Increasing pal-catalytic protein ligation efficiency by cascade enzymatic method

Abstract

The present invention relates generally to enzymatic peptide or protein ligation. Specifically, the present invention provides an improved enzymatic peptide or protein ligation method comprising coupling a peptidyl asparaginyl ligase (PAL) catalyzed ligation with a glutamine cyclase (QC) catalyzed pyroglutamyl formation to increase the yield of the ligation product.

Description

Technical Field

[0001] The present invention generally relates to enzymatic peptide or protein ligation. Specifically, the present invention provides an improved enzymatic peptide or protein ligation method, which comprises coupling a peptidyl asparaginyl ligase (PAL)-catalyzed ligation with a glutamine cyclase (QC)-catalyzed pyroglutamyl formation to increase the yield of the ligation product. Technical Background

[0002] Site-specific protein ligation facilitates the study of protein function and promotes the development of protein therapeutics (Spicer, CD and Davis, BG, Nat. Commun. 5:4740 (2014); Hoyt, EA et al., Nat. Rev. Chem. 3, 147–171 (2019); Zhang, G. et al., Chem. Soc. Rev. 44, 3405-3717 (2015); Sletten, EM and Bertozzi, CR, Angew. Chem. Int. Ed. 48, 6974-6998 (2009); Rosen, CB and Francis, MB, Nat. Chem. Biol. 13, 697-705 (2017)). In addition to mature chemical ligation methods, peptide ligases have become increasingly used in protein modification in recent years (Zhang, Y. et al., Chem. Soc. Rev. 47, 9106-9136 (2018); Lotze, J. et al., Mol Biosyst. 12, 1731–1745 (2016); Weeks, AM and Wells, JA, Chem. Rev. 120, 3127-3160 (2020); Pishesha, NL et al., Annu. Rev. Cell Dev. Biol. 34, 163–88 (2018)). For example, a popular peptide ligase is Sortase A, which recognizes the sorting sequence LPXTG and ligates after threonine (Thr) (Pishesha, NL et al., Annu. Rev. Cell Dev. Biol. 34, 163–88 (2018)). However, the catalytic efficiency of Sortase A is low, and a stoichiometric amount of the enzyme is required for a viable ligation reaction. In contrast, the recently discovered peptidyl asparaginyl ligase (PAL), represented by butelase-1 (Nguyen, GKT et al., Nat Chem Biol. 10, 732–738 (2014)), has a catalytic efficiency that is 10 times that of Sortase A. 4times (Nguyen, GKT et al., "Nat. Protoc." 11, 1977-1988 (2016); Tam, JP et al., "Science China Chemistry" 63, 296-307 (2020)). In addition, PAL requires a minimal asparagine tripeptide recognition motif Asn-P1′-P2′ for connection after asparagine (Asn), where P1′ can tolerate a wide range of amino acid residues and P2′ is usually a large hydrophobic residue (Nguyen, GKT et al., "Nat. Protoc." 11, 1977-1988 (2016); Tam, JP et al., "Science China Chemistry" 63, 296-307 (2020)). As a member of the asparaginyl endopeptidase (AEP) family, PAL utilizes catalytic cysteinyl thiol to cleave the Asn-P1′ peptide bond in the acyl donor substrate, and the resulting asparaginyl thioester intermediate is then decomposed by the amine nucleophile of the acyl acceptor substrate. Therefore, the ligation product is formed by a transpeptidase reaction. For example, butelase-1, VyPAL2 and OaAEP1b-C247A are two other PALs with excellent transpeptidase activity (Hemua, X. et al., Proc. Natl. Acad. Sci. 116, 11737-11746 (2019); Harris, KS et al., Nat. Commun. 6, 10199 (2015); Yang, R. et al., J. Am. Chem. Soc. 2017, 139, 5351-5358 (2017)). However, since the transpeptidation reaction is reversible, an excess of nucleophile is required to carry out high-yield intermolecular ligation reactions.

[0003] Several methods have been developed to shift the PAL-mediated ligation equilibrium to the product side (Nguyen, GKT et al., Angew. Chem. Int. Ed. 54, 15694-15698 (2015); Cao, Y. et al., Bioconj. Chem. 27, 2592-2596 (2016); Rehm, FBH et al., J. Am. Chem. Soc. 141 (43), 17388-17393 (2019); Tang, TMS et al., Chem. Sci. 11, 5881-5888 (2020); Rehm, FBH et al., Angew. Chem. Int. Ed. 60, 4004-4008 (2021)). For the first time, a thiodepsipeptide method was developed, which uses asparaginyl thioester peptides as acyl donor substrates (peptide-Asn-thioglc-Xaa), thereby making the ligation reaction irreversible (Nguyen, G.K.T. et al., Angew. Chem. Int. Ed. 54, 15694-15698 (2015); Cao, Y. et al., Bioconj. Chem. 27, 2592-2596 (2016)). However, the limitation of this method is that the thioester substrate needs to be prepared. In the second method developed by Rehm et al., a glycine valine acyl acceptor substrate is used to connect with an acyl donor containing NGL, because the resulting Asn-Gly-Val (NGV) motif is more stable than Asn-Gly-Leu (NGL) to OaAEP-C247A, which reduces the reversibility of product hydrolysis and ligation reactions. However, NGV is only relatively more stable than NGL. As a poor receptor substrate, the amount of GV peptide used must far exceed the acyl receptor (>20 times). No protein-protein connection was found. In another method developed by Tang et al., Asn-Cys-Leu was used as the P1-P1′-P2′ tripeptide motif of OaAEP1-C247A (Tang, TMS et al., "Chem. Sci." 11, 5881-5888 (2020)). Quenching the N-terminal cysteine ​​of the Cys-Leu leaving group with the aldehyde compound 2-formylphenylboronic acid (FPBA) reduced the reversibility of the connection reaction. However, FPBA significantly slowed the connection reaction, probably because aldehydes can form imines with amine substrates and / or enzymes. Finally, the method reported by Rehm et al. uses Ni 2+The leaving group of the GLH tripeptide is chelated to mask the nucleophilicity of its N-terminal amine (Rehm, FBH et al., Angew. Chem. Int. Ed. 60, 4004-4008 (2021)). This method greatly improves the yield of protein N-terminal and C-terminal labeling reactions with small synthetic peptides. However, for protein-to-protein connections, only a modest increase in yield was observed—from 24% to 36% in one case and from 28% to 39% in another (Rehm, FBH et al., Angew. Chem. Int. Ed. 60, 4004-4008 (2021)). Therefore, all currently known methods have certain limitations, especially when it comes to protein-protein connections.

[0004] Therefore, it is necessary to provide an improved protein ligation method to overcome or at least improve one or more of the above-mentioned disadvantages. Summary of the invention

[0005] The present invention relates to a method for enzymatic peptide ligation, wherein PAL-mediated intermolecular ligation is coupled with pyroglutamyl formation catalyzed by glutamine cyclase (QC) to significantly increase the yield of ligation products.

[0006] In one aspect, a method for enzymatic peptide ligation is provided, the method comprising providing

[0007] i) peptidyl asparaginyl ligase (PAL) and glutamine cyclase (QC);

[0008] ii) a first peptide or protein having a P1-P1′-P2′ tripeptide PAL motif as an acyl donor, wherein

[0009] P1 is Asn or Asp, P1′ is Gln or Glu, and P2′ is a hydrophobic amino acid or a β-branched amino acid;

[0010] iii) the second peptide or protein is identical or different from the first peptide or protein and has a P1"-P2" motif at the N-terminus as an acyl acceptor, wherein P1" is any amino acid and P2" is a hydrophobic amino acid

[0011] or β-branched-chain amino acids;

[0012] iv) contacting the peptidyl asparaginyl ligase (PAL) and glutamine cyclase (QC) with the first and second peptides / proteins;

[0013] Among them, PAL cuts the first peptide / protein after P1 in the tripeptide PAL motif and connects the first peptide / protein to the P1"-P2" motif of the second peptide / protein, and QC cyclizes P1' in the released P1'-P2' dipeptide motif to pyroglutamyl (pGlu).

[0014] It will be appreciated that peptides are generally shorter than proteins, comprise amino acids, and are suitable for linkage according to the present invention.

[0015] In some embodiments, P2' is selected from the group consisting of Leu, Met, Phe, Tyr, Trp, Val, He, and Thr.

[0016] In some embodiments, P2" is selected from the group consisting of Leu, Phe, Tyr, Trp, Val, He, and Thr.

[0017] In some embodiments, the PAL is selected from the group consisting of butelase 1 comprising the amino acid sequence shown in SEQ ID NO: 1, butelase-2 comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, VyPAL2 comprising the amino acid sequence shown in SEQ ID NO: 4, VyPAL3 comprising the amino acid sequence shown in SEQ ID NO: 5, OaAEP1b-C247A comprising the amino acid sequence shown in SEQ ID NO: 6, HeAEP3 comprising the amino acid sequence shown in SEQ ID NO: 7, AtLEGγ comprising the amino acid sequence shown in SEQ ID NO: 8, VuPAL1 comprising the amino acid sequence shown in SEQ ID NO: 9, HaPAL1 comprising the amino acid sequence shown in SEQ ID NO: 10, and OaAEP1b comprising the amino acid sequence shown in SEQ ID NO: 11.

[0018] In some embodiments, QC is selected from the group consisting of a human glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 12, a mouse glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 13, a Drosophila glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 14, an Arabidopsis thaliana glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 15, a Conus glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 16, a Sistrurus glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 17, and a bacterial glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 18.

[0019] In some embodiments, the second peptide or protein further comprises a spacer of at least one amino acid between the P1"-P2" acyl acceptor and the second peptide / protein.

[0020] In some embodiments, the ratio of QC:PAL:first peptide or protein is in the range of 0.1:1:1000 to 1:1:50, respectively, preferably, in the range of 0.1:1:1000 to 0.1:1:50, respectively.

[0021] In some embodiments, the first and second peptides or proteins are identical and form a dimer when linked.

[0022] In some embodiments, one of the first and second peptides or proteins is an epitope binding peptide or protein and the other peptide or protein comprises a payload.

[0023] In some embodiments, the payload further includes a payload-release link.

[0024] In some embodiments, the epitope-binding peptide or protein is selected from the group consisting of an antibody or a functional fragment thereof, an affinity body such as Z EGFR or Z EGFR -Fc and DARPin.

[0025] In some embodiments, the antibody or functional fragment thereof is selected from the group consisting of a mini-antibody, a diabody, a scFv, a nanobody and a F(ab′) 2 .

[0026] In some embodiments, the payload is an imaging agent or a therapeutic agent.

[0027] In some embodiments, the imaging agent is a radiolabeled chelator or an optical label.

