Improving the efficiency of PAL-catalyzed protein ligation by a cascade enzymatic scheme

Abstract

The present invention relates generally to enzymatic peptide or protein ligation. In particular, the present invention provides an improved method for enzymatic peptide or protein ligation, comprising combining peptidyl asparaginyl ligase (PAL)-catalyzed ligation with glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation to improve the yield of the ligation product.

Description

[Technical field]

[0001] The present invention relates generally to enzymatic peptide or protein ligation. In particular, the present invention provides an improved method for enzymatic peptide or protein ligation, comprising combining peptidyl asparaginyl ligase (PAL)-catalyzed ligation with glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation to improve the yield of the ligation product. [Background technology]

[0002] Site-specific protein ligation facilitates the study of protein function and enables 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 well-established chemical ligation methods, recent years have seen an increased use of peptide ligases for protein modification (Zhang, Y. et al., Chem. Soc. Rev. 47, 9106-9136 (2018); Lotze, J. et al., Mol Biosyst. 12, 1731-1745 (2016); Weeks, A. M. and Wells, J. A., Chem. Rev. 120, 3127-3160 (2020); Pishesha, N. et al., L. Annu. Rev. Cell Dev. Biol. 34, 163-88 (2018)). For example, a common peptide ligase is sortase A, which recognizes the sorting sequence LPXTG and ligates after Thr (Pishesha, N. et al., L. 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 practical ligation reactions. Conversely, the recently discovered peptidyl asparaginyl ligase (PAL), typified by buterase-1 (Nguyen, GKT et al., Nat Chem Biol. 10, 732-738 (2014)), has a catalytic efficiency 10 times that of sortase A. 4It has a catalytic efficiency 2-fold higher than that of the IL-10 ... As a member of the asparaginyl endopeptidase (AEP) family, PAL utilizes a 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. Thus, the ligation product is formed via transpeptidation. Similar to 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, K.S. et al., Nat. Commun. 6, 10199 (2015); Yang, R. et al., J. Am. Chem. Soc. 2017, 139, 5351-5358 (2017)). However, because transpeptidation is reversible, the introduction of excess amounts of nucleophile is necessary for high-yield intermolecular ligation reactions.

[0003] Several methods have been developed to shift the equilibrium of PAL-mediated ligation towards the product (Nguyen, GKT et al., Angew. Chem. Int. Ed. 54, 15694-15698 (2015); Cao, Y. et al., Bioconj. Chem. 27, 2592-2596 (2016); Rehm, F.B.H. et al., J. Am. Chem. Soc. 141(43), 17388-17393 (2019); Tang, T.M.S. et al., Chem. Sci. 11, 5881-5888 (2020); Rehm, F.B.H. et al., Angew. Chem. Int. Ed. 60, 4004-4008 (2021)). The thiodepsipeptide method was first developed, which utilizes an asparaginyl thioester peptide as an acyl donor substrate (peptide-Asn-thioglc-Xaa) to undergo an irreversible ligation reaction (Nguyen, GKT et al., Angew. Chem. Int. Ed. 54, 15694-15698 (2015); Cao, Y. et al., Bioconj. Chem. 27, 2592-2596 (2016)). However, a limitation of this method is the need to prepare a thioester substrate. In the second method developed by Rehm et al., a glycinyl-valinyl acyl acceptor substrate was used for ligation with the acyl donor with NGL because the resulting Asn-Gly-Val (NGV) motif was more stable for OaAEP-C247A than Asn-Gly-Leu (NGL), which reduced both the hydrolysis of the product and the reversibility of the ligation reaction. However, NGV is only relatively more stable than NGL. The GV-peptide must be used in large excess (>20-fold) over the acyl donor because of its poorer acceptor substrate. Protein-protein ligation has not been demonstrated. Another method developed by Tang et al. used Asn-Cys-Leu as the P1-P1'-P2' tripeptide motif for 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 ligation reaction. However, FPBA was found to significantly slow down the ligation reaction, presumably because the aldehyde can form imines with amine substrates and / or enzymes. Finally, the method reported by Rehm et al. 2+ was used to chelate the GLH tripeptide leaving group and mask the nucleophilicity of its N-terminal amine (Rehm, FBH et al., Angew. Chem. Int. Ed. 60, 4004-4008 (2021)). This approach resulted in a large increase in yield for N- and C-terminal labeling reactions of proteins with small synthetic peptides. However, only a small increase in yield was observed for protein-protein ligation, 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)). Thus, all these currently known methods have certain limitations, especially when it comes to protein-protein ligation. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need to provide improved protein ligation methods that overcome, or at least ameliorate, one or more of the above-mentioned disadvantages. [Means for solving the problem]

[0005] The present invention relates to a method of enzymatic peptide ligation in which PAL-mediated intermolecular ligation is combined with glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation to greatly increase the yield of the ligation product.

[0006] In one aspect, i) peptidyl asparaginyl ligase (PAL) and glutaminyl cyclase (QC); ii) a first peptide or protein having a P1-P1'-P2' tripeptide PAL motif as an acyl donor, where 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 or different to the first peptide or protein, having a P1″-P2″ motif as an N-terminal acyl acceptor, where P1″ is any amino acid and P2″ is a hydrophobic amino acid or a β-branched amino acid; providing a iv) contacting said first and second peptides / proteins with peptidyl asparaginyl ligase (PAL) and glutaminyl cyclase (QC); A method for enzymatic peptide ligation comprising: PAL cleaves a first peptide / protein after P1 in a tripeptide PAL motif, ligates 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). The method is provided.

[0007] It will be appreciated that these peptides are generally shorter than proteins, contain amino acids, and are both suitable for ligation according to the present invention. In some embodiments, P2' is selected from the group including Leu, Met, Phe, Tyr, Trp, Val, Ile, and Thr.

[0008] In some embodiments, P2'' is selected from the group including Leu, Phe, Tyr, Trp, Val, Ile, and Thr. In some embodiments, the PAL is selected from the group including butelase1 comprising the amino acid sequence set forth in SEQ ID NO:1, butelase2 comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3, VyPAL2 comprising the amino acid sequence set forth in SEQ ID NO:4, VyPAL3 comprising the amino acid sequence set forth in SEQ ID NO:5, OaAEP1b-C247A comprising the amino acid sequence set forth in SEQ ID NO:6, HeAEP3 comprising the amino acid sequence set forth in SEQ ID NO:7, AtLEGγ comprising the amino acid sequence set forth in SEQ ID NO:8, VuPAL1 comprising the amino acid sequence set forth in SEQ ID NO:9, HaPAL1 comprising the amino acid sequence set forth in SEQ ID NO:10, and OaAEP1b comprising the amino acid sequence set forth in SEQ ID NO:11.

[0009] In some embodiments, the QC is selected from the group comprising human glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 12, mouse glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 13, Drosophila glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 14, Arabidopsis glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 15, Conus glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 16, Rattlesnake glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 17, and bacterial glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0010] 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 said second peptide / protein.

[0011] 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.

[0012] In some embodiments, the first and second peptides or proteins are identical and form a dimer upon ligation. 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.

[0013] In some aspects, the payload further comprises a payload release linkage. In some embodiments, the epitope-binding peptide or protein is an antibody or functional fragment thereof, Z EGFR or Z EGFR -Fc, and DARPins.

[0014] In some embodiments, the antibody or functional fragment thereof is selected from the group comprising a minibody, a diabody, an scFv, a nanobody, and an F(ab')2. In some aspects, the payload is an imaging agent or a therapeutic agent.

[0015] In some aspects, the imaging agent is a radiolabeled chelator or an optical label. In some embodiments, the radiolabeled chelator is selected from the group including 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), and / or the optical label is, for example, HRP or GFP.

[0016] In some embodiments, the therapeutic agent is monomethylauristatin E (MMAE) or radiolabeled DOTA. Advantageously, the methods of the present disclosure allow protein-protein ligation, protein-peptide ligation, and peptide-peptide ligation to be performed with exceptional efficiency, achieving nearly equal quantitative yields even when the molar ratio between the two ligation partners is equal. These and other advantages of the present disclosure will become readily apparent to those skilled in the art from the detailed description that follows.

