Site-specific conjugation of antibody lysine residues using both solid-phase immobilized microorganism transglutaminase MTG and MTG in solution

Abstract

This application relates to site-specific conjugation of antibody lysine residues using solid phase immobilized microbial transglutaminase MTG and MTG in solution. Site-specific modification of proteins using microbial transglutaminase (MTG) is a powerful and versatile strategy for controlled modification of proteins under physiological conditions. We present evidence that solid phase microbead immobilization can be used to site-specifically and efficiently link different functional molecules important for further downstream applications to therapeutically relevant proteins, including scFV, Fab fragments and antibodies. We demonstrate that MTG remains firmly immobilized and there is no detectable column bleed and the enzyme activity is maintained during continuous operation, which allows for convenient recycling of the enzyme, thus outperforming solution phase MTG conjugation. Furthermore, it is shown that immobilized MTG exhibits an enhanced selectivity for certain residues in the presence of several reactive residues, all of which are targeted if conjugation is performed in solution. Site-specific lysine conjugation of antibodies using immobilized and solution MTG with a highly efficient glutamine-containing peptide is also reported. In addition, the generation of bis-site-specific conjugated IgGl with site-specific conjugation of both glutamine and lysine residues of an IgGl antibody using immobilized and solution MTG is reported. Site-specific glutamine conjugation with small peptides containing lysine residues and functional moieties is also described.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780045202.X entitled "Site-specific conjugation of antibody lysine residues by solid-phase immobilized microbial transglutaminase MTG and MTG in solution". The original application was PCT international application PCT / EP2017 / 067403 filed on July 11, 2017, which entered the Chinese national phase on January 21, 2019.

[0002] This invention relates to a method for conjugating organic molecules with proteins to produce fusion proteins using microbead-immobilized MTG (microbial transglutaminase MTG) and / or MTG polymer conjugates in solution and / or free MTG in solution.

[0003] In recent years, the site-specific functionalization of proteins via enzymes has attracted considerable interest in the field of bioconjugation. Bioconjugation reactions mediated by enzymes typically exhibit rapid kinetics, high conversion efficiency at low reagent concentrations (sub-millimolar), and can be carried out under physiological conditions.

[0004] Despite these benefits, the enzyme must subsequently be removed from the mixture to avoid any downstream interference and thus loss. With solid-phase immobilization, this can be avoided through simple enzyme recovery. To date, enzyme immobilization has been applied almost exclusively to the transformation of small compounds, and it has been reported to enhance enzyme stability and may also lead to increased activity, selectivity, or specificity. Tunable properties (i.e., enhanced selectivity for certain residues) of immobilized enzymes that perform site-specific modifications to large molecular weight substrates such as proteins through successive rounds of conjugation in different protein forms have not been reported.

[0005] Recently, Policarpo et al. demonstrated the conjugation of enzymes to Ni-NTA agarose beads. Unfortunately, this conjugation is non-covalent and shows a high risk of column efflux. Summary of the Invention

[0006] Therefore, the object of the present invention is to provide a method for use in an active flow reactor column or a rotating column to achieve high-rate, robust immobilization of enzymes and / or enzymes in solution for the desired conjugation of organic molecules with proteins, while avoiding any significant column loss of the enzymes.

[0007] According to the present invention, this objective is achieved by using immobilized and / or non-immobilized forms of MTG (microbial transglutaminase) to conjugate organic molecules with target proteins to produce fusion proteins, comprising the following steps:

[0008] a) Immobilizing the MTG by attaching it to the polymer through exposing the cross-reactive groups of the polymer;

[0009] b) Adsorbing the MTG polymer conjugate onto microbeads or making the MTG polymer conjugate into a solution;

[0010] c) Pack an active flow reactor column with microbeads adsorbed with the MTG polymer conjugate and / or a solution containing the MTG polymer conjugate and / or a solution containing the MTG.

[0011] d) Under defined conditions, the target protein and the organic molecule are provided in a fluid and the fluid is passed through a packed active flow reactor column or the fluid is mixed with a solution containing the MTG polymer conjugate and / or a solution containing MTG, thereby conjugating the organic molecule to the protein under the catalysis of the MTG;

[0012] e) Extract protein-organic molecular conjugates from the fluid.

[0013] This method offers, for the first time, the opportunity to efficiently conjugate organic molecules to proteins using high conversion of the precipitate to form protein-organic molecular conjugates. Due to the covalent binding of MTG to the polymer, the loss of MTG can be suppressed to a favorable degree. Through enzyme immobilization, this method unexpectedly leads to enhanced selectivity for one (or more) desired enzyme-reactive residues on the protein, peptide, or other biomolecule to be conjugated in the presence of multiple reactive residues, whereas if conjugation were performed in solution with a non-immobilized enzyme, all of these residues would be targeted. This would result in an undesirable mixture of molecules conjugated to varying degrees or with all residues completely conjugated. Therefore, using an immobilized form of MTG allows for the targeting and conjugation of only one (or more) desired residues, individually. Of course, MTG can also be used as its free form in solution and / or as an MTG polymer conjugate in solution. Regarding MTG, those skilled in the art will understand that MTG is preferably derived from the organism *Streptomyces mobaraensis*.

[0014] The binding of the polymer to the microspheres can be achieved in a stable manner when the polymer undergoes ionic and / or covalent bonding with the microspheres. A preferred example of the polymer is a second-generation dendritic polymer (de-PG2).