[0028] In some embodiments, the radiolabel chelator is selected from the group consisting of 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclo-1-pentanedioic acid-4,7-diacetic acid (NODAGA); and / or the optical label is HRP or GFP or the like.

[0029] In some embodiments, the therapeutic agent is monomethyl auristatin E (MMAE) or radiolabeled DOTA.

[0030] Advantageously, the methods disclosed herein allow for protein-protein, protein-peptide and peptide-peptide ligation with greater efficiency and near quantitative yields even when the molar ratios of the two ligation partners are equal. These and other advantages of the present invention will be readily apparent to those skilled in the art through the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings illustrate the disclosed embodiments and are used to explain the principles of the disclosed embodiments. However, it should be understood that the accompanying drawings are only for illustration purposes and are not intended to limit the scope of the present invention.

[0033] Figure 1 A model study of the proof-of-concept of the cascade enzymatic reaction scheme using the ligation reaction between Ac-SYRNQL and GIGGIR is shown. (a) Product yields over time in the absence (solid triangles) or presence (solid circles for 0.1 eq of QC vs. PAL, solid squares for 1 eq of QC vs. PAL) of QC in the reaction. Reaction conditions: 5 mM acyl acceptor and 5 mM acyl donor OaAEP1b-C247A (0.0005 eq) dissolved in 20 mM PBS (pH 7) at 37 °C, with or without QC (0.0005 or 0.00005 eq). Yields were determined by HPLC (UV absorbance at 220 nm). Yields are presented as mean ± SEM of three replicates. (b) HPLC traces. Upper trace: Ac-SYRNQL and GIGGIR alone; middle trace: ligation reaction without QC; lower trace: ligation reaction with QC. Reaction time = 32 minutes.

[0034] Figure 2 The use of the PAL-QC coupling scheme for protein-peptide ligation is shown. (a) Ligation between ubiquitin-NQL-His6 and GIGGIRK (biotin); (b) Ligation between biotin-GRSNQL and GI-ubiquitin. Both reactions were performed at 37°C using 500 μM ubiquitin, 1.2 eq of peptide, 0.001 eq of OaAEP1b-C247A, with or without 0.0001 eq of QC in 20 mM PBS (pH 7) for 1 h. Both reactions were monitored using HPLC (middle panel) and the labeled products were characterized by ESI-MS (bottom panel).

[0035] Figure 3 The PAL-QC coupling cascade protocol for the preparation of affibody protein Z is shown. EGFR C-terminal conjugate of EGFR - Linkage between NQL and GIGGGK[Fe(DOTA)]; (b) Z EGFRThe reaction was carried out at 23 °C using 500 μM Z EGFR -NQL and 1.5eq of nucleophilic substrate and 0.001eq of OaAEP1b-C247A were added to 20mM PBS (pH 7) with or without 0.0001eq of QC and reacted for 2h. (c) Preparation of Z EGFR At 23°C, 1.1 mM Z EGFR -NQL was reacted with 500 μM of the divalent peptide (GISGGRG) 2KGC, 2 μM of aAEP1b-C247A, with or without 0.2 μM QC for 2 h. The reaction was monitored by HPLC and ESI-MS.

[0036] Figure 4 The use of VyPAL2 and QC conjugates to improve protein-protein ligation efficiency is described. (a) HPLC monitoring of VyPAL2-mediated ligation between DARPin-NQL and GI-ubiquitin with or without QC. (b) Yields of various protein-protein ligations (i: DARPin-NQL and GI-ubiquitin, ii: DARPin-NQL and GI-DARPin, iii: Z EGFR -NQL and GI-ubiquitin, iv: DARPin-QL and GI-GFP, v: DARPin-NQL and GI-GGGSGGGS-GFP). All reactions were performed at 37°C using 400 μM acyl donor protein, 1.8 eq acyl acceptor protein, 0.001 eq VyPAL2, with or without 0.0001 eq QC in 20 mM PBS (pH 7) for 3-4 h. The reactions were monitored using HPLC and ESI-MS ( Figure 13-18 The yields in b were calculated based on the integrated areas of the reactant and product peaks in HPLC and are presented as the mean ± SEM of three replicate experiments.

[0037] Figure 5 Describes Z EGFR -Linkage between Fc-NQL and GI-GGGSGGGS-GFP. (a) 0.4 μM VyPAL2 in 20 mM PBS (pH 7) with or without 0.04 μM QC was used to treat Z EGFR -Fc-NQL (200 μM) and GI-GGGSGGGS-GFP (500 μM) ligation reaction was subjected to SDS-PAGE. The reaction mixture was treated with 50 mM DTT and analyzed using SDS-PAGE gel. The bands at ~63 kDa and 34 kDa corresponded to the ligation product and Z, respectively.EGFR - Reduced form of Fc-NQL. After reduction with DTT, the ligation product was characterized using LC-MS (ESI). (b) Z EGFR -Confocal microscopy images of Fc-GFP binding to A431 cells. (c) Z EGFR -Fc-GFP and GFP flow cytometric analysis.

[0038] Figure 6 Shows Z EGFR - Connection between Fc-NQL and GVA-PABC-MMAE. Left: without QC. Right: with QC.

[0039] Figure 7 QC catalyzes pyroglutamate (pGlu) formation of 4 substrates (QFGSA, QLGSA, QIGSA and QVGSA) is shown. Using 5mM QXGSA, 0.0001eq of QC was reacted in 20mM PBS (pH 7) at 37°C for 15 minutes and monitored using RP-HPLC. All bar graphs represent the mean ± SEM of three replicates.

[0040] Figure 8 The yield of the ligation between Ac-SYRNQL and GIGGIR catalyzed by different PALs (OaAEP1b-C247A, VyPAL2 or butelase-1) in the absence or presence of QC is shown. The ligation reaction was performed at 37°C in 20 mM PBS pH 7 (OaAEP1b-C247A) or PBS pH 6.5 (VyPAL2, butelase-1) using 5 mM Ac-SYRNQL, 5 mM GIGGIR, 0.0005 eq of PAL, with or without 0.00005 eq of QC, for 30 minutes. All bar graphs represent the mean ± SEM of three replicates.

[0041] Fig. 9 Describes the use of RP-HPLC to monitor Z at 2 h EGFR Ligation reaction between -NQL and GIGGGK[Fe(DOTA)]. The ligation product was analyzed by ESI-MS. The ligation reaction was carried out at 37°C with 500 μM Z EGFR -NQL, 1.5 eq of GIGGGK[Fe(DOTA)], 0.001 eq of VyPAL2, in the absence or presence of 0.0001 eq of QC, were reacted in 20 mM PBS (pH 7) for 2 h.

[0042] Fig.10 Describes the use of RP-HPLC to monitor ZEGFR Ligation reaction between -NQL and GIGKVA-PABC-MMAE. The ligation products were analyzed by ESI-MS. 500 μM Z EGFR -NQL, 1.5eq of GIGKVA-PABC-MMAE, and 0.001eq of VyPAL2 were reacted in 20mM PBS (pH 7) at 37°C for 2h.

[0043] Fig.11 Describes Z EGFR RP-HPLC monitoring of the ligation reaction between -NQL and the divalent peptide (GISGGRAG) 2KGC. The ligation product was analyzed using ESI-MS. 1.1 mM Z EGFR -NQL, 500 μM (GISGGRAG)2KGC, 2 μM VyPAL2, in 20 mM PBS (pH 7) at 37°C for 2 h.

[0044] Fig.12 Shows the use of Z EGFR -After 3 days of PABC-MMAE treatment, Z EGFR IC values ​​of -MMAE conjugates on A431 cells and MCF7 cells 50 IC for A431 cells 50 About 12.9nM; IC for MCF7 cells 50 >100nM. To detect the effects of A431 and MCF-7 cells on Z EGFR To test the activity of the MMAE conjugates, approximately 5000 A431 or MCF-7 cells were seeded in 98-well plates and incubated overnight at 37°C in 5% CO2 medium. EGFR -MMAE and continued incubation for 3 days at 37°C, 5% CO2. Cell viability was measured using the MTT assay according to the recommended protocol.

[0045] Fig.13 RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-ubiquitin at different time points is described. The ligation products were analyzed using ESI-MS. The ligation reactions were performed at 37°C in 20 mM PBS (pH 7) using 400 μM DARPin-NQL, 1.8 eq GI-ubiquitin, 0.001 eq VyPAL2, in the absence or presence of 0.0001 eq QC.

[0046] Fig.14RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-GFP at different time points is described. The ligation product was analyzed using ESI-MS. The ligation reaction was performed at 37°C using 400 μM DARPin-NQL, 1.8 eq GI-GFP, 0.001 eq OaAEP1b-C247A, 0.0001 eq QC in 20 mM PBS (pH 7). The reaction produced about 15% of the hydrolysis product DARPin-N-OH (labeled as H).

[0047] Fig.15 RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-DARPin at different time points is described. The ligation products were analyzed using ESI-MS. The ligation reaction was performed at 37°C in 20 mM PBS (pH 7) using 400 μM DARPin-NQL, 1.8 eq GI-DARPin, 0.001 eq VyPAL2, in the absence or presence of 0.0001 eq QC.

[0048] Fig.16 Shows Z at different time points EGFR RP-HPLC monitoring of the ligation reaction between -NQL and GI-ubiquitin. The ligation product was analyzed using ESI-MS. The ligation reaction was carried out at 37°C using 400 μM Z EGFR -NQL, 1.8 eq GI-ubiquitin, 0.001 eq VyPAL2, in the absence or presence of 0.0001 eq QC in 20 mM PBS pH 7. * Peak = by adding an extra Z at the N-terminus of the desired product EGFR The formation of [Z EGFR ]2-ubi byproduct.

[0049] Fig.17 RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-GFP at different time points is shown. The ligation product was analyzed using ESI-MS. The ligation reaction was performed at 37°C in 20 mM PBS (pH 7) using 400 μM DARPin-NQL, 1.8 eq GI-GFP, 0.001 eq VyPAL2, in the absence or presence of 0.0001 eq QC. H peak = hydrolysis product DARPin-N-OH.

[0050] Fig.18RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-Linker-GFP at different time points is shown. The ligation products were analyzed using ESI-MS. The ligation reaction was performed at 37°C in 20 mM PBS (pH 7) using 400 μM DARPin-NQL, 1.8 eq GI-GGGSGGGS-GFP, 0.001 eq VyPAL2, in the absence or presence of 0.0001 eq QC.

[0051] Fig.19 Non-reducing SDS-PAGE gels are shown to monitor Z at different time points. EGFR -Results of the ligation reaction between Fc-NQL and GI-GGGSGGGS-GFP. The ligation reaction was carried out at 37°C using 200 μM Z EGFR -Fc-NQL, 500 μM GI-GGGSGGGS-GFP, 0.4 μM VyPAL2, in the absence or presence of 0.04 μM QC in 20 mM PBS (pH 7).

