8).

[0091] Example 5 CD11b+Gr-1+ cells from Example 4 were incubated with 100 μg / mL eGFP for 30 min at 4°C to detect binding of eGFP to lipidated VHH ENH inserted into the cell membrane. Flow cytometry for eGFP fluorescence was performed on a FACSAria II (Beckton, Dickinson and Company, Franklin Lakes, NJ, USA), and results were analyzed by FlowJo (Tree Star, V.10.0.8).

[0092] Example 6 For cell-cell interaction experiments, T cells were isolated from the spleens of C57BL / 6j mice maintained under specific pathogen-free conditions in the animal facility at the University of Heidelberg and euthanized under registered protocol T47 / 16. Spleens were crushed and purified using mouse CD8a + CD8 T cells were isolated after lysing red blood cells (ACK lysing buffer, #A1049201, Thermo Fisher Scientific) using a T cell isolation kit (#130-104-075, Miltenyi Biotec, Bergisch-Gladbach, Germany) and magnetic cell isolation (LS columns, #130-042-401, Miltenyi Biotec) according to the manufacturer's instructions. + Cells were isolated and 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. Stained T cells (1.65 x 10 8 cells / mL) were incubated with 650 nM of native or lipidated VHH DC13 and VHH ENH for 1 h at 4°C. VHH-labeled T cells were washed twice with FACS buffer and 3 × 10 7 1.1 x 10 T cells 7 The cells were incubated with MDSCs (obtained as described in Example 4) for 1 hour at 4°C. +For magnetic bead isolation of T cells and co-purification of T cell-bound MDSCs, T cells and MDSCs were loaded onto an LS column. 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.

[0093] Example 7 Native or lipidated VHH ENH (Batch #3 from Example 1) was mixed with eGFP in DPBS (pH 7.4) (1x, Sigma, #D1408) in various variations of ThermoFisher Polysorp 96-well plates (# Nunc 475094). The variations included: PBS, PBS with eGFP (50 nM), PBS with eGFP (50 nM) + native VHH ENH (50 nM), and PBS with eGFP (50 nM) + lipidated VHH ENH (5–50 nM). The plates were incubated at 60 rpm and 37°C for 1.5 hours on an orbital shaker. The plates were then centrifuged at 300 g for 1 minute to collect all liquid at the bottom of the wells. The plates were then measured using a TecanReader Spark (ThermoFisher) at excitation of 485 nm and emission of 525 nm. The liquid in the wells was then replaced five times. Fluorescence was measured after each of the 1, 2, 3, and 5 exchange steps.

[0094] Surprisingly, it was found that a compound consisting of an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety (structure 2 in Figure 1) can be dissolved in aqueous buffer up to 10 mg / mL (4 mM) (Example 1). 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 when analyzed by reverse-phase HPLC ( Figure 3 , exemplary chromatogram of VHH ENH utilized as Structure 1). Additionally, no aggregation or precipitation of the lipid adduct was observed during the reaction as analyzed via visual inspection (Example 1).

[0095] Mass spectrometry confirmed the expected molecular mass of two different single domain antibodies (VHH ENH (Figure 4), VHH DC13 (Figure 5)) (Example 1). The reaction bulk was purified by collecting the column effluent of the described 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 pellet thus obtained could be dissolved in water to a protein content of approximately 1 mg / mL (Example 1). The purity of the lipidated single domain antibodies thus obtained was analyzed via reversed-phase HPLC coupled with UV detection at 214 nm. Purity (based on UV area) of >95% was found for the following batches (Figure 6, Table 1) (Example 1):

[0096] Table 1 [Table 3]

[0097] Yields were calculated based on the mass and concentration of the recovered protein solution and the mass of the target product (320 μg protein per batch). Good yields of >50% were obtained, except for VHH ENH Lot #3, which was prepared without cooling the column effluent (Example 1). Biological activity via reversed-phase HPLC-purified conjugates was analyzed by the ability of lipidated VHH ENH to increase eGFP fluorescence upon binding (Example 2) [1]. 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 Lot #1 and Lot #2 (Figure 7) (Example 2). Lot #3, prepared without cooling the column effluent, showed a slight decrease in eGFP fluorescence enhancement (Example 2).

[0098] Lipidated and isolated single-domain antibodies VHH ENH and VHH DC13 were incubated with an FITC-labeled liposomal drug delivery system (Example 3) at different VHH to phospholipid ratios. The modified liposomes were then incubated with cultured mouse CD11b+Gr-1+ cells via this post-insertion process. VHH DC13 binds to the cell surface receptor CD11b, and VHH DC13-modified liposomes demonstrated clear cell association with CD11b+Gr-1+ cells during flow cytometry analysis (Figure 8 shows data for VHH DC13 surface density on liposomes at 2 nM / µM phospholipid; the percentage in each dot plot indicates the number of positive cells in the marked gate. FSC: forward scatter) (Example 3).

[0099] When liposomes were incubated with different ratios of lipidated VHH DC13 to phospholipid concentration, an optimum for binding to cells was observed at 0.5 nM VHH DC13 per 1 μM phospholipid (FIG. 8) (Example 3). 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 were stained with FITC-anti-llama antibody and then the presence of VHHs on the cell surface was confirmed by flow cytometry (Example 4, Figure 10). Lipidated VHHs clearly increased the fluorescent signal obtained from the cells by flow cytometry (Example 4). When VHH DC13 was incubated with CD11b+ cells, no difference was detected between the lipidated and non-lipidated forms, because direct binding of VHH DC13 to CD11b abolished the hydrophobic intercalation effect (Example 4).

[0100] 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 washing, the cells were incubated with the VHH ENH-corresponding antigen eGFP. Flow cytometry revealed selective capture of eGFP by cells treated with lipidated VHH ENH (Example 5, Figure 11).