[0015] According to a preferred embodiment of the invention, the target protein may be selected from the group consisting of antibodies or fragments thereof in the form of IgG, IgM, IgA or IgE, and is preferably monoclonal, optionally selected as chimeric, humanized, human or bispecific, deglycosylated or non-glycosylated, containing N297 mutations (e.g. N297Q or N297A) (EU numbering scheme), and preferably also containing other reactive glutamine residue mutations in the antibody backbone capable of MTG-mediated conjugation.

[0016] According to another preferred embodiment of the invention, the target protein is a peptide, such as Fab, Fab', F(ab)'2, F(ab)'3, Dab, Fv, single-chain Fv (scFv) fragment scFv-Fc (scFv)2, wherein other possible proteins and / or peptides include proteins and peptides involved in the recognition of other proteins and peptides, including but not limited to protein kinases, such as mitogen-activated protein (MAP) kinase, and kinases that directly or indirectly phosphorylate MAP kinase, Januse kinase (JAKI), and cyclin-dependent kinases, epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, fibroblast-derived growth factor (FGF) receptor, insulin receptor, and insulin-like growth factor (IGF) receptor. Factor (IGF), engineered proteins such as darpin, affinity / nanobody or fibronectin fragments, or carrier proteins, or haptens that elicit an immune response and are therefore important for vaccination, such as diphtheria toxin mutant CRM197, or GBS67 (an accessory protein of PI-2a); in addition, it preferably includes conjugations to non-protein structures, such as mono- or poly-dextrans, such as dextran.

[0017] Regarding enzymes, enzymes can be modified with organic molecules to add one or more reactive glutamine residues (e.g., Q295 and N297Q in antibodies) or one or more reactive lysine residues (e.g., K288, K290, K340 in antibodies); wherein said residues are endogenous or artificially introduced by genetic methods or combinations thereof.

[0018] Preferred embodiments of the organic molecule to be conjugated with the target protein may be selected from the group consisting of: fluorescent dyes / labels (e.g., Alexa488, Alexa647), cellular cytotoxic or influencing moieties such as toxins or cell regulators, immune cell immunomodulatory / stimulatory compounds, metal chelators suitable for SPECT / PET or MRI (e.g., NODA-GA), functional peptides (e.g., α-defensin NP-1), chemical moieties suitable for click reactions such as strain-promoted azide-alkyne click chemistry (SPAAC) or tetrazine-olefin linkages, including azides and cyclooctyne derivatives (e.g., DIFO, BCN, DIBAC, DIBO, ADIBO), and tetrazine and trans-cyclooctene derivatives having a primary amine for MTG-mediated conjugation and a C n >20 spacer portion.

[0019] Furthermore, organic molecules can be selected from the group consisting of: those with (C+N) conjugated to the functional moiety. n Peptides with a concentration >20, wherein the functional moiety is, for example, a cytotoxic moiety, a fluorescent dye, a metal chelating agent, or a chemical moiety suitable for SPAAC click reactions (e.g., an azide or DBCO- group) or a tetrazine and trans-cyclooctene group or derivative thereof. In particular, the peptide may contain lysine (e.g., KNAA or KAYA) or glutamine residues (e.g., FGLQPRY) and be targeted by MTG (i.e., a substrate of MTG), optionally containing C n >20 spacer portions (e.g., polyethylene glycol, alkyl groups) and / or via primary amine conjugation.

[0020] Furthermore, the organic molecule may be selected from the group consisting of: peptides containing lysine residues at any position (e.g., KNAAGGG, KDAAGGG, KAYAGGG, or AKETAA) or glutamine residues at any position (e.g., FGLQPRY, SLLQGR), and targeted by MTG (i.e., substrates of MTG), and optionally containing enzymatically cleavable peptide sequences (e.g., valine-citrulline (VC), KNAAGGG-VC); the size (length) of the lysine peptide is (C+N). n >20 and the size (length) of the glutamine peptide is 1 < (C+N) n <200.

[0021] Preferred embodiments of microbeads or microbead resins may be selected from the group consisting of: glass, nickel, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polyacrylate, polyethylene terephthalate, rayon, nylon, poly(vinyl butyrate), polyvinylidene fluoride (PCDF), silicone, polyoxymethylene, cellulose, cellulose acetate, nitrocellulose, etc. Other solid supports include gelatin, glass, agarose beads, cross-linked dextran 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; polyacrylaldehyde; polydimethylsiloxane; polyvinyl alcohol; polymethacrylate; perfluorocarbon; inorganic compounds, such as silica, glass, diatomaceous earth, alumina, gold, iron oxides, graphene and graphene oxides or other metal oxides; or copolymers of any combination of two or more naturally occurring polymers, synthetic polymers, or inorganic compounds. Bead sizes can range from 1 nm to 100 nm, 100 nm to 1000 nm, 1 μm to 10 μm, or 10 μm to 1000 μm.

[0022] Suitable examples of fluids may be selected from the group consisting of: water containing a suitable buffer (e.g., Tris) and a salt additive (e.g., NaCl), the buffered aqueous solution may also contain up to 60% glycerol and other organic solvents such as ethanol, propanol, isopropanol, 1-propanol, DMSO, methanol, acetonitrile.

[0023] 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) (zoline), polyvinylpyrrolidone, polylysine and polyglutamic acid, poly(ethyl) (Azoline), polymethacrylic acid and polypropacrylic acid, or mixtures thereof and dendritic structures. Also includes sugar residue-based polymers, poly-N-isopropylacrylamide (polyNIPAM), poly(glycidyl methacrylate), polytetrafluoroethylene (PTFE) and poly(ethylene-alternating-tetrafluoroethylene) (ETFE), poly(oligoethylene glycol) methacrylate (POEGMA), poly(2-methyl-2- (PMOXA), poly(vinyl alcohol) (PVA), and poly(ethylene imine) and their derivatives.

