Glycosynthase variants for the engineering of antibody-drug conjugates

Abstract

This disclosure relates to novel glycosynthase enzymes for the engineering of antibody-drug conjugates (ADCs). The enzyme variants, designated EndoSd-D232M and EndoSz-D234M, contain glycan conjugation and / or modification activity at the conserved N297 glycosylation site in the Fc region of exemplary antibodies. The glycosynthase activity of EndoSd-D232M and EndoSz-D234M was demonstrated to be applicable to various mAbs targeting various receptors, including, but not limited to, Globo H, SSEA-4, and SSEA-3 series receptors (OBI-888; Globo H ganglioside), Herceptin (Her 2 receptor), Perjeta (Her 2 receptor), and Vectibix (EGFR receptor). Both mAb-GlcNAc and mAb-GlucNAc(F) were found to be suitable substrates for EndoSd-D232M and EndoSz-D234M.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 382,951, filed November 9, 2022, the entirety of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically under Rule ST.26 in ASCII XML format and is incorporated herein by reference in its entirety. The ASCII copy was created on September 21, 2023, is named G3004-01800PCT_20230921_SequenceListing.xml, and is 12,350 bytes in size.

[0003] The present disclosure relates to glycosylated antibody conjugates and methods for preparing the same. [Background technology]

[0004] Numerous surface carbohydrates are expressed on malignant tumor cells. For example, the carbohydrate antigen Globo H (Fucα1→2 Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc) was first isolated and identified as a ceramide-linked glycolipid from breast cancer MCF-7 cells in 1984 (Bremer EG et al. (1984) J Biol Chem 259:14773-14777). Previous studies have also shown that Globo H and stage-specific embryonic antigen 3 (Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glcβ1) (SSEA-3, also known as Gb5) are observed on breast cancer cells and breast cancer stem cells (WW Chang et al. (2008) Proc Natl Acad Sci USA 105(33):11667-11672). Furthermore, SSEA-4 (stage-specific embryonic antigen-4) (Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1) is commonly used as a cell surface marker for pluripotent human embryonic stem cells and has been used to isolate mesenchymal stem cells and enrich for neural progenitor cells (Kannagi et al. (1983) EMBO J, 2:2355-2361). These findings suggest that the Globo series of antigens (Globo H, SSEA-3, and SSEA-4) are unique targets for cancer therapy and may be used as direct therapeutic agents that effectively target cancer cells.

[0005] Programmed death 1 (PD-1) is an inhibitory receptor expressed on T cells, B cells, and monocytes (Ishida et al. (1992) EMBO J. 11:3887-2895; Agata et al. (1996) Int. Immunol. 8:765-772). PD-L1 and PD-L2 are ligands of PD-1 that have been identified to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8). Engagement of PD-1 with PD-L1 or PD-L2 results in downregulation of immune responses. Therefore, blocking the PD-1 or PD-L1 antigen pathway has been proposed to attenuate central and peripheral immune responses against cancer. Targeting the PD-1 and PD-L1 antigen pathways has been shown to have clinical efficacy in over 15 cancer types, including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, and Hodgkin's lymphoma (Sharma et al. (2015) Science 348(6230):56-61). However, many patients still do not successfully respond to PD-1 or PD-L1 antigen therapy. In some cases, patients initially respond but develop resistance over time. Therefore, there is an urgent need to identify the mechanisms of resistance to PD-1 or PD-L1 antigen combination therapy.

[0006] Therapeutic monoclonal antibodies (mAbs) have been developed for the treatment of many diseases, such as cancer, autoimmune diseases, and infections (Adams, GP and Weiner, LM (2005) Nat. Biotechnol. 23:1147-1157; Aggarwal, SR (2012) Nat. Biotechnol. 30:1191-1197; Aggarwal, SR (2014) Nat. Biotechnol. 32:323-330). Several mAbs have already been approved for cancer therapy. These mAbs recognize specific biomarkers on the surface of tumor cells and enhance cellular apoptosis by various mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), or by blocking signaling pathways. The Her2 receptor is the most well-known biomarker overexpressed in breast cancer, leading to the development of two related mAbs by Roche: Herceptin (trastuzumab) and Perjeta (pertuzumab). The EGFR receptor is also a well-known cancer target for the development of mAbs. For example, Vectibix (panitumumab) and Erbitux (cetuximab), targeted at the treatment of metastatic colorectal cancer, were developed by Amgen and Merck, respectively. Furthermore, Rituxan (rituximab, Roche) and Arzerra (ofatumumab, GSK), designed to recognize the CD20 receptor, are commonly used to treat non-Hodgkin's B lymphoma and chronic lymphocytic leukemia. Recently, OBI Pharma. Inc. has developed the OBI-888 and OBI-898 antibodies based on the ganglioside biomarkers Globo H and SSEA-4, which are found in breast, lung, ovary, stomach, and small cell lung (Hakomori, S. I. (2008) Biochim. Biophys. Acta. 1780:325-346; Hakomori, S. and Zhang, Y. (1997) Chem. Biol. 4:97-104; Zhang, S. et al. (1997) Int. J. Cancer 73:42-49; Zhang, S. et al. (1997) Int. J. Cancer 73:50-56), but are not detectable in normal cells.Erbitux, Rituxan, Arzerra, OBI-888, and OBI-898 kill cancer cells through cytotoxicity associated with ADCC and CDC. Furthermore, several mAbs have been developed to block protein-protein interactions. For example, Humira (adalimumab, AbbVie) blocks the signaling pathway mediated by the TNF-α receptor in rheumatoid arthritis, an autoimmune disease. Keytruda (pembrolizumab, Merck) blocks the PD-1 receptor, disrupting the protective mechanisms of cancer cells and treating metastatic melanoma. Compared with small molecule drugs, mAbs have greater specificity for target cells and cause fewer side effects in patients. These two important features make them powerful tools for treating a variety of diseases.