[0052] Fig. 20 Depicted are untreated A431 and MCF7 cells (ctrl), shown as dark grey fill (blank), and cells treated with GFP (100 nM, 30 min) or Z EGFR - Fluorescence intensity of cells (arrows) treated with Fc-GFP (100 nM, 30 minutes) (measured using flow cytometry, λex=488 nm). Forward scatter (FSC-A) and side scatter (SSC-A) were used to sort intact cells.

[0053] Fig.21 Shows the Z EGFR -Confocal fluorescence microscopy images of EGFP-positive A431 cells after incubation with Fc-GFP or GFP. Compare the upper left image of each area, Z EGFR - Membrane staining of Fc-GFP is brighter than that of GFP. The plasma membrane was stained using PKH26 red fluorescent dye as shown in the lower left and merged images of each region. Scale bar = 20 μm.

[0054] Fig. 22 The mass spectra of the protein substrates of the present invention are described.

[0055] Fig.23 The structure of Gly-Val-Ala-PABC-MMAE (or GVA-PABC-MMAE) is depicted.

[0056] Fig.24 Shown are structures of examples of payload drugs where the amine groups can be replaced with e.g. Fig.25 , 26 and the PAL-linked linker modification shown in 29.

[0057] Fig.25 Shown are structures of examples of linker-payload compounds that are substrates for PAL-mediated acyl acceptor attachment to monoclonal antibodies or other proteins. In these compounds there is a payload-release linkage.

[0058] Fig.26 Described are structures of examples of linker-payload compounds that are substrates for PAL-mediated attachment of acyl acceptors to monoclonal antibodies or other proteins. Compounds In these compounds there is no payload-release linkage.

[0059] Fig. 27 Describes the structure of an example of a payload drug, where the hydroxyl group can be modified by linker attachment to PAL, such as Fig.28 and 30 shown.

[0060] Fig.28 Shown are structures of example linker-payload compounds that are acyl acceptor substrates for attachment to proteins and monoclonal antibodies via PAL.

[0061] Fig.29 The structures of bivalent drug-linker compounds as acyl acceptor substrates for attachment to proteins and monoclonal antibodies via PAL are described. The drug payload is attached to the bivalent linker via the amine group in the drug via PABC.

[0062] Fig.30 The structures of bivalent drug-linker compounds as acyl acceptor substrates for attachment to proteins and monoclonal antibodies via PAL are described. The drug payload is attached to the bivalent linker via its hydroxyl group.

[0063] Fig.31 The ligation reaction between Ac-SYRNQL and GIGGIR using mouse QC and OaAEP1b is described. Reaction conditions: 5 mM acyl acceptor and 5 mM acyl donor OaAEP1b-C247A (0.0005 eq or 0.05 mol%) were dissolved in 20 mM PBS (pH 7) at 37°C in the presence of QC (0.00005 eq). In the absence of QC, the reaction produced a ligation product with a yield of approximately 45% (see Figure 1 The yield was determined by HPLC (UV absorption at 220 nm).

[0064] Fig.32The schematic diagram of the method of the present invention is shown. (A) The intermolecular ligation catalyzed by PAL is a reversible reaction; (B) QC and PAL are coupled to form a cascade enzymatic reaction scheme, which overcomes the reversibility problem of PAL-mediated ligation. Specific implementation plan

[0065] The following detailed description has mentioned specific details and embodiments of the present invention by way of example. The detailed description of these embodiments is enough to enable those skilled in the art to implement the present invention. Without departing from the scope of the present invention, other embodiments can also be used, and structural and logical changes can be carried out. Various embodiments are not necessarily mutually exclusive, because some embodiments can be combined with one or more other embodiments to form new embodiments.

[0066] For convenience, the references mentioned in this specification are listed in the form of a reference list and added at the end of the examples. The entire contents of these references are incorporated by reference, but the mention in this specification does not mean that they constitute part of the common general knowledge.

[0067] definition

[0068] For convenience, certain terms used in the specification, example embodiments, and appended claims are collected here.

[0069] In general, the technical, scientific and medical terms used herein have the same meanings as those understood by those skilled in the art to which the present invention belongs. In addition, the following technical notes and definitions are provided. These definitions should not limit the scope of the present invention to these terms, but are provided for a better understanding of the following description.

[0070] As used herein, "a" or "an" means one or more than one, unless otherwise specified or obvious from the context. The inventors have found that the more enzyme used, the faster the reaction proceeds.

[0071] As used herein, the term "amino acid" may refer to natural and / or non-natural or synthetic amino acids, including D and L optical isomers, amino acid analogs (e.g., norleucine is an analog of leucine) and peptide mimetics. As in the context of this application, the term "amino acid" generally refers to 20 naturally occurring L-amino acids, which are Gly, Ala, Val, Leu, He, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr and Trp.

[0072] As used herein, the terms "comprising" or "including" should be interpreted as specifying the presence of the features, integers, steps or components mentioned, but do not exclude the presence or use of one or more features, integers, steps or components or groups thereof. However, in the context of the present disclosure, the terms "comprising" or "including" also include "consisting of". Variants of the word "comprising", such as "including" and "comprising", and "including", such as "including" and "comprising", have corresponding different meanings.

[0073] As used herein, the term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., biological activity or function, such as enzymatic activity) of the full-length protein, such as the ability to catalyze a ligation reaction between two peptides. A functional fragment can be of any size as long as the fragment retains the activity / function of the full-length protein / enzyme.

[0074] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a polymer composed of at least two amino acids covalently linked by amide bonds. While peptides are considered short amino acid chains and polypeptides are long amino acid chains, proteins tend to have a stable structure and may contain modifications (e.g., glycosylation and phosphorylation). The term "protein" can include naturally occurring and artificially synthesized (e.g., engineered or variant) full-length proteins as well as functional fragments of proteins. It should be understood that for the purposes of the present invention, any combination of peptides, polypeptides, or proteins can be linked in a reaction using PAL and QC, as long as one of them has a PAL acyl donor and the other has an acyl acceptor.

[0075] As used herein, the term "QC" refers to glutamine cyclase (QC) and QC-like enzymes. QC and QC-like enzymes have identical or similar enzymatic activities, i.e., catalyzing the intramolecular cyclization of glutamine groups and glutamyl residues at the N-terminal ends of peptides and proteins to form pyroglutamyl residues (pGlu). In this respect, QC-like enzymes may be fundamentally different from QC in molecular structure.

[0076] As used herein, the term "variant" refers to an amino acid sequence formed by changing one or more amino acids in a non-variant reference sequence, but retains the ability to recognize its target and affect its function. For example, a QC peptide variant formed by changing one or more amino acids in a non-variant QC peptide reference sequence, but retains the ability to catalyze the intramolecular cyclization of glutamine and glutamyl residues at the N-terminus of peptides and proteins to form pyroglutamyl residues (pGlu). The variant may have "conservative" changes, in which the substituted amino acids have similar structures or chemical properties (e.g., replacing leucine with isoleucine). More rarely, variants may have "non-conservative" changes (e.g., replacing glycine with tryptophan). Similar small variants may also include amino acid deletions, insertions, or combinations thereof. Using computer programs well known in the art, for example Software (DNASTAR, Inc., Madison, WI, USA) can provide guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity.

[0077] Exemplary, non-limiting embodiments of the present invention will now be described.

[0078] The present invention provides an improved peptide ligation method. In this regard, the present invention is based in part on the inventors' discovery that coupling QC and PAL to form a cascade enzyme reaction scheme overcomes the reversibility problem of PAL-mediated ligation (see Fig.32 ).

[0079] As disclosed herein, the acyl donor substrate of PAL in the present invention preferably has asparagine (Asn / N) at the P1 position and glutamine (Gln / Q) at the P1' position of the P1-P1'-P2' tripeptide PAL recognition motif. After connection with the acyl acceptor substrate, the acyl donor substrate releases a leaving group, wherein the exposed N-terminal glutamine is cyclized by QC to quench the Gln N in the lactam. α -amine.

[0080] Without being limited by theory, it is believed that after the Asn-Gln peptide bond is cleaved, QC cyclizes the exposed Gln to form a pyroglutamyl residue (pGlu), thereby quenching the nucleophilic N in the lactam. α Therefore, coupling PAL-mediated ligation with QC-catalyzed pGlu formation can effectively overcome the reversibility problem of transpeptide ligation reactions and improve the yield of ligation products.

[0081] To this end, one aspect of the present invention provides an enzymatic peptide ligation method, the method comprising providing i) a peptidyl asparaginyl ligase (PAL) and a glutamine cyclase (QC);

[0082] ii) a first peptide or protein having a P1-P1′-P2′ tripeptide PAL motif as an acyl donor, wherein P1 is Asn or Asp, P1′ is Gln or Glu, and P2′ is a hydrophobic amino acid or a β-branched amino acid;

[0083] iii) a second peptide or protein, which may be identical to or different from the first peptide or protein, having a P1″-P2″ motif at the N-terminus as an acyl acceptor, wherein P1″ is any amino acid and P2″ is a hydrophobic amino acid or a β-branched amino acid;

[0084] iv) contacting the peptidyl asparaginyl ligase (PAL) and glutamine cyclase (QC) with the first or second peptide / protein;

[0085] Among them, PAL cuts the first peptide / protein after P1 in the tripeptide PAL motif and connects the first peptide / protein to the P1"-P2" motif of the second peptide / protein, and QC cyclizes P1' in the released P1'-P2' dipeptide motif to pyroglutamyl (pGlu).

[0086] It should be understood that PAL requires a minimal tripeptide recognition motif, P1-P1′-P2′, for the site-specific ligation reaction after P1, where P1 is usually Asn or Asp, and P1′ and P2′ can be any of the naturally occurring amino acids Gly, Ala, Val, Leu, He, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr and Trp.

[0087] For the purposes of the present invention, P1 is preferably Asn or Asp, P1′ is preferably Gln or Glu, and P2′ is preferably a hydrophobic amino acid or a β-branched amino acid. In some embodiments, P1 is preferably Asn, and P1′ is preferably Gln. It is well known that Glu can replace Gln on the acyl donor P1′ (Seifert, F. et al., Biochemistry 48, 11831–11833 (2009)), and Asp can replace Asn on the acyl donor P1′ (Zhang, D. et al., (2021) Journal of the American Chemical Society 143 (23): 8704-8712). Therefore, in some embodiments, P1 can be Glu. In various embodiments, P1′ can be Asp.