[0024] In some embodiments, the conjugation of the MTG and the polymer involves a joint (spacer) in the polymer and the MTG, said joint being selected from the group consisting of: bifunctional joint systems S-HyNic (succinimide-6-hydrazinoamide, S-4FB (4-formylbenzoate) or derivatives thereof, or SMCC (succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate) or derivatives thereof, and homo- or hetero-bifunctional spacers having a YSZ-like structure (Y can also be Z, or vice versa), wherein Y and Z are the following Groups or their derivatives: tetrazine, trans-cyclooctene, azide, cyclooctene (e.g., dibenzylcyclooctyne or bicyclononyne), n-hydroxysuccinimide, maleimide, isothiocyanate, aldehyde, epoxide, alcohol, amine, thiol, phosphonate, alkyne, potassium acyltrifluoroborate, α-keto-hydroxylamine, O-acylhydroxylamine, carboxylic acid, hydrazine, imine, norbornene, nitrile, and cyclopropene, and S is a spacer entity as a polymer or its derivative, amino acid, or peptide derivative, such as oligomers or poly(ethylene glycol) (PEG), dextran, which are composed of alkyl moieties.

[0025] Suitable conditions for conjugation can be achieved when the determined conditions include the following details: a temperature of 0°C to 50°C, a contact time of several seconds to 168 hours (or 7 days), a flow rate / rate in the active flow reactor column of less than 1 μL / min or 1 μL / min to 10 ml / min, a protein concentration of 1 μM to 1 mM, a molar ratio of organic molecules to target protein of 0.5 to 50× or 50 to 500× or 500 to 10,000×, and an MTG concentration of 0.001 mg / ml to 0.01 mg / ml or 0.01 mg / ml to 10 mg / ml per ml of resin or microbeads or conjugation solution. Preferably, the conjugation efficiency of the organic molecules to the target protein is at least 30% and up to 100% conversion, and the flow pressure is 0.1 bar to 20 bar.

[0026] Furthermore, the functionalized microbeads can be placed in a suitable rotating column apparatus, wherein the reaction mixture is incubated with the beads for a specified period of time, from 1 second to 60 seconds, from 1 minute to 60 minutes, or from 1 hour to 168 hours. The mixture is then removed from the microbeads again by centrifugation and the removal of the supernatant, or the solution is pushed through a suitable filtration device during centrifugation, which retains the microbeads but not the desired mixture. Attached Figure Description

[0027] Preferred embodiments of the present invention will now be described in more detail with reference to the accompanying drawings, which are described below:

[0028] Figure 1The immobilization process of microbial transglutaminase MTG on de-PG2 polymer and its subsequent adsorption on microbeads are schematically illustrated.

[0029] Figure 2. Characterization and quantification of MTG conjugates on de-PG2 polymer. The activity of MTG and MTG-polymer conjugates was normalized to 1 μM MTG.

[0030] Figure 3 schematically illustrates how MTG immobilized with microbeads allows functional molecules to be conjugated to antibody-like backbones such as scFV and Fab fragments.

[0031] Figure 4 schematically illustrates the use of microbead-immobilized MTG to conjugate functional molecules with (non-glycosylated) antibodies (N297S or N297Q mutants); and the detection of MTG attached to the polymer by anti-MTG antibody; and

[0032] Figure 5 Compared to conjugation in solution, immobilized MTG exhibits enhanced selectivity in the presence of a variety of reactive amino acids.

[0033] Figure 6 Peptides containing glutamine that are conjugated with deglycosylated IgG1 (Herceptin) were screened using different pH conditions (sequences shown in the table on the right). Peptide 2 was found to be most effective at pH 7.6, producing ≥50% conjugation.

[0034] Figure 7 The structure of peptide 2 (left) and its azide derivative (right).

[0035] Figure 8 Deglycosylated IgG1 was conjugated to peptide FGLQRPY (peptide 2) using a solid-phase immobilized MTG.

[0036] Figure 9 Peptide 2 was conjugated to IgG1 (containing the N297S mutation) using fixed MTG (left) and in solution MTG (right), resulting in >70% conjugation.

[0037] Figure 10 Using fixed MTG (left) and in-solution MTG (right), the peptide 2-azide derivative was conjugated with lysine 340 and lysine 288 / 290 of the IgG1N297S antibody, resulting in >70% conjugation.

[0038] Figure 11Using immobilized (left) and in-solution MTG (right) to specifically conjugate IgG1 N297S antibody at two sites, approximately 40% of IgG1 was specifically modified at two sites. The substrate TCO-PEG3-NH2 was used for glutamine Q295 conjugation, and peptide 2 azide derivatives were used for lysine 340 (and 288 / 290) conjugation.