[0017] The accompanying drawings illustrate disclosed aspects and serve to explain the principles of the disclosed aspects, but it is understood that the drawings are designed for purposes of illustration only and not as a definition of the limits of the invention. [Brief description of the drawings]

[0018] [Figure 1] Figure 1. Model study using ligation reaction between Ac-SYRNQL and GIGGIR as proof of concept of cascade enzymatic reaction scheme. (a) Product yield versus time when reaction was performed in the absence (filled triangles) or presence (filled circles for 0.1 equivalent of QC to PAL, filled squares for 1 equivalent of QC to PAL) of QC. Reaction conditions: 5 mM acyl acceptor and 5 mM acyl donor; OaAEP1b-C247A (0.0005 equivalents) in 20 mM PBS (pH 7) at 37 °C; with or without QC (0.0005 or 0.00005 equivalents). Yields were determined by HPLC (UV absorbance at 220 nm). Yields are presented as mean ± SEM of triplicate experiments. (b) HPLC traces. Top trace: Ac-SYRNQL and GIGGIR only, middle trace: ligation reaction without QC, bottom trace: ligation reaction with QC. Reaction time = 32 min. [Diagram 2] Figure 1 shows a combined PAL and QC scheme for protein-peptide ligation. (a) Ligation between ubiquitin-NQL-His6 and GIGGIRK (biotin). (b) Ligation between biotin-GRSNQL and GI-ubiquitin. Both reactions were performed at 37°C in 20 mM PBS (pH 7) for 1 h using 500 μM ubiquitin, 1.2 equivalents of peptide, 0.001 equivalents of OaAEP1b-C247A with or without 0.0001 equivalents of QC. Both reactions were monitored using HPLC (middle panel) and the labeled products were characterized by ESI-MS (lower panel). [Diagram 3]Figure 1 shows the use of a cascade scheme combining PAL and QC to prepare a C-terminal conjugate of the affibody protein ZEGFR. (a) Ligation of ZEGFR-NQL with GIGGGK[Fe(DOTA)]. (b) Ligation of ZEGFR-NQL with GIGKVA-PABC-MMAE. The reaction was carried out at 23°C in 20 mM PBS (pH 7) for 2 hours using 500 μM ZEGFR-NQL, 1.5 equivalents of nucleophile substrate, and 0.001 equivalents of OaAEP1b-C247A, with or without 0.0001 equivalents of QC. (c) Preparation of the C-terminal dimer of ZEGFR. 1.1 mM ZEGFR-NQL was reacted with 500 μM of the bivalent peptide (GISGGRG)2KGC, 2 μM of OaAEP1b-C247A, with or without 0.2 μM of QC, in 20 mM PBS (pH 7) at 23° C. for 2 h. The reaction was monitored using HPLC and ESI-MS. [Figure 4] Figure 2: Enhancement of the efficiency of protein-protein ligation by the combined use of VyPAL2 and QC: (a) HPLC monitoring of VyPAL2-mediated ligation between DARPin-NQL and GI-ubiquitin with or without QC, (b) Yields of different protein-protein ligations in the presence or absence of QC (i: DARPin-NQL and GI-ubiquitin, ii: DARPin-NQL and GI-DARPin, iii: ZEGFR-NQL and GI-ubiquitin, iv: DARPin-QL and GI-GFP, v: DARPin-NQL and GI-GGGSGGGS-GFP). All reactions were carried out at 37°C in 20 mM PBS (pH 7) using 400 μM acyl donor protein, 1.8 equivalents of acyl acceptor protein, 0.001 equivalents of VyPAL2 with or without 0.0001 equivalents of QC for 3-4 hours. Reactions were monitored using HPLC and ESI-MS (Figures 13-18). Yields in b were calculated based on the integrated areas of reactant and product peaks in HPLC and are expressed as the mean ± SEM of triplicate experiments. [Diagram 5]Ligation between ZEGFR-Fc-NQL and GI-GGGSGGGS-GFP. (a) SDS-PAGE of the ligation reaction between ZEGFR-Fc-NQL (200 μM) and GI-GGGSGGGS-GFP (500 μM) using 0.4 μM VyPAL2 with or without 0.04 μM QC in 20 mM PBS (pH 7). The reaction mixture was treated with 50 mM DTT and analyzed by SDS-PAGE gel. The bands at approximately 63 kDa and 34 kDa correspond to the reduced form of the ligation product and ZEGFR-Fc-NQL, respectively. The ligation product was also characterized by LC-MS (ESI) after reduction with DTT. (b) Confocal microscopy images of ZEGFR-Fc-GFP binding to A431 cells. (c) Flow cytometry analysis of ZEGFR-Fc-GFP and GFP targeting to A431 cells. [Figure 6] Ligation between ZEGFR-Fc-NQL and GVA-PABC-MMAE. Left panel: without QC. Right panel: with QC. [Figure 7] Figure 1 shows QC-catalyzed pyroglutamate (pGlu) formation with four substrates (QFGSA, QLGSA, QIGSA, and QVGSA). Reactions were carried out in 20 mM PBS (pH 7) with 5 mM QXGSA, 0.0001 equivalents of QC at 37°C for 15 min and monitored by RP-HPLC. All bars represent the mean ± SEM of triplicate determinations. [Figure 8] Figure 1 shows the yield of ligation between Ac-SYRNQL and GIGGIR catalyzed by different PALs (OaAEP1b-C247A, VyPAL2, or butelase-1) in the absence or presence of QC. Ligation reactions were carried out at 37°C for 30 min in 20 mM PBS at pH 7 (OaAEP1b-C247A) or pH 6.5 (VyPAL2, butelase-1) using 5 mM Ac-SYRNQL, 5 mM GIGGIR, 0.0005 equivalents of PAL with or without 0.00005 equivalents of QC. All bars represent the mean ± SEM of triplicate determinations. [Figure 9] RP-HPLC monitoring of the ligation reaction between ZEGFR-NQL and GIGGGK[Fe(DOTA)] at 2 hours. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37° C. in 20 mM PBS (pH 7) with 500 μM ZEGFR-NQL, 1.5 equivalents of GIGGGK[Fe(DOTA)], 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC. [Figure 10] RP-HPLC monitoring of the ligation reaction between ZEGFR-NQL and GIGKVA-PABC-MMAE. The ligation product was analyzed using ESI-MS. The ligation reaction was carried out in 20 mM PBS (pH 7) with 500 μM ZEGFR-NQL, 1.5 equivalents GIGKVA-PABC-MMAE, 0.001 equivalents VyPAL2 in the absence or presence of 0.0001 equivalents QC at 37° C. for 2 hours. [Figure 11] RP-HPLC monitoring of the ligation reaction between ZEGFR-NQL and the bivalent peptide (GISGGRAG)2KGC. The ligation product was analyzed using ESI-MS. The ligation reaction was carried out at 37° C. for 2 hours in 20 mM PBS (pH 7) with 1.1 mM ZEGFR-NQL, 500 μM (GISGGRAG)2KGC, 2 μM VyPAL2 in the absence or presence of 0.2 μM QC. [Figure 12]Figure 1 shows the IC50 of ZEGFR-MMAE conjugates for A431 and MCF7 cells under treatment with ZEGFR-PABC-MMAE for 3 days. The IC50 for A431 cells is about 12.9 nM, and the IC50 for MCF7 cells is >100 nM. To test the viability of A431 and MCF-7 cells for ZEGFR-MMAE conjugates, about 5000 A431 or MCF-7 cells were separately seeded in a 98-well plate and incubated overnight in culture medium at 37°C under 5% CO2. Different concentrations of ZEGFR-MMAE were added, and the incubation was continued for 3 days at 37°C under 5% CO2. The viability of cells was measured by MTT assay according to the recommended protocol. [Figure 13-1] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-ubiquitin at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents of GI-ubiquitin, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC. [Figure 13-2] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-ubiquitin at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents of GI-ubiquitin, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC. [Figure 14]Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-GFP at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents of GI-GFP, 0.001 equivalents of OaAEP1b-C247A and 0.0001 equivalents of QC. The reaction resulted in about 15% hydrolysis product DARPin-N-OH (labeled H). [Figure 15-1] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-DARPin at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents of GI-DARPin, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC. [Figure 15-2] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-DARPin at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents of GI-DARPin, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC. [Figure 16-1] Figure 1 shows RP-HPLC monitoring of the ligation reaction between ZEGFR-NQL and GI-ubiquitin at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C using 400 μM ZEGFR-NQL, 1.8 equivalents of GI-ubiquitin, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC in 20 mM PBS (pH 7). *Peak = [ZEGFR]2-ubiquitin by-product formed by the addition of excess ZEGFR to the N-terminus of the desired product. [Figure 16-2] Figure 1 shows RP-HPLC monitoring of the ligation reaction between ZEGFR-NQL and GI-ubiquitin at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C using 400 μM ZEGFR-NQL, 1.8 equivalents of GI-ubiquitin, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC in 20 mM PBS (pH 7). *Peak = [ZEGFR]2-ubiquitin by-product formed by the addition of excess ZEGFR to the N-terminus of the desired product. [Figure 17-1] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-GFP at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C with 400 μM DARPin-NQL, 1.8 equivalents of GI-GFP, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC in 20 mM PBS (pH 7). Peak H = hydrolysis product DARPin-N-OH. [Figure 17-2] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-GFP at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C with 400 μM DARPin-NQL, 1.8 equivalents of GI-GFP, 0.001 equivalents of VyPAL2 in the absence or presence of 0.0001 equivalents of QC in 20 mM PBS (pH 7). Peak H = hydrolysis product DARPin-N-OH. [Figure 18-1] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-Linker-GFP at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents GI-GGGSGGGS-GFP, 0.001 equivalents VyPAL2 in the absence or presence of 0.0001 equivalents QC. [Figure 18-2] Figure 2 shows RP-HPLC monitoring of the ligation reaction between DARPin-NQL and GI-Linker-GFP at different time points. The ligation products were analyzed using ESI-MS. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 400 μM DARPin-NQL, 1.8 equivalents GI-GGGSGGGS-GFP, 0.001 equivalents VyPAL2 in the absence or presence of 0.0001 equivalents QC. [Figure 19-1] Figure 1 shows the results of non-reducing SDS-PAGE gel monitoring of the ligation reaction between ZEGFR-Fc-NQL and GI-GGGSGGGS-GFP at different time points. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 200 μM ZEGFR-Fc-NQL, 500 μM GI-GGGSGGGS-GFP, and 0.4 μM VyPAL2 in the absence or presence of 0.04 μM QC. [Figure 19-2] Figure 1 shows the results of non-reducing SDS-PAGE gel monitoring of the ligation reaction between ZEGFR-Fc-NQL and GI-GGGSGGGS-GFP at different time points. The ligation reaction was carried out at 37°C in 20 mM PBS (pH 7) with 200 μM ZEGFR-Fc-NQL, 500 μM GI-GGGSGGGS-GFP, and 0.4 μM VyPAL2 in the absence or presence of 0.04 μM QC. [Figure 20-1] Fluorescence intensity (measured by flow cytometry, λex=488 nm) (indicated by arrows) of untreated A431 and MCF7 cells (control) (blank), shown in dark grey fill, and cells treated with either GFP (100 nM, 30 min) or ZEGFR-Fc-GFP (100 nM, 30 min), respectively. Intact cells were gated using forward scatter (FSC-A) versus side scatter (SSC-A). [Figure 20-2]Fluorescence intensity (measured by flow cytometry, λex=488 nm) (indicated by arrows) of untreated A431 and MCF7 cells (control) (blank), shown in dark grey fill, and cells treated with either GFP (100 nM, 30 min) or ZEGFR-Fc-GFP (100 nM, 30 min), respectively. Intact cells were gated using forward scatter (FSC-A) versus side scatter (SSC-A). [Figure 20-3] Fluorescence intensity (measured by flow cytometry, λex=488 nm) (indicated by arrows) of untreated A431 and MCF7 cells (control) (blank), shown in dark grey fill, and cells treated with either GFP (100 nM, 30 min) or ZEGFR-Fc-GFP (100 nM, 30 min), respectively. Intact cells were gated using forward scatter (FSC-A) versus side scatter (SSC-A). [Figure 21] Confocal fluorescence microscopy images of EGFR-positive A431 cells after incubation with ZEGFR-Fc-GFP or GFP. Comparing the top left panel of each block, the membrane ZEGFR-Fc-GFP staining is much brighter than that of GFP. The plasma membrane was stained with PKH26 red fluorescent dye, as shown in the bottom left panel of each block and in the merged images. Scale bar = 20 μm. [Figure 22-1] FIG. 2 shows the mass spectrum of a protein substrate of the present invention. [Figure 22-2] FIG. 2 shows the mass spectrum of a protein substrate of the present invention. [Figure 22-3] FIG. 2 shows the mass spectrum of a protein substrate of the present invention. [Figure 23] FIG. 1 shows the structure of Gly-Val-Ala-PABC-MMAE (or GVA-PABC-MMAE). [Figure 24] 25, 26, and 29 show structures of example payload drugs whose amine groups may be modified with linkers for ligation with PAL. [Diagram 25] 1 shows structures of example linker-payload compounds as acyl acceptor substrates for PAL-mediated ligation to monoclonal antibodies or other proteins. These compounds have a payload-releasing linkage. [Figure 26] 1 shows structures of example linker-payload compounds as acyl acceptor substrates for PAL-mediated ligation to monoclonal antibodies or other proteins. These compounds do not have a payload-releasing linkage. [Figure 27] 28 and 30 show structures of example payload drugs in which hydroxyl groups may be modified with linkers for ligation with PAL. [Figure 28] FIG. 1 shows structures of example linker-payload compounds as acyl acceptor substrates for ligation to proteins and monoclonal antibodies by PAL. [Figure 29] FIG. 1 shows the structure of a bivalent drug-linker compound as an acyl acceptor substrate for ligation to proteins and monoclonal antibodies by PAL. The drug payload is attached to the bivalent linker via an amino group in the drug via PABC. [Diagram 30] 1 shows the structure of a bivalent drug-linker compound as an acyl acceptor substrate for ligation to proteins and monoclonal antibodies by PAL. The drug payload is attached to the bivalent linker via its hydroxyl group. [Diagram 31]Figure 1 shows the ligation reaction between Ac-SYRNQL and GIGGIR using mouse QC and OaAEP1b. Reaction conditions: 5 mM acyl acceptor and 5 mM acyl donor, OaAEP1b-C247A (0.0005 equivalents or 0.05 mol%), QC (0.00005 equivalents) in 20 mM PBS (pH 7) at 37°C. In the absence of QC, the reaction gave rise to the ligation product in about 45% yield (see Figure 1). The yield was determined by HPLC (UV absorbance at 220 nm). [Diagram 32] Schematic diagram of the method of the present invention: (A) PAL-catalyzed intermolecular ligation is a reversible reaction; (b) The combination of QC and PAL forms a cascade enzymatic reaction scheme, which overcomes the reversibility problem of PAL-mediated ligation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following detailed description, by way of example, relates to specific details and embodiments in which the present invention may be implemented. These embodiments are described in sufficient detail to enable those skilled in the art to implement the present invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the present invention. Various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

