Engineer next-generation antibody-drug conjugates using orthogonal bioconjugation methods

Abstract

The present invention relates to providing more efficient methods of conjugating two or more peptides. For example, the invention provides more efficient methods of producing antibody-drug conjugates. More particularly, the methods can be used to efficiently produce ADCs with at least two unique payloads. One approach involves the use of peptidyl asparaginyl ligases (PALs) for two consecutive ligation reactions at the N-terminal and C-terminal ends of a protein and the other approach involves the use of a chemical protein modification reaction and a PAL-mediated enzymatic ligation reaction, both able to introduce two different payloads to an antibody at specific sites.

Description

[0001] ENGINEER NEXT-GENERATION ANTIBODY-DRUG CONJUGATES USING ORTHOGONAL BIOCONJUGATION METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority from Singapore Patent Application No. 10202401918Y filed on 28 June 2024, the contents of which are incorporated herein in their entirety by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to providing more efficient methods of conjugating two or more peptides. For example, the invention provides more efficient methods of producing antibodydrug conjugates. More particularly, the methods can be used to efficiently produce antibodydrug conjugates (ADCs) with at least two unique payloads. One approach involves the use of peptidyl asparaginyl ligases (PALs) for two consecutive ligation reactions at the N-terminal and C-terminal ends of a protein and the other approach involves the use of a chemical protein modification reaction and a PAL-mediated enzymatic ligation reaction, both able to introduce two different payloads to an antibody at specific sites.

[0006] BACKGROUND OF THE INVENTION

[0007] Antibody-drug conjugates (ADCs) are a class of precision medicines that may be employed for cancer eradication. Loaded with a cytotoxic agent through a covalent linker, the antibody component guides the ADC to tumor sites and delivers the payload inside cancerous cells for their selective killing. With this targeted approach, highly potent cytocidal compounds that are otherwise too toxic to use in chemotherapy can serve as ADC warheads, expanding the repertoire of chemical agents for cancer treatment. Although ADCs have proven to be a powerful drug modality in oncology, resistance is a big challenge that can lead to treatment failures. To address this issue, ADCs that contain two or more than two unique payloads are designed, a concept that is similar to that of traditional combination chemotherapy in which combinations of chemo-therapeutic agents work together to provide synergistic effects for optimal treatment outcomes and slow down the development of drug resistance. However, preparing homogeneous dual- or multi-payload ADCs is challenging because of the structural complexity of an antibody and the large number of reactive groups it has on its surface. For instance, the preparation of a dual-payload ADC involves the use of two different bioconjugation reactions to attach two distinct toxic warheads to the antibody. The two bioconjugation reactions must be site-specific and orthogonal to one another to allow the generation of homogeneous dual-payload ADCs with defined antibody-to-drug ratios. Site-specific protein modification is a frequently used strategy for advancing protein biological function studies and their pharmaceutical applications (Hoyt et al., Nat. Rev. Chem. 2019; Krall et al., Nat. Chem. 2016; Chen et al., Front. Chem. 2021 ). Common chemoselective reactions can be used to derivatize and conjugate proteins via their lysine side-chain amines or cysteine side-chain thiols (Nguyen et al., Angew. Chem. Int. Ed. Engl. 2015; Spicer et al., Nat. Common. 2014). However, the presence of multiple Lys or Cys residues in a protein typically leads to the formation of heterogeneous mixtures, presenting challenges in product characterization and drug development (Sornay et al., R. Soc. Open. Set. 2022; Vught et al., Struct. Biotechnol. J. 2014). For example, an lgG1 or lgG4 typically has more than 80 lysine residues and 32 cysteines that form 16 disulfide bonds (Liu et al., MAbs. 2012). Compared to chemical modification, enzymatic methods offer superior specificity and operate under mild reaction conditions (Rehm et al., J. Am. Chem. Soc. 2019). Enzymes like microbial transglutaminase, sortase and subtiligase have been harnessed to site-specifically modify proteins with remarkable precision (Zhang et al., Chem. Soc. Rev. 2018). The potential applications of enzymatic bioconjugation are vast and varied, ranging from protein labeling to drug delivery and to the creation of bioconjugates with customized properties (Zhang et al., Chem. Soc. Rev. 2018; Debon et aL, JACS. Au. 2023).

[0008] Peptidyl asparaginyl ligases (PALs) belong to a family of cysteine proteases called asparaginyl endopeptidases (AEPs). PALs recognize a minimal tripeptide motif, Asx-P1 '-P2', where P1 ’ can be almost any amino acid and P2’ should be a relatively large and hydrophobic one and catalyze transpeptidation at the Asx (Asn or Asp) peptide bond. With their high efficiency and specificity, PALs are very useful enzymes for protein modifications (Nguyen et al., Nat. Protoc. 2016; Cao et al., Chem. comm. 2015; Nguyen et al., Nat. Chem. Biol. 2014; Harris et al., Nat. Commun. 2015). Among the PALs, butelase 1 , extracted from the plant Clitoria ternatea, is the first PAL identified for ligation at Asx-specific junctions (Nguyen et al., Nat. Chem. Biol. 2014; Hemu et al., RSC. Advances. 2021 ). Butelase 1 has a catalytic efficiency that is more than 10,000 times higher than that of sortase A (Wang et al., Angew. Chem. Int. Ed. Engl. 2017; Li et al., Sci. Rep. 2017; Dorr et al., Proc. Natl. Acad. Sci. U.S.A. 2014). In addition to butelase 1 , other potent enzymes in this family include OaAEP1 b-C247A from Oldenlandia affinis and VyPAL2 from Viola yedonesis. Both have highly efficient transpeptidase activities, making them powerful tools for bioconjugation (Hemu et al., Proc. Natl. Acad. Sci. U.S.A. 2019; Harris et al., Nat. Commun. 2015; Nguyen et al., J. Am. Chem. Soc. 2015). In a PAL-catalyzed transpeptidation process, the Asx-PT peptide bond in the acyl donor substrate is attacked by the cysteinyl thiol in the enzyme's active site. This action cleaves the Asx-PT peptide bond and forms an acyl-enzyme thioester intermediate, which is then aminolyzed by the N-terminal amino group of an incoming nucleophile peptide substrate, resulting in the formation of a new Asx-Xaa peptide bond (Nguyen et al., Nat. Protoc. 2016).

[0009] PAL-catalyzed ligation exhibits strict specificity for substrates containing a P1 -asparaginyl or aspartyl residue or a close analog (Nguyen et al., Nat. Protoc. 2016; Hu et al., Plant Cell 2022; Hemu et al., Proc. Natl. Acad. Sci. U.S.A. 2019; Zhang et al., J. Am. Chem. Soc. 2021 ; Xia et al., Angew. Chem. Inti. Ed. 2021 ). Additionally, during the catalytic process, the released dipeptide P1'-P2' can be re-engaged by the PAL in the reverse reaction (Cao et al., Chem. comm. 2015), reducing the efficiency of the ligation reaction (Morgan et al., Chem. Soc. Rev. 2022). To overcome the reversibility problem, Xia et al. developed a PAL-QC (glutaminyl cyclase) coupled cascade enzymatic scheme in which a PT-GIn undergoes, upon cleavage of the Asn-GIn bond, QC-mediated cyclization to form a pyroglutamyl residue, quenching the a-amine of glutamine (Xia et al., J. Am. Chem. Soc. 2023). Tang et al. used Asn-Cys-Leu as the tripeptide recognition motif for OaAEP1 b-C247A and 2-formyl phenylboronic acid (FPBA) to quench the Cys residue of the Cys-Leu dipeptide leaving group as a stable thiazolidine ring (Tang et al., Chem. Sci. 2020). Rehm et a / , reported enhanced efficiency of OaAEP1 b-C247A- catalyzed ligation by using Ni2+-complexed quenching of the Gly-Leu-His leaving group (Rehm et al., Angew. Chem. Int. Ed. Engl. 2021 ). Although these methods reduce the reversibility of the reaction, they require the addition of other substances, and Asn is an indispensable recognition site. In previous studies (Nguyen et al., Nat. Chem. Biol. 2014), thiodepsipeptides were used as the acyl donor substrates (Asn-thioglc-Xaa) to attain an irreversible intermolecular ligation. Although the addition of other substances is not necessary, the use of a P1 -Asn continues to be a limiting element.

[0010] Thus, there is still a need to provide a method of preparing a conjugate, especially a conjugate with two payloads attached at specific positions, with improved efficiency.

[0011] SUMMARY OF THE INVENTION

[0012] In the present invention, the inventors developed an Asx-independent ligation method based on the use of PAL enzymes. Based on this method, two approaches were further developed, one involves the use of peptidyl asparaginyl ligases (PALs) for two consecutive ligation reactions at the N-terminal and C-terminal ends of a protein and the other involves use of a chemical protein modification reaction and a PAL-mediated enzymatic ligation reaction, both allowing the introduction of two different payloads to an antibody at specific sites.

[0013] In a first aspect, there is provided a method of preparing a conjugate, said method comprising i) providing a first molecule comprising a first peptide and a chemical group acting as an acyl donor, wherein the sulfaneylacetamide group is attached to the C-terminus of the first peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid; and iii) contacting a peptidyl asparaginyl ligase (PAL) with the first and second molecules to form a conjugate of the first and second peptides.

[0014] In some embodiments, in the chemical group of formula I, n is any integer from 1 to 3, preferably n is 1 . In some embodiments, X is N, and R is H or an amino acid, preferably R is Gly or Ala. In some embodiments, X is O, and R is a Ci-Cg alkyl group, preferably R is a methyl, ethyl or propyl group, more preferably R is a methyl group. In some embodiments, the chemical group of formula I is selected from a group consisting of a sulfaneyl-acetamide, a methyl thioglycolate, a sulfanyl-ethanamide, a sulfanyl-propanamide, a sulfanyl- acetylglycinamide and a sulfanyl-acetylalaninamide. More preferably, the chemical group of formula I is selected from a group consisting of a sulfaneyl-acetamide, a methyl thioglycolate, a sulfanyl-acetylglycinamide or a sulfanyl-acetylalaninamide.

[0015] In a second aspect, there is provided a method of producing a conjugate comprising two payloads comprising ligating one payload using the method of the first aspect of the invention, and then ligating the other payload using the known Asx-P1 '-P2' tripeptide in the presence of PAL and optionally QC. Specifically, this method comprises the following steps: i) providing a first molecule comprising a first payload, a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to a C-terminal residue of the first peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide, a X1 -X2 motif acting as an acyl acceptor and a P1 -P1 ’-P2’ tripeptide motif as a second acyl donor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide and the P1 -P1’-P2’ tripeptide motif is attached to the C-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid, wherein P1 is Asn or Asp, P1’ is Gly, Ser, Ala, Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid; ill) providing a third molecule comprising a second payload and a P1 "-P2" motif as an acyl acceptor, wherein PT is any amino acid and P2" is a hydrophobic amino acid or a p- branched amino acid; iva) contacting a first peptidyl asparaginyl ligase (PAL) with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) in the presence of a second PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) in the presence of a first PAL to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in the presence of a second PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

[0016] In a third aspect, there is provided a method of producing a conjugate comprising two payloads comprising ligating one payload using the method of the first aspect of the invention, and then ligating the other payload using a chemical protein modification reaction based on a cysteine thiol group. Specifically, this method comprises the following steps: i) providing a first molecule comprising a first payload, a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to a C-terminal residue of the first peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the second peptide comprises at least one cysteine thiol group for chemical conjugation with a maleimide-functionalized moiety, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid; ill) providing a third molecule comprising a second payload and a maleimide-functionalized moiety; and iva) contacting a peptidyl asparaginyl ligase (PAL) with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

[0017] In a fourth aspect, there is provided a conjugate produced by the method of the first, second and third aspect of the invention. Preferably, the conjugated is an antibody drug conjugate (ADC).

[0018] In a fifth aspect, there is provided a first molecule of the first aspect of the invention. Said molecule comprises a peptide and a chemical group, wherein the chemical group is attached to a C-terminal residue of the peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid. Preferably, it further comprising a payload attached to the N-terminus of the peptide, preferably through a payload-releasing linker.

[0019] Advantageously, the method of the present disclosure, which is Asx independent and PAL- mediated, allows conjugation of any two peptides to be conducted in a much greater efficiency compared to the Asx dependent PAL-mediated ligation. These and other advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description.

[0020] In another aspect, there is also provided a method of producing a conjugate comprising two payloads comprising ligating one payload using a Asx-dependent PLA-dependent ligation, and then ligating the other payload using a chemical protein modification reaction based on a cysteine thiol group. Specifically, this method comprises the following steps: i) providing a first molecule comprising a second payload and a P1"-P2" motif as an acyl acceptor, wherein P1 " is any amino acid and P2" is a hydrophobic amino acid or a p-branched amino acid; ii) providing a second molecule comprising a second peptide and a P1-PT-P2’ tripeptide motif as a acyl donor, the P1 -PT-P2’ tripeptide motif is attached to the C-terminus of the second peptide, wherein the second peptide comprises at least one cysteine thiol group for chemical conjugation with a maleimide-functionalized moiety, wherein P1 is Asn or Asp, PT is Gly, Ser, Ala, Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid; iii) providing a third molecule comprising a second payload and a maleimide-functionalized moiety; and iva) contacting a peptidyl asparaginyl ligase (PAL) and a QC with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step II) with the third molecule of step iii) to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a PAL and a QC to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0023] Figure 1 shows a synthesis scheme of DOTA-GVYSAYGPRALG-SCH2CONH2.

