Solid-phase immobilized microbial transglutaminase MTG and site-specific conjugation of antibodies to lysine residues by MTG in solution

Abstract

To provide a method for conjugation of organic molecules to proteins, and a method for creation of fusion proteins.SOLUTION: A method for conjugating a peptide linker comprising a lysine residue and / or a glutamine residue to an antibody or an antigen-binding fragment thereof by means of a microbial transglutaminase (MTG) comprises: a) mixing solutions containing the antibody or the antigen-binding fragment thereof, the peptide linker and the MTG within a fluid under predetermined conditions, thereby conjugating the peptide linker to the antibody or antigen-binding fragment thereof under a catalyzing effect of the MTG, where the peptide linker is mixed with the antibody or antigen-binding fragment thereof at a molar ratio of 0.5 to 50 times; and b) extracting the conjugate obtained in the step (a) from the fluid.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for conjugating an organic molecule to a protein and a method for producing a fusion protein using microbead-immobilized MTG (microbial transglutaminase MTG) and / or MTG polymer conjugate in solution and / or free MTG in solution.

[0002] In recent years, site-specific functionalization of proteins by enzymes has attracted considerable interest in the field of bioconjugation. Bioconjugation reactions carried out by enzymes usually exhibit fast reaction rates at low reagent concentrations (submillimolar), high conversion efficiencies, and this can be carried out under physiological conditions.

[0003] Despite these advantages, the enzyme must then be removed from the mixture to avoid any downstream interference, and thus the enzyme is lost. This could be avoided by the simple recovery of the enzyme through immobilization on a solid phase. Immobilization of enzymes has so far been applied almost only to the conversion of small compounds, and it has been reported that it enhances the stability of the enzyme and furthermore leads to enhanced activity, selectivity or degree of selectivity. The tunable properties of immobilized enzymes (i.e., enhanced selectivity for specific residues) for site-specific modification of high molecular weight substrates such as proteins under continuous operation for conjugating various protein formats several times have not yet been reported.

[0004] Policarpo et al. have recently shown the conjugation of an enzyme onto Ni-NTA agarose beads. Unfortunately, this conjugation has a non-covalent nature and a high risk of column bleed.

[0005] Accordingly, an object of the present invention is to provide a firmly immobilized enzyme and / or a method for providing an enzyme in solution for use in an active flow reactor column or spin column that reaches high speeds for the desired conjugation of organic molecules to proteins while substantially avoiding any column bleed of the enzyme.

[0006] This object is achieved by a method for conjugating an organic molecule to a target protein and producing a fusion protein using MTG (microbial transglutaminase) in immobilized and / or non-immobilized form according to the present invention, the method comprising the following steps: a) immobilizing MTG by binding the MTG to the polymer by exposing cross-reactive groups of the polymer; b) adsorbing the MTG-polymer conjugate onto microbeads or placing the MTG-polymer conjugate in solution; c) filling an active flow reactor column with the MTG-polymer conjugate-adsorbed microbeads and / or the solution containing the MTG-polymer conjugate and / or the solution containing MTG; d) supplying the target protein and the organic molecule into the fluid and passing the fluid through the filled active flow reactor column under predetermined conditions or mixing with the solution containing the MTG-polymer conjugate and / or the solution containing MTG, thereby conjugating the organic molecule to the protein under the catalytic action of MTG; e) extracting the protein-organic molecule conjugate from the fluid is achieved by a method comprising.

[0007] This method provides, for the first time, an opportunity to conjugate organic molecules to proteins with a high conversion rate of the free form, efficiently forming protein-organic molecule conjugates. For covalent bonding to the polymer of MTG, the breeding of MTG can be suppressed to an advantageous extent. This method surprisingly results in enhanced selectivity for one (or more desired) enzyme-reactive residues on a protein, peptide, or other biomolecule to be conjugated in the presence of multiple reactive residues that would all be targeted if conjugation were performed using an immobilized enzyme in solution, which would result in an undesirable mixture of conjugated molecules to varying degrees or complete conjugation of all residues. When using MTG in its immobilized form in this way, only one (or more desired) residue is each targeted and conjugated. Of course, MTG can also be used in its free form in solution and / or as an MTG polymer conjugate introduced into solution. Regarding MTG, those skilled in the art understand that MTG is preferably derived from the organism Streptomyces mobaraensis.

[0008] The binding of the polymer to the microbeads can be brought about in a stable manner when the polymer ionically and / or covalently binds to the microbeads. A preferred example of the polymer can be a second-generation dendritic polymer (de-PG2).