[0020] The bibliographic references referred to in this specification, which are listed in the form of a list of references and added at the end of the examples, are for convenience only, and the entire contents of such bibliographic references are incorporated herein by reference, but their mention in the specification does not imply that they form part of the common general knowledge.

[0021] definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0022] Generally, the technical, scientific and medical terms used herein have the same meaning as understood by those skilled in the art to which the present invention belongs. In addition, the following technical comments and definitions are provided. These definitions are in no way intended to limit the scope of the present invention to these terms alone, but are presented for a better understanding of the following description.

[0023] As used herein, "a" or "an" can mean one or more than one, unless otherwise indicated or clear from the context. The inventors have found that the more enzymes used, the faster the reaction proceeds.

[0024] As used herein, the term "amino acid" may refer to natural and / or unnatural or synthetic amino acids, including both D and L optical isomers, amino acid analogs (e.g., norleucine is an analog of leucine), and peptidomimetics. As used in the context of this application, the term "amino acid" typically refers to the 20 natural L-amino acids, namely, Gly, Ala, Val, Leu, Ile, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr, and Trp.

[0025] As used herein, the term "comprising" or "including" is to be construed as specifying the presence of the stated features, integers, steps, or components as stated, but does not exclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. However, in the context of this disclosure, the term "comprising" or "including" also includes "consisting of." Variations of the word "comprising," such as "comprise" and "comprises," and variations of the word "including," such as "include" and "includes," have correspondingly varied meanings.

[0026] 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, e.g., 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, so long as the fragment retains the activity / functionality of the full-length protein / enzyme.

[0027] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds. Peptides are considered to be short amino acid chains, whereas polypeptides are long amino acid chains, and proteins tend to have stable structures and may include modifications (e.g., glycosylation or phosphorylation). The term "protein" may encompass natural and artificial (e.g., engineered or variant) full-length proteins, as well as functional fragments of said proteins. For purposes of the present invention, it will be understood that any combination of peptides, polypeptides, or proteins may be ligated in a reaction using PAL and QC, provided that one has a PAL acyl donor and the other has an acyl acceptor.

[0028] As used herein, the term "QC" refers to glutaminyl cyclase (QC) enzyme and QC-like enzyme. QC enzyme and QC-like enzyme have the same or similar enzymatic activity, i.e., catalyze the intramolecular cyclization of glutaminyl and glutamyl residues at the N-terminus of peptides and proteins to form pyroglutamyl residues (pGlu). In this regard, QC-like enzyme can fundamentally differ from QC in its molecular structure.