[0024] Figure 2 shows a synthesis scheme of ValCit-PABC-MMAE. PABC: paraaminobenzyloxycarbonyl. MMAE: Monomethyl auristatin E.

[0025] Figure 3 shows a synthesis scheme of [Ga(DOTA)]-GVYSAYGPRALG-GI-ZEGFR.

[0026] Figure 4 shows (A) a mechanism of PAL-catalyzed ligation using a peptidyl- sulfaneylacetamide as the acyl donor substrate; and (B) peptide cyclization and intermolecular ligation of a peptidyl-sulfaneylacetamide. POI: protein of interest. G: Glycine. I: Isoleucine.

[0027] Figures 5A-5F shows model study on PAL-mediated cyclization and intermolecular ligation of peptides with different substrates. (A) Ligation reaction based on sulfaneyl-acetamide substrate. The reaction was conducted with 200 pM peptide of interest (POI), H2N- GVYSAYGPRALG-SCH2CONH2, and 0.005 eq of VyPAL2 in 20 mM sodium phosphate buffer (pH 7) at room temperature for 1 h. (B) Product yields at different time points of the intermolecular ligation reactions of the thioester substrate H2N-GVYSAYGPRALG- SCH2CONH2 and the conventional substrate Ac-GVYSAYGPRALGNGL, respectively, with the acyl acceptor substrate GIGGIR. The yields are average values ± SD from triplicate experiments. The reaction was conducted with 200 pM acyl donor, 2 eq of acyl acceptor and 0.01 eq of VyPAL2 in 20 mM PBS (pH 7) at room temperature for 0.5-8 h. (C) Examples of different substrates. Substrate 1 : carbamoyl-methoxy group (-OCH2CONH2); Substrate 2: sulfaneyl-acetamide (-SCH2CONH2); Substrate 3: methyl thioglycolate (-SCH2COOCH3); Substrate 4: sulfanyl-ethanamide (-SCH2CH2CONH2); Substrate 5: sulfanyl-propanamide (- SCH2CH2CH2CONH2); Substrate 6: sulfanyl-acetylglycinamide (-SCH2CO-Gly-NH2); substrate 7: sulfanyl-acetylalaninamide (-SCH2CO-Ala-NH2). (D) Reaction monitoring of the peptide ester substrate 1 and sulfaneylacetamide thioester substrate 2 by the catalysis of OaAEPI b-C247A (0.01 equivalent) in phosphate buffer (pH7) at 37 °C for 3h. (E) Comparison of sulfaneylacetamide thioester substrate 2 with substrate 3, substrate 4 and substrate 5 in the cyclization reactivity under the catalysis of OaAEPI b-C247A. (F) Cyclization reactivity of substrate 6 and substrate 7. Both substrates worked similarly, and results are shown with substrate 7 only.

[0028] Figure 6 shows PAL-mediated cyclization of a D-peptide thioester substrate. (A) Sequence and reaction scheme of the D-peptide substrates (D-amino acids by low-case letters. For clarity purpose, the achiral Gly is also written in upper-case letters in the D-peptide sequence). (B) The rate of cyclic product formation from the D-peptide substrate by the catalysis of OaAEPI b-C247A or VyPAL2 in phosphate buffer (pH7) at room temperature for 20 min - 5 h.

[0029] Figure 7 shows effect of the C-terminal residue attached to the thioester substrate (i.e., amino acid at position 1 ) on PAL-mediated cyclization reaction. (A) Scheme of the PAL-mediated cyclization reaction. (B) HPLC monitoring of the cyclization reactions. The reaction was conducted with 250 pM of substrate containing sulfaneyl acetamide and 0.01 eq of VyPAL2 in 20 mM PBS (pH 7) at room temperature for 0.5-1 h.

[0030] Figure 8 shows effect of the penultimate amino acid residue (i.e., amino acid at position 2) on PAL-mediated cyclization reaction. (A) Scheme of the PAL-mediated cyclization reaction. (B) Yields of the cyclic products as a function of time for the thioester substrates containing XG motifs as catalyzed by 0.01 eq of VyPAL2 in phosphate buffer (pH7) at room temperature for 0-2h. (C) The HPLC profiles of the reactions.

[0031] Figure 9 shows effect of the N-terminal amino acid residue (amino acid at position 3) on PAL- mediated cyclization. (A) Scheme of the PAL-mediated cyclization reaction. (B) The HPLC profiles of the reactions. Yields of the cyclic products as a function of time for the thioester substrates containing different motifs as catalyzed by 0.01 eq of VyPAL2 in phosphate buffer (pH7) at room temperature for 1 h.

[0032] Figure 10 shows pH scan and kinetic studies on PAL-catalyzed cyclization of the peptide substrate containing C-terminal sulfaneyl-acetamide. The kinetic studies were performed to plot the Michaelis-Menten graphs. Error bars represent standard deviations. The average yields were calculated from experiments performed in triplicate ± SDs. (A) pH-scan results. The cyclization reactions were performed at room temperature in 20 mM PBS (pH 4.5-8) for 30 min, with 0.01 eq of OaAEPI b-C247A or 0.005 eq of VyPAL2. (B) Kinetics of the cyclization reaction at pH 7. Cyclization reactions were conducted with different concentrations of the linear peptide thioester substrate in 20 mM PBS (pH 7) at room temperature, with durations ranging from 10 to 30 minutes. These reactions included 0.01 eq OaAEP1 b-C247A or 0.005 eq of VyPAL2. Reactions were monitored by analytical RP-HPLC.

[0033] Figure 1 1 shows examples of protein N-terminal conjugation using PAL-mediated ligation at non-Asx junctions. A variety of payloads were attached to GI-ZEGFR affibody (1 -3) or to the Gl- DARPin affibody (4-6), respectively, where GRLG or GVYSAYGPRALG is a peptide linker. The products were characterized by ESI-MS. (1) FAM-GRLG-GI-ZEGFR; (2) PNU-GRRLG-GI- ZEGFR, (3) [Ga(DOTA)]-GVYSAYGPRALG-GI-ZEGFR; (4) FAM-GRLG-GI-DARPin; (5) MMAE- linker-GRRLG-GI-DARPin; (6) Biotin-GRRLG-GI-DARPin.

[0034] Figure 12 shows N-terminal labeling of GI-ZEGFR with FAM-GRLG-SCH2CONH2, where GRLG is a peptide linker. (A) Ligation between GI-ZEGFR and FAM-GRLG-SCH2CONH2. The reaction mixture containing GI-ZEGFR (250 pM) and FAM-GRLG-SCH2CONH2(2 eq) in phosphate buffer at pH 7 was treated with VyPAL2 (0.05 eq) and incubated at room temperature for 30 min. (B) ESI-MS analysis of the labelled product. (C) The HPLC profile of the labelled product.

[0035] Figure 13 shows N-terminal labeling of GI-ZEGFR with PNU-GRRLG-SCH2CONH2. (A) Preparation of PNU-GRRLG-SCH2CONH2. To a suspension of PNL) (125mg, 0.2 mmol) in methanol (2.5 mL, 0.04 M) and water (1 mL, 0.1 M) was added a solution of sodium periodate (47 mg, 0.22 mmol) in water. The mixture was stirred at room temperature for 4h in the dark. The solvent was removed in vacuo, and the oxidized form was used for the next step. Then, the oxidized PNU was dissolved in dry DMF. 1 .5 eq of HATL), 1 .3 eq of GRRLG-SCH2CONH2 and 3 eq of DIEA was added. The mixture was stirred at room temperature overnight. The product PNU-GRRLG-SCH2CONH2 was conformed in ESI-MS. (B) Ligation between GI-ZEGFR and PNU-GRRLG-SCH2CONH2. The reaction mixture containing GI-ZEGFR (250 pM) and PNU- GRRLG-SCH2CONH2(3 eq) in phosphate buffer at pH 7 was treated with VyPAL2 (0.05 eq) and incubated at room temperature for 1 .5 h. (C) ESI-MS analysis of the labelled product. (D) The HPLC profile of the labelled product.

[0036] Figure 14 shows N-terminal labelling of GI-ZEGFR with [Ga(DOTA)]-GVYSAYGPRALG- SCH2CONH2. (A) Preparation of labelled product. Peptide DOTA-GVYSAYGPRALG- SCH2CONH2was prepared as the standard Boc-SPPS method described in Figure 1 . 00 pM of protein GI-ZEGFR was ligated with 5 eq of DOTA-GVYSAYGPRALG-SCH2CONH2by the peptide-protein ligation method described above. After that, 10 eq of Ga (NO2)3 was dissolved in water, and the pH of this solution was adjusted to pH 6 using 0.2M phosphate buffer (pH 7). The mixture was then left to react at 37°C overnight, resulting in the formation of [Ga (DOTA)]- GVYSAYGPRALG-GI-ZEGFR. (B) ESI-MS analysis of the labelled product. (C) The HPLC profile of the labelled product.

[0037] Figure 15 shows N-terminal labeling of GI-DARPin with FAM-GRLG-SCH2CONH2. (A) Preparation of labelled product. 150 pM of protein GI-DARPin was ligated with 3 eq of FAM- GRLG-SCH2CONH2 in phosphate buffer at pH 7, in the presence VyPAL2 (0.05 eq), at room temperature for 2 h. (B) ESI-MS analysis of the labelled product. (C) The HPLC profile of the labelled product.

[0038] Figure 16 shows N-terminal labeling of GI-DARPin with MMAE-PABC-CitVal-CO(CH2)3CO- GRRLG-SCH2CONH2. (A) Preparation of labelled product. 150 pM of protein GI-DARPin was ligated with 3 eq of MMAE-PABC-CitVal-CO(CH2)3CO-GRRLG-SCH2CONH2in phosphate buffer at pH 7, in the presence VyPAL2 (0.05 eq), and at room temperature for 2 h. (B) ESI- MS analysis of the labelled product. (C) The HPLC profile of the labelled product.

[0039] Figure 17 shows N-terminal labeling of GI-DARPin with Biotin-GRRLG-SCH2CONH2. (A) Preparation of labelled product. 150 pM of protein GI-DARPin was ligated with 10 eq of Biotin- GRRLG-SCH2CONH2 in phosphate buffer at pH 7, in the presence VyPAL2 (0.05 eq), at room temperature for 2 h. (B) ESI-MS analysis of the labelled product. (C) The HPLC profile of the labelled product.

[0040] Figure 18 shows the stability of conjugate FAM-GRLG-ZEGFR and FAM-GRLGN-ZEGFR in the presence of human Legumain. (A) The HPLC profiles of the reactions. (B) The residual conjugation for the substrates containing Asn or that without Asn.

[0041] Figure 19 shows the analysis of specific binding of FAM-GRLG-GI-DARPin with BT474 cells (over-expression HER2) using FACS.

[0042] Figure 20 shows the cytotoxicity of MMAE-PABC- CitVal-CO(CH2)3CO-GRRLG-GI-DARPin towards MCF-7 cells and BT474 cells.

[0043] Figure 21 shows PAL-catalyzed N-terminal labeling of the antibody light chain. (A) Preparation of the MMAE labelled antibody 2 (ADC 2). 12 pM of antibody 1 was conjugated with 10 eq of MMAE-PABC-CitVal-CO(CH2)3CO-GRRLG-SCH2CONH2in 20 mM phosphate buffer at pH 7, in the presence VyPAL2 (0.1 eq), and incubated at room temperature overnight. (B) ESI-MS analysis of the ADC2. Figure 22 shows the cytotoxicity of MMAE-PABC-CitVal-CO(CH2)3CO-GRRLG-antibody towards MCF-7 cells and BT474 cells.

[0044] Figures 23A-23B shows PAL-catalyzed sequential labeling of the antibody. (A) ADC2 was obtained based on the method disclosed in Figure 21. Then, 0.1 eq of OaAEP1 b-C247A and 0.01 eq of human QC were added to a solution containing ADC 2 (12 pM) and 100 eq of the payload GIG-MMAF-OCH3in 20 mM PBS with 0.25 mM TCEP to identify the C-terminus of the antibody's heavy chain. (B) ESI-MS analysis of the ADC3.

[0045] Figure 24 shows (A) HPLC profile of the ADC3; and (B) cytotoxicity of the ADC3 towards MCF-7 cells and BT474 cells.

[0046] Figures 25A-25D show preparation of an ADC with two payloads using a chemoselective conjugation method followed by the Asx-independent PAL-mediated conjugation. (A) Reaction scheme. (B) HPLC analysis of the products of the two-step reactions. (C) Characterization of the LC and HC by ESI-MS. (D) In vitro cytotoxicity results for ADC5 and ADC6 towards MCF- 7 cells and BT474 cells. LC: light chain; HC: heavy chain.

[0047] Figures 26A-26C show reparation of a dual-payload ADC by a chemo-enzymatic sequential conjugation scheme. (A) Reaction scheme. (B) HPLC analysis of the products of the two-step reactions. (C) In vitro cytotoxicity results for ADC9 towards MCF-7 cells and BT474 cells. LC: light chain; HC: heavy chain.

[0048] Figure 27 shows HPLC analysis of (A) ADC10, (B) ADC1 1 and (C) ADC12.

[0049] Figure 28 shows in vitro cytotoxicity results for (A) ADC10, (B) ADC1 1 and (C) ADC12 towards MCF-7 cells and BT474 cells.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art.