[0101] To assess 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. The modified cells were then incubated with CD11b+Gr-1+ cells. This cell mixture was first separated by magnetic bead-assisted cell sorting specific for the antigen CD8. CD8 + The retentate was stained for CD11b and Gr-1 and analyzed by flow cytometry (Figure 14) (Example 6). Cell populations preincubated with lipidated VHH DC13 elicited a higher signal for MDSCs in the CD8-positive column retentate, indicating that the VHH promoted cell interactions between MDSCs and T cells (Figure 13) (Example 6).

[0102] To assess whether lipidated VHHs can be immobilized on hydrophobic surfaces such as hydrophobic polystyrene, lipidated or native VHH ENHs were incubated in the presence of 50 nM eGFP (the corresponding antigen of VHH ENHs) in Polysorp® 96-well plates (Example 7). After several washing steps, significant coating and retention of eGFP was observed in wells coated with 25 nM and 50 nM eGFP (Figure 14). The data demonstrate the utility of lipidated VHHs for antigen or cell capture or immobilization on cells (Example 7). They can also be used for protein purification.

Claims

1. 1. A process for the preparation of a conjugate comprising: wherein the conjugate comprises a biomolecule, an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety; wherein the process comprises enzymatic coupling of a component comprising an enzyme tag, a hydrophilic spacer, a linker, and a lipophilic moiety to a biomolecule in an aqueous medium, and purification of the conjugate; wherein the process comprises the following steps: a) preparing an aqueous dispersion of a component having formula (II); 【Chemistry 1】 where: m is any integer from 15 to 60; n is any integer from 3 to 27, and p is any integer from 0 to 9, b) adding a transpeptidase and a biological molecule which is a polypeptide and is selected from an antigen, a cell adhesion protein, a peptide hormone, a cytokine, or a receptor for 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 form conjugates with biomolecules; and d) purifying the conjugate obtained in step c), where purification is a process step that reduces the amount of extraneous elements, including side reaction products, that are not conjugates and may be present in the medium after carrying out the enzymatic reaction; The process comprising:

2. The component represented by formula (II) 【Chemistry 2】 2. The process of claim 1, wherein

3. 3. The process of claim 1 or 2, wherein the biomolecule is an integrin, cadherin, growth factor, or interleukin.

4. 3. The process of claim 1 or 2, wherein the antibody is a monoclonal antibody or fragment thereof; a camelid- or cartilaginous fish-derived heavy chain-only antibody or fragment thereof, or the artificial antibody is a DARPin®, Adnectin™, Anticalin™, or Affibody™.

5. The process of claim 4, wherein the fragment of the monoclonal antibody is a single chain variable fragment (scFv), a variable fragment (Fv), or a Fab, Fab', or F(ab')2.

6. 5. The process of claim 4, wherein the camelid- or cartilaginous-derived heavy chain-only antibody fragment is a single variable neoantigen receptor (NAR) domain antibody fragment (vNAR).

7. 5. The process of claim 1, 2 or 4, wherein the biomolecule is a single domain antibody derived from the variable domain of a camelid heavy chain only antibody (VHH).

8. 8. The process according to any one of claims 1 to 7, wherein the biomolecule has a C-terminal motif for enzymatic conjugation by a transpeptidase prior to its coupling with the building block according to formula (II).

9. 9. The process of claim 8, wherein the biomolecule has a C-terminal motif for enzymatic conjugation with sortase A prior to its coupling with the building block according to formula (II).

10. 10. The process of claim 9, wherein the C-terminal motif consists of the amino acid sequence "Leucine-Proline-X-Threonine-Glycine" (LPXTG), where "X" can be any proteinogenic amino acid.

11. 11. The process of claim 10, wherein the proteinogenic amino acids present in the LPXTG motif are glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, or histidine.

12. 12. The process of claim 11, wherein the proteinogenic amino acid present in the LPXTG motif is glutamic acid.

13. 13. The process of any one of claims 1 to 12, wherein the enzyme in step b) is a sortase.

14. 14. The process of claim 13, wherein the sortase is sortase A.

15. m is any integer from 25 to 45; n is any integer from 7 to 19, and The process of any one of claims 1 to 14, wherein p is any integer from 2 to 7.

16. 16. The process of any one of claims 1 to 15, wherein the side reaction products include fragments of the enzyme substrate that inevitably appear during conjugation, the enzyme itself or a cofactor required for the reaction, or unreacted substrate.

17. The process of any one of claims 1 to 16, wherein the purification comprises one or more chromatography steps.

18. The process according to any one of claims 1 to 17, wherein the purification involves reverse phase HPLC.

19. A method for modifying a lipid-based drug delivery system comprising a solid lipid nanoparticle, a nanoemulsion, a micelle, or a liposome, comprising: Preparing the conjugate by the process of any one of claims 1 to 18, and Use of the resulting conjugates in the modification of lipid-based drug delivery systems, including solid lipid nanoparticles, nanoemulsions, micelles, or liposomes. The method comprising:

20. A method for modifying the membrane of a living cell, including a T cell, comprising: Preparing a conjugate by the process of any one of claims 1 to 14, and Using the resulting conjugate for membrane modification of living cells, including T cells. The method comprising:

21. A method for modifying a surface to have an affinity for a hydrophobic material, including hydrophobic polystyrene, comprising: Preparing a conjugate by the process of any one of claims 1 to 14, and Using the resulting conjugate to modify surfaces with affinity for hydrophobic materials, including hydrophobic polystyrene. The method comprising:

22. A method for modifying the membrane of an exosome, comprising: Preparing a conjugate by the process of any one of claims 1 to 14, and Using the resulting conjugate for modifying the membrane of exosomes. The method comprising:

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