[0029] As used herein, the term "variant" refers to an amino acid sequence in which one or more amino acids of a non-variant reference sequence have been altered, but which retains the ability to recognize its target and affect the function of said target. For example, a QC peptide variant has one or more amino acids of a non-variant QC peptide reference sequence altered, but retains the ability to catalyze the intramolecular cyclization of glutaminyl and glutamyl residues at the N-terminus of peptides and proteins to form pyroglutamyl residues (pGlu). A variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "non-conservative" changes (e.g., replacement of glycine with tryptophan). Similar minor changes may also include amino acid deletions or insertions, or both. Guidance for determining which amino acid residues may be substituted, inserted, or deleted without losing biological activity can be found in computer programs well known in the art, for example, DNASTAR® software (DNASTAR, Inc., Madison, Wis., USA).

[0030] A description of exemplary, non-limiting aspects of the present invention follows. The present invention provides an improved peptide ligation method, based in part on the inventors' discovery that the combination of QC and PAL forms a cascade enzymatic reaction scheme that overcomes the reversibility problem of PAL-mediated ligation (see FIG. 32).

[0031] As disclosed herein, the acyl donor substrates for PAL of the present invention are designed to preferably have an asparagine (Asn / N) at the P1 position and a glutamine (Gln / Q) at the P1' position of the P1-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group where the exposed N-terminal glutamine is cyclized by QC to form the Gln N in the lactam. α - Quenching the amine.

[0032] Without being bound by theory, it is believed that upon cleavage of the Asn-Gln peptide bond, QC cyclizes the exposed Gln to form a pyroglutamyl residue (pGlu), thereby catalyzing its nucleophilic N in the lactam. α -amine. The combination of PAL-mediated ligation with QC-catalyzed pGlu formation thus advantageously overcomes the problem of reversibility of the ligation reaction by transpeptidation, leading to increased yields of the ligation product.

[0033] To this end, in one aspect of the present disclosure, i) peptidyl asparaginyl ligase (PAL) and glutaminyl cyclase (QC); ii) a first peptide or protein having a P1-P1'-P2' tripeptide PAL motif as an acyl donor, where 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 or different to the first peptide or protein, having a P1″-P2″ motif as an N-terminal acyl acceptor, where P1″ is any amino acid and P2″ is a hydrophobic amino acid or a β-branched amino acid; providing a iv) contacting said first and second peptides / proteins with peptidyl asparaginyl ligase (PAL) and glutaminyl cyclase (QC); A method for enzymatic peptide ligation comprising: PAL cleaves a first peptide / protein after P1 in a tripeptide PAL motif, ligates 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). The method is provided.

[0034] It is understood that PAL performs a site-specific ligation reaction and requires a minimal tripeptide recognition motif P1-P1'-P2' for ligation after P1, where P1 is typically Asn or Asp, and P1' and P2' can be any of the natural amino acids Gly, Ala, Val, Leu, Ile, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gln, Asp, Asn, His, Lys, Arg, Tyr, and Trp.

[0035] 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 known that Glu can act as a substituent for Gln at P1' of an acyl donor (Seifert, F. et al., Biochemistry 48, 11831-11833 (2009)), and that Asp can act as a substituent for Asn at P1 of an acyl donor (Zhang, D. et al., (2021) Journal of the American Chemical Society 143 (23): 8704-8712). Thus, in some embodiments, P1 can be Glu. In various embodiments, P1' can be Asp.

[0036] 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.

[0037] In some embodiments, P2' may be selected from the group including Leu, Met, Phe, Tyr, Trp, Val, Ile, and Thr. In various embodiments, P2'' may be selected from the group including Leu, Phe, Tyr, Trp, Val, Ile, and Thr.

[0038] In some embodiments, the P1-P1'-P2' tripeptide PAL motif of the acyl donor can be Asn-Gln-Leu. Those skilled in the art will appreciate that different PALs and variants thereof having the desired protein ligase activity may be suitable for practicing the present invention. Thus, in some embodiments, the PAL may be butelase-1, butelase-2, VyPAL2, VyPAL3, OaAEP1b-C247A, HeAEP3, AtLEGγ, VuPAL1, HaPAL1, OaAEP1b, or a functional fragment or variant thereof.

[0039] In certain embodiments, the PAL may be selected from the group including butelase1 comprising the amino acid sequence set forth in SEQ ID NO:1, butelase2 comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3, VyPAL2 comprising the amino acid sequence set forth in SEQ ID NO:4, VyPAL3 comprising the amino acid sequence set forth in SEQ ID NO:5, OaAEP1b-C247A comprising the amino acid sequence set forth in SEQ ID NO:6, HeAEP3 comprising the amino acid sequence set forth in SEQ ID NO:7, AtLEGγ comprising the amino acid sequence set forth in SEQ ID NO:8, VuPAL1 comprising the amino acid sequence set forth in SEQ ID NO:9, HaPAL1 comprising the amino acid sequence set forth in SEQ ID NO:10, OaAEP1b comprising the amino acid sequence set forth in SEQ ID NO:11, and functional fragments or variants thereof.

[0040] It is also envisioned that various QCs with desired QC enzyme activity can be suitable for use in the implementation of the present invention.Thus, in some embodiments, QC can be human glutaminyl cyclase, mouse glutaminyl cyclase, Drosophila glutaminyl cyclase, Arabidopsis glutaminyl cyclase, Conus glutaminyl cyclase, Rattus rapae glutaminyl cyclase, bacterial glutaminyl cyclase, or their functional fragments or variants.

[0041] In some embodiments, the QC may be selected from the group comprising human glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 12, mouse glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 13, Drosophila glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 14, Arabidopsis glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 15, Conus glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 16, Rattlesnake glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 17, bacterial glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 18, and functional fragments or variants thereof.

[0042] As will be appreciated by those skilled in the art, the function of a protein / enzyme is directly related to its structure and sequence, and there is a positive relationship between sequence identity and functional similarity. In this regard, methods for determining protein sequence identity are known in the art.

[0043] Thus, the sequences of the enzymes of the present disclosure may be varied sufficiently so long as the enzymes maintain their functionality and are capable of exhibiting the required activity (e.g., QC variants are capable of catalyzing the intramolecular cyclization of N-terminal glutaminyl and glutamyl residues of peptides and proteins to form pyroglutamyl residues (pGlu)).

[0044] In some embodiments, the PAL is selected from the group consisting of butelase-1, which comprises 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 set forth in SEQ ID NO:1; butelase-2, which comprises 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 set forth in SEQ ID NO:2 or 3; VyPAL2, which comprises 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 set forth in SEQ ID NO:4; VyPAL3, which comprises 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 set forth in SEQ ID NO:5; OaAEP1b-C247A comprising an amino acid sequence having 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 set forth in SEQ ID NO:7; HeAEP3 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 set forth in SEQ ID NO:8; AtLEGγ comprising an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:9; VuPAL1 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 set forth in SEQ ID NO:10;Alternatively, it may be HaPAL1 comprising an amino acid sequence having at least 99% identity, or OaAEP1b 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 set forth in SEQ ID NO:11.

[0045] In some embodiments, the QC is a human glutaminyl 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 set forth in SEQ ID NO: 12; a mouse glutaminyl 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 set forth in SEQ ID NO: 13; a Drosophila glutaminyl 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 set forth in SEQ ID NO: 14; the amino acid sequence shown in SEQ ID NO:16; a Conus glutaminyl 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:16; a Typha glutaminyl 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:17; or a bacterial glutaminyl 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:18.

[0046] 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 said second peptide or protein. As described herein, the introduction of a spacer between the P1"-P2" acyl acceptor and said second peptide or protein may improve the accessibility of PAL to catalyze ligation, and therefore improve the yield, especially in cases where the second protein is large enough to hinder the accessibility of PAL.

[0047] The reaction rate of the disclosed method can be controlled by varying the ratio of enzyme to the substrate of interest. In this regard, the inventors have found that the more enzyme used, the faster the reaction proceeds. In some embodiments, a small amount of enzyme (e.g., 0.005% equivalent of QC to substrate and 1 / 10 equivalent of QC to PAL) may be sufficient to carry out the present invention. For some difficult protein substrates, such as antibodies, a high enzyme-to-substrate ratio may be required, such as 0.1:1:100 or 0.1:1:50 (QC:PAL:first peptide / protein). The enzyme-to-substrate ratio used is highly dependent on the substrate and the specific application, and can be easily determined using standard techniques known to those skilled in the art, or can be estimated by referring to the relevant literature.