[0009] According to a preferred embodiment of the present invention, the target protein can be selected from the group consisting of antibodies or fragments thereof in IgG, IgM, IgA, or IgE format, preferably monoclonal, optionally containing a mutation at N297 (e.g., N297Q or N297A) (EU numbering scheme), and selected to be chimeric, humanized, or bispecific, deglycosylated or non-glycosylated monoclonal, and also preferably further containing other reactive glutamine residue mutations in the antibody backbone that allow conjugation via MTG.

[0010] According to another preferred embodiment of the present invention, the target protein is a peptide, for example, Fab, Fab’, F(ab)’2, F(ab)’3, Dab, Fv, single-chain Fv (scFv) fragment scFv-Fc, (scFv)2, where the further possible proteins and / or peptides are proteins and peptides involved in the recognition of other proteins and peptides, for example, but not limited to, protein kinases, such as mitogen-activated protein (MAP) kinases, and kinases that directly or indirectly phosphorylate MAP kinases, Janus kinases (JAK1) and cyclin-dependent kinases, epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, fibroblast growth factor (FGF) receptor, insulin receptor and insulin-like growth factor (IGF), artificial proteins, such as darpin, affibody / nanobody or fibronectin fragment, or carrier proteins or haptens, such as CRM197, a mutant of diphtheria toxin or GBS67 (accessory protein of PI-2a) that induce an immune response and are thus important for vaccination; furthermore, the target protein preferably also includes conjugation to a non-protein structure, such as a monomeric or multimeric dextran, such as glucan.

[0011] Regarding the enzyme, the enzyme may modify either one or more reactive glutamine residues (such as Q295 and N297Q of the antibody) or one or more reactive lysine residues (such as K288 or K290, K340 of the antibody) on the target protein with an organic molecule; here, the residues are introduced endogenously or artificially by genetic means or a combination thereof.

[0012] Preferred embodiments of the organic molecule to be conjugated to the target protein include fluorescent dyes / labels (e.g., Alexa488, Alexa647), cytotoxic or effector moieties such as toxins or cell modulators, immunocyte immunomodulatory / stimulatory compounds, metal chelating agents suitable for SPECT / PET or MRI (e.g., NODA-GA), functional peptides (e.g., alpha defensin NP-1), chemical moieties suitable for click reactions such as strain-promoted azide-alkyne click chemistry (SPAAC) or tetrazine-alkene ligation, e.g., azide and cyclooctyne derivatives (e.g., DIFO, BCN, DIBAC, DIBO, ADIBO), and primary amines and C n It may be selected from the group consisting of tetrazine and trans-cyclooctene derivatives having a spacer moiety of >20.

[0013] Furthermore, the organic molecule is conjugated to a functional moiety such as a cytotoxic moiety with a (C+N) n >20 peptide, fluorescent dye, metal chelating agent, or chemical moiety suitable for SPAAC click reaction (e.g., azide group or DBCO group) or tetrazine and trans-cyclooctene group or its derivatives. In particular, the peptide may contain lysine (e.g., KNAA or KAYA) or glutamine residues (e.g., FGLQPRY), which are targeted by MTG (i.e., are substrates for MTG), optionally with a C n >20 spacer moiety (e.g., polyethylene glycol, alkyl group) and / or conjugated via a primary amine.

[0014] Furthermore, the organic molecule can be selected from the group consisting of a peptide containing lysine at any position (e.g., KNAAGGG or KDAAGGG or KAYAGGG or AKETAA) or a peptide containing a glutamine residue at any position (e.g., FGLQPRY, SLLQGR), which is targeted by MTG (i.e., is a substrate for MTG) and contains a peptide sequence that is optionally enzymatically cleavable (e.g., valine-citrulline (VC), KNAAGGG-VC); the lysine peptide has a size (length) of (C+N) n >20, and the glutamine peptide has a size (length) of 1 < (C+N) n <200.