[0088] In some embodiments, P2′ and / or P2″ may be a hydrophobic amino acid or a β-branched amino acid. Examples of hydrophobic amino acids may include Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, Tyr, and Trp. Examples of β-branched amino acids may include Thr, Val, and Ile.

[0089] In some embodiments, P2' can be selected from the group consisting of Leu, Met, Phe, Tyr, Trp, Val, Ile, and Thr. In various embodiments, P2" can be selected from the group consisting of Leu, Phe, Tyr, Trp, Val, Ile, and Thr.

[0090] In some embodiments, the acyl donor P1-P1'-P2' tripeptide PAL motif can be Asn-Gln-Leu.

[0091] It will be appreciated by those skilled in the art that different PALs and variants thereof having the desired protein ligase activity may be suitable for the practice of the present invention. Thus, in some embodiments, the PAL may be butyrate enzyme-1, butyrate enzyme-2, VyPAL2, VyPAL3, OaAEP1b-C247A, HeAEP3, AtLEGγ, VuPAL1, HaPAL1, OaAEP1b, or a functional fragment or variant thereof.

[0092] In certain embodiments, the PAL can be selected from the group consisting of butyrate enzyme-1 comprising the amino acid sequence shown in SEQ ID NO: 1, butyrate enzyme-2 comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, VyPAL2 comprising the amino acid sequence shown in SEQ ID NO: 4, VyPAL3 comprising the amino acid sequence shown in SEQ ID NO: 5, OaAEP1b-C247A comprising the amino acid sequence shown in SEQ ID NO: 6, HeAEP3 comprising the amino acid sequence shown in SEQ ID NO: 7, AtLEGγ comprising the amino acid sequence shown in SEQ ID NO: 8, VuPAL1 comprising the amino acid sequence shown in SEQ ID NO: 9, HaPAL1 comprising the amino acid sequence shown in SEQ ID NO: 10, OaAEP1b comprising the amino acid sequence shown in SEQ ID NO: 11, and functional fragments or variants thereof.

[0093] It is also contemplated that various QCs having the desired QC enzymatic activity may be suitable for the practice of the present invention. Thus, in some embodiments, the QC may be human glutamine cyclase, mouse glutamine cyclase, Drosophila glutamine cyclase, Arabidopsis thaliana glutamine cyclase, cono glutamine cyclase, pygmy rattlesnake glutamine cyclase, bacterial glutamine cyclase, or a functional fragment or variant thereof.

[0094] In some embodiments, QC can be selected from the following group: human glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 12, mouse glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 13, Drosophila glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 14, Arabidopsis thaliana glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 15, Cono glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 16, Dwarf rattlesnake glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 17, bacterial glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 18, and functional fragments or variants thereof.

[0095] Those skilled in the art will appreciate that the function of a protein / enzyme is directly related to its structure and sequence, and that there is a positive correlation between sequence identity and functional similarity. In this regard, methods for determining protein sequence identity are known in the art.

[0096] Thus, the sequence of the enzymes of the present disclosure can vary substantially as long as the enzymes retain their functionality and exhibit the desired activity (e.g., QC variants can catalyze the intramolecular cyclization of N-terminal glutamine groups and glutamyl residues of peptides and proteins to form pyroglutamyl residues (pGlu)).

[0097] In some embodiments, the PAL can be butyrate enzyme-1 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1; butyrate enzyme-2 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 2 or 3; VyPAL2 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 4; VyPAL3 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5; NO:6; OaAEP1b-C247A comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:7; HeAEP3 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:7; AtLEGγ comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:8; VuPAL1 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9; A HaPAL1 comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:10; or an OaAEP1b comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:11.

[0098] In some embodiments, QC can be human glutamine cyclase, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 12; mouse glutamine cyclase, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 13; Drosophila glutamine cyclase, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 14; Arabidopsis thaliana glutamine cyclase, comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 15. NO:15; a conus snail glutamine cyclase comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:16; a pygmy rattlesnake amino acid cyclase comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:17; or a bacterial glutamine cyclase comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:18.

[0099] In some embodiments, the second peptide or protein may include a spacer of at least one amino acid between the P1"-P2" acyl acceptor and the second peptide or protein. As used herein, the introduction of a spacer between the P1"-P2" acyl acceptor and the second peptide or protein can increase the accessibility of PAL-catalyzed ligation, thereby increasing the yield, particularly when the second protein is large enough to hinder the accessibility of PAL.

[0100] The reaction rate of the disclosed method can be controlled by changing the ratio of enzyme to relevant substrate. In this regard, the inventors found that the more enzyme used, the faster the reaction is carried out. In some embodiments, a small amount of enzyme (such as QC of 0.005%eq relative to substrate and QC of 1 / 10eq relative to PAL) may be sufficient to implement the present invention. For some difficult protein substrates such as antibodies, a higher enzyme to substrate ratio is required, such as 0.1:1:100 or 0.1:1:50 (QC:PAL: first peptide / protein). The ratio of enzyme to substrate depends to a great extent on substrate and specific application, and can be easily determined according to standard techniques known to those skilled in the art, or can be inferred by reference to relevant literature.

[0101] In some embodiments, the ratio of QC:PAL:first peptide or protein ranges from 0.1:1:2000 to 1:1:50, preferably from 0.1:1:1000 to 0.1:1:50, respectively. In some embodiments, the ratio of QC:PAL:first peptide or protein is 0.1:1:20, respectively.

[0102] As described herein, the methods of the present disclosure are suitable for protein-protein connection, and acyl acceptor peptides and acyl donor substrates can also be appropriately selected and modified according to the methods of the present disclosure for the preparation of antibody-drug conjugates, etc. Therefore, within the scope of the present invention, the methods of the present disclosure can also be used for the effective connection of monoclonal antibodies and other proteins with a variety of linker-payload drug compounds (e.g., with linker-payload drug compounds as acyl acceptor substrates). Therefore, the modified peptides used in the present invention can be prepared by standard techniques known to those skilled in the art of organic chemical synthesis, or can be inferred by reference to relevant literature.

[0103] In some embodiments, according to the present application, the first and second peptides or proteins to be linked can be further modified to carry a labeling component. The labeling component can be any molecule, such as, but not limited to, an affinity tag, a detectable label, a therapeutic agent, a framework molecule, an epitope binding peptide, a ubiquitin molecule, a biotin molecule, a His6 tag, a green fluorescent protein (GFP), an epitope binding peptide, and an affinity body such as Z EGFR , Z EGFR -Fc and DARPin.

[0104] In some embodiments, the first and second peptides or proteins can be identical and can form a dimer when linked.

[0105] It should be understood that the key components of an antibody-drug conjugate may include an antibody, a linker, and a payload. Thus, in some embodiments, one of the first and second peptides or proteins may be an epitope binding peptide or protein, and the other peptide or protein may comprise a payload. In some embodiments, the payload may include a payload-release linkage.

[0106] In some embodiments, the payload is an imaging agent or a therapeutic agent. Typically, the imaging agent can be a radiolabeled chelator or an optical label.

[0107] In some embodiments, the radiolabeled chelator is selected from the group consisting of 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclo-1-pentanedioic acid-4,7-diacetic acid (NODAGA); and / or the optical label is HRP or GFP or an analog. In some embodiments, the therapeutic agent may be monomethyl auristatin E (MMAE) or radiolabeled DOTA.

[0108] In various embodiments, the epitope binding peptide or protein can be selected from the group consisting of antibodies or functional fragments thereof, affibodies such as Z EGFR , Z EGFR -Fc and DARPin.

[0109] In certain embodiments, the antibody or functional fragment thereof is selected from the group consisting of a minibody, a diabody, a scFv, a nanobody and a F(ab′) 2 .

[0110] Unless otherwise specified, or obvious from the context and understanding of a person of ordinary skill in the art, in various embodiments, the value expressed in a range can be any specific value or sub-range assumed in the range, up to one tenth of the unit of the lower limit of the range, unless otherwise clearly specified in the context. Unless otherwise specified or obvious from the context, the "about" referring to a numerical value generally refers to a range falling within ±10% of the value, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%. In any embodiment with "about" as a numerical prefix, an embodiment describing an exact numerical value is provided. In an embodiment without "about" as a numerical prefix, an embodiment with "about" as a numerical prefix is ​​also provided. If there is "about" in front of the range, an embodiment of "about" applicable to the lower and upper limits of the range or to the lower or upper limits is provided, unless otherwise clearly specified in the context. If there is "at least", "at most", "not more than" or similar phrases before a group of numbers, it should be understood that the phrase is applicable to each number in the list in different embodiments (it should be understood that, according to the context, 100% of the value, such as a value expressed as a percentage, may be an upper limit), unless otherwise clearly specified in the context. For example, in various embodiments, "at least 1, 2, or 3" should be understood as "at least 1, at least 2, or at least 3." It should also be understood that any and all reasonable lower and upper limits are expressly contemplated.

[0111] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0112] Example

[0113] Standard molecular biology techniques known in the art but not specifically described generally follow those described by Green and Sambrook in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York (2012)).

[0114] Materials and general methods

[0115] All solvents and reagents were purchased from commercial suppliers and used without further purification. Human glutamine cyclase (QC) was purchased from Abcam (ab206806) and aliquoted and stored at -80°C.

[0116] Peptides were synthesized according to the standard Fmoc solid phase synthesis process. The synthesized peptides were purified using semi-preparative RP-HPLC. Semi-preparative RP-HPLC was performed using a Shimadzu HPLC system equipped with a Phenomenex-C18 RP column (10×250 mm, 5 μm) at a flow rate of 2.5 mL / min, using buffer B (90% acetonitrile, 10% H2O, 0.045% TFA) in buffer A (H2O, 0.045% TFA) for gradient elution. All synthesized compounds were stored at 4°C or -20°C.

[0117] The protein was produced by recombinant DNA method. For protein purification, immobilized metal affinity chromatography (IMAC), protein A affinity chromatography and size-exclusion chromatography (SEC) were used. HiLoadTM 16 / 600SuperdexTM 200pg column was used for purification. SEC was performed on FPLC UPC-900. HiTrap5ml MabSelectTM column or HisTrap HP5ml column was used in Protein A affinity chromatography and NiNTA affinity chromatography were performed on .

[0118] During analysis, peptide mass spectra were obtained using a Bruker Ultraflex Extreme matrix-assisted laser desorption (MALDI) tandem TOF or electrospray ionization (ESI) mass spectrometer (Thermo Fisher LTQ XL). MALDI data were analyzed using Data Explorer software, and ESI data were analyzed using Thermo Xcalibur QualBrowser and Magtran software. Protein mass spectra were deconvoluted using MagTran. The chromatographic column was equipped with a Phenomenex-C18 RP column (4.6×150 mm, 55 μm, ) or Phenomenex Jupiter-C4 column (4.6×150mm, 3.6μm, ) on a Shimadzu HPLC (RP-HPLC) system at a flow rate of 1.0 mL per minute, with gradient elution using buffer B (90% ACN, 10% H2O, 0.045% TFA) in buffer A (H2O, 0.045% TFA).