[0048] In some embodiments, the ratio of QC:PAL:first peptide or protein ranges from 0.1:1:2000 to 1:1:50, respectively, and preferably ranges 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.

[0049] As described herein, the method of the present disclosure is suitable for protein-protein ligation, and can be adapted to, for example, the preparation of antibody-drug conjugates by appropriately selecting and modifying acyl acceptor peptides and acyl donor substrates according to the method of the present invention.Therefore, it is also within the scope of the present invention that the method of the present disclosure can be adapted to the effective ligation of monoclonal antibodies and other proteins with a wide range of linker-payload drug compounds (e.g., linker-payload drug compounds as acyl acceptor substrates).Therefore, the modified peptides for use in the present invention can be prepared using standard techniques known to those skilled in the art of synthetic organic chemistry, or can be deduced by referring to relevant literature.

[0050] In some embodiments, the first and second peptides or proteins ligated according to the present application may be further modified to include a labeling moiety. The labeling moiety may be any molecule, including but not limited to, affinity tags, detectable labels, therapeutic agents, scaffolding molecules, epitope-binding peptides, ubiquitin molecules, biotin molecules, His6 tags, green fluorescent protein (GFP), epitope-binding peptides, and other molecules, such as Z EGFR , Z EGFR -Fc, and affibodies such as DARPins.

[0051] In some embodiments, the first and second peptides or proteins may be identical and may form a dimer upon ligation. It is understood that the important components of antibody-drug conjugates can include antibody, linker, and payload.Thus, in some embodiments, one of the first and second peptides or proteins can be an epitope-binding peptide or protein, and the other peptide or protein can include a payload.In some embodiments, the payload can include a payload-releasing bond.

[0052] In some aspects, the payload can be an imaging agent or a therapeutic agent. In particular, the imaging agent can be a radiolabeled chelator or an optical label. In some embodiments, the radiolabeled chelator may be selected from the group including 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), and / or the optical label is HRP or GFP, etc. In some embodiments, the therapeutic agent may be monomethylauristatin E (MMAE) or radiolabeled DOTA.

[0053] In various embodiments, the epitope-binding peptide or protein is an antibody or a functional fragment thereof, Z EGFR or Z EGFR -Fc, and DARPins.

[0054] In certain embodiments, the antibody or functional fragment thereof is selected from the group comprising a minibody, a diabody, an scFv, a nanobody and an F(ab')2. Unless otherwise indicated or otherwise evident from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges may assume any specific value or subrange within the stated range in various embodiments, to the tenth of the lower limit of the range, unless otherwise clearly indicated from the context. "About" when referring to a numerical value generally refers to a range of values ​​within ±10%, in some embodiments within ±5%, in some embodiments within ±1%, and in some embodiments within ±0.5%, unless otherwise indicated or otherwise evident from the context. In any embodiment in which a numerical value is preceded by "about", an embodiment is provided in which the exact value is recited. When an embodiment is provided in which a numerical value is not preceded by "about", an embodiment is also provided in which the value is preceded by "about". When a range is preceded by "about", an embodiment is provided in which "about" applies to the lower and upper limits, or either the lower or upper limit, of the range, unless otherwise clearly indicated from the context. When expressions such as "at least," "up to," "less than or equal to," or similar expressions precede a series of numbers, it is understood that the expression applies to each number in the list in various embodiments (with the understanding that 100% of a value, e.g., a value expressed as a percentage, may be an upper limit, depending on the context). For example, "at least 1, 2, or 3" should be understood to mean "at least 1, at least 2, or at least 3" in various embodiments. It is also understood that any and all reasonable lower and upper limits are expressly contemplated.

[0055] The invention will now be generally described, but the same will be more readily understood by reference to the following examples, which are offered by way of illustration and are not intended to be limiting of the invention. EXAMPLES

[0056] Generally, standard molecular biology techniques known in the art but not specifically described were followed, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0057] Materials and general methods All solvents and reagents were purchased from commercial suppliers and used without further purification. Human glutaminyl cyclase (QC) was purchased from Abcam (ab206806), aliquoted and stored at -80°C.

[0058] Peptides were synthesized according to standard Fmoc solid phase synthesis protocols. 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, and eluted using a gradient of buffer B (90% acetonitrile, 10% HO, 0.045% TFA) in buffer A (HO, 0.045% TFA). All synthesized compounds were stored at 4° C. or −20° C.

[0059] Proteins were produced using recombinant DNA methods. Protein purification involved immobilized metal affinity chromatography (IMAC), Protein A affinity chromatography, and size exclusion chromatography (SEC). SEC was performed on an AKTA FPLC UPC-900 using a HiLoad™ 16 / 600 Superdex™ 200pg column. Protein A and NiNTA affinity chromatography were performed on an AKTAstart using a HiTrap 5ml MabSelect™ column or a HisTrap HP 5ml column, respectively.

[0060] For analysis, peptide mass spectra were obtained using a Bruker Ultraflex Extreme matrix-assisted laser desorption / ionization (MALDI) tandem TOF or electrospray ionization (ESI) mass spectrometry (Thermo Fisher LTQ XL). MALDI data were analyzed using Data Explorer software, and ESI data were analyzed using Thermo Xcalibur Qual Browser and Magtran software. Protein mass spectral deconvolution was performed using MagTran. Analytical reversed-phase HPLC (RP-HPLC) was performed on a Shimadzu HPLC system equipped with a Phenomenex-C18 RP column (4.6 × 150 mm, 55 μm, 100 Å) or a Phenomenex Jupiter-C4 column (4.6 × 150 mm, 3.6 μm, 200 Å) at a flow rate of 1.0 mL per minute and eluted with a gradient of buffer B (90% ACN, 10% HO, 0.045% TFA) in buffer A (HO, 0.045% TFA).

[0061] Solid Phase Peptide Synthesis (SPPS) All peptides were synthesized as C-terminal amides using Rink amide MBHA resin by standard Fmoc chemistry using a Liberty Blue peptide synthesizer or 2-chlorotrityl chloride resin. For conjugation of 5(6)-carboxyfluorescein and biotin to the Lys(MTT) side chain, the MTT protecting group was first removed using TFA / TIS / DCM (2.5% / 2.5% / 95%), followed by conjugation of 5(6)-carboxyfluorescein (or biotin) to the Lys side chain amine using 2.5 equivalents of 5(6)-carboxyfluorescein (or biotin), 2.5 equivalents of Oxyma, 2.5 equivalents of DIC in NMP for 3 h. For peptide cleavage from the resin and deprotection of the side chain protecting group at the end of the SPPS, the peptidyl-resin was treated with a cocktail of TFA / HO / TIS (95% / 2.5% / 2.5%) for 1–3 h. The cleavage solution was separated from the resin by filtration, and the cleaved peptide was precipitated in cold Et2O. The crude product was isolated by centrifugation and purified by RP-HPLC. The peptide fraction after HPLC purification was lyophilized to obtain the peptide in powder form.

[0062] Synthesis of GIGKVA-PABC-MMAE (Compound 4)

[0063] [ka]

[0064] Compound 1 was synthesized using standard SPPS chemistry. 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), EEDQ (84 mg, 0.34 mmol) and 4-aminobenzyl alcohol (27.8 mg, 0.226 mmol) were added. 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 compound 2 (70 mg, 58%) as an off-white solid.

[0065] FmocGIGK(ivDde)VA-PAB-PNP-carbonate 3 was prepared by adding DIPEA (60 μL, 0.33 mmol) and bis(p-nitrophenyl)carbonate (100 mg, 0.33 mmol) to a solution of compound 2 (120 mg, 0.11 mmol) in anhydrous DMF and stirring the mixture at room temperature for 18 h under N2 atmosphere. After removing the solvent by rotary evaporation, the residue was subjected to column chromatography (2–4% MeOH in DCM) to give PNP-carbonate 3 (110 mg, 79%) as an off-white solid.

[0066] 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 subjected to reverse-phase HPLC purification (Buffer A: 0.045% TFA in H2O, Buffer B: 90% acetonitrile, 0.045% TFA in 10% H2O). The fractions containing the product were pooled and lyophilized to give compound 4 as an off-white powder. MS (ESI): m / z [M / H] + Calculated value 1392.9, observed value 1393.0.

[0067] Synthesis of GIGGGK[Fe(DOTA)] (compound 6)

[0068] [ka]

[0069] Compound 5 was synthesized using standard SPPS chemistry. One equivalent of compound 5 (10 mM) was mixed with one equivalent of FeCl3 in water and the pH of the solution was adjusted to pH 6 with 2 M NaOH. The mixture was left at 37°C overnight to give GIGGGK[Fe(DOTA)], which was used without purification. MS(ESI): m / z[M / H] + Calculated value 926.6, observed value 926.5.