[0052] A. Definitions

[0053] For convenience, certain terms employed in the specification, examples and appended claims are collected here. In general, technical, scientific and medical terminologies used herein has the same meaning as understood by those skilled in the art to which this invention belongs. Further, the following technical comments and definitions are provided. These definitions should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

[0054] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0055] As used herein, the term “comprising” may include the embodiments “consisting of and “consisting essentially of”. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named features / steps and permit the presence of other features / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of” the enumerated features / steps, which allows the presence of only the named features / steps, along with any impurities that might result therefrom, and excludes other features / steps.

[0056] As used herein, the term "amino acid" may refer to natural and / or unnatural or synthetic amino acids, including both the D and L optical isomers, amino acid analogs (for example norleucine is an analog of leucine) and peptidomimetics. As used in the context of the present application, the term “amino acid” typically refers to the 20 naturally occurring L-amino acids, namely Gly, Ala, Vai, Leu, He, Phe, Cys, Met, Pro, Thr, Ser, Glu, Gin, Asp, Asn, His, Lys, Arg, Tyr, and Trp.

[0057] As used herein, the terms “peptide” is used to denote a polymer of at least two amino acids covalently linked by an amide bond. It encompasses short amino acid chains, long amino acid chains (such as polypeptides) and proteins tend to have a stable structure and may comprise modifications (e.g., glycosylation or phosphorylation). The term “protein” may encompass a naturally-occurring as well as artificial (e.g., engineered or variant) full-length protein as well as a functional fragment of the protein. It would be understood that, for the purpose of the invention, any combination of peptides such as short amino acid chains, polypeptides or proteins (such as antibodies) may be ligated in a reaction using the method of the present invention. As used herein, the term "variant", refers to an amino acid sequence that is altered by one or more amino acids of the non-variant reference sequence, but retains the ability to recognize its target and affect its function. For example, a QC peptide variant is altered by one or more amino acids of the non-variant QC peptide reference sequence, but retains the ability to catalyse the intramolecular cyclization of N-Terminal glutaminyl and glutamyl residues of peptides and proteins to form pyroglutamyl residue (pGlu). The variant may have "conservative" changes, wherein a 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). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNASTAR® software (DNASTAR, Inc. Madison, Wisconsin, USA).

[0058] 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 or antigen binding activity) of the full-length protein, such as, e.g., the ability to catalyse a ligation reaction between two peptide (for an enzyme) or the ability to bind a specific antigen (for an antibody). The functional fragment can be any size, provided that the fragment retains the activity / functionality of the full-length protein. For example, functional fragments of a classical IgG antibody may include but not limited to Fab, Fab’, F(ab’)2, Fv, scFv, diabody, minibody, and nanobody.

[0059] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0060] It should be understood that any and all embodiments of the present disclosure can be combined with technical features in any other embodiment or multiple other embodiments to obtain additional embodiments under the premise of no conflict. The invention includes such combinations resulting in further embodiments.

[0061] B. Conjugation of two peptides

[0062] The present invention provides an improved method of peptide ligation at specific sites. In this regard, the present invention is based, in part, on the inventors’ discovery that using a specific chemical group having formula I as the acyl donor in a PAL-mediated ligation removes the need of an Asx recognition site and overcomes the reversibility problem.

[0063] To this end, provided in one aspect of the present disclosure is a method of preparing a conjugate, said method comprising i) providing a first molecule comprising a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to the C-terminus of the first peptide and has the following formula I wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid; and iii) contacting a peptidyl asparaginyl ligase (PAL) with the first and second molecules to form a conjugate of the first and second peptides.

[0064] It would be appreciated that PALs perform a site-specific ligation reaction and results in a conjugate in the form of the first peptide-X1 -X2-the second peptide, wherein the sulfaneylacetamide group is cut off during the ligation reaction.

[0065] One skilled in the art may choose suitable conditions for PALs to function properly and efficiently in step iii) to mediate the ligation between the first and second molecule. For example, the ratio of the PAL and first / second molecules, duration of the ligation, and the temperatures can be adjusted by one skilled in the art as needed. In some embodiments, the step iii) of contacting is performed at a pH selected in the range from 5 to 8, preferably at a pH around 7.

[0066] 1. The first molecule

[0067] The first molecule comprises a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to the C-terminus of the first peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid. This acyl donor reacts with the acyl acceptor in the second molecule to mediate the ligation.

[0068] In some embodiments, in the chemical group of formula I, n is any integer from 1 to 3, preferably n is 1 . In some embodiments, X is N, and R is H or an amino acid, preferably R is H, Gly or Ala. In some embodiments, X is O, and R is a CrCs alkyl group, preferably R is a methyl, ethyl or propyl group, more preferably R is a methyl group. In some embodiments, the chemical group of formula I is selected from a group consisting of a sulfaneyl-acetamide, a methyl thioglycolate, a sulfanyl-ethanamide, a sulfanyl-propanamide, a sulfanyl- acetylglycinamide and a sulfanyl-acetylalaninamide. More preferably, the chemical group of formula I is selected from a group consisting of a sulfaneyl-acetamide, a methyl thioglycolate, a sulfanyl-acetylglycinamide or a sulfanyl-acetylalaninamide.

[0069] In some embodiments, the first peptide has a C-terminal residue attach to the chemical group selected from the group consisting of Gly, Ala and Ser, preferably Gly. In some embodiments, the first peptide has a penultimate residue at the C-terminus selected from the group consisting of Leu, Ala, Arg, Ser, Pro and Gly. In some embodiments, the first peptide has an N-terminal residue selected from the group consisting of Gly, Ala, Arg and Ser. In some embodiments, the first or the second molecule further comprises a payload. For example, the first molecule may further comprise a payload attached to the N-terminus of the first peptide, and the second peptide is an epitope-binding peptide; or the first peptide is an epitope-binding peptide, and the second molecule further comprises a payload attached to the C-terminus of the second peptide. In these embodiments, the conjugation of the first and second peptides results in an epitope-binding peptide conjugated with a payload, such as an antibody drug conjugate (ADC).

[0070] In some embodiments, the epitope-binding peptide is selected from the group consisting of an antibody or functional fragment thereof, an affibody, adnectin, anticalin, fynomer, Kunitz domain and designed ankyrin repeat protein (DARPin). Preferably, the antibody or functional fragment thereof is selected from the group consisting of IgG, Fab, Fab’, F(ab’)2, Fv, scFv, diabody, minibody, and nanobody. Preferably, the affibody is ZEGFR or ZEGFR-FC, and the DARPin is an anti-HER2 DARPin.

[0071] In embodiments where the first peptide is an antibody comprising light chains and heavy chains, the sulfaneylacetamide group may be attached to the C-terminus of the light chains or the heavy chains. In embodiments wherein the second peptide is an antibody comprising light chains and heavy chains, the X1 -X2 motif may be attached to the N-terminus of the light chains or the heavy chains.

[0072] In some embodiments, the payload is an imaging agent or a therapeutic agent. Preferably, the imaging agent is a reporter or a radioisotope linked to a chelator. For example, the imaging agent may be a reporter selected from the group consisting of horseradish peroxidase (HRP), a fluorescent protein (such as GFP, RFP, YFP, CFP), chloramphenicol acetyltransferase (CAT), ere recombinase (Cre), LacZ, monomeric cherry (mCherry), biotin, a fluorescent dye (such as rhodamine, FAM); or may be a radioisotope linked to a chelator, wherein the chelator is selected from the group consisting of 1 ,4,7,10-tetraazacyclododecane-1 , 4, 7, 10-tetraacetic acid (DOTA), 1 ,4,7-triazacyclononane-triacetic acid (NOTA), 1 ,4,7-triazacyclononane-1 ,4,7- tris[methyl(2-carboxyethyl)phosphinic acid] (TRAP), desferrioxamine (DFO) and 1 ,4,7- triazacyclononane-1 -glutaric acid-4, 7-diacetic acid (NODAGA), and / or the radioisotope is selected from a group consisting of18F,67Ga,68Ga,44Sc,47Sc,11 1ln,64Cu,86Y,89Zr,177Lu, and225Ac. Examples of therapeutic agent include but not limited to Monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Deruxtecan (Dxd), DM1 (also known as mertansine), SN-38, PDB, PDB dimer, tubulysin, cryptophycin, eribulin, carmaphycin, Proteolysis Targeting Chimeras (PROTAC), or a therapeutic radioisotope such as177Lu,90Y,212Pb,225Ac, and213Bi.

[0073] In some embodiments, the payload further comprises a payload-releasing linker. Selection of such linker is within the knowledge of one skilled in the art based on the specific payload. For example, Val-Cit-PABC linker is a known linker for the release of MMAE.

[0074] The first molecule per se described above is also one aspect of the present invention.

[0075] 2. The second molecule

[0076] The second molecule comprises a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid.

[0077] In some embodiments, X1 may be any amino acid except Pro. For example, X1 may be selected from the group consisting of Gly, Ser, Ala, Cys, Gin, His, Leu, Phe, Tyr, preferably Gly. In some embodiments, X2 may be a hydrophobic amino acid or a p-branched amino acid. Examples of a hydrophobic amino acid may include Ala, Vai, Leu, lie, Met, Phe, Tyr, Trp, Gly, and Cys. Examples of a p-branched amino acid may include lie, Leu and VaL In some embodiments, X2 may be selected from a group consisting of Ala, Vai, Leu, lie, Met, Phe, Tyr, Trp, Gly, and Cys, and preferably selected from a group consisting of Leu, Phe, Tyr, Trp, Vai, and He, and more preferably X2 is He. In a preferred embodiment, the X1 -X2 motif is Gly-lle.

[0078] In some embodiments, the X1 -X2 may be indirectly attached to the N-terminus of the second peptide through a spacer. Such spacer may be at least one amino acid. As described herein, introducing a spacer between the X1 -X2 acyl acceptor and said second peptide may improve accessibility for the PAL to catalyze the ligation and consequently improve its yield, especially in cases where the second protein is large enough to hinder accessibility of PAL.

[0079] As described herein, in embodiments wherein the second peptide is an antibody comprising light chains and heavy chains, the X1 -X2 motif may be attached to the N-terminus of the light chains or the heavy chains.

[0080] 3. The PALs It would be appreciated by a person 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. Accordingly in some embodiments, the PAL may be a butelase-1 , butelase-2, VyPAL2, VyPAL3, OaAEPI b-C247A, HeAEP3, AtLEGy, VuPALI , HaPALI , OaAEPIb or a functional fragment or variant thereof.

[0081] In certain embodiments, the PAL may be selected from the group comprising 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 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, OaAEPI b-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, AtLEGy comprising the amino acid sequence set forth in SEQ ID NO: 8, VuPALI comprising the amino acid sequence set forth in SEQ ID NO: 9, HaPALI comprising the amino acid sequence set forth in SEQ ID NO: 10, OaAEPIb comprising the amino acid sequence set forth in SEQ ID NO: 1 1 and a functional fragment or a variant thereof.

[0082] As those skilled in the art would appreciate, a protein / enzyme’s function is directly related to its structure and sequence, and that there is a positive relationship between sequence identity and function similarity. In this regard, methods of determining a protein sequence identity are known in the art. Accordingly, the sequences of the enzymes of the present disclosure may be sufficiently varied so long as the enzymes maintain their functionality and can exhibit the required activity.

[0083] In some embodiments, the PAL may be a butelase-1 comprising an amino acid sequence with 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 , a butelase-2 comprising an amino acid sequence with 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 set forth in SEQ ID NO: 2 or 3, a VyPAL2 comprising an amino acid sequence with 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, a VyPAL3 comprising an amino acid sequence with 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, a OaAEPI b-C247A comprising an amino acid sequence with 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: 6, a HeAEP3 comprising an amino acid sequence with 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: 7, a AtLEGy comprising an amino acid sequence with 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, a VuPALI comprising an amino acid sequence with 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: 9, a HaPALI comprising an amino acid sequence with 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 or a OaAEPI b comprising an amino acid sequence with 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 .

[0084] C. Preparation of conjugates with two payloads

[0085] The Asx-independent ligation mediated by PALs and based on the chemical group of formula I of the present invention is especially useful in producing conjugates with two kinds of payloads (same or different), in particularly producing ADCs with two different payloads.

[0086] Thus, the present invention also provides a method of producing a conjugate comprising two payloads by two consecutive ligations, wherein one ligation is achieved by the method of the first aspect of the invention, and the other additional ligation is achieved by either a PAL- mediated enzymatic ligation reaction or a chemical protein modification reaction. Based on the selection of this additional ligation, the second molecule may be modified to comprise more components that are required for such ligation.