[0119] Solid Phase Peptide Synthesis (SPPS)

[0120] All peptides were synthesized as C-terminal amides using Rin amide MBHA resin by standard Fmoc chemistry using a Liberty Blue peptide synthesizer or 2-chlorotrityl resin. For 5(6)-carboxyfluorescein and biotin coupled to Lys (MTT) side chains, the MTT protecting group was first removed using TFA / TIS / DCM (2.5% / 2.5% / 95%), followed by coupling to the Lys side chain amine using 2.5 eq of 5(6)-carboxyfluorescein (or biotin), 2.5 eq of Oxyma, and 2.5 eq of DIC in NMP for 3 h. To cleave the peptides from the resin and deprotect the side chain protecting groups at the SPPS termini, the peptidyl resin was treated with a mixture of TFA / H2O / TIS (95% / 2.5% / 2.5%) for 1-3 h. The cleaved solution was separated from the resin by filtration and the cleaved peptides were precipitated in cold Et2O. The crude product was separated by centrifugation and purified by RP-HPLC. The peptide fraction purified by HPLC was lyophilized to obtain a powdered peptide.

[0121] Synthesis of GIGKVA-PABC-MMAE (Compound 4)

[0122]

[0123] Compound 1 was synthesized by standard SPPS chemistry.

[0124] FmocGIGK(ivDde)VA-PAB-OH (2): To a suspension of compound 1 (110 mg, 0.113 mmol) in MeOH (2.5 mL) and DCM (5.0 mL) were added EEDQ (84 mg, 0.34 mmol) and 4-aminobenzyl alcohol (27.8 mg, 0.226 mmol). The mixture was stirred at room temperature in the dark for 36 h. After evaporation of the solvent, the residue was subjected to column chromatography (2-6% MeOH in DCM) to give off-white solid compound 2 (70 mg, 58%).

[0125] FmocGIGK(ivDde)VA-PAB-PNP carbonate 3 was prepared by adding DIPEA (60 μL, 0.33 mmol) and bis(p-nitrobenzene) carbonate (100 mg, 0.33 mmol) to anhydrous DMF solution of compound 2 (120 mg, 0.11 mmol), and stirring the mixture under N2 atmosphere at room temperature for 18 h. After removing the solvent by rotary evaporation, the residue was subjected to column chromatography (2-6% MeOH in DCM) to obtain PNP-carbonate 3 (110 mg, 79%) as an off-white solid.

[0126] MMAE·HCl (66 mg, 0.088 mmol) was added to a solution of compound 3 (100 mg, 0.081 mmol), HOAt (5.5 mg, 0.04 mmol), and DIPEA (0.07 mL, 0.405 mmol) in anhydrous DMF. The resulting reaction mixture was stirred at room temperature for 18 h. Hydrazine hydrate (0.5 mL) was then added and the mixture was stirred for 4 h. After the solvent was removed by rotary evaporation, the residue was purified by reverse phase HPLC (buffer A: 0.045% TFA in H2O, buffer B: 0.045% TFA in 90% acetonitrile and 10% H2O). The fractions containing the product were pooled and freeze-dried to obtain compound 4 as an off-white powder. MS (ESI): m / z [M+H] + Calculated value (calc.) 1392.9, found value (obvs.) 1393.0.

[0127] Synthesis of GIGGGK[Fe(DOTA)](Compound 6)

[0128]

[0129] Compound 5 was synthesized by standard SPPS chemistry. 1 eq of compound 5 (10 mM) and 1 eq of FeCl3 were mixed in water, and the pH of the solution was adjusted to 6 using 2 M NaOH. The mixture was placed at 37 °C overnight to give GIGGGK[Fe(DOTA)], which was used without purification. MS (ESI): m / z[M+H] + calc.926.6,obvs.926.5.

[0130] OaAEP1 b -C247A expression, purification and activation

[0131] OaAEP1 b -C247A was cloned into the vector pET28a (Genscript) and expressed in T7 Shuffle E. coli. Pro-OaAEP1 was activated in acetate buffer (0.1 M NaCl, 0.5 mM TCEP) at pH 4. b -C247A for 2 h at 37° C. After activation, the activated enzyme was purified by size exclusion chromatography (SEC) at pH 7 (20 mM PBS, 0.1 M NaCl). The purified enzyme was stored at -80° C. in 5% glycerol at pH 7 (20 mM PBS, 0.5 mM TCEP).

[0132] VyPAL2 expression, purification and activation

[0133] Use sf9 insect cells to express VyPAL2. Use 100 mL of viral vector containing the VyPAL2 gene at 2.5 × 10 6 The cell density of cells / mL was used to infect sf9 cells. The MOI of infection was set between 1-10 for protein expression. The culture was incubated at a speed of 135 rpm in a 27°C shaker for 3 days (72 hours). Protein purification was performed in three steps: immobilized metal affinity chromatography (IMAC), ion exchange chromatography (IEX), and size exclusion chromatography (SEC). Pro-VyPAL2 was activated for 2-3h at 37°C in 50mM sodium citrate buffer (0.1M NaCl, 1mM DTT, 0.5Mm LS) at pH 4.5. After activation, the activated enzyme was purified by SEC at pH 6.5 (20mM PBS, 0.1M NaCl, 1mM DTT). The purified enzyme was stored at -80°C, 5% glycerol at pH 7 (20mM PBS, 0.5mM TCEP).

[0134] DARPin9_26, GFP, ubiquitin, Z EGFR and Z EGFR -Fc expression and purification

[0135] All protein genes were cloned into pET28a, pET3a, pTxB1 or pETDuet (Genscript) and expressed in E. coli (DE3) or T7 Shuffle. The bacterial OD was 2.0447 W / v at 37°C or 30°C in LB medium (Luria Bertani broth) (Kana or Amp). 600 After reaching 0.4-0.6, 0.1-0.4Mm IPTG was used to induce expression (except GI-ubi). After induction, cells were incubated at 16°C for 18h. Cells were harvested by centrifugation (5000x g, 10 minutes) and then resuspended in lysis buffer (50mM PBS, 0.1M NaCl, 10mM imidazole, 0.01% 100X triton, pH 7.5). Ultrasonic probe (Vibra cell TM) The solution mixture was lysed on ice for 15-30 minutes with alternating cycles of 3-second pulses every 8 seconds. The protein solution was then centrifuged at 15,000 x g (20 minutes) at 4°C, filtered with a 0.2 μm filter, and bound to NiNTA magnetic beads or protein A magnetic beads at 4°C for 1 hour. The Ni magnetic beads were washed with 20 mM imidazole, 0.1 M NaCl, 20 mM PBS buffer (pH 7.5), and the protein was eluted with 500 mM imidazole, 0.1 M NaCl, 20 mM PBS buffer (pH 7.5). The protein A magnetic beads were washed with 20 mM PBS (pH 7.5), and the protein was eluted with 30 mM citrate buffer (pH 3.5). For GI-ubi, it was expressed as a C-terminal intein fusion protein in E. coli (DE3) and the protein solution was bound to chitin magnetic beads and incubated overnight at RT in 50mM DTT, 20mM PBS (pH 8) to cleave GI-ubi from bound enterin. All proteins were exchanged into 20mM PBS (pH 7) and stored at 4°C for short term storage and -20°C for long term storage.

[0136] Ligation of model peptides

[0137] Enzyme-mediated ligation reactions were performed in 20 mM PBS buffer (pH 6.5 or pH 7) at 37°C in the presence or absence of QC for different time courses. The ratio of QC, ligase and substrate (NQL peptide) was 0.1:1:2000. The reaction was quenched by 10% TFA and monitored by analytical RP-HPLC. The ligation products were characterized by MALDI-MS or ESI-MS.

[0138] Protein N- or C-terminal tagging

[0139] The ligation reaction was carried out at pH 7, 37°C for different time periods in the presence or absence of QC. The ratio of QC / ligase / protein substrate was 0.1 / 1 / 1000. The reaction was quenched by 6M guanidine-HCl (pH 3) and monitored by analytical RP-HPLC. The ligation product was characterized by ESI-MS.

[0140] Protein-Protein Linkage

[0141] Ligation reactions were performed at pH 7, 37°C for different time courses in the presence or absence of QC. The ratio of QC / ligase / protein substrate was 0.1 / 1 / 1000 (500). The reaction was quenched with 6M guanidine-HCl (pH 3) and monitored by analytical RP-HPLC. The ligation products were characterized by ESI-MS. Z was analyzed by SDS-PAGE under reducing or non-reducing conditions (reducing conditions: 50 mM DTT, pH 8.8, reaction time 20 min). EGFR -Analysis of the ligation reaction between Fc-NQL and GI-GGGSGGGS-GFP.

[0142] Purification of ligated proteins

[0143] The linked Z was analyzed by size exclusion chromatography (SEC) at pH 7 (20 mM PBS, 0.1 M NaCl). EGFR -Fc-GFP protein was purified. The purified protein was stored at -20°C. Immobilized metal affinity chromatography (IMAC) was used to quantify the Z EGFR -MMAE was purified and stored in pH 7 buffer (20 mM PBS, 0.1 M NaCl).

[0144] Flow cytometry

[0145] To study Z EGFR -Fc-GFP, A-431 (ATCC, USA) and MCF-7 (ATCC, USA) living cells were washed three times with PBS (HyClone, USA), trypsinized with 0.05% trypsin-EDTA, and then resuspended in cold DMEM (Gibco, USA) containing 10% FBS (Gibco, USA). One million cells of each cell line were incubated with Z EGFR -Fc-GFP (100 nM) and GFP (100 nM) were incubated on ice for 30 min. After incubation, cells were washed three times with cold PBS and analyzed using a Fortessa X-20 flow cytometer (BD, USA). The cytometer was set to record 10,000 events per sample, excite the fluorophore with a 488 nm laser, and collect the emitted fluorescence signal at 530 / 30 nm. The fluorescence intensity was measured using Flowjo TM 10 (BD, USA) to analyze the generated raw data.