[0070] OaAEP1 b Expression, purification, and activation of -C247A OaAEP1 b -C247A was cloned into vector pET28a (Genscript) and expressed using T7 SHuffle E. coli. b -C247A was activated in acetate buffer (0.1 M NaCl, 0.5 mM TCEP) at pH 4 for 2 hours 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, pH 7 (20 mM PBS, 0.5 mM TCEP).

[0071] Expression, purification, and activation of VyPAL2 VyPAL2 was expressed using sf9 insect cells. 100 mL of viral vector carrying the VyPAL2 gene was used to infect 2.5×10 sf9 cells. 6Cells were infected at a cell density of 10 ...

[0072] DARPin9_26, GFP, ubiquitin, Z EGFR , and Z EGFR -Fc expression and purification All protein genes were cloned into pET28a, pET3a, pTxB1, or pETDuet (Genscript) and expressed in E. coli (DE3) or T7 SHuffle. The bacterial OD 600After reaching 0.4-0.6, expression (except GI-ubiquitin) was induced with 0.1-0.4 mM IPTG. After induction, cells were incubated at 16°C for 18 h. Cells were harvested by centrifugation (5000×g, 10 min) and resuspended in lysis buffer (50 mM PBS, 0.1 M NaCl, 10 mM imidazole, 0.01% 100X Triton, pH 7.5). The solution mixture was lysed on ice for 15-30 min using a sonicator probe (Vibra cell™) with alternating cycles of 3 s pulses after each 8 s interval. Protein solutions were then centrifuged at 15000×g (20 min) at 4°C, filtered using a 0.2 μm membrane, and bound to NiNTA or Protein A beads for 1 h at 4°C. The Ni beads were washed with 20 mM imidazole, 0.1 M NaCl, 20 mM PBS buffer (pH 7.5), and then the proteins were eluted using 500 mM imidazole, 0.1 M NaCl, 20 mM PBS buffer (pH 7.5). The Protein A beads were washed with 20 mM PBS (pH 7.5), and then the proteins were eluted using 30 mM citrate buffer (pH 3.5). For GI-ubiquitin, which is expressed as a C-terminal intein fusion protein in E. coli (DE3), the protein solution was bound to chitin beads, and GI-ubiquitin was cleaved from the bound intein by incubating overnight at RT in 50 mM DTT, 20 mM PBS (pH 8). All proteins were transferred to 20 mM PBS (pH 7) and stored at 4°C for short periods or at -20°C for long periods.

[0073] Ligation of model peptides Enzyme-mediated ligation reactions were carried out with or without QC in 20 mM PBS buffer (pH 6.5 or pH 7) at 37°C for various time courses. The ratio of QC to ligase to substrate (NQL peptide) is 0.1:1:2000. Reactions were quenched by 10% TFA and monitored by analytical RP-HPLC. Ligation products were characterized by MALDI-MS or ESI-MS.

[0074] Labeling of the N- or C-terminus of proteins Ligation reactions were performed with or without QC at pH 7, 37°C, with various time courses. The ratio of QC / ligase / protein substrate is 0.1 / 1 / 1000. The reaction was quenched by 6M guanidine-HCl (pH 3) and the reaction was monitored by analytical RP-HPLC. The ligation products were characterized by ESI-MS.

[0075] Protein-protein ligation Ligation reactions were performed with or without QC at pH 7, 37° C. for various time courses. The ratio of Qc / ligase / protein substrate is 0.1 / 1 / 1000 (500). Reactions were quenched with 6 M guanidine-HCl (pH 3) and the completed reaction was monitored by analytical RP-HPLC. Ligation products were characterized by ESI-MS. EGFR The ligation reaction of -Fc-NQL and GI-GGGSGGGS-GFP was analyzed by SDS-PAGE under reducing or non-reducing conditions (reducing conditions: 50 mM DTT, pH 8.8 for 20 min).

[0076] Purification of ligated proteins Ligated Z EGFR The -Fc-GFP protein was purified by size exclusion chromatography (SEC) at pH 7 (20 mM PBS, 0.1 M NaCl). The purified protein was stored at -20°C. EGFR -MMAE was purified by immobilized metal affinity chromatography (IMAC) and stored in pH 7 buffer (20 mM PBS, 0.1 M NaCl).

[0077] Flow cytometry assay Z EGFRTo examine the binding ability of -Fc-GFP, A-431 (ATCC, USA), and MCF-7 (ATCC, US), live cells were washed three times with PBS (HyClone, USA), trypsinized with 0.05% trypsin-EDTA (Gibco, USA), and then resuspended in chilled DMEM (Gibco, USA) with 10% FBS (Gibco, USA). One million cells of each cell line were then transferred to Z EGFR The cells were incubated with -Fc-GFP (100 nM) and GFP (100 nM) for 30 minutes on ice. After incubation, the cells were washed three times with chilled PBS and analyzed by a Fortessa X-20 flow cytometer (BD, USA). The cytometer was set to record 10000 events per sample, excite the fluorophores with a 488 nm laser, and collect the emission fluorescent signal at 530 / 30 nm. The resulting raw data was analyzed by Flowjo™ 10 (BD, USA).

[0078] Confocal imaging of live cells Z EGFR To visualize the binding activity of -Fc-GFP, A431 and MCF-7 cells were seeded in 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) for 10 min at 37°C. The stained cells were then transferred to a 50-well plate and incubated for 1 h at 37°C for 1 h. EGFR The cells were incubated with -Fc-GFP (100 nM) and GFP (100 nM) for 30 min on ice. After incubation, the cells were washed three times with chilled PBS and fixed with cold 4% formaldehyde for 15 min. Fixed cells were imaged by an LSM980 confocal microscope (Zeiss, Germany). The key settings of the microscope were: 1) excitation laser wavelength: 488 nm and 561 nm, 2) emission filters: 507-552 nm and 575-620 nm, 3) imaging mode: Z-stack. The 3D Z-stack images were processed into 2D images by the technique MIP (maximum intensity projection images) using Zen software (Zeiss, Germany).

[0079] Cell viability assay Z EGFR To examine the cytotoxicity of Z-MMAE, approximately 5000 A431 cells and MCF-7 cells were seeded individually in 96-well plates and incubated overnight at 37°C under 5% CO2. EGFR -MMAE was added to the wells at different concentrations and incubated at 37° C. under 5% CO2 for 3 days. Then, 0.5 mg / mL MTT was added and incubation was continued for 1 hour at 37° C. Cell viability was determined based on the absorbance at 570 nm.

[0080] Peptide and protein sequences and mass spectrometry data

[0081] [Table 1-1]

[0082] [Table 1-2]

[0083] [Table 1-3]

[0084] [Table 1-4]

[0085] [Table 2-1]

[0086] [Table 2-2]

[0087] [Table 2-3]

[0088] [Table 2-4]

[0089] [Table 2-5]

[0090] [Table 3-1]

[0091] [Table 3-2]

[0092] [Table 3-3]

[0093] [Table 4-1]

[0094] [Table 4-2]

[0095] Example 1: Demonstration of the PAL-QC cascade scheme in a ligation reaction of a model peptide. By catalyzing N-terminal pGlu formation, QC is involved in the maturation of many biologically active peptides and proteins (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 from different organisms in catalyzing unimolecular lactamization reactions ranges from about 10 5 M -1 ·S -1 (Seifert, F. et al., Biochemistry 48, 11831-11833 (2009)), however, the efficiency of PAL in catalyzing biomolecular ligation reactions is approximately 10 4 M -1 ·S -1 (Wang, Z. et al., Theranostics 11, 5863-5875 (2021)). This makes QC a particularly attractive enzyme for capturing released glutaminyl leaving groups. We first showed that QC efficiently converted the N-terminal Gln to pGlu in four synthetic peptides of the sequence QXGSA (X = L, I, F, or V, which is often used as the P2' amino acid by PAL) (Figure 7). Consistent with previous studies, the presence of a large hydrophobic residue such as X at position 2 of the glutaminyl peptide did not negatively affect the ability of QC to catalyze the lactamization reaction. Indeed, at 0.0001 equivalents relative to the substrate, QC was able to drive the reaction to completion in less than 30 minutes (pH 7). The reactions of the QI-, QL-, and QF-peptides had similar kinetics, whereas the reaction rate of the QV-peptide was about 30% slower (Figure 7). Because Leu is the most commonly used P2' residue in PAL, we chose Asn-Gln-Leu as the tripeptide recognition motif in all acyl donor substrates used in this study.