[0087] In one aspect, there is provided a method of producing a conjugate comprising two payloads by two consecutive ligation, wherein one ligation is achieved by the method of the first aspect of the invention, and the other additional ligation is achieved by an Asx dependent enzymatic ligation reaction mediated by a PAL and optionally a QC. Specifically, this method may comprise the following steps: i) providing a first molecule comprising a first payload, a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to a C-terminal residue of the first peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide, a X1 -X2 motif acting as an acyl acceptor and a P1 -P1 ’-P2’ tripeptide motif as a second acyl donor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide and the P1 -P1’-P2’ tripeptide motif is attached to the C-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid, wherein P1 is Asn or Asp, P1’ is Gly, Ser, Ala, Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid; ill) providing a third molecule comprising a second payload and a P1 "-P2" motif as an acyl acceptor, wherein PT is any amino acid and P2" is a hydrophobic amino acid or a p- branched amino acid; and iva) contacting a first peptidyl asparaginyl ligase (PAL) with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) in presence of a second PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) in presence of a first PAL to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a second PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

[0088] In another aspect, there is provided a method of producing a conjugate comprising two payloads by two consecutive ligations, wherein one ligation is achieved by the method of the first aspect of the invention, and the other additional ligation is achieved by a chemical protein modification reaction based on cysteine. Specifically, this method may comprise the following steps: i) providing a first molecule comprising a first payload, a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to a C-terminal residue of the first peptide and has the following formula I: wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the second peptide comprises at least one cysteine thiol group for chemical conjugation with a maleimide-functionalized moiety, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid; ill) providing a third molecule comprising a second payload and a maleimide-functionalized moiety; and iva) contacting a first peptidyl asparaginyl ligase (PAL) with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a first PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

[0089] The definitions of the formula I, X1 -X2 motif, PALs and payloads are as described above.

[0090] In some embodiments, the first PAL and the second PAL may be the same. In some embodiments, the first PAL and the second PAL may be different. Specifically, the first and second PAL may be independently selected from a group consisting of butelase-1 , butelase- 2, VyPAL2, VyPALS, OaAEP1 b-C247A, HeAEP3, AtLEGy, VuPALI , HaPALI, OaAEPIb and a functional fragment or variant thereof.

[0091] In some embodiments, the first and second payloads may be the same or different, and may be an imaging agent or a therapeutic agent. Preferably, the imaging agent is a reporter or a radioisotope linked to a chelator. For example, the imaging agent may be a reporter selected from the group consisting of horseradish peroxidase (HRP), a fluorescent protein (such as GFP, RFP, YFP, CFP), chloramphenicol acetyltransferase (CAT), ere recombinase (Cre), LacZ, monomeric cherry (mCherry), biotin, a fluorescent dye (such as rhodamine, FAM); or may be a radioisotope linked to a chelator, wherein the chelator is selected from the group consisting of 1 ,4,7,10-tetraazacyclododecane-1 , 4, 7, 10-tetraacetic acid (DOTA), 1 ,4,7- triazacyclononane-triacetic acid (NOTA), 1 ,4,7-triazacyclononane-1 ,4,7-tris[methyl(2- carboxyethyl)phosphinic acid] (TRAP), desferrioxamine (DFO) and 1 ,4,7-triazacyclononane- 1 -glutaric acid-4, 7-diacetic acid (NODAGA), and / or the radioisotope is selected from a group consisting of18F,67Ga,68Ga,44Sc,47Sc,111In,64Cu,86Y,89Zr,177Lu, and225Ac. Examples of therapeutic agent include but not limited to Monomethyl auristatin E (MMAE), Dxd, DM1 , SN- 38, PDB, PDB dimer, tubulysin, cryptophycin, eribulin, carmaphycin, Proteolysis Targeting Chimeras (PROTAC), monomethyl auristatin F (MMAF) or a therapeutic radioisotope such as177Lu,90Y,212Pb,225Ac, and213Bi.

[0092] In some embodiments, P1 " is selected from the group consisting of Gly, Ser, Ala, Gin, His, and Cys. In some embodiments, P2’ and P2" are independently selected from the group consisting of Leu, Phe, Tyr, Trp, Vai, and lie, preferably lie.

[0093] In some embodiments, the P1 -PT-P2’ tripeptide motif may be indirectly attached to the N- terminus of the second peptide through a spacer. Such spacer may be at least one amino acid. As described herein, introducing a spacer between the P1 -PT-P2’ tripeptide motif and said second peptide may improve accessibility for the PAL to catalyze the ligation and consequently improve its yield, especially in cases where the second protein is large enough to hinder accessibility of PAL.

[0094] In some embodiments, the second peptide is an epitope-binding peptide. Preferably, the epitope-binding peptide is selected from the group consisting of an antibody or functional fragment thereof, an affibody, and designed ankyrin repeat protein (DARPin). More preferably, the antibody or functional fragment thereof is selected from the group consisting of IgG, Fab, Fab’, F(ab’)2, Fv, scFv, diabody, minibody, and nanobody.

[0095] In embodiments wherein the second peptide is an antibody comprising light chains and heavy chains in the second aspect of the invention, (i) the X1 -X2 motif is attached to the N-terminus of the light chain and the P1 -PT-P2’ tripeptide motif is attached to the C-terminus of the heavy chain, or (ii) the X1 -X2 motif is attached to the N-terminus of the heavy chain and the P1 -PT- P2’ tripeptide motif is attached to the C-terminus of the light chain, or (iii) the X1 -X2 motif is attached to the N-terminus of the light chain and the P1-PT-P2’ tripeptide motif is attached to the C-terminus of the light chain, or (iv) the X1 -X2 motif is attached to the N-terminus of the heavy chain and the P1-PT-P2’ tripeptide motif is attached to the C-terminus of the heavy chain.

[0096] In some embodiments, in step iva), the first conjugate is contacted with the third molecule of step iii) in presence of the second PAL and a glutaminyl cyclase (QC) to form the second conjugate; in step ivb), wherein the second molecule of step ii) is contacted with the third molecule of step iii) in presence of a first PAL and a QC to form the first conjugate, and P1 ’ of the second molecule is Gin or Glu. The presence of QC helps reduce the reverse reaction and thus improve the yield.

[0097] It is also envisaged that various QCs having the desired QC enzymatic activity may be suitable for use in the practice of the present invention. Accordingly in some embodiments, the QC may be a Human glutaminyl cyclase, a Mouse glutaminyl cyclase, a Drosophila glutaminyl cyclase, an Arabidopsis glutaminyl cyclase, a Conus glutaminyl cyclase, a Sistrurus glutaminyl cyclase, a Bacterial glutaminyl cyclase or a functional fragment or variant thereof.

[0098] 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:

[0099] 15, Conus glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO:

[0100] 16, Sistrurus glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO:

[0101] 17, Bacterial glutaminyl cyclase comprising the amino acid sequence set forth in SEQ ID NO: 18 and a functional fragment or a variant thereof.

[0102] In some embodiments, the QC may be a Human glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 12, a Mouse glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 13, a Drosophila glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 14, an Arabidopsis glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 15, a Conus glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 16, a Sistrurus glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 17, or a Bacterial glutaminyl cyclase comprising an amino acid sequence with 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 SEQ ID NO: 18.

[0103] In embodiments wherein the second peptide is an antibody comprising light chains and heavy chains in the third aspect of the invention, (i) the X1 -X2 motif is attached to the N-terminus of the light chain and the heavy chain comprises the at least one cysteine thiol group, or (ii) the X1 -X2 motif is attached to the N-terminus of the heavy chain and the light chain comprises the at least one cysteine thiol group, or (iii) the X1 -X2 motif is attached to the N-terminus of the light chain and the light chain comprises the at least one cysteine thiol group, or (iv) the X1 - X2 motif is attached to the N-terminus of the heavy chain and the heavy chain comprises the at least one cysteine thiol group.

[0104] The present disclosure also provides a method of producing a conjugate comprising two payloads based on a Asx-dependent PLA-dependent ligation and a chemical protein modification reaction based on a cysteine thiol group. Specifically, this method comprises the following steps: i) providing a first molecule comprising a second payload and a P1"-P2" motif as an acyl acceptor, wherein P1 " is any amino acid and P2" is a hydrophobic amino acid or a -branched amino acid; ii) providing a second molecule comprising a second peptide and a P1-PT-P2’ tripeptide motif as a acyl donor, the P1 -PT-P2’ tripeptide motif is attached to the C-terminus of the second peptide, wherein the second peptide comprises at least one cysteine thiol group for chemical conjugation with a maleimide-functionalized moiety, wherein P1 is Asn or Asp, PT is Gly, Ser, Ala, Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid; iii) providing a third molecule comprising a second payload and a maleimide-functionalized moiety; and iva) contacting a peptidyl asparaginyl ligase (PAL) and a QC with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a PAL and a QC to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

[0105] EXAMPLES

[0106] The following examples are intended to exemplify the present disclosures and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples and other sections of the specification, figures, and claims form part of the disclosure of the invention.

[0107] Example 1. Materials and methods

[0108] 1.1 Materials

[0109] All solvents, reagents and resins were obtained from commercial sources (Sigma-Aldrich, Alfa Aesar and Acros Organics) and without further purification. Pro-Human legumain was purchase from Prospec (ENZ-923) and stored at -20°C. VyPAL2 (SEQ ID NO: 4) and OaAEPI b-C247A (SEQ ID NO: 6) were prepared as previously reported in Yang et aL, J. Am. Chem. Soc., 2017; Xia et al., J. Am. Chem. Soc., 2023; and Hemu et al., Proc Natl Acad Sci U.S.A, 2019.

[0110] For purification of the synthesized peptides, semi-preparative Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) was employed. Analytical HPLC was utilized to monitor the reactions. MS data acquisition and analysis were carried out using Electrospray Ionization Mass Spectrometry (ESI-MS) and the Bruker Ultraflex Extreme Matrix Assisted Laser Desorption / lonization (MALDI) Tandem Time-of-Flight (5800 MALDI-TOF / TOF) system.

[0111] 1.2 HPLC

[0112] Analytical Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) was conducted using a SHIMADZU Prominence LC-20AT system, equipped with a Grace Vydac "Protein C4" analytical column (4.6 x 250 mm, 5 pm), maintaining a flow rate of 1.0 mL / min. UV absorption at 220 nm and 254 nm was used to monitor the elution during analytical HPLC. For semi-preparative RP-HPLC, a Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC) system was utilized, featuring a Phenomenex Jupiter-C18 RP column (10 x 250 mm, 5 pm). This process was conducted at a flow rate of 2.5 mL / min, employing a gradient elution with buffer B (90% ACN, 10% H2O, 0.045% TFA) in buffer A (H2O, 0.045% TFA).

[0113] 1 .3 Mass spectrometry

[0114] ESI mass spectrometry data of peptides and proteins were obtained from a Thermo Finnigan LCQ DECA XP MAX (ESI ion source, positive mode) or ABI 5800 MALDI-TOF / TOF system (Applied Biosystems, Framingham, MA, USA). The instrument was equipped with a solid-state laser (diode pumped Nd: YAG laser), pulsing at a repetition rate of 200 Hz. The MALDI matrix was an 8 mg / mL solution of a-cyano-4-hyroxycinnamic acid (CHCA) in 60% acetonitrile and 0.05% TFA. The samples of interest were combined in a 1 :1 (v / v) ratio, and 0.5 pL of the mixture was spotted onto a target plate. The mass spectrum was analyzed to determine the mass.

[0115] 1.4 Solid phase peptide synthesis (SPPS) of DOTA-GVYSAYGPRALG-SCH?CONH?

[0116] All the peptide thioesters used were synthesized using 4-methylbenzylhydrylamine (MBHA) resin, employing standard tert-butyloxycarbonyl (Boc) chemistry (see Figure 1).

[0117] Initially, the MBHA resin was pre-swollen in dimethylformamide (DMF) for 10 minutes. The first thiol-linker coupling involved dissolving the linker compound Trt-SCH2COOH (3 equivalents) and benzotriazol-1 -yloxytripyrrolidinophosphonium hexafluorophosphate (PyBop) (3 equivalents) in DMF within a 15 mL tube, followed by the addition of N,N- diisopropylethylamine (DIEA) (8 equivalents). This mixture was then introduced to the resin and shaken for 40 min at ambient temperature. After this, the resin underwent sequential washing with DMF and dichloromethane (DCM). The trityl protecting group attached to the linker was removed using a mixture of 49% DCM, 49% TFA, and 2% TIS for 15 mins to give HSCH2CO-NH-MBHA resin.

[0118] Boc-SPPS was carried out to assemble the peptide chain on the above-obtained HSCH2CO- NH-MBHA resin. A Boc-protected amino acid (Boc-AA(n)-OH, 3 equivalents) and PyBop (3 equivalents) were initially dissolved in a DMF / DCM (1 :1 ) solution. This solution was then added to the resin, followed by adding 6 equivalents of DIEA to activate the amino acid. The mixture was shaken for one hour at room temperature. Then the resin was washed again with DMF and DCM. The coupling efficiency was evaluated using the ninhydrin test, except for the first coupling step. For Boc group deprotection, the resin was treated with a 50% trifluoroacetic acid (TFA) solution in DCM for 15 minutes. At the last step, DOTA was coupled to the peptidyl resin using the same couple reagent PyBOP to obtain DOTA-GVYSAYGPRALG-SCH2CONH- MBHA resin (with protecting groups on DOTA and certain amino acid side chains such as Tyr, Ser and Arg).

[0119] To perform the final cleavage of the peptide from the resin, 100 mg of peptidyl resin was mixed with 200 pL of thioanisole and 100 pL of 1 ,2-ethanedithiol (EDT) for 10 minutes at 0°C. This mixture was then combined with 1 mL of TFA and maintained at 0°C for an additional 20 minutes. Subsequently, 100 pL of trifluoromethanesulfonic acid (TFMSA) was slowly added to the mixture, drop by drop, and the stirring continued for another 30 minutes at 0°C. Following this period, the reaction mixture was taken out of the ice bath and left to stir at ambient temperature for 2 hours. The cleaved product DOTA-GVYSAYGPRALG- SCH2CONH2 was then separated from the resin by filtration and precipitated into 20 mL of cold diethyl ether (Et2O). The resulting crude peptide was collected via centrifugation and further purified using semi-preparative Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). The peptides were then freeze-dried to obtain them in powder form. Electrospray Ionization Mass Spectrometry (ESI-MS) was employed to characterize all the synthesized peptides.