[0015] Preferred embodiments of the microbeads or microbead resins can be selected from the group consisting of glass, nickel, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polyacrylate, polyethylene terephthalate, rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PCDF), silicone, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other solid supports include gelatin, glass, Sepharose macrobeads, Sephadex beads or dextran microcarriers, such as CYTODES® (Pharmacia, Uppsala, Sweden), polysaccharides such as agarose, alginate, carrageenan, chitin, cellulose, dextran or starch, polycaprolactone (PCL), polyacrylamide, polystyrene, polyacrolein, polydimethylsiloxane, polyvinyl alcohol, polymethyl acrylate, perfluorocarbon, inorganic compounds such as silica, glass, diatomaceous earth, alumina, gold, iron oxide, graphene and graphene oxide or other metal oxides, or copolymers consisting of any combination of two or more naturally occurring polymers, synthetic polymers or inorganic compounds. The size of the beads can be 1 to 100 nm, or 100 to 1000 nm, or 1 μm to 10 μm, or 10 μm to 1000 μm.

[0016] Suitable examples of the fluid can be selected from the group consisting of water containing a suitable buffer (e.g., Tris) and a salt additive (e.g., NaCl), and the buffer aqueous solution may further contain glycerol and other organic solvents, such as ethanol, propanol, isopropanol, 1-propanol, DMSO, methanol, and acetonitrile up to 60%.

[0017] In addition to first-generation, second-generation, and higher-generation dendritic polymers (de-PG2), suitable polymers can be selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylene oxide, poly(alkyl oxazoline), polyvinyl pyrrolidone, polylysine and polyglutamate, poly(ethyl oxazoline), polymethacrylic acid and polypropacrylic acid, or mixtures and dendrimer structures thereof. Also, examples of polymers based on sugar residues include poly-N-isopropylacrylamide (polyNIPAM), poly(glycidyl methacrylate), polytetrafluoroethylene (PTFE), and poly(ethylene-alt-tetrafluoroethylene) (ETFE), poly(oligoethylene glycol) methacrylate (POEGMA), poly(2-methyl-2-oxazoline) (PMOXA), poly(vinyl alcohol) (PVA), and poly(ethyleneimine) and their derivatives.

[0018] In some embodiments, the conjugation of MTG and the polymer includes a linker (spacer) between the polymer and MTG, and the linker can be selected from the group consisting of bifunctional linker systems S-HyNic (succinimidyl-6-hydrazino-nicotinamide), S-4FB (4-formylbenzoate) or derivatives thereof, or SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) or derivatives thereof, homobifunctional or heterobifunctional spacers having a structure such as Y-S-Z (where Y can also be Z and vice versa), where Y and Z are from the following group or derivatives thereof: tetrazine, trans-cyclooctene, azide, cyclooctene (e.g., dibenzylcyclooctyne or bicyclononene), n-hydroxysuccinimide, maleimide, isothiocyanate, aldehyde, epoxide, alcohol, amine, thiol, phosphonate, alkyne, acylpotassium trifluoroborate, α-keto acid-hydroxylamine, O-acylhydroxylamine, carboxylic acid, hydrazine, imine, norbornene, nitrile and cyclopropene, and S is a spacer unit that is a polymer or a derivative thereof, such as oligo or poly(ethylene glycol) (PEG), dextran made with an alkyl moiety, an amino acid or a peptide derivative.

[0019] Conditions suitable for the progress of conjugation can be brought about when certain conditions include the following details: a temperature range of 0°C to 50°C, a contact time of several seconds to 168 hours (or 7 days), a flow rate / speed of less than 1 μL / min or 1 μL / min to 10 ml / min in an active flow reactor column, a protein concentration of 1 μM to 1 mM, where the molar ratio of the organic molecule to the target protein is 0.5 to 50-fold or 50 to 500-fold or 500 to 10,000-fold, and the MTG concentration is 0.001 mg / ml to 0.01 mg / ml or 0.01 mg / ml to 10 mg / ml per 1 ml of resin or microbeads or conjugation solution, more preferably using an organic molecule with a conjugation efficiency to the target protein of at least 30% and up to 100% and a flow pressure of 0.1 bar to 20 bar.

[0020] Furthermore, the functionalized microbeads can also be placed in a device suitable for a spin column, where the reaction mixture is incubated with the microbeads for a certain period of time of 1 second to 60 seconds, 1 minute to 60 minutes, 1 hour to 168 hours. Then, by centrifugation and removing the supernatant, the mixture is removed from the microbeads again, or during centrifugation, the microbeads are retained while the solution is pushed through a suitable filter device without retaining the mixture / conjugate required for the reaction.