[0146] Live cell confocal imaging

[0147] To observe Z EGFR-Fc-GFP binding activity, A431 and MCF-7 cells were seeded on 8-well chamber slides (ibidi, USA) and incubated overnight at 37°C under 5% CO2. The cells were stained with 2 μM PKH26 red fluorescent dye (Sigma, USA) at 37°C for 10 minutes. The stained cells were then incubated with Z EGFR -Fc-GFP (100nM) and GFP (100nM) were incubated on ice for 30 minutes. After incubation, the cells were rinsed three times with cold PBS and fixed with cold 4% formaldehyde for 15 minutes. Fixed cells were imaged using an LSM 980 confocal microscope (Zeiss, Germany). The key settings of the microscope are as follows: 1) Excitation laser wavelength: 488nm and 561nm; 2) Emission filler: 507-552nm and 575-620nm; 3) Imaging mode: Z-stack. The three-dimensional Z-stack images were processed into two-dimensional images using Zen software (Zeiss, Germany) using MIP (maximum intensity projection) technology.

[0148] Cell viability assay

[0149] To detect Z EGFR -MMAE cytotoxicity, approximately 5000 A431 and MCF-7 cells were seeded in 96-well plates and incubated overnight at 37°C under 5% CO2. EGFR -MMAE was added to the wells and incubated at 37°C 5% CO2 for 3 days. Then 0.5 mg / mL of MTT was added and incubated for 1 h at 37°C. Cell activity was measured based on the absorbance at 570 nm.

[0150] Peptide and protein sequence and mass spectrometry data

[0151] Table 1. Peptides used in this study

[0152]

[0153]

[0154]

[0155]

[0156] Table 2. Peptidyl asparaginyl ligase / asparaginyl endopeptidase and its amino acid sequence

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] (The grey area corresponds to the catalytically active core domain that is prepared from the zymogen after activation at acetic acid pH; the underlined grey sequences can be further processed during activation. Expression markers or mutations are underlined)

[0163] Table 3. Glutamine cyclase and its amino acid sequence

[0164]

[0165]

[0166]

[0167] Table 4. Protein sequences and their associated mass spectrometry data

[0168]

[0169]

[0170]

[0171] Example 1: Demonstration of the PAL-QC cascade protocol in a model peptide ligation reaction.

[0172] QC participates in the maturation of many bioactive peptides and proteins by catalyzing the formation of N-terminal pGlu (Busby, Jr. et al., J. Biol. Chem. 262, 8532–8536 (1987); Schilling, S. et al., Biochemistry 41, 10849–10857 (2002); Seifert, F. et al., Biochemistry 48, 11831–11833 (2009)). The efficiency of QC catalyzing the unimolecular lactamization reaction of different organisms is about 10 5 M -1 ·S -1 (Seifert, F. et al., Biochemistry 48, 1831–11833 (2009)), while the efficiency of PAL-catalyzed bimolecular ligation reaction is about 10 4 M -1 ·S -1(Wang, Z. et al., Ranostics 11, 863–5875 (2021)). This makes QC a particularly attractive enzyme to capture the released glutamine leaving group. We first found that among four synthetic peptides with the sequence QXGSA (X = L, I, F, or V, with preference for PAL as the P2′ amino acid), QC efficiently converted the N-terminal Gln to pGlu( Figure 7 ). Consistent with previous studies, the presence of a large hydrophobic residue such as X at the second position of the glutamine peptide had no negative impact on the ability of QC to catalyze the lactamization reaction. In fact, QC was able to complete the reaction within 30 min (pH 7) with 0.0001 eq of substrate. The reaction rates of QI-, QL-, and QF-peptides were similar, while the reaction rate of QV-peptide was approximately 30% slower ( Figure 7 ). Since Leu is the most preferred P2′ residue of PAL, we chose Asn-Gln-Leu as the tripeptide recognition motif for all acyl donor substrates in this study.

[0173] We then used Ac-SYRNQL (5 mM) as the acyl donor and GIGGIR (1 eq) as the acyl acceptor in a model ligation reaction to test the cascade enzymatic scheme of the present invention. At a molar ratio of PAL to substrate of 0.0005:1, the reaction catalyzed by OaAEP1b-C247A at pH 7 gave a product yield of about 45%, and the reaction reached equilibrium in about 30 minutes ( Figure 1 a). In contrast, the addition of 0.00005 eq of QC increased the ligation yield to >95% in 32 minutes ( Figure 1 a and 1b). The substantial increase in yields suggests that very low amounts of QC (0.005 mol% of substrate and 1 / 10 eq of PAL) are sufficient to quench the released QL dipeptide by forming pGlu. This provides clear validation of the PAL-QC coupling reaction scheme. When 0.05 mol% of QC was used in the reaction (e.g., QC:PAL = 1:1), the reaction reached equilibrium faster, but the final yield was almost the same ( Figure 1 a). Since the reaction rate of a QC to PAL ratio of 1 / 10 was sufficiently fast, this ratio was used in all subsequent studies. Under the same conditions, ligation reactions were performed using VyPAL2 or butyrate enzyme-1. A significant increase in yield was also observed when QC was present in the reaction, indicating that QC is compatible with the other three most commonly used PALs ( Figure 8 ).

[0174] Example 2: Cascade enzymatic protocol for protein N- and C-terminal labeling.

[0175] Then, ubiquitin was used as a model protein to demonstrate the application of this method in protein labeling reactions. Two recombinant ubiquitin variants, GI-ubiquitin and ubiquitin-NQL-His6, were prepared using two biotin synthetic peptides, biotin-GRSNQL and GIGGIRK (biotin), for N- and C-terminal labeling. 500 μM of ubiquitin substrate protein and 1.2 eq of biotin peptide were used in both ligation reactions, and the reactions were carried out at 37 ° C, pH 7, and 0.5 μM OaAEP1b-C247A (0.001 eq).

[0176] For the ligation reaction of ubiquitin-NQL-His6 and GIGGIRK (biotin), when 0.0001eq of QC was added, the yield increased from 40 to 94% ( Figure 2 ). Similarly, after adding QC, the yield of biotin-GRSNQL and GI-ubiquitin ligation reaction increased from 49% to 88%. QC greatly improved the labeling efficiency of protein C- and N-termini, further verifying the effectiveness of this method in protein-peptide ligation reactions.

[0177] Next, the anti-EGF affibody protein Z EGFR ( S. et al., Trends Biotechnol. 35, 691–712 (2017)) were labeled with functional molecules with potential diagnostic and therapeutic significance at the C-terminus. Two specific peptides, GIGGGK[Fe(DOTA)] and GIGKVA-PABC-MMAE, were prepared and used as acyl acceptor substrates to react with Z EGFR -NQL connection Figure 3 a and 3b). As the most powerful chelating agents, DOTA and its derivatives can form very stable compounds with certain metal ions (Viola-Villegas, N. et al., Coordination Chemistry Reviews 2009, 253, 1906–1925 (2009)). Disease-targeting proteins can be coupled to DOTA compounds containing radionuclides for diagnostic or therapeutic applications (Sgouros, G. et al., Nat. Rev. Drug Dis. 19, 589–608 (2020)). In this study, DOTA compounds only contain non-radioactive Fe 3+ ions for demonstration purposes. MMAE or monomethyl auristatin E is a potent cytotoxic agent and a common drug payload in antibody-drug conjugates (Chen, H. et al., Molecules 22, 1281 (2017)). It is usually linked to the antibody via PABC, a self-cleaving linker that releases the payload (Doronina, S. et al., Bioconjugate Chem. 17, 114–124 (2006)). Figure 3 As shown, the cascade enzymatic method makes Z EGFR -[Fe(DOTA)] and Z EGFR The yields of the ZEGFR-[Fe(DOTA)] conjugates reached 90% and 88%, respectively, while when there was no QC, the yield was only about 50%. During HPLC purification and ESI-MS measurement, the [Fe(DOTA)] compound was stable because the intact molecular ions of the GIGGGK[Fe(DOTA)] peptide and the ZEGFR-[Fe(DOTA)] conjugate could be observed. EGFR The -MMAE conjugates showed high cytotoxicity to A431 cells overexpressing EGFR, but relatively low cytotoxicity to MCF-7 cells with extremely low EGFR expression. Clearly, these two examples show that the PAL-QC conjugation cascade protocol can provide a safer production process for bioconjugates containing radioactive or toxic payloads, because high-yield reactions do not require excess reactants, thereby minimizing the risk of exposure to these hazardous substances.

[0178] By adding Z EGFR Protein was linked to an acceptor containing two Gly-Ile dipeptidyl groups to prepare a C-terminally linked dimer Z EGFR protein( Figure 3 c) This is a more stringent test of the method, as both nucleophilic sites in the bivalent peptide need to be attached to the protein. Fortunately, only a slight excess of Z EGFR (1.1 effective equivalents of divalent peptide), the cascade protocol yielded approximately 80% of the desired C-terminal dimer protein product. In contrast, without QC, only approximately 46% of the C-terminal dimer was obtained, and a large amount of single-linked intermediates ( Figure 3 c) This parallel protein dimer synthesis experiment illustrates the utility of this method in preparing this particular protein conjugate.

[0179] Example 3: Use of cascade enzymatic reactions in protein-protein ligation.

[0180] Then, several proteins were selected—GFP, ubiquitin, DARPin9-26 (Steiner, D. et al.; J. Mol. Biol. 382, ​​1211–1227 (2008); Jost, C. et al.; Structure 21, 1979–1991 (2013)) and anti-EGFR affinity body Z EGFR , to determine whether the method of the present invention can be further extended to protein-protein ligation reactions. First, DARPin-NQL (400 μM) and GI-ubiquitin (1.8 eq) were ligated using VyPAL2 (0.001 eq) at pH 7 and 37°C. Without QC, the yield of the product obtained in 3 h reaction was 39%, while the yield increased to 91% when QC was added ( Figure 4 a). Interestingly, we found that VyPAL2 was more effective than OaAEP1b-C247A in the DARPin-ubiquitin ligation reaction, as the latter produced a small amount of the hydrolysis product DARPin-N-OH ( Fig.14 ). This phenomenon was not observed in either peptide-peptide or peptide-protein ligation reactions. It appears that OaAEP1b-C247A has residual hydrolase activity that may be exacerbated in difficult, entropically demanding protein-protein ligation reactions. Therefore, VyPAL2 was used for all subsequent protein-protein ligation reactions.