[0120] To prepare the N-terminal acetylated peptide Ac-GVYSAYGPRALG-SCH2CONH2, the resinbound peptide, i.e., H2N-GVYSAYGPRALG-SCH2CONH-MBHA resin (with side chain protecting groups on Tyr, Ser and Arg) was treated with 5 equivalents of acetic anhydride and 8 equivalents of DIEA in DMF and shaken for 30 minutes, then washed three times each with DMF and DCM. Final cleavage of the N-terminal acetylated peptide from the resin was conducted using the same procedure described in the above paragraph.

[0121] 1 .5 Cyclization assays

[0122] Cyclization reactions were carried out in 50-pL reaction mixtures, which included 20 mM phosphate buffer at pH 7, the PAL enzyme, and the substrate at concentrations ranging from 25 to 800 pM. The enzyme-to-substrate ratio was maintained either at 1 :100 or 1 :200. To quench the reaction, 6 M Guanidine-HCI (pH 3) was added, and the progress of the reaction was tracked using RP-HPLC. The proportion of the cyclized product to the residual linear precursor in the mixture was determined by comparing their respective peak areas in the HPLC data. The identity of the cyclized product was verified using either Electrospray Ionization Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption / lonization Mass Spectrometry (MALDI-MS).

[0123] 1 .6 Ligation of model peptide Enzyme-meditated ligation reactions were performed in 20 mM PBS buffer (pH 7) at room temperature for various time courses. The progress of these reactions was observed using analytical RP-HPLC. The ligated products were then characterized using ESI-MS.

[0124] 1.7 pH canning assays

[0125] To assess the impact of pH on enzyme activity, reactions were conducted across various pH levels using suitable buffer systems, following the methodology described earlier. Solutions of 0.2 M citrate, 0.2 M sodium acetate, and 0.2 M phosphate were prepared to establish the required pH levels in these buffer systems.

[0126] 1 .8 Enzymatic kinetic studies

[0127] The kinetic parameters for PALs-catalysed cyclization were determined using the initial reaction rates. The catalytic efficiencies of VyPAL2 and OaAEP1b-C247A were assessed with peptide 1 (SEQ ID NO: 19). Various substrate concentrations were used in the reaction (25 pM, 50 pM, 100 pM, 200 pM, 400 pM, and 800 pM). The reactions involved mixing the peptide with either the OaAEP1 b-C247A or VyPAL2 enzyme, maintaining enzyme-to-substrate ratios of either 1 :100 or 1 :200. These reactions were conducted in phosphate buffer (pH7) at room temperature for durations ranging from 10 to 30 minutes. To ensure that the reaction rate measured corresponded to the initial velocity, the reaction was quenched with 6 M Guanidine- HCI (pH 3) once less than 25% of the substrate had been converted. These experiments were performed in triplicates and the data were analysed using GraphPad Prism software (GraphPad Software, San Diego). Kinetic parameters, specifically Kcatand Km, were derived from the Michaelis-Menten curve calculated using GraphPad 7.0 software.

[0128] 1.9 Protein N-terminal labelling

[0129] The ligation reactions were conducted at room temperature and monitored at various time points. 200 pM or 250 pM of protein was incubated with 1 -5 equivalents of substrate (not containing Asx) in 20 mM PBS (pH 7), in the presence of 0.005 equivalents of VyPAL2. The reactions were quenched by 6 M Guanidine-HCI (pH 3) and monitored by analytical RP-HPLC. The ligated products were characterized by ESI-MS.

[0130] 1.10 Antibodv-druo conjugation by PAL-mediated ligation

[0131] An example of producing a ADC comprising two payloads using two PAL mediated sequential ligation is shown in Figure 23.

[0132] For N-terminal conjugation, the reaction was conducted at room temperature overnight. 12 pM of antibody 1 (Table 2) was incubated with 10 equivalents of the labelling agent containing the payload in 20 mM PBS (pH 7), in the presence of 0.1 equivalents of VyPAL2. The reaction mixture was treated with 50 mM dithiothreitol (DTT) and analysed by analytical RP-HPLC. The peaks from HPLC were collected for analysis by ESI-MS (ESI). After purification by a Protein A column, the concentration of the product ADC 2 was determined using a Nanodrop.

[0133] For sequential antibody labelling, ADC 2 was modified at the C-terminus of the light chains to introduce the 2ndpayload. ADC 2 (12 pM) was treated with a solution containing 100 equivalents of a GIG-MMAF-OCH3, 0.1 equivalents of OaAEP1b-C247A and 0.01 equivalents of human QC (SEQ ID NO: 12) with a 6xHis tag at the C-terminus for purification in 20 mM PBS supplemented with 0.25 mM tris(2-carboxyethyl)phosphine (TCEP). To monitor the reaction, the mixture was treated with 50 mM DTT and characterized by RP-HPLC and ESIMS. After 16 h, the product ADC 3 was isolated by purification using a protein A column.

[0134] 1.1 1 Human leaumain stability assay

[0135] Pro-legumain was activated using the activation buffer (100 mM citric acid, 100 mM NaCI, 5 mM DTT, pH 4) at 37 °C overnight (Dall et la., Proc. Natl. Acad. Sci. U.S.A., 2013; Miller et al., Bioconjug Chem, 2021 ). Activated legumain was diluted using the reaction buffer (100 mM citric acid, 100 mM NaCI, 2 mM DTT, pH 5.5) to yield a 0.05 mg / mL enzyme concentration. Then, 250 pM of a substrate was incubated with 0.0002 equivalents of activated-legumain at 37 °C for 3-48 h. The reaction was analysed by RP-HPLC.

[0136] 1.12 Cell viability assay

[0137] MCF-7 human breast cancer cells or BT474 cells were cultivated in 96-well plates with a density of 4500 cells / well. Subsequently, the cells were exposed to ten-fold serial dilutions of various samples at 37°C for 3 days in an atmosphere containing 5% CO2. After that, 10 pL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg / mL) was added to each well and incubated under the same conditions (37°C and 5% CO2) for 2 hours. The absorbance of each well was measured at 575 nm.

[0138] 1.13 OaAEP1 b-C247A expression and purification

[0139] The production of OaAEP1b-C247A in E. coll was conducted using the T7 Shuffle system from New England Biolabs, USA, in accordance with a previously established protocol. Selected bacterial colonies were transferred into a sterile culture flask containing liquid LB medium. This flask was agitated in an incubator maintained at 30°C until the optical density (OD) of the culture reached 0.4. At this point, protein expression was initiated by adding 0.1 mM IPTG and maintaining the culture at 16°C for 48 hours. Post induction, the bacterial cells were harvested and subjected to sonication in a lysis solution composed of 50 mM sodium phosphate and 100 mM NaCI, 0.01% TritonX-100, at pH 8.0. Following centrifugation, the supernatant was applied to a column containing Ni-NTA beads (Bio Rad) and incubated at 4°C for one hour. The protein was then eluted from the column using an elution buffer that included 500 mM imidazole, after the beads had been washed thrice with the wash buffer. The final step in the purification process involved size-exclusion chromatography (SEC). The purified protein was stored at - 80°C.

[0140] Pro-OaAEP1 b-C247A underwent activation in an acetic buffer composed of 20 mM PBS, 0.1 M NaCI and 0.5 mM TCEP at a pH of 4, at a temperature of 37°C for 2 hours. Following this activation phase, the enzyme was then stored at -80°C in a stabilizing cocktail containing 5% glycerol, 20 mM PBS, and 0.5 mM TCEP, adjusted to a pH of 7.

[0141] 1.14 VyPAL2 expression and purification

[0142] The expression and purification of VyPAL2 was performed as described in Xia et al., J. Am. Chem. Soc., 2023.

[0143] Briefly, the pFB-Sec-NH (Amp+) donor vector encoding VyPAL2 was transformed into E. coli DHIOBac cells (Invitrogen). Following X-gal blue / white screening at 37 °C for 48 h, white colonies were selected for colony PCR with M13 primers and sequence verification. Positive clones were used to generate bacmid via cell resuspension and extraction. The resulting bacmids were transfected into Sf9 insect cells using Cellfectin™ in Grace’s insect medium (Gibco). After 72 h, P0 virus-containing supernatant was harvested. The virus was then amplified through three successive passages, and 1 L of Sf9 culture (2.5 10scells / mL) was infected with 25 mL of P3 virus and incubated at 27 °C, 120 rpm for 72 h. Cells were removed by centrifugation (4 000 g, 30 min) to collect the secreted protein. Protein purification was performed using size-exclusion chromatography (SEC). Pro-VyPAL2 activation was carried out at pH 4.5 in 50 mM sodium citrate buffer (0.1 M NaCI, 1 mM DTT, 0.5 mM LS) for 2-3 h at 37 °C. The activated enzyme was then polished by SEC in 20 mM PBS (pH 6.5) containing 0.1 M NaCI and 1 mM DTT. Finally, purified VyPAL2 was stored at -80 °C in 20 mM PBS (pH 7.0) with 0.5 mM TCEP and 5 % glycerol.

[0144] 1.15 GI-ZFGFR and GI-DARPin expression and purification

[0145] The protein genes were encoded into vectors pET28a or pETDuet, and then expressed in E. coli strains. The expression was induced using 0.1 mM isopropyl p-d-1 -thiogalactopyranoside (IPTG) after the ODeoo of the bacterial culture in Luria Bertani broth (supplemented with either Kanamycin or Ampicillin) reached 0.4-0.6. This was done at temperatures of either 37°C or 30°C. Following induction, the cultures were further incubated at 16°C for 18 hours. The cells were then collected via centrifugation (5000 x g for 10 minutes) and resuspended in a lysis buffer containing 50 mM PBS, 0.1 M NaCI, 0.01 % TritonX-100, at pH 7.5. The cells were lysed using an ultrasonicator probe (Vibra cell TM), applying alternative cycles of 4 seconds pulse after every 6 seconds interval for 15-20 minutes on ice. After this, the protein solution was centrifuged at 15000 x g for 20 minutes at 4°C, then filtered through a 0.2 pm membrane, and subsequently bound to NiNTA beads for 1 hour at 4°C. The Ni beads were washed using a buffer of 20 mM imidazole, 0.1 M NaCI, 20 mM PBS at pH 7.5, and proteins were eluted with a buffer containing 500 mM imidazole, 0.1 M NaCI, 20 mM PBS at pH 7.5. All proteins were buffer-exchanged into 20 mM PBS (pH 7) and stored at -20°C.

[0146] 1.16 Flow cytometry assay

[0147] To study the binding affinity of the affibody 5(6)-FAM-GRLG-GI-DARPin with BT474 cells (HER2-over expression), one million of the cells were incubated with different concentrations of 5(6)-FAM-GRLG-GI-DARPin and 5(6)-FAM-GRLG on ice for 30 min, respectively. Then, the cells were washed with PBS three times. After that, the mixture was analyzed by the Fortessa X-20 flow cytometer (BD, USA). (Excitation wavelength: 504 nm, emission wavelength: 523 nm).

[0148] 1.17 Synthesis of ValCit-PABC-MMAE

[0149] The synthesis scheme of ValCit-PABC-MMAE is shown in Figure 2.

[0150] To a solution of compound 1 (1 mmol) in a 1 :1 (v / v) mixture of acetonitrile (ACN) and water (10 mL) was added L-citrulline (1 mmol) and NaHCO3(3 mmol). The reaction mixture was stirred at room temperature for 16 h, after the solvent was evaporated, and the residue was redissolved in H2O (50 mL). The aqueous phase was washed with diethyl ether (2 x 30 mL), acidified with 4 M HCI, and extracted with ethyl acetate (3 x 30 mL). The combined organic extracts were dried over MgS04and concentrated under reduced pressure to afford compound 2 as an off-white solid, which was used in the next step without further purification.

[0151] To a suspension of compound 2 (496 mg, 1 .0 mmol) in MeOH (5.0 mL) and DCM (5.0 mL) were added N-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ) (740 mg, 3.0 mmol) and 4-aminobenzyl alcohol (245 mg, 2.0 mmol). The mixture was stirred under the dark at room temperature for 36 h. After evaporation of the solvent, the residue was subjected to column chromatography (2-6% MeOH in DCM) to yield compound 3 (450 mg, 75%) as off-white solid. FmocVCit-PABC-PNP carbonate (compound 4) was prepared by adding DIEA (260 pL, 1 .50 mmol) and bis(p-nitrophenyl) carbonate (304 mg, 0.10 mmol) to a solution of compound 3 (300 mg, 0.50 mmol) in anhydrous DMF, and the mixture under N2atmosphere was stirred at room temperature for 16h. After solvent removal by rotary evaporation, the residue was subjected to column chromatography (2-4% MeOH in DCM) to yield compound 4 (330 mg, 86%) as off- white solid.