[0039] Figure 12 SDS-PAGE and Coomassie and fluorescence analysis of lysine-conjugated antibodies. Only the heavy chain was selectively conjugated, while the light chain was not. Detailed Implementation Plan

[0040] Formation and characterization of MTG-polymer-conjugates

[0041] The conjugation of N-succinimide-4-formylbenzamide (4FB) with microbial transglutaminase (MTG) Figure 1 Different linker excesses were investigated, with the aim of one linker per MTG. Linker ratios were quantified using LC-MS and UV-VIS spectrophotometry, and the two methods showed good correlation (Fig. 2a). At equal linker amounts of MTG and 4FB, a conjugation ratio of approximately 0.4 was obtained, while for an excess of 2, it increased to approximately 0.8 (Fig. 2a). Although a ratio of 0.8 seemed ideal, we chose to use 1.5 equivalents of 4FB to achieve a linker ratio of approximately 0.5 because we observed during experiments that an excessively high linker ratio of 0.8 could lead to the enzyme-polymer conjugate being unable to be purified by ultracentrifugation due to sample precipitation. This can be attributed to the fact that at 2 equivalents, the amount of bilinked MTG (MTG-(4FB)2) was >20%, while at 1.5 equivalents, it remained <10% (Fig. 2b). Excessive amounts of MTG-(4FB)2 resulted in undesirable over-crosslinking on the same or other polymer chains, decreased MTG activity, and reduced solubility of the enzyme-polymer conjugate. For these reasons, it is particularly emphasized that MTG should not be over-conjugated and that a 1.5 excess of 4FB to MTG ratio should be selected to produce single-conjugated MTG with >90% purity.

[0042] Then the polymer de-PG2, which is conjugated with N-succinimide-6-hydrazine nicotinate (S-HyNic)-connector, is... 500 ( Figure 1 The enzyme was incubated with MTG-4FB. The mixing of these two linked compounds resulted in the formation of a characteristic peak and an increase in absorption at 354 nm, as shown by UV-VIS (Fig. 2c), which corresponds to the formation of the bis-aryl-hydrazone (BAH) bond and the successful generation of the enzyme-polymer conjugate.

[0043] The activity of the polymer-enzyme conjugate in solution was determined using a colorimetric hydroxylamine-amine assay, revealing that MTG still possessed catalytic activity, albeit significantly reduced compared to native MTG (Figure 2d). This decrease in activity can be explained by the reduced rotational freedom and arrangement possibilities of polymer-immobilized MTG compared to unbound native MTG, making it more difficult for hydroxylamine to enter the enzyme's active site. Similar observations of reduced activity in proteinase K immobilized on the same polymer in solution have been reported. Nevertheless, the most important finding is that MTG retains catalytic activity after immobilization.

[0044] Calculate the amount of MTG-polymer conjugates adsorbed on the surface of the microsphere glass.

[0045] At 354 nm (29000 M) -1 cm -1 The quantifiable bis-aryl-hydrazone bonds in the UV-VIS allowed for estimation of the amount of MTG immobilized on the beads from the elution volume. After 1 hour of incubation, the concentration in the elution volume was determined to be 1.6 ± 0.15 μM. Considering the initial concentration of 5 μM, approximately 70% of the conjugate had been adsorbed onto the beads. Since the MTG is primarily monocrosslinked with the polymer, we can estimate the adsorbed mass to be approximately 300 ng / cm³. 2 Or approximately 7.8 pmol MTG / cm 2 These values ​​are consistent with previously published results using proteinase K or horseradish peroxidase immobilized on denpol polymers, as well as other enzymes covalently immobilized on silica polymer surfaces.

[0046] Microbead-immobilized MTG is used for site-specific conjugation of functional molecules to proteins.

[0047] To ensure stable immobilization of the MTG-polymer conjugate, glass microspheres were chosen because the positively charged denpol-amine has a strong affinity for the negatively charged glass surface, and because the beads can be readily assembled into a flow-based microreactor. This assembly allows for well-controlled and repeatable sample bead overflow, thus enabling the driving of the reaction. Furthermore, the microspheres can be easily washed after the conjugation process to recover the immobilized MTG for the next round of conjugation.

[0048] Smaller antibody-like backbones of therapeutically relevant proteins (including scFV, nanobodies, or Fab fragments) are of considerable interest due to their increased tumor penetration, ease of production, and faster clearance compared to larger antibodies. Therefore, in the first attempt to functionalize the protein, the aim was to conjugate Fab fragments and scFV, both of which have previously been shown to be effectively conjugated to MTG in solution using biotin-cadherin as an amine via its C-terminal myc-labeled glutamine 2 ('Q2'). Amines are ideal substrates for further downstream applications, including immobilization on streptavidin-coated surfaces. While flexible loops and terminal labels on glutamine-containing proteins are known to be preferentially targeted by MTGs, the presence of globular structures and accessible terminal labels allows for conjugation to surface-immobilized MTGs, and access to the enzyme's active site is less challenging compared to the loop structures on bulky antibodies such as glutamine 295. Therefore, biotin-cadherin was mixed with C-terminal myc-labeled Fab fragments and scFV, and the solution was flowed through microbeads immobilized with MTG in a microreactor. Figure 1 (and 3a). The solution was pumped onto a microreactor for 30 minutes and analyzed by LC-MS. ≥95% complete conversion to the desired biotin-conjugated Fab-fragment was found, with no unconjugated material detected (Fig. 3b). Similarly, under the same conditions, c-myc-labeled scFV was effectively conjugated to produce ≥95% of the desired conjugate in just 30 minutes (Fig. 3b).

[0049] Dansyl cadaverine (the fluorescent amine donor of MTG) was also conjugated to Fab and scFV in an excess of only 8 equimolar amounts (Fig. 3a), which produced ≥95% dansyl-labeled Fab fragments and ≥90% scFV within 30 minutes (Fig. 3b).