[0021] Preferred embodiments of the present invention are described in more detail below with reference to the accompanying drawings:

Brief Description of the Drawings

[0022]

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[0023] Formation and Characterization of MTG-Polymer-Conjugates Regarding the conjugation of N-succinimidyl-4-formylbenzamide (4FB) to microbial transglutaminase (MTG) (Figure 1), various linker excesses were investigated targeting one linker per MTG. When the linker ratio was quantified by LC-MS and spectrophotometry using UV-VIS, it was found that both methods were in good correlation (Figure 2a). At an equal amount of linker of MTG to 4FB, a conjugation ratio of about 0.4 was obtained, and when it exceeded 2, this ratio increased to about 0.8 (Figure 2a). Although a ratio of 0.8 seemed desirable, the inventors chose to use 1.5 equivalents of 4FB, which resulted in a linker ratio of about 0.5. This is because during the experiment, when the linker ratio was 0.8 and too high, although the enzyme-polymer conjugate was generated, it was observed that the sample precipitated and could not be purified by ultracentrifugation. This may be due to the fact that in the case of 2 equivalents, the amount of doubly bound MTG (MTG-(4FB)2) exceeded 20%, while in contrast, in the case of 1.5 equivalents, this amount remained below 10% (Figure 2b). If the amount of MTG-(4FB)2 is too high, it can lead to unwanted over-crosslinking with the same or another polymer strand, a decrease in MTG activity, and a decrease in the solubility of the enzyme-polymer conjugate. For these reasons, it was particularly emphasized to select 1.5 equivalents of excess 4FB relative to MTG without over-conjugating MTG and to generate singly bound MTG with a purity of over 90%.

[0024] Polymer de-PG2 conjugated to N-succinimidyl 6-hydrazinonicotinate (S-HyNic)-linker 500 (Figure 1) was then incubated with MTG-4FB. When these two bound compounds were mixed, a characteristic peak was formed at 354 nm and the absorption increased as can be seen from UV-VIS (Figure 2c). This indicates that a bis-aryl-hydrazone (BAH) bond was formed and the enzyme-polymer conjugate was successfully generated.

[0025] When the activity of the polymer-enzyme conjugate was assayed in solution using a colorimetric hydroxylamine-amine assay, it was found that MTG still had catalytic activity, although it was clearly reduced compared to native MTG (Figure 2d). This reduction in activity could be explained by the reduced rotational freedom and configurational possibilities of the polymer-immobilized MTG compared to non-immobilized native MTG, which made it more difficult for hydroxylamine to access the enzyme's active site. Similar reductions in activity have been reported for proteinase K immobilized on the same polymer in solution. Nevertheless, the most important thing was that MTG still showed catalytic activity after immobilization.

[0026] Calculation of the amount of MTG-polymer conjugate adsorbed on the microbead glass surface 354 nm (29,000 M -1 cm -1 ) The amount of bead-immobilized MTG could be estimated from the elution volume by a bis-aryl-hydrazone bond quantifiable by UV-VIS at. After 1 hour of incubation, the concentration in the elution volume was determined to be 1.6 ± 0.15 μM, and when the starting concentration was 5 μM, approximately 70% of the conjugate was adsorbed onto the beads. Since most of the MTG was singly cross-linked to the polymer, the adsorbed mass could be estimated to be about 300 ng / cm 2 or about 7.8 pmol MTG / cm 2 These values are consistent with previously published results using proteinase K immobilized on a dendrimer polymer (denpol-polymer) or horseradish peroxidase and other enzymes covalently immobilized on the silica polymer surface.

[0027] Microbead-immobilized MTG for site-specific conjugation of functional molecules to proteins For the stable immobilization of the MTG-polymer conjugate, the use of glass microbeads was selected. This is because the positively charged dendrimer amine (denpol-amine) shows a strong affinity for the negatively charged glass surface and the beads can be easily attached to a flow-based microreactor. Such a setup allows the sample beads to be repeatedly overflowed in a well-controlled manner and thus it can be used to drive the reaction to completion. Furthermore, the microbeads can be easily washed after the conjugation process, thereby enabling the recovery of the immobilized MTG for the next conjugation.