[0152] MMAE HCI (66 mg, 0.088 mmol) was added to a solution of compound 4 (190 mg, 0.25 mmol), 1-hydroxy-7-azabenzotriazole (HOAt) (13.6 mg, 0.1 mmol) and DIEA (0.13 mL, 0.75 mmol) in anhydrous DMF. The resulting reaction mixture was stirred at room temperature for 16h. 20% piperidine in DMF (0.5 mL) was then added, and the mixture was stirred for 4h. After removing solvent by rotary evaporation, the residue was subjected to reverse-phase HPLC purification (Buffer A: 0.045% TFA in H2O, Buffer B: 0.045% TFA in 90% acetonitrile, 10% H2O). The fractions containing the product were pooled and freeze dried to afford compound 6 as off- white powder. MS (ESI): m / z [M+H]+ calc.1 123.71 , found 1 123.75.

[0153] 1.18 Synthesis of [Ga(DOTA)l-GVYSAYGPRALG-GI-ZEGFR

[0154] The synthesis scheme of [Ga(DOTA)]-GVYSAYGPRALG-GI-ZEGFR is shown in Figure 3.

[0155] Peptide DOTA-GVYSAYGPRALG-SCH2CONH2 was prepared as using the method described in section 1 .4 above. The protein GI-ZEGF was ligated with DOTA-GVYSAYGPRALG- SCH2CONH2 by the peptide-protein ligation method described in section 1.9. After that, 10 equivalents of Ga(NOs)3 were dissolved in water, and the pH of this solution was adjusted to pH 6 using 0.2 M phosphate buffer (pH 7). The mixture was then left to react at 37°C overnight, resulting in the formation of [Ga (DOTA)]-GVYSAYGPRALG-GI-ZEGFR. MS (ESI): m / z, [M+H]+calc.10183, found 10191.

[0156] Example 2. PAL-catalysed ligation reaction on small model peptides

[0157] After screening several ester and thioester constructs, we found a unique peptidyl- sulfaneylacetamide thioester to be an effective acyl donor substrate of PALs. Hypothetically, in the absence of a C-terminal Asx residue, the thioester linkage is reactive enough to engage the active-site cysteinyl thiol of the enzyme in a trans-thioesterification reaction to form the acyl-enzyme thioester intermediate which is subsequently resolved by a nucleophilic amine substrate, forming a non-Asx peptide bond in the cyclization or ligation product (Figure 4). The reaction is irreversible because the newly formed bond at the ligation site does not have an Asx residue for PAL recognition. Therefore, this method not only solves the reversibility problem but also broadens the substrate scope of PAL-mediated ligation. This method can be used to modify a protein at the N-terminus as long as it has a suitable nucleophilic a-amine. Moreover, since the ligation product is no longer recognized as a substrate by the PAL enzyme, it is possible to conduct a second PAL-mediated ligation at the C-terminal end of the same protein. With this orthogonality, we can conduct sequential ligation reactions on an antibody to attach two different drug payloads to the N- and C-termini of its polypeptide chains, yielding a dual-payload ADC. This advancement further broadens the applications of PALs, underpinning their role as powerful bioconjugation tools for biologies drug development.

[0158] We first demonstrated this new PAL-catalyzed ligation reaction on small model peptides. The first model peptide substrate comprising a sulfaneylacetamide group at the C-terminus (H2N- GVYSAYGPRALG-SCH2CONH2, SEQ ID NO: 19) was designed for cyclization. When treated with 0.005 equivalents of VyPAL2 in 20 mM phosphate buffer (pH 7) at room temperature, the linear thioester peptide was almost completely converted to the cyclic form in just 1 h (Figure 5A). This result indicates that the sulfaneylacetamide-containing peptide is a good substrate of VyPAL2. Next, the 2nd model peptide Ac-GVYSAYGPRALG-SCH2CONH2(SEQ ID NO: 34) (200 pM), acetylated according to the method described in 1.4 above, was used in an intermolecular ligation reaction with an acyl acceptor peptide, GIGGIR (SEQ ID NO: 35) (400 pM), which was compared with the ligation reaction between the conventional substrate without a sulfaneylacetamide group Ac-GVYSAYGPRALGNGL (SEQ ID NO: 36) (200 pM) and GIGGIR (400 pM). The results showed that, although the conventional substrate with the NGL tripeptide motif at the C-terminus gave a faster reaction initially, the yield plateaued at around 59%. In contrast, the thioester substrate achieved a yield exceeding 95%, owing to the irreversibility of the reaction (Figure 5B).

[0159] Other chemical substrates for Asx-independent ligation were also tested, including Substrate 1 : carbamoyl-methoxy group (-OCH2CONH2); Substrate 3: methyl thioglycolate (- SCH2COOCH3); Substrate 4: sulfanyl-ethanamide (-SCH2CH2CONH2); Substrate 5: sulfanyl- propanamide (-SCH2CH2CH2CONH2); Substrate 6: sulfanyl-acetylglycinamide (-SCH2CO- Gly-NH2); and Substrate 7: sulfanyl-acetylalaninamide (-SCH2CO-Ala-NH2). The results showed that substrate 1 was unable to mediate the ligation, while substrate 3 exhibited similar reactivity compared to substrate 2. In comparison, substrates 4 and 5 exhibited significantly reduced reactivity, affording cyclized products in only 10.2% and 4.2% yield, respectively, after 2 hours of enzymatic catalysis by OaAEP1 b-C247A. Remarkably, when the sulfaneylacetamide group was further substituted on the amide nitrogen with a Gly or Ala amide (corresponding to substrates 6 or 7 respectively), efficient cyclization of the linear peptide was still achieved. These findings suggest that sulfaneyl-acetamide (substrate 2) and methyl thioglycolate (substrate 3) thioesters represent the most favorable groups for PAL-mediated cyclization. A D-peptide thioester substrate was also prepared, which has the same sequence as the 1 st model peptide but with all L-amino acids replaced with D-amino acids (H2N-GvysayGpralG- SCH2CONH2, SEQ ID NO: 20). We found that, while both OaAEP1 b-C247A and VyPAL2 could cyclize the all-D peptide substrate, VyPAL2 exhibited notably higher catalytic activity than OaAEPI b-C247A (Figure 6). Furthermore, we studied the effect of the C-terminal and N- terminal residues on VyPAL2-mediated cyclization. Specifically, we synthesized various mutants of the 1stmodel peptide, wherein the C-terminal residue was mutated to A (SEQ ID NO: 21 ), or S (SEQ ID NO: 22), , the penultimate residue at the C-terminus was mutated to A (SEQ ID NO: 23), R (SEQ ID NO: 24), S (SEQ ID NO: 25), P (SEQ ID NO: 26), or G (SEQ ID NO: 27), or the N-terminal residue was mutated to A (SEQ ID NO: 28), R (SEQ ID NO: 29), S (SEQ ID NO: 30) or P (SEQ ID NO: 31 ). We found that a C-terminal Gly is the most preferred amino acid (Figure 7), while the penultimate position tolerates a broad range of amino acids (Figure 8). Moreover, glycine is also the most preferred N-terminal amino acid for PAL- mediated cyclization reaction (Figure 9).

[0160] Table 1 . MS data of the tested peptides.

[0161] Example 3. Cyclization reactions at different pHs

[0162] We further examined the cyclization reactions catalysed by two PAL enzymes, OaAEPI b- C247A and VyPAL2, at different pHs (e.g., pH4.5, pH5, pH5.5, pH6, pH6.5, pH7, pH7.5, pH8). As shown in Figure 10A, both enzymes exhibit a notable pH dependency for cyclizing the peptidyl-sulfinyl-acetamide thioester substrate, with optimal activity at neutral or weakly alkaline pHs (pH7-8). A significant reduction in activity was observed in mildly acidic conditions. This finding underscores the importance of maintaining a specific pH to maximize the enzymatic activity of OaAEP1 b-C247A and VyPAL2 in these types of reactions. It also provides valuable insights into the biochemical properties of these enzymes, which could be pivotal in designing and optimizing processes for peptide and protein modification, especially in contexts where the precise control of reaction conditions is vital.

[0163] The catalytic efficiency of the cyclization reaction is 1517 M1S1for OaAEP1 b-C247A and 2568 M1S'1for VyPAL2, respectively (Figure 10B). While these kinetics are moderate compared to a typical PAL-mediated cyclization reaction of conventional P1 -Asn substrates, they are still approximately 200-fold faster than those of sortase A-mediated cyclization of substrates with a C-terminal LPXTG motif.

[0164] Example 4. Stability of conjugates with GI-ZEGFR and GI-DARPin

[0165] The N-terminus is a good site for protein modification (Deng et al., Commun. Chem. 2020; Rosen et al., Nat. Chem. Biol. 2017). Peptidyl-sulfaneylacetamide is a suitable reagent for protein N-terminal labelling. An anti-EGFR affibody ZEGFR and an anti-HER2 DARPin were selected as the model proteins (Rosen et al., Nat. Chem. Biol. 2017; Garousi et al., Int. J. Oncol. 2016; Orlova et aL, Cancer. Res. 2006; Stahl et al., Trends. Biotechnol. 2017). As shown in Figure 1 1 , various payload entities, such as a fluorescent dye (e.g., 5(6)- carboxyfluorescein, also known as FAM), small-molecule drugs (e.g., PNU-159682 carboxylic acid (also known as PNU), MMAE), metal chelators (e.g. DOTA), and biotin, were conjugated to the N-terminus of GI-ZEGF (SEQ ID NO: 35), which has a 6xHis tag at the C-terminus or GI-DARPin (SEQ ID NO: 36), when they were functionalized as the sulfaneyl-acetamide thioester and linked to different peptides such as GVYSAYGPRALG (SEQ ID NO: 19), GRRLG (SEQ ID NO: 37) and GRLG (SEQ ID NO: 38). The labelling reaction was performed using 200 pM or 250 pM of the protein and 1-5 eq of the sulfaneyl-acetamide thioester substrate in 20 mM PBS (pH 7) and with 0.005 eq of VyPAL2 to afford the desired products as characterized by ESI-MS (Figures 12-17). The non-Asx ligation linkage in the obtained protein conjugates was stable towards human legumain, an AEP-type protease that hydrolyzes asparaginyl and aspartyl peptide bonds (Dall et aL, Biochimie. 2016). Indeed, the FAM-GRLG- GI-ZEGFR conjugate remained intact when incubated with human legumain for 48 h (Figure 18). The resistance to hydrolysis by this cysteine endopeptidase can provide the conjugate with enhanced stability in vivo. In contrast, a very similar ZEGF conjugate FAM-GRLG-NGI-ZEGFR, prepared by the conventional Asn-dependent PAL ligation, underwent significant hydrolysis, approximately 50%, after 48 h incubation with human legumain (Figure 18).

[0166] FACS analysis showed that the conjugate, FAM-GRLG-GI-DARPin, maintained the specific binding ability of the DARPin protein to BT474 cells, which overexpress HER2 (Figure 19). In addition, the MMAE-conjugated DARPin exhibited selective cytotoxic activity against the HER2-overexpressing BT474 cells (IC50 at 12.8 nM), while having negligible cytotoxicity towards MCF-7 cells (Figure 20). It is noteworthy that the MMAE-DARPin conjugate contains the Val-Cit-PABC linker for the release of MMAE as it is commonly used in antibody-drug conjugates (Chuprakov et al., Bioconjug. Chem. 2021). These findings underscore the potential of our Asx-independent PAL ligation scheme in the development of diagnostic and therapeutic protein conjugates.

[0167] Example 5. PAL-catalysed ligation reaction on antibodies

[0168] This new PAL ligation scheme was also applied for conjugating cytotoxic drugs to therapeutic antibodies at a non-Asn junction. Antibody-drug conjugates (ADCs) are composed of tumortargeting monoclonal antibodies linked to cytotoxic payloads through designed chemical linkers. This combination allows for both precise targeting and potent efficacy in cancer eradication, thereby broadening the therapeutic index (Dumontet et al., Nat. Rev. Drug Discov. 2023; Fu et al., Signal. Transduct. Target. Ther. 2022). As a case study, we selected trastuzumab, a pioneering antibody first approved in 1998 for the treatment of HER2-positive breast cancer (Shak et al., Semin Oncol. 1999). This antibody serves as an exemplary backbone for ADC development. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), well-known for their potent cytotoxicity, are frequently utilized as effective payloads in ADCs (Fu et al., Signal. Transduct. Target. Ther. 2022; Wang et al., Cancers (Basel). 2020; Yao et al., Aaps. j. 2022). In our experiments, 12 pM of antibody 1 (Table 2), which was genetically engineered to have a Gl dipeptide at the N-terminus of the light chains linked by a spacer LS (SEQ ID NO: 39) and a DQL tripeptide at the C-terminus of the heavy chains of trastuzumab (SEQ ID NO: 40) as the nucleophile substrate for PALs, was reacted with 10 eq of the sulfaneylacetamide thioester-functionalized payload (MMAE in this case) in the presence of 0.1 eq of VyPAL2 for overnight reaction (Figures 21 A and 21 B). A near quantitative yield (97%) was observed for the conjugation reaction as monitored by reverse-phase analytical HPLC (Figure 24A). The high yield was attributed to the irreversibility of the reaction and to the stability of the newly formed linkage towards the PAL enzyme. After purification, the resulting single-payload ADC 2 exhibited potent cytotoxicity (IC50 = 0.612± 0.04 nM) against HER2-overexpressing breast cancer BT474 cells, Conversely, its cytotoxicity against MCF-7 cells, which are HER2 antigen-negative, was lower by at least two orders of magnitude (IC50 > 50 nM), showing high selectivity of the ADC towards cancer cells (Figure 22).