[0050] In some cases, the direct conjugation of bulky primary amine-containing substrates with MTG-reactive glutamine occasionally leads to incomplete product conversion, leaving unconjugated material. This is particularly true for substrates containing highly hydrophilic groups, such as carboxyl groups, on the metal chelating agent. This problem can be avoided and quantitative conjugation can be achieved using a two-step approach, where a "clickable" moiety is first attached to the protein, followed by click conjugation of the desired molecule. Therefore, the possibility of conjugating myc-labeled scFV with an amine-PEG3-azide (Fig. 3c), suitable for SPAAC (strain-promoted azidide-alkyne cycloaddition) click chemistry, was investigated. Myc-labeled scFV was flown through a column, and after 30 minutes, the sample was retrieved and analyzed by LC-MS, yielding 82% conversion of the desired scFV-N3 (Fig. 3c). Continuous bead overflow for 90 minutes resulted in 97% conversion, and unconjugated material was no longer detectable after continuous overnight flow (14 hours) (Fig. 3c). These results clearly demonstrate that even with low reagent excess, bead-immobilized MTG can quantitatively conjugate a variety of substrates to myc-labeled proteins in less than 90 minutes.

[0051] Based on these results, the possibility of intact antibodies also being conjugated via bead-fixed MTG was explored. It was previously noted that glutamine 295 (Q295) on the flexible C'E ring of the Fc domain of deglycosylated antibodies is the only MTG target site in the antibody backbone; therefore, introducing an N297Q point mutation to remove the glycosylation site yields well-defined antibody conjugates with two or four attachment sites. Antibody conjugation on surfaces may be more challenging than in solution because the bulk of the antibody limits its orientation possibilities, making MTG entry of Q295 and Q297 into the ring, respectively, potentially more difficult.

[0052] Therefore, the immobilized MTG was subjected to an IgG1 antibody containing an N297S point mutation to eliminate deglycosylation and biotin-cadaverine (Fig. 4a). LC-MS analysis of the reduced antibody showed 37% heavy chain conversion after 30 minutes at room temperature, and 72% after 1 hour, increasing to ≥95% during overnight incubation (Fig. 4a). Compared to conventional nonspecifically conjugated antibodies with the same drug load, the site-specific modification of the antibody with a high drug-to-antibody ratio (DAR) of 4 to 6 was considered to efficiently deliver the conjugated drug to the tumor target site, making such ADCs very attractive as improved cancer therapies. Therefore, the ability of the immobilized MTG to maintain conjugation to the two very close bifunctional linkers responsible for SPAACs of the N297Q antibody was investigated (Fig. 4b). It was found that the bead-immobilized MTG could indeed quantitatively and efficiently conjugate the two linkers, producing an N297Q antibody with a ratio of 4 linkers per antibody (Fig. 4b).

[0053] These studies clearly demonstrate that MTG maintains its specificity and effective conjugation ability after fixation, and can even modulate the residue specificity of proteins.

[0054] Stability study of MTG-polymer conjugates and MTG activity on microbeads

[0055] Previous studies have shown that the polycationic nature of denpol-polymers provides stable surface anchoring on anion exchange glass surfaces for several weeks. Since solid-state enzyme immobilization is important for downstream applications, particularly for the intended therapeutic proteins, enzyme leakage was addressed using slit blot assays and an anti-MTG antibody. Slit blots were chosen because they allow for the application of large sample volumes and due to their sensitivity. The MTG-polymer conjugate, as a positive control, showed a strong signal (Fig. 4c, lane 1 I), and unconjugated MTG was also detectable (lane 1, ii), while the HyNic-polymer produced no signal (lane 1, iii), demonstrating that the antibody specifically recognizes MTG upon conjugation with the polymer. After 14 hours of continuous operation, the MTG remained firmly attached (lanes 2, i and ii), and after an additional 40 hours of operation with several conjugations, the MTG remained firmly attached to the polymer without any enzyme detection (lane 2, iii). Under the latter condition, the immobilized MTG was still able to conjugate >90% of BC with N297SIgG1 after 3 hours. These data lead to the conclusion that MTG not only maintains tight attachment to glass beads, but also remains active over extended periods and after multiple rounds of conjugation. Therefore, it is a very promising tool for conjugating proteins of similar size to MTG.

[0056] Immobilized MTG enhances residue selectivity in the presence of multiple MTG-reactive amino acids.

[0057] The immobilized enzyme also showed enhanced selectivity for its substrate, suggesting its potential application to immobilized MTG. In solution, MTG was used to conjugate ZQG-Tamra-cadaverine (ZQG-TC) to avidin (used as a model protein) with multiple reactive lysine residues, resulting in two main peaks in deconvolution LC-MS. These peaks corresponded to avidin with one ZQG-TC, avidin with two conjugated ZQG-TC, and some unmodified avidin. Figure 5 (Left figure). Using a fixed MTG, the same experiment was unexpectedly found to target almost exclusively one lysine residue of avidin, with only limited conjugation to the second residue. Figure 5(See right figure), possibly due to reduced flexibility of the rotating MTG. These data show that a fixed MTG can indeed be used to modulate residue selectivity, which is not possible with solution-phase conjugation where two residues are significantly conjugated.

[0058] Small peptides containing glutamine specifically conjugate to lysine residues of deglycosylated and non-glycosylated IgG1.