[0028] Therapeutic-related proteins with relatively small antibody-like scaffolds, including scFv, nanobodies or Fab fragments, are quite interesting due to their increased tumor penetration ability, their easy production and their faster clearance compared to larger antibodies. In a first attempt to functionalize the proteins, it was thus aimed to conjugate Fab fragments and scFv, both of which are ideal substrates for further downstream applications involving immobilization on streptavidin-coated surfaces via glutamine 2 (‘Q2’) at their C-terminal myc-tags, using biotin-cadaverine as an amine and being efficiently conjugated by MTG in solution, as has already been shown. Mobile loops and terminal tags on proteins containing glutamine are known to be preferentially targeted by MTG, but the presence of a globular structure and an accessible terminal tag enables conjugation by surface-immobilized MTG and it is not so difficult for the enzyme's active site to enter compared to loop structures such as glutamine 295 on bulky antibodies. Thus, biotin-cadaverine was mixed with C-terminally myc-tagged Fab fragments and scFv, and the solution was flowed over microbead-immobilized MTG in a microreactor (Figures 1 and 3a). This solution was pumped through the microreactor for 30 minutes and LC-MS analysis was performed. It was found that more than 95% complete conversion to the desired biotin-conjugated Fab fragments occurred and non-conjugated material was not detected (Figure 3b). Also, c-myc-tagged scFv was efficiently conjugated within just 30 minutes under the same conditions, yielding more than 95% of the desired conjugate (Figure 3b).

[0029] Dansylcadaverine, a fluorescent amine donor for MTG, was also conjugated to Fab and scFv at a slight 8 equimolar excess (Figure 3a), yielding more than 95% of dansyl-labeled Fab fragments and more than 90% of scFv within 30 minutes (Figure 3b).

[0030] In some cases, directly conjugating a bulky primary amine-containing substrate to MTG-reactive glutamine may result in incomplete product conversion with residual unconjugated material. This occurs particularly with those substrates containing highly hydrophilic groups such as carboxy groups on metal chelating agents. Using a two-step approach where a "clickable" moiety is first installed on the protein followed by click conjugation of the desired molecule can avoid this problem and result in quantitative conjugation. Therefore, it was investigated whether an amine-PEG3-azide suitable for strain-promoted alkyne-azide cycloaddition (SPAAC) click chemistry (Figure 3c) could be conjugated to myc-tagged scFV. When the myc-tagged scFV was flowed through the column and samples were taken out after 30 minutes and subjected to LC-MS analysis, already up to 82% of the desired scFV-N3 conversion had occurred (Figure 3c). Continuing to overflow the beads for 90 minutes gave a 97% conversion, and after flowing continuously overnight (14 hours), unconjugated material was no longer detected (Figure 3c). These results clearly demonstrate that microbead-immobilized MTG was able to quantitatively conjugate various substrates to myc-tagged proteins in less than 90 minutes with a small reagent excess.

[0031] Based on these results, it was investigated whether full antibodies could also be conjugated by bead-immobilized MTG. Previously, one specific glutamine 295 (Q295) of a deglycosylated antibody was on the mobile C’E loop of the Fc domain as the only MTG target site within the antibody backbone. Thus, introducing an N297Q point mutation that excises the glycosylation site results in defined antibody conjugates with two or four binding sites. Antibody conjugation on the surface may be more difficult than in solution due to the bulkiness of the antibody, which limits the possible orientations. Therefore, access of MTG to Q295 and Q297 in the loop is likely to be more difficult.

[0032] Therefore, to avoid deglycosylation and biotin-cadaverine, immobilized MTG was provided to an IgG1 antibody containing the N297S point mutation (Figure 4a). After 30 minutes, LC-MS analysis of the reduced antibody already showed a 37% heavy chain conversion rate, and after 1 hour at room temperature it showed 72%, which increased to over 95% during an overnight incubation (Figure 4a). Site-specifically modified antibodies with a high drug-to-antibody ratio (DAR) of 4 to 6 are thought to effectively deliver the conjugated drug to the tumor target site compared to conventional non-specific conjugated antibodies with the same amount of drug, and thus such ADCs are very attractive as improved cancer therapies. Therefore, it was investigated whether immobilized MTG would maintain its ability to conjugate two SPAAC-sensitive bifunctional linkers in very close proximity to the N297Q antibody (Figure 4b). It was found that bead-immobilized MTG could actually conjugate both linkers quantitatively and efficiently, resulting in an N297Q antibody with a ratio of 4 linkers per antibody (Figure 4b).

[0033] These studies revealed that the specific and efficient conjugation ability of MTG is maintained upon immobilization and can even be adjusted for protein residue specificity.