[0169] As stated before, the Asx-independent ligation schemes is orthogonal to conventional Asx- dependent ligation by the PALs. This makes it possible to prepare dual-payload ADCs through a sequential ligation scheme using the PALs (Figures 23A and 23B). As indicated above, antibody 1 (Table 2) was also modified with a DQL tripeptide motif at the C-terminus of the heavy chains, which serves as a PAL recognition tag. With a P1 -Asp residue, the DQL tripeptide is stable to VyPAL2 at neutral to weakly basic pH as shown previously (Xia et al., Angew. Chem. Inti. Ed. 2021 ). So, during the first-step ligation reaction at pH 7, this DQL was not affected by VyPAL2. At the second ligation step, ADC 2 was conjugated to a different payload, an MMAF derivative linked to GIG (with Gl being the P1"-P2" motif, and the 3rdG being a spacer), by OaAEP1b-C247A, which has been shown to be able to process P1 -Asp substrates at neutral pH. So, to label the C-terminus of the antibody's heavy chains, 0.1 eq of OaAEP1 b-C247A and 0.01 eq of QC were added to a solution containing 100 eq of the labelling agent containing MMAF-OCH3 with a linker GIG in 20 mM PBS supplemented with 0.25 mM TCEP. QC was used to catalyze pyroglutamyl formation of the Gin residue in the released QL dipeptide, therefore overcoming the reversibility problem of the transpeptidative ligation reaction (Xia et al., J. Am. Chem. Soc. 2023). The second-step ligation reaction afforded the dual-payload ADC 3 in very good yields (Figure 24A). Excitingly, the dual-payload ADC product generated from the sequential ligation scheme exhibited excellent selectivity and potent activity towards BT474 (IC50 = 0.129 ± 0.02 nM, Figure 24C). In each step, the conjugation products were easily purified to homogeneity and analyzed using RP-HPLC and ESI-MS. These results firmly establish a novel PAL-based sequential ligation scheme for the preparation of dual-payload ADCs.

[0170] As shown in the above sequential ligation scheme, we can attach two different payloads at the N- and C-termini of an antibody using two PAL enzymes. Alternatively, a PAL-mediated ligation reaction can also be used in combination with a chemoselective conjugation method to introduce two different payloads onto an antibody. To demonstrate this, we used the well- established maleimide-thiol Micheal addition reaction as the chemoselective conjugation method. Again, trastuzumab was used as the model antibody. Of the 16 disulfide bonds in the antibody, the 4 interchain S-S bonds are more exposed and hence can be selectively reduced under well-controlled conditions to give the free Cys thiols for chemical conjugation. Therefore, at the first step, the antibody 4, which comprises light chains engineered to have a Gl dipeptide linked to the N-terminal of light chains through a spacer LS (SEQ ID NO: 39) and unmodified heavy chains (SEQ ID NO: 41 ), was treated with a certain amount of the reducing agent, TCEP, and subsequently treated with a maleimide-functionalized deruxtecan derivative (maleimidocaproyl-GGFG-aminomethoxy-Dxd), affording the single-payload ADC 5 with a DAR ~ 8. Next, ADC 5, which has a Gl dipeptide motif at the N-terminal ends of the light chains, was conjugated with MMAE-linker (Val-Cit-PABC)-CO-SCH2CONH2 using VyPAL2 to yield the desired dual-payload ADC 6 (Figure 25A). Specifically, a solution of the antibody 4 (20 pM) was first incubated with 5 equivalents of TCEP at 37 °C for 1 hour to reduce interchain disulfides. Subsequently, 10 equivalents of maleimide-linker-deruxtecan were added in the presence of 5 mM EDTA in HEPES buffer (pH 7.0). The conjugation was completed after 40 minutes, as shown by HPLC analysis on an aliquot of the reaction mixture which was reduced using 50 mM DTT before loading to HPLC column (Figure 25B). The resulting conjugate ADC5, predominantly with a drug-to-antibody ratio (DAR) of 8, was purified by size-exclusion chromatography (SEC). In the subsequent PAL conjugation step, 0.04 equivalent of VyPAL2 was added to a solution containing 50 equivalents of the linker-payload reagent (payload drug = MMAE) in 20 mM PBS (pH 7.0). After 2 hours of incubation, an aliquot of the reaction mixture was treated with 50 mM DTT and subjected to HPLC analysis, which showed completion of the ligation reaction (Figure 25B). The final dual-payload ADC6 was purified via SEC, analysed by ESI-MS (Figure 25C), and evaluated for their in vitro anticancer activity against MCF-7 and BT474 cell lines.

[0171] As expected, this dual-payload ADC exhibited potent and selective anti-cancer activity in cellbased assays, with an IC50 = 0.034 ± 0.010 nM against HER2-postive BT474 cells (Figure 25D).

[0172] Example 5. PAL-catalysed ligation reaction on antibodies

[0173] ADCs with two different kinds of payloads were also prepared based on a chemo conjugation method and a Asx-dependent PAL ligation method. As shown in Figure 26A, a first payload (Dxd) was conjugated to the antibody 7 ( trastuzumab comprising a NQL tripeptide linked to C-terminus of the light chains through a spacer SGGSGGSGS (SEQ ID NO: 42) and unmodified heavy chains (SEQ ID NO: 41 ) ) using a maleimide-thiol Micheal addition reaction, and then the obtained ADC8 was further conjugated to a second payload (MMAE) using a PLA-mediated ligation in presence of QC. Specifically, 20 pM of antibody 7 was treated with 5 eq of TCEP at 37 °C for 1 h. 10 eq of maleimidocaproyl-linker (GGFG tetrapeptidyl- aminomethoxy linkerj-deruxtecan was then added to the mixture in the presence of 5 mM EDTA in HEPES buffer at pH7. After 40 min, the mixture was reduced with 50 mM DTT and checked by HPLC. The ADC8 (DAR8) was purified by SEC (Size-Exclusion Chromatography). Subsequently, 5 (12 uM) was reacted with 50 eq of the linker-MMAE labeling reagent in the presence of 0.04 eq of VyPAL2 and 0.004 eq of QC in 20 mM PBS at pH7. After 1 h, the reaction mixture was treated with 50 mM DTT and the obtained ADC9 was analyzed by HPLC (Figure 26B). In vitro anticancer activity assay showed that ADC9 has potent and selective anti-cancer activity in cell-based assays, with an IC50 = 0.024 ± 0.010 nM against HER2- postive BT474 cells (Figure 26C).

[0174] Using the same method, ADC10-12 were prepared, wherein the first payload Dxd was conjugated to the Cys residue of the heavy chains and light chains, and the second payload (DM1 with a GIG linker for ADC10 and ADC12, and MMAE for ADC11 ) was conjugated to the C-terminus of either the heavy chains or the light chains (Figures 27A-C).

[0175] All three ADCs showed very strong activity towards BT474 cells. ICso for ADC10, ADC1 1 and ADC12 were 0.077 ± 0.010 nM, 0.041 ± 0.010 nM, 0.085 ± 0.010 nM respectively (Figures 28A-C). To sum up, the orthogonal bioconjugation methods described herein further demonstrate the usefulness of PALs as powerful tools for protein modification. And these methods hold great promise in the development of next-generation ADCs and other bioconjugates for disease

[0176] While the subject matter of this disclosure has been described and shown in considerable detail with reference to certain illustrative aspects, including various combinations and sub- combinations of features, those skilled in the art will readily appreciate other aspects and variations and modifications thereof as encompassed within the scope of the present disclosure. Moreover, the descriptions of such aspects, combinations, and sub-combinations is not intended to convey that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of this disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims. Bibliography

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Claims

1. Claims1 . A method of preparing a conjugate, said method comprising i) providing a first molecule comprising a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to the C-terminus of the first peptide and has the following formula I:, wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid; and iii) contacting a peptidyl asparaginyl ligase (PAL) with the first and second molecules to form a conjugate of the first and second peptides.

2. The method of claim 1 , wherein in the chemical group of formula I, n is any integer from 1 to 3.

3. The method of claim 1 or 2, wherein in the chemical group of formula I, X is N, and R is H or Gly or Ala.

4. The method of claim 1 or 2, wherein in the chemical group of formula I, X is O, and R is a C C6alkyl group.

5. The method of claim 1 or 2, wherein in the chemical group of formula I is selected from a group consisting of a sulfaneyl-acetamide, a methyl thioglycolate, a sulfanyl-ethanamide, a sulfanyl-propanamide, a sulfanyl-acetylglycinamide and a sulfanyl-acetylalaninamide.

6. The method of any one of claims 1 to 5, wherein the first peptide has a C-terminal residue attached to the sulfaneylacetamide group selected from the group consisting of Gly, Ala and Ser.

7. The method of any one of claims 1-6, wherein the first peptide has a penultimate residue at the C-terminus selected from the group consisting of Leu, Ala, Arg, Ser, Pro and Gly.

8. The method of any one of claims 1 to 7, wherein the first peptide has an N-terminal residue selected from the group consisting of Gly, Ala, Arg and Ser.

9. The method of any one of claims 1 to 8, wherein X1 is selected from the group consisting of Gly, Ser, Ala, Cys, Gin, His, Leu, Phe, Tyr.

10. The method of any one of claims 1 to 9, wherein X2 is selected from the group consisting of Leu, Phe, Tyr, Trp, Vai, and lie.11 . The method of any one of claims 1 to 10, wherein the X1 -X2 motif is Gly- He.

12. The method of any one of claims 1 to 11 , wherein the PAL is selected from a group consisting of butelase 1 , butelase 2, VyPAL2, VyPAL3, OaAEP1b-C247A, HeAEP3, AtLEGy, VuPALI , HaPALI , OaAEPI b and a functional fragment or a variant thereof.

13. The method of any one of claim 12, wherein the PAL is selected from a 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, OaAEPI b-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, AtLEGy comprising the amino acid sequence set forth in SEQ ID NO: 8, VuPALI comprising the amino acid sequence set forth in SEQ ID NO: 9, HaPALI comprising the amino acid sequence set forth in SEQ ID NO: 10, OaAEPI b comprising the amino acid sequence set forth in SEQ ID NO: 1 1 and a functional fragment or a variant thereof.

14. The method of any one of claims 1 to 13, wherein:(i) the first molecule further comprises a payload attached to the N-terminus of the first peptide, and / or the second peptide is an epitope-binding peptide; or(ii) the first peptide is an epitope-binding peptide, and / or the second molecule further comprises a payload attached to the C-terminus of the second peptide.

15. The method of claim 14, wherein the epitope-binding peptide is selected from the group consisting of an antibody or functional fragment thereof, an affibody, adnectin, anticalin, fynomer, Kunitz domain and designed ankyrin repeat protein (DARPin).

16. The method of claim 15, wherein the antibody or functional fragment thereof is selected from the group consisting of IgG, Fab, Fab’, F(ab’)2, Fv, scFv, diabody, minibody, and nanobody.

17. The method of claim 16, wherein the affibody is ZEGFR or ZEGFR-FC, and the DARPin is an anti-HER2 DARPin.

18. The method of claim 17, wherein the second peptide is an antibody comprising light chains and heavy chains, and the X1 -X2 motif is attached to the N-terminus of the light chain or the heavy chain.

19. The method of any one of claims 14 to 18, wherein the payload further comprises a payload-releasing linker.

20. The method of any one of claims 14 to 19, wherein the payload is an imaging agent or a therapeutic agent.21 . The method of claim 20, wherein the imaging agent is a reporter or a radioisotope linked to a chelator.

22. The method of claim 21 , wherein the imaging agent is a reporter selected from the group consisting of horseradish peroxidase (HRP), a fluorescent protein (such as GFP, RFP, YFP, CFP), chloramphenicol acetyltransferase (CAT), ere recombinase (Cre), LacZ, monomeric cherry (mCherry), biotin, a fluorescent dye (such as rhodamine, FAM).

23. The method of claim 21 , wherein the imaging agent is a radioisotope linked to a chelator, wherein the chelator is selected from the group consisting of 1 ,4,7,10-tetraazacyclododecane- 1 , 4, 7, 10-tetraacetic acid (DOT A), 1 ,4,7-triazacyclononane-triacetic acid (NOTA), 1 ,4,7- triazacyclononane-1 ,4,7-tris[methyl(2-carboxyethyl)phosphinic acid] (TRAP), desferrioxamine (DFO) and 1 ,4, 7-triazacyclononane-1 -glutaric acid-4, 7-diacetic acid (NODAGA); and / or the radioisotope is selected from a group consisting of18F,67Ga,68Ga,44Sc,47Sc,1 11In,64Cu,86Y,89Zr,177Lu, and225Ac.

24. The method of claim 20, wherein the therapeutic agent is Monomethyl auristatin E (MMAE), Dxd, DM1 , SN-38, PDB, PDB dimer, tubulysin, cryptophycin, eribulin, carmaphycin, Proteolysis Targeting Chimeras (PROTAC), monomethyl auristatin F (MMAF) or a therapeutic radioisotope such as177Lu,90Y,212Pb,225Ac, and213Bi.

25. The method of any one of claims 1 to 24, wherein the step iii) of contacting is performed at a pH selected in the range from 5 to 8.