[0096] We then decided to test our cascade enzyme scheme in a model ligation reaction using Ac-SYRNQL (5 mM) as the acyl donor and GIGGIR (1 equivalent) as the acyl acceptor. At a molar ratio of PAL to substrate of 0.0005:1, the reaction with OaAEP1b-C247A at pH 7 gave the product in 45% yield when the reaction reached equilibrium in about 30 minutes (FIG. 1a). Conversely, the addition of 0.00005 equivalents of QC increased the ligation yield to >95% in 32 minutes (FIGS. 1a and 1b). This large increase in yield indicated that a very small amount of QC (0.005 mol% for substrate and 1 / 10 equivalent for PAL) was sufficient to quench the released QL dipeptide via pGlu formation. This provided a clear validation of the combined PAL-QC reaction scheme. When ligation reactions were performed using 0.05 mol% QC (i.e., QC:PAL=1:1), the reactions reached equilibrium faster, but the final yields were nearly identical (Figure 1a). The reactions were fast enough with 1 / 10 QC / PAL, so this ratio was used in all subsequent studies. Ligation reactions were also performed using VyPAL2 or butelase-1 under identical conditions. A similar large increase in product yield was observed when the reactions were performed in the presence of QC, indicating that QC is compatible with all three of the most useful PALs (Figure 8).

[0097] Example 2: Use of a cascade enzymatic scheme for protein N- and C-terminal labeling. Next, ubiquitin was used as a model protein to demonstrate the method in protein labeling reactions. Two recombinant ubiquitin variants, GI-ubiquitin and ubiquitin-NQL-His6, were prepared for N- and C-terminal labeling with two biotinylated synthetic peptides, biotin-GRSNQL and GIGGIRK (biotin), respectively. 500 μM of ubiquitin substrate protein and 1.2 equivalents of biotin peptide were used in both ligation reactions, which were carried out at pH 7 and 37° C. with 0.5 μM of OaAEP1b-C247A (0.001 equivalents).

[0098] In the ligation reaction between ubiquitin-NQL-His6 and GIGGIRK (biotin), the yield increased from 40 to 94% when 0.0001 equivalents of QC was added (Figure 2). Similarly, the yield of the ligation between biotin-GRSNQL and GI-ubiquitin increased from 49 to 88% with the addition of QC. The large enhancement of the labeling efficiency of the C- and N-termini of proteins by QC further validated the method in the ligation reaction of proteins and peptides.

[0099] Next, anti-EGFR affibody protein Z EGFR (Stahl, S. et al., Trends Biotechnol. 35, 691-712 (2017)) were labeled at the C-terminus with functional moieties of interest for diagnostic and therapeutic potential. Two special peptides, GIGGGK[Fe(DOTA)] and GIGKVA-PABC-MMAE, were prepared and Z EGFRDOTA was used as an acyl acceptor substrate for ligation with -NQL (Figures 3a and 3b). As the most powerful chelators, DOTA and its derivatives form very stable complexes with certain metal ions (Viola-Villegas, N. et al., Coordination Chemistry Reviews 2009, 253, 1906-1925 (2009)). Disease-targeting proteins can be conjugated with DOTA complexes bearing radionuclides for diagnostic or theranostic applications (Sgouros, G. et al., Nat. Rev. Drug Dis. 19, 589-608 (2020)). In this work, the DOTA complexes were conjugated with non-radioactive Fe, for demonstration purposes only. 3+ ions. MMAE, or monomethyl auristatin E, a highly potent cytotoxic agent, is a common drug payload in antibody-drug conjugates (Chen, H. et al., Molecules 22, 1281 (2017)), which is often attached to antibodies via PABC, a self-immolative linker for payload release (Doronina, S. et al., Bioconjugate Chem. 17, 114-124 (2006)). As can be seen in Figure 3, the cascade enzymatic method is EGFR -[Fe(DOTA)] conjugates and Z EGFR The results of the synthesis of the GIGGGK[Fe(DOTA)] peptide and Z-MMAE conjugates were shown in 90% and 88% yield, respectively, whereas in the absence of QC, the yield was only about 50%. EGFR The [Fe(DOTA)] complex was stable during HPLC purification and ESI-MS measurement, as intact molecular ions of the -[Fe(DOTA)] conjugate were observed. EGFRIt was shown that the -MMAE conjugate has high cytotoxicity against A431 cells overexpressing EGFR, but relatively low cytotoxicity against MCF-7 cells expressing very low levels of EGFR (Figure 12). Clearly, these two examples demonstrate that our combined PAL and QC cascade scheme may provide a safer manufacturing process for bioconjugates with radioactive or toxic payloads, since no large excess of reactants is required for high-yield reactions, which minimizes the risk of exposure to these hazardous substances.

[0100] Z EGFR A C-terminally linked dimer of the protein was also prepared by ligating it to a bivalent peptide substrate bearing two Gly-Ile dipeptide acyl acceptors (Figure 3c). This is a more stringent test of our method since it requires that both nucleophilic sites in the bivalent peptide be ligated to the protein. Satisfyingly, a slight excess of Z EGFR Using only 1.1 effective equivalents per bivalent peptide, this cascade scheme yielded about 80% of the desired C-terminal dimer protein product. Conversely, without QC, only about 46% C-terminal dimer was obtained, with a significant amount of mono-ligated intermediates (Figure 3c). Such parallel synthesis of protein dimers demonstrates the utility of our method in the preparation of such unusual protein conjugates.

[0101] Example 3: Use of a cascade enzymatic reaction scheme for protein-protein ligation To determine whether the method of the present invention can be further extended to protein-protein ligation reactions, several proteins, namely, 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 affibody Z were ligated to the GFP-Ubiquitin complex.EGFR (Stahl, S. et al., Trends Biotechnol. 35, 691-712 (2017)) was then selected. We first performed the ligation of DARPin-NQL (400 μM) with GI-ubiquitin (1.8 equiv.) at pH 7 and 37 °C using VyPAL2 (0.001 equiv.). Without QC, the reaction yielded 39% product in 3 h, but the addition of QC increased the yield to 91% (Figure 4a). Interestingly, we found that VyPAL2 was a better ligase than OaAEP1b-C247A in this ligation reaction of DARPin with ubiquitin, since OaAEP1b-C247A yielded a small amount of the hydrolysis product DARPin-N-OH (Figure 14). This phenomenon was not observed in either peptide-peptide or peptide-protein ligation reactions. OaAEP1b-C247A appears to have residual hydrolase activity, which can be amplified in difficult, entropically demanding protein-protein ligation reactions. Therefore, VyPAL2 was used for all subsequent protein-protein ligation reactions.

[0102] Similarly, ligation of DARPin-NQL (1 equiv.) with GI-DARPin (1.8 equiv.) yielded the tandemly linked DARPin-NGI-DARPin in 47% (without QC) and 95% (with QC) (Figure 4b). EGFRLigation of DARPin-NQL with GI-ubiquitin gave rise to products in 37% (without QC) and 76% (with QC). DARPin-NQL was also ligated with GI-GFP, a much larger GFP protein, to give DARPin-NGI-GFP in 42% (without QC) and 74% (with QC). The proximity of the N-terminal GI nucleophile to the rigid β-barrel structure of the GFP protein may hinder its accessibility, resulting in slightly lower ligation yields and some hydrolysis in this reaction (Figure 17). The addition of a longer spacer after the GI dipeptide improved the accessibility and thus the ligation yield between DARPin and GI-GGGSGGGS-GFP to 50% (without QC) and 85% (with QC). In all cases, the use of QC with VyPAL2 improved product yields by 33-53%. This improvement is remarkably comparable to that seen in model peptide ligation, making the method of the present invention clearly advantageous over 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)).

[0103] Next, Affibody Z EGFR and Z, a high molecular weight dimeric fusion protein consisting of the Fc domain of IgG. EGFR -Fc-NQL (MW approximately 68 kDa) was used to ligate with GI-GGGSGGGS-GFP (29 kDa) to yield a very high molecular weight protein product with a mass of approximately 126 kDa. EGFRThe ligation reaction between -Fc-NQL (200 μM) and GFP protein (500 μM) reached a yield of about 90% in the presence of QC (Figure 5a). This is due to the large size of the protein and the fact that the acceptor GFP protein substrate is a dimer of the donor substrate Z, which has two reactive sites. EGFR This is noteworthy considering that only 1.25 molar equivalents of QC was used relative to -Fc-NQL. Furthermore, as seen in the non-reducing SDS-PAGE gel (Figure 19), in the presence of QC, the final dual ligation product was predominant, whereas in the absence of QC, the monoligation product was predominant. Figures 5b and 5c show the dual ligation product Z to A431 cells overexpressing EGFR. EGFR This shows the specific binding of Z-Fc-GFP. EGFR We show that the receptor binding activity of VyPAL2 and the fluorogenicity of GFP are preserved after the ligation reaction. This example demonstrates that the VyPAL2-QC combination method is also suitable for the preparation of macromolecular therapeutic protein conjugates.