[0034] Investigation of the stability of MTG-polymer conjugates and MTG activity on microbeads It has already been shown that the polycationicity of the dendritic polymer results in stable surface immobilization on anionic glass surfaces over several weeks. Since solid enzyme immobilization is important especially for downstream applications of promising therapeutic proteins, slot blot assays and countermeasures against enzyme leakage using anti-MTG antibodies have been made. The slot blot was selected because a large number of samples can be applied and because of its sensitivity. The MTG-polymer conjugate as a positive control showed a strong signal (Figure 4c, lane 1, i), and unconjugated MTG could also be detected (lane 1, ii), but the HyNic polymer did not generate a signal (lane 1, iii). This demonstrates that the antibody specifically recognized MTG even when MTG was conjugated to the polymer. After 14 hours of continuous operation, MTG remained firmly bound (lane 2, i and ii), and even after 40 hours of operation with multiple conjugations, MTG remained firmly bound to the polymer and the enzyme was not detected (lane 2, iii). Under the latter conditions, immobilized MTG was able to conjugate BC to N297S IgG1 to over 90% even after 3 hours. From these data, it was concluded that MTG not only remains firmly bound to glass microbeads, but is also active during long periods and multiple conjugations, and is therefore a very promising tool for conjugating proteins in the same size range as MTG.

[0035] Increased residue selectivity in the presence of multiple MTG-reactive amino acids of immobilized MTG Since immobilized enzymes have been reported to exhibit enhanced selectivity towards substrates, it was thought that this could also be applicable to immobilized MTG. The conjugation of ZQG-Tamra-cadaverine (ZQG-TC) in solution to avidin (functioning as a model protein) having several reactive lysine residues using MTG showed two major peaks in the deconvoluted LC-MS spectrum. These peaks corresponded not only to avidin having one ZQG-TC and avidin having two conjugated ZQG-TCs, but also to several unmodified avidin (Figure 5, left panel). The same experiment was performed, but using immobilized MTG instead. Surprisingly, it was found that, probably due to the reduced rotational freedom of MTG, MTG almost exclusively targeted one lysine residue of avidin and conjugation to the second residue was limited (Figure 5, right panel). These data indicate that immobilized MTG can actually be used to tune residue selectivity that is inaccessible in liquid-phase conjugation which mainly conjugates two residues.

[0036] Site-specific conjugation of small glutamine-containing peptides to lysine residues of deglycosylated and non-glycosylated IgG1 Functionalization of antibodies via MTG through lysine side chains using ZQG derivatives has been reported, but the conjugation yield was not satisfactory (i.e., <20%), and the modified lysine sites were not reported. Therefore, further studies aim to target the lysine residues of non-glycosylated and deglycosylated IgG1 using MTG in solution and immobilized MTG. It was inferred that various glutamine peptides might conjugate more efficiently to the lysine residues on IgG1 than the commonly applied ZQG or its derivatives. Therefore, a small library of glutamine-containing peptides with high reported MTG activity in solution was first screened under various pH conditions, which was then desired to be applied to immobilized MTG. Indeed, after incubating at room temperature for 16 hours, sequences with favorable conjugation ratios to deglycosylated IgG1 could be identified, which showed higher reactivity than ZQG using LC-MS for analysis (Figure 6). In particular, the peptide sequence NH2-FGLQRPY-COOH showed a conjugation efficiency of nearly 50%, which was nearly twice as high as that of ZQG in a solution at pH 7.6 for deglycosylated IgG1 (Figure 6). When deglycosylated IgG1 and the peptide FGLQRPY (Peptide 2, for the peptide structure, see the left of Figure 7) were subjected to immobilized MTG overnight at room temperature, 30% conjugation was found (Figure 8). Surprisingly, when using the IgG1 non-glycosylated N297S mutant and FGLQRPY, the conjugation rate increased to 71% by immobilized MTG and MTG in solution (the left and right of Figure 9, respectively). The same results were obtained using the FGLQRPY azide derivative (for the peptide structure, see the right of Figure 7) with immobilized MTG and MTG in solution and using LC-MS for analysis (the left and right of Figure 10, respectively). Using LC-MS for analysis, mainly a single modified species and a minor second conjugation were found, both of which were exclusively on the heavy chain. Peptide mapping confirmed two modification sites at Lys288 or Lys290 and at Lys340.

[0037] Site-Specific Dual Conjugation of Immobilized and Solution MTG to Non-Glycosylated IgG1 (N297S Variant) Since conjugation of glutamine and lysine was established using immobilized and solution MTG, it was inferred whether site-specific dual modification using immobilized and solution MTG could be achieved by modifying Q295, K340, and K288 / K290 of N297S IgG1. Such dual-modified antibodies, for example, dual-modified antibodies having two imaging probes, would be very suitable for, for example, non-invasive and / or intraoperative / postoperative tissue imaging. Also, two different toxic payloads showing a synergistic effect could be conjugated. When Q295 was first modified with NH2-PEG3-TCO by more than 95% and then with a peptide-2 azide derivative, a dual-site-specific modified IgG1 with a slightly lower yield of 38% was obtained (Figure 11, left), and similar results were obtained with dual-site-specific conjugation using solution MTG (Figure 11, right). SDS-PAGE confirmed heavy-chain-specific conjugation to lysine residues, and LC-MS results were also confirmed for the dual-conjugate antibody (Figure 12).