[0059] Although MTG-mediated antibody functionalization via lysine side chains using ZQG derivatives has been reported, conjugation yields have been unsatisfactory (i.e., <20%), and no modified lysine sites have been reported. Therefore, the aim of further investigation was to target lysine residues of deglycosylated and deglycosylated IgG1 in solution and with immobilized MTG. It was inferred that different glutamine peptides could conjugate more effectively to lysine residues on IgG1 compared to commonly used ZQG or its derivatives. Therefore, a small library of glutamine-containing peptides with reported high MTG activity was first screened in solution under different pH conditions, which we subsequently intended to apply to immobilized MTG. Indeed, after 16 hours of incubation at room temperature, sequences with favorable conjugation ratios to deglycosylated IgG1 were identified, showing higher reactivity than ZQG using LC-MS analysis. Figure 6 In particular, in a solution at pH 7.6, the peptide sequence NH2-FGLQRPY-COOH showed almost twice the conjugation efficiency with deglycosylated IgG1, reaching nearly 50%. Figure 6 At room temperature, deglycosylated IgG1 and peptide FGLQRPY (peptide 2, Figure 7 The peptide structure on the left was subjected to immobilized MTG overnight, and 30% conjugation was found. Figure 8 Notably, using the IgG1 glycosylated N297S mutant and FGLQRPY, the conjugation ratio was increased to 71% with immobilized and in-solution MTG (respectively). Figure 9 Left and right). Using FGLQRPY azide derivatives ( Figure 7 The peptide structure on the right), using immobilized MTG and MTG in solution (respectively). Figure 10 (Left and right), the same results were obtained using LC-MS analysis. LC-MS analysis mainly revealed single-modified substances and only a small number of second conjugates, both located only on the heavy chain. Peptide mapping confirmed two modification sites at Lys288 or Lys 290 and Lys340.

[0060] Site-specific double conjugation of fixed and in-solution MTG with glycosylated IgG1 (N297S mutant)

[0061] The feasibility of site-specific dual modification with fixed and in-solution MTG has been established by modifying Q295 and K340, K288 / K290 of N297S IgG1. Antibodies with such dual modification, for example, two imaging probes, would be well-suited for, for example, non-invasive and / or intraoperative / postoperative tissue imaging. Alternatively, two different toxic payloads exhibiting synergistic effects can be linked. First, Q295 was modified with NH2-PEG3-TCO to ≥95%, then modified with a peptide-2 azide derivative, resulting in a slightly lower yield of 38% dual-site-specific modified IgG1. Figure 11 (Left). Similar results were obtained by dual-site specific conjugation using MTG in solution ( Figure 11 (Right). SDS-PAGE confirmed heavy chain-specific conjugation to lysine residues, and LC-MS results also confirmed the double conjugation antibody. Figure 12 ).

[0062] Conjugation of functionalized lysine peptides with deglycosylated antibodies

[0063] The study also investigated whether peptides containing lysine residues could be used for site-specific modification of deglycosylated antibodies at the glutamine 295 position using MTG in solution, a process not yet described in the literature. In the first case, equipping these peptides with functional groups such as N3- groups (KAYA-GGG-N3) or metal chelators (e.g., NODAGA) would allow subsequent linking of another moiety via SPAAC click chemistry at low molar equivalents. In the second case, the functional moiety can be directly conjugated, thus eliminating the need for a second step to facilitate further downstream processing. Furthermore, the solubility of the functional moiety (“payload”) can be increased by introducing hydrophilic amino acids into the peptide, which is highly beneficial for hydrophobic payloads. In the work of this invention, it has been shown that KAYA-GGG-N3, as well as KNAA-GK-PEG3-NODAGA and KAYA-GK-PEG3-NODAGA, can be conjugated to deglycosylated antibodies with high efficiency (>95%).

[0064] The technical solutions corresponding to the original claims of the parent application are hereby incorporated in this specification:

[0065] 1. A method for conjugating organic molecules with target proteins to produce fusion proteins using immobilized and / or non-immobilized forms of MTG (microbial transglutaminase), comprising the following steps:

[0066] a) Immobilizing the MTG by attaching it to the polymer through exposing the cross-reactive groups of the polymer;

[0067] b) Adsorbing the MTG polymer conjugate onto microbeads or making the MTG polymer conjugate into a solution;

[0068] c) Pack an active flow reactor column with microbeads adsorbed with the MTG polymer conjugate and / or a solution containing the MTG polymer conjugate and / or a solution containing the MTG.

[0069] d) Under defined conditions, the target protein and organic molecules are provided in a fluid and the fluid is passed through a packed active flow reactor column or the fluid is mixed with a solution containing the MTG polymer conjugate and / or a solution containing MTG, thereby conjugating the organic molecules to the protein under the catalysis of the MTG;

[0070] e) Extract protein-organic molecular conjugates from the fluid.

[0071] 2. The method according to Project 1, wherein the polymer undergoes ionic and / or covalent bonding with the microspheres.

[0072] 3. The method according to Project 1 or 2, wherein the polymer is a second-generation dendritic polymer (de-PG2).

[0073] 4. The method according to any one of the preceding items, wherein the target protein is selected from the group consisting of antibodies or fragments thereof in the form of IgG, IgM, IgA or IgE, and preferably monoclonal, optionally selected as chimeric, humanized, human or bispecific, deglycosylated or non-glycosylated, comprising a mutation of residue N297 (e.g. N297Q or N297A) (EU numbering scheme), and preferably also comprising other reactive glutamine residue mutations in the antibody backbone that enable MTG-mediated conjugation.