[0181] Similarly, the ligation of DARPin-NQL (1 eq) to GI-DARPin (1.8 eq) gave tandem DARPin-NGI-DARPin in yields of 47% (without QC) and 95% (with QC) ( Figure 4 b) Z EGFR Ligation of DARPin-NQL to GI-ubiquitin gave product in 37% (without QC) and 76% (with QC). Ligation of DARPin-NQL to the larger GFP protein (GI-GFP) gave DARPin-NGI-GFP in 42% (without QC) and 74% (with QC). The proximity of the N-terminal GI nucleophile to the rigid β-tubular structure of the GFP protein may have hindered its access, resulting in slightly lower ligation yields in this reaction and some hydrolysis ( Fig.17Adding a longer spacer after the GI dipeptide improved accessibility, so the yield of the connection between the DARPin and GI-GGGSGGGS-GFP was 50% (without QC) and 85% (with QC). In all cases, the use of QC and VyPAL2 increased product yields by 33-53%. This improvement is comparable to that observed in model peptide ligation, making the method of the present invention significantly superior to all previous methods (Nguyen, GKT et al.; Angew. Chem. Int. Ed. 54, 15694–15698 (2015); Cao, Y. et al.; Bioconj. Chem. 27, 2592–2596 (2016); Rehm, FBH et al.; J. Am. Chem. Soc. 141 (43), 17388–17393 (2019); Tang, TMS et al.; Chem. Sci. 11, 5881–5888 (2020); Rehm, FBH et al.; Angew. Chem. Int. Ed. 2021, 60, 4004–4008 (2021)).

[0182] Next, Z EGFR -Fc-NQL was linked to GI-GGGSGGGS-GFP (29 kDa) to obtain a large protein product with a mass of about 126 kDa, in which Z EGFR -Fc-NQL is composed of affimer Z EGFR A large bivalent fusion protein (MW ~ 68 kDa) consisting of the Fc domain of IgG. EGFR -The ligation reaction between Fc-NQL (200 μM) and GFP protein (500 μM) obtained about 90% yield in the presence of QC ( Figure 5 a) Considering the large size of the protein and the size of the acceptor GFP protein substrate and the donor substrate Z EGFR The molar equivalent of -Fc-NQL is only 1.25, while Z EGFR -Fc-NQL, as a dimer, has two reaction sites, so the results are significant. In addition, it can be seen from the non-reducing SDS-PAGE gel ( Fig.19 ), when QC is present, the major product is the final double-ligation product, while when QC is absent, the major product is the single-ligation product. Figure 5 b and c show the double ligation product Z EGFR -Fc-GFP specific binding to A431 cells overexpressing EGFR, indicating that Z EGFR The receptor binding activity and GFP fluorescence of the GFP conjugate were retained. This example shows that the VyPAL2-QC method is also suitable for preparing large therapeutic protein conjugates.

[0183] In addition, Z EGFR -Fc-NQL is also linked to Gly-Val-Ala-PABC-MMAE ( Fig.23 MMAE or monomethyl auristatin E is an effective antimitotic agent. Val-Ala-para-carbamic acid benzyl ester (ValAla-PABC) is a linker that can be cleaved by intracellular proteases, and Gly-Val dipeptide is a good acyl acceptor nucleophilic substrate for PAL enzymes. Z was catalyzed by VyPAL2 (0.002 eq) at pH 7 and 23 °C. EGFR -Fc-NQL (200 μM) was linked to Gly-Val-Ala-PABC-MMAE (800 μM). Again, the absence or presence of QC (0.0002 eq) resulted in significant differences in the ligation efficiency ( Figure 6 ). When QC was present, the yield of the ligation product obtained after 4 h was about 80%, while in the absence of QC, the yield was only 42%. EGFR -Fc-NQL is a dimer, so the chemical valency of the MMAE nucleophilic substrate is 2. EGFR The conjugate of the -Fc fusion protein with MMAE is similar to an antibody-drug conjugate or ADC. This result fully demonstrates that the PAL-QC combination method of the present invention can be used to prepare ADCs for the treatment of cancer and other diseases, and does not require the use of a large amount of acyl acceptor substrate in the ligation reaction. It is expected that this method can efficiently ligate monoclonal antibodies and other proteins, and use a wide range of linker-payload compounds as acyl acceptor substrates ( Figure 24-30 ).

[0184] Example 4: Demonstration of the PAL-QC cascade protocol in a model peptide ligation reaction using mouse QC.

[0185] The peptide ligation reaction between Ac-SYRNQL and GIGGIR was tested using mouse QC and OaAEP1b-C247A.

[0186] Reaction conditions: 5 mM acyl acceptor and 5 mM acyl donor OaAEP1b-C247A (0.0005 eq or 0.05 mol%) were dissolved in 20 mM PBS (pH 7) at 37°C in the presence of QC (0.00005 eq). In the absence of QC, the reaction yielded about 45% of the ligation product (see Figure 1 The yield was determined by HPLC (UV absorption wave at 220 nm).

[0187] The results showed that mouse QC had the same effect as human QC in overcoming the reversibility of PAL-only ligation and increasing the yield of PAL-mediated ligation reactions.

[0188] Summarize

[0189] As the most powerful transpeptidase known to date, PAL has been shown to be able to catalyze the cyclization reactions of peptides and proteins very efficiently (Xia, Y. et al.; Angew. Chem. Int. Ed. 60, 22207–22211 (2021); Zhang, D. et al.; J. Am. Chem. Soc. 143, 8704–8712 (2021)). cat / K m At least greater than the k of the intermolecular ligation reaction cat / K m An order of magnitude higher. This is due to the favorable nature of the intermolecular reaction entropy. In addition, the rigid conformation of the cyclized product tends to make it resistant to PAL. Therefore, except as a transpeptidation reaction, PAL-catalyzed cyclization reactions are generally irreversible. This is different from the case of bimolecular ligation reactions. When the ratio of the two reactants is 1:1, their reversibility generally limits the product yield to ≤50%. We show that the use of P1′Gln in the acyl donor substrate can overcome this problem, because its α-amine can be quenched by lactamization after cleavage of the Asn-Gln peptide bond. Pyroglutamyl can be formed spontaneously, but this is a slow process. The rate constant for the spontaneous formation of pGlu from N-terminal Gln at pH 6 has been reported to be 1.7x 10 -6 s -1 , the corresponding half-life is about 4.7 days (Seifert, F. et al., Biochemistry 48, 11831–11833 (2009)). cat 30s -1, the rate was increased by seven orders of magnitude (Seifert, F. et al., Biochemistry 48, 11831–11833 (2009)). The high efficiency of QC makes it suitable for use with PAL. In this cascade enzymatic protocol, the amount of QC used was one-tenth of that of PAL enzyme, and the molar ratio of PAL enzyme to substrate was 1:1000 or 1:2000. Using this protocol, the yield of intermolecular ligation was greatly improved when the molar equivalents of the acyl acceptor substrate and the acyl donor substrate were equal or slightly higher. Our method is generally applicable to all PALs and substrates of various volumes, from small peptides to large recombinant proteins. Compared with existing methods that use metal ions, synthetic chemicals or non-natural elements in the substrate to solve the reversibility problem, this method uses a harmless enzyme. Although the use of another enzyme may lead to cost-related issues, this is not a big problem because QC can be easily expressed in E. coli and yeast systems. The high reaction yield and extremely low enzyme dosage also facilitate product purification, making the process cost-effective. In conclusion, this robust cascade enzymatic protocol according to the present invention greatly improves the applicability of PAL-mediated ligation for the precise fabrication of large protein conjugates.

[0190] References

[0191] Bi, X.; Yin, J.; Nguyen, G.K.T.; Rao, C.; Halim, N.B.A.; Hemu, X.; Tam, J.P.; Liu, C.-F., “Enzymatic engineering of the cell surface of living bacteria using butyrate enzyme 1.” Angew. Chem. Int. Ed. Engl. 2017, 56, 7822–7825.

[0192] Bi, X.; Yin, J.; Hemu, X.; Rao, C.; Tam, JP; Liu, C.-F. "Immobilization and intracellular delivery of cyclic proteins by modifying genetically incorporated unnatural amino acids." Bioconjugate Chem. 2018, 29, 2170–2175.

[0193] Bi, X.; Yin, J.; Zhang, D.; Zhang, X.; Balamkundu, S.; Lescar, J.; Dedon, P.C.; Tam, J.P.; Liu, C.-F. "Labeling transferrin receptor with disulfide FRET probe to measure the redox state of endosomal compartments." Anal. Chem. 2020, 92, 12460–12466.

[0194] Busby, Jr., WH; Quackenbush, GE; Humm, J.; Youngblood, WW; Kizer, JS. "An enzyme that converts glutaminyl peptide to pyroglutaminyl peptide is present in the pituitary, brain, adrenal medulla, and lymphocytes." J. Biol. Chem. 1987, 262, 8532–8536.

[0195] Cao, Y.; Nguyen, G.K.T.; Tam, JP; Liu, C.-F. "Butyrase-mediated protein thioester synthesis and its application in tandem chemical ligation." Chem. Commun. 2015, 51, 17289–17292.

[0196] Cao, Y.; Nguyen, GKT; Chuah, S.; Tam, JP; Liu, CF. “Butyrase-mediated ligation is an efficient bioligation method for synthetic peptide dendrimers.” Bioconj. Chem. 2016, 27, 2592–2596.

[0197] Chen, H.; Lin, Z.; Arnst, K.; Miller, D.; Li, W., “Tubulin inhibitor-based antibody-drug conjugates for cancer therapy.” Molecules, 2017, 22, 1281

[0198] Doronina, SO, Mendelsohn, BA, Bovee, TD, Cerveny, CG, Alley, SC, Meyer, DL, Oflazoglu, E., Toki, BE, Sanderson, RJ, Zabinski, RF. "Enhancing the activity of monomethyl auristatin F via monoclonal antibody delivery: Effects of conjugation technology on efficacy and toxicity." Bioconjugate Chem. 2006, 17, 114–124.

[0199] Harris, KS; Durek, T.; Kaas, Q.; Poth, AG; Gilding, EK; Conlan, BF; Saska, I.; Daly, NL; Van der Weerden, NL; Craik, DJ; Anderson, MA. "Efficient backbone cyclization of linear peptides by recombinant asparagine endopeptidase." Nat. Commun. 2015, 6, 10199.

[0200] Hemu, X.; Sahili, A.E.; Hu, S.; Wong, K.; Chen, Y.; Wong, Y.H.; Zhang, X.; Serra, A.; Goh, B.C.; Darwis, D.A.; Chen, M.W.; Sze, S.K.; Liu, C.F.; Lescar, J.; Tam, J.P. "Structural determinants of peptide bond formation by asparaginyl ligase." Proc. Natl. Acad. Sci. 2019, 116, 11737–11746.

[0201] Hoyt, EA; Cal, PMSD; Oliveira, BL; Bernardes, GL. Modern methods for site-selective protein modification. Nat. Rev. Chem. 2019, 3, 147–171.

[0202] Jost, C.; Schilling, J.; Tamaskovic, R.; Schwill, M.; Honegger, A.; and Pluckthun, A. "Structural basis for cytotoxic effects in HER2-overexpressing cancer cells via binding to the HER2 extracellular domain." Structure, 2013, 21, 1979–1991.

[0203] Lotze, J.; Reinhardt, U.; Seitz, O.; Beck-Sickinger, AG. "Peptide tags for site-specific protein labeling in vitro and in vivo." Mol Biosyst. 2016, 12, 1731–1745.