[0038] Conjugation of Functional Lysine Peptides to Deglycosylated Antibodies It was also investigated whether peptides containing lysine residues could also be used to site-specifically modify deglycosylated antibodies at glutamine 295 using solution-phase MTG, which has not been described in the literature to date. By equipping peptides with functional groups such as such N3-groups (KAYA-GGG-N3) or metal chelators (e.g., NODAGA), in the first case, for example, another moiety could be subsequently conjugated by SPAAC-click chemistry at low molar equivalents. In the second case, since the functional moiety can be directly conjugated, the second step is not required, which facilitates further downstream processing. Furthermore, by incorporating hydrophilic amino acids into the peptide, the solubility of the functional moiety (“the payload”) can be increased, which is highly beneficial for hydrophobic payloads. In this study, it was shown that KAYA-GGG-N3 can be conjugated to deglycosylated antibodies with high efficiency (>95%) similar to KNAA-GK-PEG3-NODAGA and KAYA-GK-PEG3-NODAGA.

Claims

**Claim 1** A method for conjugating a peptide linker containing lysine residues and / or glutamine residues to an antibody or an antigen-binding fragment thereof using microbial transglutaminase (MTG), comprising: a) mixing in a fluid a solution containing the antibody or the antigen-binding fragment thereof, the peptide linker, and MTG, thereby conjugating the peptide linker to the antibody or the antigen-binding fragment thereof under the catalytic action of MTG, the step of mixing the peptide linker with the antibody or the antigen-binding fragment thereof at a molar ratio of 0.5 to 50 times; and b) extracting the conjugate obtained in step a) from the fluid A method comprising the above steps. **Claim 2** The method according to claim 1, wherein the MTG concentration in the fluid is 0.01 mg / mL to 10 mg / mL. **Claim 3** The method according to claim 1 or 2, wherein the conjugation efficiency to the antibody or the antigen-binding fragment thereof is at least 30%. **Claim 4** The method according to any one of claims 1 to 3, wherein the peptide linker is a linker containing lysine residues. **Claim 5** The method according to any one of claims 1 to 4, wherein the fluid is a buffered aqueous solution. **Claim 6** The method according to claim 5, wherein the buffered aqueous solution contains Tris and NaCl. **Claim 7** The method according to any one of claims 1 to 6, wherein the antibody or the antigen-binding fragment thereof, the peptide linker, and the MTG are mixed at pH 7.

6. **Claim 8** The method according to any one of claims 1 to 7, wherein the antibody is an antibody or a fragment thereof in the IgG, IgM, IgA or IgE format. **Claim 9** The antigen-binding fragment may be Fab, Fab', F(ab)' ２ , F(ab)′ ３ , Dab, Fv fragment, single chain Fv (scFv) fragment or scFv-Fc (scFv) ２ 8. The method according to claim 1, wherein **Claim 10** The MTG modifies either one or more reactive glutamine residues or one or more reactive lysine residues on the antibody or the antigen-binding fragment thereof with the peptide linker, wherein the one or more reactive glutamine residues or lysine residues are a) endogenous glutamine residues or lysine residues; b) artificially introduced into the antibody or the antigen-binding fragment thereof by genetic means; or c) a combination of (a) or (b) The method according to any one of claims 1 to 9. **Claim 11** The peptide linker further includes a fluorescent dye / label, a cytotoxic or influencing moiety, a metal chelating agent, a functional peptide, a chemical moiety, and / or a spacer moiety having C ｎ >20, the method according to any one of claims 1 to 10. **Claim 12** The method according to any one of claims 1 to 11, wherein the peptide linker further comprises an enzymatically cleavable peptide sequence.

13. The method according to any one of claims 1 to 12, wherein the peptide linker comprises a sequence of KNAA, KAYA, KNAAGGG, KDAAGGG, KAYAGGG, AKETA, FGLQPRY or SLLQGR.

14. The method according to any one of claims 1 to 13, wherein the peptide linker further comprises a self-destructive group.