[0074] 5. The method according to any one of the foregoing items, wherein the target protein is a peptide, such as Fab, Fab', F(ab)'2, F(ab)'3, Dab, Fv, single-chain Fv(scFv) fragment scFv-Fc(scFv)2, wherein other possible proteins and / or peptides include proteins and peptides involved in the recognition of other proteins and peptides, including but not limited to protein kinases, such as mitogen-activated protein (MAP) kinase, and kinases that directly or indirectly phosphorylate MAP kinase, Januse kinase (JAKI), and cyclin-dependent kinases, epidermal growth factor (EGFR) EGF) receptor, platelet-derived growth factor (PDGF) receptor, fibroblast-derived growth factor (FGF) receptor, insulin receptor and insulin-like growth factor (IGF), engineered proteins such as darpin, affinity / nanobody or fibronectin fragments, or carrier proteins, or haptens that elicit an immune response and are therefore important for vaccination, such as diphtheria toxin mutant CRM197, or GBS67 (an accessory protein of PI-2a); in addition, it preferably includes conjugation with non-protein structures such as mono- or poly-dextran, such as dextran.

[0075] 6. The method according to item 4 or 5, wherein the enzyme modifies one or more reactive glutamine residues (e.g., Q295 and N297Q in antibodies) or one or more reactive lysine residues (e.g., K288 or K290, K340 in antibodies) on the target protein with an organic molecule; wherein the residues are endogenous or artificially introduced by genetic methods or a combination thereof.

[0076] 7. The method according to any one of the foregoing items, wherein said organic molecule is selected from the group consisting of: fluorescent dyes / labels (e.g., Alexa488, Alexa647), cytotoxic or influencing moieties of cells, such as toxins (e.g., MMAE, PBD-dimer) or cell regulators, immunomodulatory / stimulatory compounds of immune cells, metal chelators suitable for SPECT / PET or MRI (e.g., NODA-GA), functional peptides (e.g., α-defensin NP-1), chemical moieties suitable for click reactions such as strain-promoted azide-alkyne click chemistry (SPAAC) or tetrazine-olefin linkages, including azides and cyclooctyne derivatives (e.g., DIFO, BCN, DIBAC, DIBO, ADIBO), and tetrazine and trans-cyclooctene derivatives having a primary amine conjugated for MTG-mediated linkage, and having C n >20 spacer portion, of which (c) nPreferably, it is a substituted or unsubstituted alkyl or heteroalkyl chain and its derivatives (e.g., any carbon in the chain is optionally substituted with an alkoxy, hydroxyl or amide, or alkylamide), and n is an integer selected from the range >20.

[0077] 8. The method according to any one of items 1 to 7 above, wherein the organic molecule is selected from the group consisting of: peptides, said peptides containing lysine residues at any position (e.g., KNAAGGG or KDAAGGG or KAYAGGG or AKETAA) or glutamine residues at any position (e.g., FGLQPRY, SLLQGR), and being targeted by MTG (i.e., a substrate of MTG), and optionally containing enzymatically cleavable peptide sequences (e.g., valine-citrulline (VC), KNAAGGG-VC); said lysine peptide having a size (length) of (C+N). n >20 and the size (length) of the glutamine peptide is 1 < (C+N) n <200.

[0078] 9. The method according to Item 8, wherein the peptide contains a functional moiety, such as a cytotoxic moiety (MMAE), a fluorescent dye, a metal chelating agent, or a chemical moiety suitable for SPAAC click reactions (e.g., an azide or DBCO group, such as FGLQPRYGK (azide)) or a tetrazine and trans-cyclooctene group or a derivative thereof (e.g., KNAAGGG-PEG5-TCO), optionally containing a spacer moiety (e.g., polyethylene glycol, alkyl group) between the peptide and the functional moiety (e.g., KNAAGK (PEG3-NODAGA)) and / or optionally also containing a self-sacrificing group (e.g., p-aminobenzyloxycarbonyl (PAB)).

[0079] 10. The method according to any one of the foregoing items, wherein 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 fluoride (PCDF), silicone, polyoxymethylene, cellulose, cellulose acetate, nitrocellulose, etc.; the group further comprising: gelatin, glass, agarose beads, cross-linked dextran beads, or dextran microcarriers, such as CYTODES® (Pharmacia, Uppsala, ... The microspheres are composed of polysaccharides such as agarose, alginate, carrageenan, chitin, cellulose, dextran, or starch; polycaprolactone (PCL); polyacrylamide; polystyrene; polyacrylaldehyde; polydimethylsiloxane; polyvinyl alcohol; polymethacrylate; perfluorocarbon; inorganic compounds such as silica, glass, diatomaceous earth, alumina, gold, iron oxides, graphene and graphene oxides or other metal oxides; or copolymers of any combination of two or more naturally occurring polymers, synthetic polymers, or inorganic compounds; the size of the microspheres is 1 nm to 100 nm or 100 nm to 1000 nm or 1 μm to 10 μm or 10 μm to 1000 μm.

[0080] 11. The method according to any one of the preceding items, wherein the fluid is selected from the group consisting of: water containing a suitable buffer (e.g., Tris) and a salt additive (e.g., NaCl), the buffered aqueous solution may also contain up to 60% glycerol and other organic solvents, such as ethanol, propanol, isopropanol, 1-propanol, DMSO, methanol, acetonitrile.

[0081] 12. The method according to any one of the preceding items, wherein the polymer is selected from the group consisting of: polyethylene glycol, polypropylene glycol, polyethylene oxide, poly(alkylene oxide), etc. (zoline), polyvinylpyrrolidone, polylysine and polyglutamic acid, poly(ethyl) (Azoline), polymethacrylic acid and polyacrylic acid or mixtures thereof and dendritic structures; also including sugar residue-based polymers, poly-N-isopropylacrylamide (polyNIPAM), poly(glycidyl methacrylate), polytetrafluoroethylene (PTFE) and poly(ethylene-alternating-tetrafluoroethylene) (ETFE), poly(oligoethylene glycol) methacrylate (POEGMA), poly(2-methyl-2- (PMOXA), poly(vinyl alcohol) (PVA), and poly(ethylene imine) and their derivatives.