26. The method of any one of claims 1 to 25, wherein the ligation in step iii) is Asn or Asp independent.

27. A method of producing a conjugate comprising two payloads, comprising: i) providing a first molecule comprising a first payload, a first peptide and a chemical group of formula I acting as an acyl donor, wherein the chemical group is attached to a C-terminal residue of the first peptide and has the following formula I:, wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide, a X1 -X2 motif acting as an acyl acceptor and a P1 -PT-P2’ tripeptide motif as a second acyl donor, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide and the P1 -PT-P2’ tripeptide motif is attached to the C-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid, wherein P1 is Asn or Asp, PT is Gly, Ser, Ala, Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid; iii) providing a third molecule comprising a second payload and a P1 "-P2" motif as an acyl acceptor, wherein P1 " is any amino acid and P2" is a hydrophobic amino acid or a p- branched amino acid; and iva) contacting a first peptidyl asparaginyl ligase (PAL) with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) in presence of a second PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload; orivb) contacting the second molecule of step ii) with the third molecule of step iii) in presence of a first PAL to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a second PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

28. The method of claim 27, wherein the first PAL and the second PAL are the same.

29. The method of claim 27, wherein the first PAL and the second PAL are different.

30. The method of any one of claims 27 to 29, wherein the first PAL and the second PAL are independently selected from a group consisting of butelase 1 , butelase 2, VyPAL2, VyPAL3, OaAEPI b-C247A, HeAEP3, AtLEGy, VuPALI , HaPALI , OaAEPI b and a functional fragment or a variant thereof.

31. The method claim 30, wherein the first PAL and the second PAL are independently selected from a 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, OaAEPI b-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, AtLEGy comprising the amino acid sequence set forth in SEQ ID NO: 8, VuPALI comprising the amino acid sequence set forth in SEQ ID NO: 9, HaPALI comprising the amino acid sequence set forth in SEQ ID NO: 10, OaAEPI b comprising the amino acid sequence set forth in SEQ ID NO: 1 1 and a functional fragment or a variant thereof.

32. The method of any one of claims 27 to 31 , wherein P1 " is selected from the group consisting of Gly, Ser, Ala, Gin, His, Cys.

33. The method of any one of claims 27 to 32, wherein P2’ and P2" are independently selected from the group consisting of Leu, Phe, Tyr, Trp, Vai, and lie, preferably lie.

34. The method of any one of claims 27 to 32, wherein in step iva), the first conjugate is contacted with the third molecule of step iii) in presence of the second PAL and a glutaminyl cyclase (QC) to form the second conjugate; in step ivb), wherein the second molecule of stepii) is contacted with the third molecule of step iii) in presence of a first PAL and a QC to form the first conjugate, and P1 ’of the second molecule is Gin or Glu.

35. The method of claim 34, wherein the QC is a human QC, a mouse QC, a drosophila QC, an Arabidopsis QC, a Conus QC, a sistrurus QC, a bacterial QC or a functional fragment or a variant thereof.

36. The method of claim 35, wherein the QC is selected from a group consisting of a human QC comprising the amino acid sequence set forth in SEQ ID NO: 12, a mouse QC comprising the amino acid sequence set forth in SEQ ID NO: 13, a Drosophila QC comprising the amino acid sequence set forth in SEQ ID NO: 14, an Arabidopsis QC comprising the amino acid sequence set forth in SEQ ID NO: 15, a Conus QC comprising the amino acid sequence set forth in SEQ ID NO: 16, a Sistrurus QC comprising the amino acid sequence set forth in SEQ ID NO: 17, a bacterial QC comprising the amino acid sequence set forth in SEQ ID NO: 18 and a functional fragment or a variant thereof.

37. A method of producing a conjugate comprising two payloads, comprising: i) providing a first molecule comprising a first payload, a first peptide and a chemical group acting as an acyl donor, wherein the chemical group is attached to a C-terminal residue of the first peptide and having formula I:, wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid; ii) providing a second molecule comprising a second peptide and a X1 -X2 motif acting as an acyl acceptor, wherein the second peptide comprises at least one cysteine thiol group for chemical conjugation with a maleimide-functionalized moiety, wherein the X1 -X2 motif is attached to the N-terminus of the second peptide, wherein X1 is any amino acid and X2 is a hydrophobic amino acid or a p-branched amino acid; iii) providing a third molecule comprising a second payload and a maleimide-functionalized moiety; iva) contacting a first peptidyl asparaginyl ligase (PAL) with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third moleculeof step iii) to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a first PAL to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

38. The method of any one of claims 27 to 37, wherein X1 is selected from the group consisting of Gly, Ser, Ala, Cys, Gin, His, Leu, Phe, Tyr.

39. The method of any one of claims 27 to 38, wherein X2 is selected from the group consisting of Leu, Phe, Tyr, Trp, Vai, and lie.

40. The method of any one of claims 27 to 39, wherein the X1-X2 motif is Gly-lle.41 . The method of any one of claims 27 to 40, wherein the second peptide is an epitopebinding peptide.

42. The method of claim 41 , wherein the epitope-binding peptide is selected from the group consisting of an antibody or functional fragment thereof, an affibody, and designed ankyrin repeat protein (DARPin).

43. The method of claim 42, wherein the antibody or functional fragment thereof is selected from the group consisting of IgG, Fab, Fab’, F(ab’)2, Fv, scFv, diabody, minibody, and nanobody.

44. The method of any one of claims 27 to 36, wherein the second peptide is an antibody comprising light chains and heavy chains, wherein(i) the X1 -X2 motif is attached to N-terminus of the light chain and the P1-PT-P2’ tripeptide motif is attached to C-terminus of the heavy chain, or(ii) the X1 -X2 motif is attached to N-terminus of the heavy chain and the P1-PT-P2’ tripeptide motif is attached to C-terminus of the light chain, or(iii) the X1 -X2 motif is attached to N-terminus of the light chain and the P1-PT-P2’ tripeptide motif is attached to C-terminus of the light chain, or(iv) the X1 -X2 motif is attached to N-terminus of the heavy chain and the P1 -PT-P2’ tripeptide motif is attached to C-terminus of the heavy chain.

45. The method of claim 37, wherein the second peptide is an antibody comprising light chains and heavy chains, wherein(i) the X1 -X2 motif is attached to N-terminus of the light chain and the heavy chain comprises the at least one cysteine thiol group, or(ii) the X1 -X2 motif is attached to N-terminus of the heavy chain and the light chain comprises the at least one cysteine thiol group, or(ill) the X1 -X2 motif is attached to N-terminus of the light chain and the light chain comprises the at least one cysteine thiol group, or(iv) the X1 -X2 motif is attached to N-terminus of the heavy chain and the heavy chain comprises the at least one cysteine thiol group.

46. The method of any one of claims 27 to 45, wherein the first payload and the second payload are the same.

47. The method of any one of claims 27 to 46, wherein the first payload and the second payload are different.

48. The method of any one of claims 27 to 47, wherein the first payload and / or the second payload further comprises a payload-releasing linker.

49. The method of any one of claims 27 to 48, wherein the first payload and / or the second payload is an imaging agent or a therapeutic agent.

50. The method of claim 49, wherein the imaging agent is a reporter or a radioisotope linked to a chelator.51 . The method of claim 50, wherein the imaging agent is a reporter selected from the group consisting of horseradish peroxidase (HRP), a fluorescent protein (such as GFP, RFP, YFP, CFP), chloramphenicol acetyltransferase (CAT), ere recombinase (Cre), LacZ, monomeric cherry (mCherry), biotin, a fluorescent dye (such as rhodamine, FAM).

52. The method of claim 51 , wherein the imaging agent is a radioisotope linked to a chelator, wherein the chelator is selected from the group consisting of 1 ,4,7,1 O-tetraazacyclododecane- 1 , 4, 7, 10-tetraacetic acid (DOT A), 1 ,4,7-triazacyclononane-triacetic acid (NOTA), 1 ,4,7- triazacyclononane-1 ,4,7-tris[methyl(2-carboxyethyl)phosphinic acid] (TRAP), desferrioxamine (DFO) and 1 ,4, 7-triazacyclononane-1 -glutaric acid-4, 7-diacetic acid (NODAGA); and / or theradioisotope is selected from a group consisting of18F,67Ga,68Ga,44Sc,47Sc,1 11In,64Cu,86Y,89Zr,177Lu, and225Ac.

53. The method of claim 49, wherein the therapeutic agent is Monomethyl auristatin E (MMAE), Dxd, DM1 , SN-38, PDB, PDB dimer, tubulysin, cryptophycin, eribulin, carmaphycin,Proteolysis Targeting Chimeras (PROTAC), monomethyl auristatin F (MMAF) or a therapeutic radioisotope such as177Lu,90Y,212Pb,225Ac, and213Bi.

54. A conjugate produced by the method of any one of claims 1 to 53.

55. A conjugate comprising an antibody trastuzumab and MMAE, wherein the antibody has aGl dipeptide at the N-terminus of the light chains linked by a spacer LS and a DQL tripeptide at the C-terminus of the heavy chains, wherein the MMAE is conjugated to the antibody through the Gl dipeptide.

56. A conjugate comprising an antibody trastuzumab, MMAE and MMAF, wherein the antibody has a Gl dipeptide at the N-terminus of the light chains linked by a spacer LS and a DQL tripeptide at the C-terminus of the heavy chains, wherein the MMAE is conjugated to the antibody through the Gl dipeptide, wherein the MMAF is conjugated to the antibody through the DQL tripeptide.

57. A molecule comprising a peptide and a chemical group, wherein the chemical group is attached to a C-terminal residue of the peptide and having formula I:, wherein n is any integer from 1 to 6, X is N or O, R is H, a Ci-Ce alkyl group or an amino acid.

58. The molecule of claim 57, wherein in the chemical group of formula I, n is any integer from 1 to 3.

59. The molecule of claim 57 or 58, wherein in the chemical group of formula I, X is N, and R is H or Gly or Ala.

60. The molecule of claim 57 or 58, wherein in the chemical group of formula I, X is O, and R is a Ci-Ce alkyl group.61 . The molecule of claim 57 or 58, wherein in the chemical group of formula I is selected from a group consisting of a sulfaneyl-acetamide, a methyl thioglycolate, a sulfanyl-ethanamide, a sulfanyl-propanamide, a sulfanyl-acetylglycinamide and a sulfanyl-acetylalaninamide.

62. The molecule of any one of claims 57 to 61 , further comprising a payload attached to N- terminus of the peptide.

63. The molecule of claim 62, wherein the payload further comprises a payload-releasing linker.

64. The molecule of claim 62 or 63, wherein the payload is an imaging agent or a therapeutic agent.

65. The molecule of claim 64, wherein the imaging agent is a radioisotope linked to a chelator or a reporter.

66. The molecule of claim 65, wherein the imaging agent is a reporter selected from the group consisting of horseradish peroxidase (HRP), a fluorescent protein (such as GFP, RFP, YFP, CFP), chloramphenicol acetyltransferase (CAT), ere recombinase (Cre), LacZ, monomeric cherry (mCherry), biotin, a fluorescent dye (such as rhodamine, FAM).

67. The molecule of claim 65, wherein the imaging agent is a radioisotope linked to a chelator, wherein the chelator is selected from the group consisting of 1 ,4,7,1 O-tetraazacyclododecane- 1 , 4, 7, 10-tetraacetic acid (DOT A), 1 ,4,7-triazacyclononane-triacetic acid (NOTA), 1 ,4,7- triazacyclononane-1 ,4,7-tris[methyl(2-carboxyethyl)phosphinic acid] (TRAP), desferrioxamine (DFO) and 1 ,4, 7-triazacyclononane-1 -glutaric acid-4, 7-diacetic acid (NODAGA); and / or the radioisotope is selected from a group consisting of18F,67Ga,68Ga,44Sc,47Sc,1 11In,64Cu,86Y,89Zr,177Lu, and225Ac.

68. The molecule of claim 64, wherein the therapeutic agent is Monomethyl auristatin E (MMAE), Dxd, DM1 , SN-38, PDB, PDB dimer, tubulysin, cryptophycin, eribulin, carmaphycin, Proteolysis Targeting Chimeras (PROTAC), monomethyl auristatin F (MMAF) or a therapeutic radioisotope such as177Lu,90Y,212Pb,225Ac, and213Bi.

69. A method of producing a conjugate comprising two payloads, comprising :i) providing a first molecule comprising a second payload and a P1"-P2" motif as an acyl acceptor, wherein P1 " is any amino acid and P2" is a hydrophobic amino acid or a p-branched amino acid; ii) providing a second molecule comprising a second peptide and a P1-PT-P2’ tripeptide motif as a acyl donor, the P1 -P1’-P2’ tripeptide motif is attached to the C-terminus of the second peptide, wherein the second peptide comprises at least one cysteine thiol group for chemical conjugation with a maleimide-functionalized moiety, wherein P1 is Asn or Asp, P1 ’ is Gly, Ser, Ala, Gin or Glu and P2' is a hydrophobic amino acid or a p-branched amino acid; iii) providing a third molecule comprising a second payload and a maleimide-functionalized moiety; and iva) contacting a peptidyl asparaginyl ligase (PAL) and a QC with the first molecule of step i) and the second molecule of step ii) to form a first conjugate of the first and second peptides comprising the first payload, and then contacting the first conjugate with the third molecule of step iii) to form a second conjugate of the first and second peptides comprising the first payload and the second payload; or ivb) contacting the second molecule of step ii) with the third molecule of step iii) to form a first conjugate of the second peptide comprising the second payload, and then contacting the first conjugate with the first molecule of step i) in presence of a PAL and a QC to form a second conjugate of the first and second peptides comprising the first payload and the second payload.

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