15. The method according to claim 14, wherein the self-destructive group is p-aminobenzyloxycarbonyl (PAB).

16. The antibody is a monoclonal antibody, chimeric antibody, humanized antibody, human antibody or bispecific antibody, and / or the antibody is deglycosylated or non-glycosylated and contains a mutation at residue N297 in the EU numbering scheme. The method according to any one of claims 1 to 15.

17. The cytotoxic or affecting moiety is a toxin or an immunocyte immunomodulatory / stimulatory compound; and / or The metal chelating agent is suitable for SPECT / PET or MRI; and / or The chemical moiety contains a reactive group suitable for a click reaction; and / or The spacer moiety contains an alkyl or heteroalkyl chain or derivatives thereof, or a polyethylene glycol moiety. The method according to claim 11.

18. The toxin is MMAE; and / or The reactive group suitable for a click reaction contains an azide moiety, a cyclooctyne moiety, a tetrazine moiety, a trans-cyclooctene moiety, or derivatives thereof. The method according to claim 17.

19. The method according to any one of claims 1 to 18, wherein the fluid contains glycerol and / or an organic solvent up to 60%.

20. The lysine peptide has a size of (C + N) ｎ > 20, and the glutamine peptide has a size of 1 < (C + N) ｎ < 200, the method according to any one of claims 1 to 19.

21. The method according to any one of claims 1 to 20, wherein the MTG is conjugated to a polymer.

22. The method according to claim 21, wherein the MTG polymer conjugate is immobilized on the microbeads via a covalent bond and / or an ionic bond.

23. The microbeads are selected from the group consisting of glass, nickel, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polyacrylate, polyethylene terephthalate, rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PCDF), silicone, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, gelatin, polysaccharides, polycaprolactone (PCL), polyacrylamide, polyacrolein, polydimethylsiloxane, polyvinyl alcohol, polymethyl acrylate, perfluorocarbon, inorganic compounds, or copolymers composed of any combination of two or more naturally occurring polymers, synthetic polymers or inorganic compounds, and / or the size of the microbeads is in the range of 1 nm to 1000 μm, the method according to claim 22.

24. The polysaccharide is agarose, alginate, carrageenan, chitin, dextran or starch; and / or the inorganic compound is silica, glass, diatomaceous earth, alumina, gold, iron oxide, graphene, graphene dioxide or other metal oxides, the method according to claim 23.

25. The polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylene oxide, poly(alkyl oxazoline), polyvinyl pyrrolidone, polylysine and polyglutamate, poly(ethyl oxazoline), polymethacrylic acid and polypropacrylic acid, or mixtures and dendrimer structures thereof; further including polymers based on sugar residues, poly-N-isopropylacrylamide (polyNIPAM), poly(glycidyl methacrylate), polytetrafluoroethylene (PTFE) and poly(ethylene-alt-tetrafluoroethylene) (ETFE), poly(oligoethylene glycol) methacrylate (POEGMA), poly(2-methyl-2-oxazoline) (PMOX A), poly(vinyl alcohol) (PVA) and poly(ethyleneimine) and their derivatives, and / or the polymer is a second-generation dendritic polymer (de-PG2), the method according to any one of claims 21 to 24.

26. The MTG polymer conjugate includes a linker (spacer) between the polymer and the MTG, and the linker is a bifunctional linker system such as S-HyNic (succinimidyl-6-hydrazino-nicotinamide), S-4FB (4-formylbenzoate) or a derivative thereof, or SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or a derivative thereof, a homo-bifunctional or hetero-bifunctional spacer having a structure such as Y-S-Z (Y can also be Z, and vice versa), where Y and Z are from the following group or a derivative thereof: tetrazine, trans-cyclooctene, azide, cyclooctene (e.g., dibenzylcyclooctyne or bicyclononene), n-hydroxysuccinimide, maleimide, isothiocyanate, aldehyde, epoxide, alcohol, amine, thiol, phosphonate, alkyne, acyl potassium trifluoroborate, α-keto acid-hydroxylamine, O-acylhydroxylamine, carboxylic acid, hydrazine, imine, norbornene, nitrile and cyclopropene, and S is a spacer unit that is a polymer or a derivative thereof, such as oligo- or poly(ethylene glycol) (PEG), dextran made with an alkyl moiety, an amino acid or a peptide derivative, the method according to any one of claims 21 to 25. **Claim 27** The MTG polymer conjugate is held in an active flow reactor, the method according to any one of claims 21 to 26.

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