[0204] Nguyen, G.K.T.; Wang, S.; Qiu, Y.; Hemu, X.; Lian, Y.; Tam, JP. “Butyrase 1 is an Asx-specific ligase that enables peptide macrocyclization and synthesis.” Nat Chem Biol. 2014, 10, 732–738.

[0205] Nguyen, G.K.T.; Qiu, Y.; Cao, Y.; Hemu, X.; Liu, C.-F.; Tam, JP. “Butyrase-mediated cyclization and ligation of peptides and proteins.” Nat. Protoc. 2016, 11, 1977–1988.

[0206] Nguyen, GKT; Kam, A.; Loo, S.; Jansson, AE; Pan, LX; Tam, JP. Syringase 1: a versatile ligase for peptide and protein macrocyclization. J. Am. Chem. Soc. 2015, 137, 15398–15401.

[0207] Nguyen, GKT; Cao, Y.; Wang, W.; Liu, CF.; and Tam, JP. Site-specific N-terminal labeling of peptides and proteins using butyrate 1 and thiodepsipeptide. Angew. Chem. Int. Ed. 2015, 54, 15694–15698.

[0208] Pishesha, N.; Ingram, JR; Ploegh, HL. "Transpeptidase A: Transpeptidase Models and Their Biological Applications." Annu. Rev. Cell Dev. Biol. 2018, 34, 163–88.

[0209] Rehm, FBH; Tyler, TJ; Xie, J.; Yap, K.; Durek, T.; Craik, DJ. "Asparaginyl ligases: new enzymes in the protein engineer's toolbox." ChemBioChem, 2021, 22, 2079–2086.

[0210] Rehm, FBH; Harmand, TJ; Yap, K.; Durek, T.; Craik, DJ; Ploegh, HL. Site-specific sequence labeling of proteins catalyzed by a single recombinase. J. Am. Chem. Soc. 2019, 141(43), 17388–17393.

[0211] Rehm, FBH; Tyler, TJ; Yap, K.; Durek, T.; Craik, DJ. "Enhancing the translocation reaction catalyzed by asparaginyl ligase by selective nucleophilic quenching." Angew. Chem. Int. Ed. 2021, 60, 4004–4008.

[0212] Rosen, CB; Francis, MB. "N-terminal targeting for site-selective protein modification." Nat. Chem. Biol. 2017, 13, 697–705.

[0213] Schilling, S.; Hoffmann, T.; Rosche, F.; Manhart, S.; Wasternack, C.; Demuth, H.-U. "Heterologous expression and characterization of human glutaminyl cyclase: evidence that the disulfide bond is essential for catalytic activity." Biochemistry, 2002, 41, 10849–10857.

[0214] Seifert, F.; Schulz, K.; Koch, B.; Manhart, S.; Demuth, H.-U.; Schilling, S. "Glutaminyl cyclase has a remarkable catalytic capacity for glutamine substrates." Biochemistry, 2009, 48, 11831–11833.

[0215] Sgouros, G.; Bodei, L.; McDeitt, MR; Nedrow, JR. "Radiopharmaceutical therapy for cancer: clinical advances and challenges." Nat. Rev. Drug Dis. 2020, 19, 589–608.

[0216] Sletten, EM; Bertozzi, CR. "Bioorthogonal chemistry: searching for selectivity in a functional ocean." Angew. Chem. Int. Ed. 2009, 48, 6974–6998.

[0217] Spicer, CD; Davis, BG. Selective chemical protein modification. Nat. Commun. 2014, 5, 4740.

[0218] S.; T.; AE; Frejd, FY; Nygren, J. “Affibody molecules for biotechnology and medical applications.” Trends Biotechnol. 2017, 35, 691–712.

[0219] Steiner, D.; Forrer, P.; A., "Efficient screening of DARPins with subnanomolar affinity using SRP phage display". J. Mol. Biol. 2008, 382, ​​1211–1227. Tam, JP; Chan, N.; Liew, HT; Tan, SJ; Chen, Y., "Peptide asparaginyl ligase - a regenerator of peptide bonds". Science China Chemistry 2020, 63, 296–307. Tang, TMS; Cardella, D.; Lander, AJ; Li, X.; Escudero, JS; Tsai, YH.; Luk, LYP, "Chemical enzymatic peptide and protein labeling using asparaginyl endopeptidase". Chem. Sci. 2020, 11, 5881–5888.

[0220] Viola-Villegas, N.; Doyle, RP. “Structural Overview and Analyses on Structure–Stability Relationships of 1,4,7,10-Tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (H4DOTA). Coordination Chemistry Reviews, 2009, 253, 1906–1925.

[0221] Weeks, AM; Wells, JA. Ferrase-catalyzed peptide ligation. Chem. Rev. 2020, 120, 3127–3160.

[0222] Xia, Y.; To, J.; Chan, N.-Y.; Hu, S.; Liew, H.T.; Balamkundu, S.; Zhang, X.; Lescar, L.; Bhattacharjya, S.; Tam, J.P.; Liu, C.-F. "N-γ-Hydroxyasparagine: A versatile unnatural amino acid that is a good P1 substrate for asparaginyl-peptide ligases." Angew. Chem. Int. Ed. 2021, 60, 22207–22211.

[0223] Yang, R.; Wong, YH; Nguyen, GKT; Tam, JP; Lescar, L.; Wu, B. "Design of recombinant protein ligases with high catalytic efficiency." J. Am. Chem. Soc. 2017, 139, 5351–5358.

[0224] Zhang, D.; Wang, Z.; Hu, S.; Balamkundu, S.; To, J.; Zhang, X.; Lescar, J.; Tam, J. P.; Liu, C.-F., "pH-controlled orthogonal ligation of proteins using asparagine peptide ligase." J. Am. Chem. Soc. 2021, 143, 8704–8712.

[0225] Zhang, G.; Zheng, S.; Liu, H.; Chen, PR. "Elucidating biological processes by site-specific protein labeling." Chem. Soc. Rev. 2015, 44, 3405–3717.

[0226] Zhang, Y.; Park, KY; Suazo, KF; Distefano, MD. "Recent advances in enzyme protein labeling technology and its applications." Chem. Soc. Rev. 2018, 47, 9106–9136.

Claims

1. A method for enzymatic peptide ligation, characterized in that: The method comprises providing i) peptidyl asparaginyl ligase (PAL) and glutamine cyclase (QC); ii) a first peptide or protein having a P1-P1′-P2′ tripeptide PAL motif as an acyl donor, wherein P1 is Asn or Asp, P1′ is Gln or Glu, and P2′ is a hydrophobic amino acid or a β-branched amino acid; iii) a second peptide or protein, which may be identical to or different from the first peptide or protein, having a P1″-P2″ motif at the N-terminus as an acyl acceptor, wherein P1″ is any amino acid and P2″ is a hydrophobic amino acid or a β-branched amino acid; iv) contacting the peptidyl asparaginyl ligase (PAL) and glutamine cyclase (QC) with the first and second peptides / proteins; Among them, PAL cuts the first peptide or protein after P1 in the tripeptide PAL motif and connects the first peptide / protein to the P1"-P2" motif of the second peptide / protein, and QC cyclizes P1' in the released P1'-P2' dipeptide motif to pyroglutamyl (pGlu).

2. The method according to claim 1, characterized in that P2' is selected from the group consisting of Leu, Met, Phe, Tyr, Trp, Val, He and Thr.

3. The method according to claim 1 or 2, characterized in that P2″ is selected from the group consisting of Leu, Phe, Tyr, Trp, Val, He, and Thr.

4. The method according to any one of claims 1 to 3, characterized in that The PAL is selected from the following group: butyrate enzyme 1 (butelase1) comprising the amino acid sequence shown in SEQ ID NO:1, butyrate enzyme-2 (butelase-2) comprising the amino acid sequence shown in SEQ ID NO:2 or 3, VyPAL2 comprising the amino acid sequence shown in SEQ ID NO:4, VyPAL3 comprising the amino acid sequence shown in SEQ ID NO:5, OaAEP1b-C247A comprising the amino acid sequence shown in SEQ ID NO:6, HeAEP3 comprising the amino acid sequence shown in SEQ ID NO:7, AtLEGγ comprising the amino acid sequence shown in SEQ ID NO:8, VuPAL1 comprising the amino acid sequence shown in SEQ ID NO:9, HaPAL1 comprising the amino acid sequence shown in SEQ ID NO:10, OaAEP1b comprising the amino acid sequence shown in SEQ ID NO:11, and functional fragments or variants thereof.

5. The method according to any one of claims 1 to 4, characterized in that The QC is selected from the group consisting of a human glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 12, a mouse glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 13, a Drosophila glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 14, an Arabidopsis thaliana glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 15, a Conus glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 16, a Sistrurus glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 17, a bacterial glutamine cyclase comprising the amino acid sequence shown in SEQ ID NO: 18, and functional fragments or variants thereof.

6. The method according to any one of claims 1 to 5, characterized in that The second peptide / protein further comprises a spacer of at least one amino acid between the P1"-P2" acyl acceptor and the second peptide / protein.

7. The method according to any one of claims 1 to 6, characterized in that The ratio of QC:PAL:first peptide or protein is in the range of 0.1:1:1000 to 1:1:50, respectively, preferably, in the range of 0.1:1:1000 to 0.1:1:50, respectively.

8. The method according to any one of claims 1 to 7, characterized in that The first and second peptides / proteins are identical and form a dimer when linked.

9. The method according to any one of claims 1 to 7, characterized in that One of the first and second peptides or proteins is an epitope binding peptide or protein and the other peptide or protein comprises a payload.

10. The method according to claim 9, characterized in that The payload also includes a payload-release link.

11. The method according to claim 9 or 10, characterized in that The epitope binding peptide / protein is selected from the group consisting of antibodies or functional fragments thereof, affinity bodies such as Z EGFR or Z EGFR -Fc and DARPin.

12. The method according to claim 11, characterized in that The antibody or functional fragment thereof is selected from the group consisting of a mini-antibody, a diabody, a scFv, a nanobody and F(ab')2.

13. The method according to any one of claims 9 to 12, characterized in that The effective load is an imaging agent or a therapeutic agent.

14. The method according to claim 13, characterized in that The imaging agent is a radioactive label chelator or an optical label.

15. The method according to claim 14, characterized in that The radiolabeled chelator is selected from the group consisting of 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclo-1-pentanedioic acid-4,7-diacetic acid (NODAGA); and / or the optical label is HRP or GFP or the like.

16. The method according to claim 13, characterized in that The therapeutic agent is monomethyl auristatin E (MMAE) or radiolabeled DOTA.

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