[0104] In addition, Z EGFR -Fc-NQL was also ligated to Gly-Val-Ala-PABC-MMAE (Figure 23). MMAE, monomethyl auristatin E, is a potent antimitotic agent. Val-Ala-para-aminobenzylcarbamate (ValAla-PABC) is a linker cleavable by intracellular proteases, and the Gly-Val dipeptide is a good acyl acceptor nucleophile substrate for the PAL enzyme. EGFR Ligation of -Fc-NQL (200 μM) and Gly-Val-Ala-PABC-MMAE (800 μM) was catalyzed by VyPAL2 (0.002 equiv.) at pH 7 and 23° C. Again, the absence and presence of QC (0.0002 equiv.) resulted in a large difference in ligation efficiency (FIG. 6). In the presence of QC, the ligation product was obtained in about 80% yield after 4 h, whereas in the absence of QC, the yield was only 42%. EGFRSince -Fc-NQL is a dimer, the stoichiometric equivalents of the MMAE-containing nucleophile substrate was 2. EGFR The conjugate of -Fc fusion protein and MMAE is similar to antibody-drug conjugate, i.e., ADC. This result clearly demonstrates that the combination method of PAL-QC of the present invention can be used to prepare ADC for the treatment of cancer and other diseases, and does not require the use of a large excess of acyl acceptor substrate in the ligation reaction. It is expected that this method will enable the efficient ligation of monoclonal antibodies and other proteins with a wide range of linker-payload drug compounds as acyl acceptor substrates (Figures 24-30).

[0105] Example 4: Demonstration of the PAL-QC cascade scheme in a model peptide ligation reaction using mouse QC. The peptide ligation reaction between Ac-SYRNQL and GIGGIR was also tested using mouse QC and OaAEP1b-C247A.

[0106] Reaction conditions: 5 mM acyl acceptor and 5 mM acyl donor, OaAEP1b-C247A (0.0005 equivalents or 0.05 mol%) in 20 mM PBS (pH 7) at 37° C. with QC (0.00005 equivalents). In the absence of QC, the reaction gave rise to ligation product in about 45% yield (see FIG. 1). Yields were determined by HPLC (UV absorbance at 220 nm).

[0107] The results show that, like human QC, mouse QC has the same effect in overcoming the reversibility seen with PAL-only ligation and by increasing the yield of PAL-mediated ligation reactions.

[0108] Overview As the most potent transpeptidase known to date, PAL has previously been shown to catalyze peptide and protein cyclization reactions 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)), and its k cat / K m is at least an order of magnitude higher than that of intermolecular ligation reactions. This is due to the entropically favored nature of intramolecular reactions. Moreover, the rigid conformation of the cyclized products often makes them resistant to PAL. Thus, PAL-catalyzed cyclizations, despite also being transpeptidation reactions, are usually irreversible. This is not the case in biomolecular ligation reactions. Their reversibility usually limits the product yield to ≦50% when the ratio between the two reaction partners is 1:1. We show that this problem can be overcome by using P1′Gln in the acyl donor substrate, since its α-amine can be quenched by lactamization upon cleavage of the Asn-Gln peptide bond. Pyroglutamyl formation can occur spontaneously, but it is a slow process. The reported rate constant for spontaneous pGlu formation of an N-terminal Gln is 1.7×10 at pH 6. -6 s -1 This corresponds to a half-life of approximately 4.7 days (Seifert, F. et al., Biochemistry 48, 11831-11833 (2009)). Human QC-catalyzed pGlu formation takes 30 s -1 k cat, which corresponds to a seven-order of magnitude enhancement in rate (Seifert, F. et al., Biochemistry 48, 11831-11833 (2009)). The high QC efficiency makes it ideally suited for use in combination with PAL. In our cascade enzyme scheme, QC was used at one-tenth the equivalent relative to the PAL enzyme, which was used at a molar ratio of 1:1000 or 1:2000 relative to the substrate. Using this scheme, the yield of intermolecular ligation was greatly improved with equal or slightly higher molar equivalents of acyl acceptor substrate relative to the acyl donor substrate. Our method is generally applicable to all PALs and to substrates of various sizes, ranging from small peptides to large recombinant proteins. Compared to existing methods that utilize metal ions, synthetic chemicals, or non-natural elements in the substrate to address reversibility issues, this method uses a non-toxic enzyme. Although the use of another enzyme may bring about cost-related problems, this is not a major issue since QC can be easily expressed in E. coli and yeast systems. The high reaction yield and very low enzyme requirement also facilitate product purification, making this process cost-effective. Overall, this robust cascade enzyme scheme according to the present invention greatly increases the applicability of PAL-mediated ligation in the precision production of macromolecular protein conjugates.

[0109] References

[0110] [Table 5-1]

[0111] [Table 5-2]

[0112] [Table 5-3]

[0113]

Table 5-4

Claims

1. i) peptidyl asparaginyl ligase (PAL) and glutaminyl cyclase (QC); ii) a first peptide or protein having a P1-P1'-P2' tripeptide PAL motif as an acyl donor, where 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 or different to said first peptide or protein, having a P1″-P2″ motif as an N-terminal acyl acceptor, where P1″ is any amino acid and P2″ is a hydrophobic amino acid or a β-branched amino acid; providing a iv) contacting said peptidyl asparaginyl ligase (PAL) and said glutaminyl cyclase (QC) with said first and second peptides / proteins; A method for enzymatic peptide ligation comprising: PAL cleaves the first peptide / protein after P1 in the tripeptide PAL motif, ligates 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). The method.

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

3. 3. The method of claim 1 or 2, wherein P2'' is selected from the group including Leu, Phe, Tyr, Trp, Val, Ile, and Thr.

4. The method of any one of claims 1 to 3, wherein the PAL is selected from the group consisting of butelase 1 comprising the amino acid sequence set forth in SEQ ID NO: 1, butelase-2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or 3, VyPAL2 comprising the amino acid sequence set forth in SEQ ID NO: 4, VyPAL3 comprising the amino acid sequence set forth in SEQ ID NO: 5, OaAEP1b-C247A comprising the amino acid sequence set forth in SEQ ID NO: 6, HeAEP3 comprising the amino acid sequence set forth in SEQ ID NO: 7, AtLEGγ comprising the amino acid sequence set forth in SEQ ID NO: 8, VuPAL1 comprising the amino acid sequence set forth in SEQ ID NO: 9, HaPAL1 comprising the amino acid sequence set forth in SEQ ID NO: 10, OaAEP1b comprising the amino acid sequence set forth in SEQ ID NO: 11, and functional fragments or variants thereof.

5. The method of any one of claims 1 to 4, wherein the QC is selected from the group consisting of human glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 12, mouse glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 13, Drosophila glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 14, Arabidopsis glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 15, Conus glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 16, Rattlesnake glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 17, bacterial glutaminyl cyclase having the amino acid sequence shown in SEQ ID NO: 18, and functional fragments or variants thereof.

6. 6. The method of any one of claims 1 to 5, wherein 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. 7. The method according to any one of claims 1 to 6, wherein the ratio of QC:PAL:first peptide / 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. 8. The method of claim 1, wherein the first and second peptides / proteins are identical and form a dimer upon ligation.

9. 8. The method of claim 1, wherein one of the first and second peptides / proteins is an epitope-binding peptide / protein and the other peptide / protein comprises a payload.

10. The method of claim 9 , wherein the payload further comprises a payload release linkage.

11. The epitope-binding peptide / protein is an antibody or a functional fragment thereof, Z ＥＧＦＲ Or Z ＥＧＦＲ - An affibody such as an Fc, and a DARPin.

12. The antibody or functional fragment thereof may be a minibody, a diabody, a scFv, a nanobody, or a F(ab') ２ The method of claim 11 , wherein the compound is selected from the group comprising:

13. 13. The method of any one of claims 9 to 12, wherein the payload is an imaging agent or a therapeutic agent.

14. 14. The method of claim 13, wherein the imaging agent is a radiolabeled chelator or an optical label.

15. 14. The method of claim 13, wherein the radiolabeled chelator is selected from the group comprising 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), and / or the optical label is, for example, HRP or GFP.

16. 14. The method of claim 13, wherein the therapeutic agent is monomethylauristatin E (MMAE) or radiolabeled DOTA.

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