[0082] 13. The method according to any one of the foregoing items, wherein the polymer MTG conjugate comprises a joint (spacer) between the polymer and the MTG, the joint being a bifunctional joint system S-HyNic (succinimide-6-hydrazinoamide, S-4FB (4-formylbenzoate) or a derivative thereof, or SMCC (succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate) or a derivative thereof, having a similar YSZ (Y can also be Z, or vice versa) structure, wherein Y and Z are... The following groups or their derivatives: tetrazine, trans-cyclooctene, azide, cyclooctene (e.g., dibenzylcyclooctyne or bicyclononyne), n-hydroxysuccinimide, maleimide, isothiocyanate, aldehyde, epoxide, alcohol, amine, thiol, phosphonate, alkyne, potassium acyltrifluoroborate, α-keto-hydroxylamine, O-acylhydroxylamine, carboxylic acid, hydrazine, imine, norbornene, nitrile, and cyclopropene, and S is a spacer entity as a polymer or its derivative, amino acid, or peptide derivative, such as oligomers or poly(ethylene glycol) (PEG), dextran, which are composed of alkyl moieties.

[0083] 14. The method according to any one of the foregoing items, wherein the determined conditions include the following details: a temperature of 0°C to 50°C, a contact time of several seconds to 168 hours (or 7 days), a flow rate / rate in the active flow reactor column of less than 1 μL / min or 1 μL / min to 10 ml / min, a protein concentration of 1 μM to 1 mM, a molar ratio of organic molecules to the target protein of 0.5 to 50×, 50 to 500×, and 500 to 10,000×, and an MTG concentration of 0.001 mg / ml to 0.01 mg / ml and 0.01 mg / ml to 10 mg / ml per ml of resin or microbeads or conjugated solution, and preferably further comprising a conjugation efficiency of the organic molecules to the target protein of at least 30% and up to 100% conversion, and a flow pressure of 0.1 bar to 20 bar; furthermore, the functionalized microbeads are preferably placed in a device suitable for a rotating column, wherein the reaction mixture is incubated with the beads for 1 second to 60 seconds, 1 A certain amount of time, from minutes to 60 minutes, or from 1 hour to 168 hours, is applied; then the mixture is removed from the microbeads again by centrifugation and the supernatant is taken out, or the solution is pushed through a suitable filter during centrifugation, the filter retaining the microbeads but not the desired mixture.

Claims

1. A method for using a non-fixed form of MTG (microbial transglutaminase) to conjugate organic molecules with a target protein to produce a fusion protein, comprising the following steps: a) Provide a solution containing the MTG; b) The target protein and the organic molecule are provided in the solution containing MTG under defined conditions, thereby conjugating the organic molecule to the protein under the catalysis of the MTG; c) Extraction of protein-organic molecular conjugates; The target protein is an antibody; and the organic molecule is a peptide comprising an amino acid sequence selected from (i) FGLQRPY; (ii) KNAAGGG; (iii) KDAAGGG; (iv) KAYAGGG; and (v) AKETAA.

2. The method according to claim 1, wherein the antibody is in the form of IgG, IgM, IgA or IgE.

3. The method according to claim 1 or 2, wherein the antibody is a deglycosylated antibody or a non-glycosylated antibody mutant.

4. The method according to claim 1 or 2, wherein the MTG modifies one or more reactive glutamine residues on the target protein.

5. The method of claim 4, wherein the glutamine residue is an endogenous glutamine residue.

6. The method of claim 5, wherein the endogenous glutamine residue is Q295.

7. The method according to claim 1 or 2, wherein the organic molecule further comprises a fluorescent dye / label, a cytotoxic or influencing moiety, a metal chelator suitable for SPECT / PET or MRI, a functional peptide, a chemical moiety suitable for click reaction or tetrazine-olefin linkage, and / or a spacer moiety.

8. The method of claim 7, wherein the chemical portion suitable for the click reaction is a chemical portion suitable for strain-promoted azide-alkyne click chemistry.

9. The method of claim 7, wherein the cytotoxic or influencing portion of the cell is a toxin, a cell regulator, or an immunomodulatory / stimulatory compound for immune cells; and / or wherein the chemical portion suitable for click reaction or tetrazine-olefin linkage comprises an azide, a cyclooctyne derivative, a tetrazine, or a trans-cyclooctene derivative; and / or wherein the spacer portion has C n Size >20, (C) n n is a substituted or unsubstituted alkyl or heteroalkyl chain and its derivatives, and n is an integer >20.

10. The method according to claim 1 or 2, wherein the peptide comprises an amino acid sequence selected from KNAAGGG, KDAAGGG, KAYAGGG, and AKETAA.

11. The method according to claim 1 or 2, wherein the peptide further comprises an enzymatically cleavable peptide sequence.

12. The method according to claim 1 or 2, wherein the peptide further comprises a self-sacrificing group.

13. The method of claim 12, wherein the self-sacrificing group is p-aminobenzyloxycarbonyl (PAB).

14. The method according to claim 1 or 2, wherein the fluid is a buffered aqueous solution.

15. The method of claim 14, wherein the buffer aqueous solution comprises Tris and NaCl.

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