[0007] Monoclonal antibodies (mAbs) have a molecular weight of approximately 150 kDa and consist of two heavy chains (approximately 50 kDa) and two light chains (approximately 25 kDa), which form three domains separated by a flexible hinge region. The two Fab domains contain variable complementarity-determining regions (CDRs) that identify antigens. The Fc domain is a constant region containing N-glycans that mediate cytotoxicity through ADCC and CDC (Jefferis, R. (2009) Nat. Rev. Drug Discoy. 8:226-234). Amino acid N297 in the Fc domain is a conserved N-glycosylation site that attaches different glycan types, such as biantennary (M3, GOF, GIF, G2F, GO, G1, and G2 complex types) and triantennary (high-mannose and hybrid types), when expressed in various cell lines. X-ray structural analysis showed that the fucose in the core of the Fc glycan interferes with specific carbohydrate-carbohydrate interactions between Fc and FcγRIIIa, reducing the binding constant by approximately one hundred-fold (Ferrara, C. et al. (2011) Proc. Natl. Acad. Sci. USA 108:12669-12674), and decreasing cell-killing efficiency. The most common cell line used in the biopharmaceutical industry is CHO cells. CHO cells generally produce mAbs containing glycan compositions primarily of the G0F, G1F, and G2F forms. These glycan forms limit the ADCC activity of mAbs due to the reduced ADCC binding efficiency caused by fucose. To reduce the glycan complexity of mAbs, engineered CHO cell lines with FUT8 (α-1,6-fucosyltransferase 8) gene knockout (Yamane-Ohnuki, N. and Satoh, M. (2009) MAbs, 1:230-236; Yamane-Ohnuki, N. et al. (2004) Biotechnol. Bioeng. 87:614-622) or upregulation of the bisecting GlcNAc (N-acetylglycosamine) transferase GnT-III (Umana, P. et al. (1999) Nat. Biotechnol. 17:176-180) are now available, but there is still great interest in finding better and more general methodologies for obtaining desired N-glycans in mAbs.Furthermore, it has been reported that removal of Fc glycans results in loss of ADCC activity (Kurogochi, M. et al. (2015) PLoS One 10:e0132848). All these data suggest that the cytotoxicity of mAbs can be effectively controlled by the type of N-glycans attached to the Fc region.

[0008] Enzymatic modification of the Fc region is a solution to establishing a homogeneous mAb. Lai-Xi Wang and coworkers attempted chemoenzymatic remodeling by removing mixed glycans and conjugating homogeneous glycans (Huang, W. et al. (2012) J. Am. Chem. Soc. 134:12308-12318). Several endo-β-N-acetylglycosaminidases (ENGases) have also been reported to remove mixed glycans on mAbs. For example, EndoD (Tai, T. et al. (1975) J. Biol. Chem. 250: 8569-8575), EndoH (Tarentino, A. L. et al. (1974) J. Biol. Chem. 249: 818-824), EndoLL (Kurogochi, M. et al. (2015) PLoS One 10: e0132848), and EndoM (Kadowaki, S. et al. (1990) Agric. Biol. Chem. 54: 97-106) can hydrolyze high-mannose or terminal-mannose glycans. EndoS (Collin M and Olsen A. (2001) EMBO J. 20:3046-3055) and EndoSd (Shadnezhad, A. et al. (2016) Future Microbiol 11:721-736) are capable of hydrolyzing both nonfucosylated and fucosylated N-glycans on the Fc domain, but not high-mannose N-glycans. Currently, no single endoglycosidase can completely hydrolyze all glycan types on mAbs. The crystal structure of EndoS was recently elucidated, revealing that it contains five functional domains (Trastoy, B. et al. (2014) Proc. Natl. Acad. Sci. USA 111:6714-6719), among which the endoglycosidase domain is highly conserved with a rigid β-barrel structure suitable for site-directed mutagenesis studies. Meanwhile, glycosynthases for antibody Fc have also been reported.EndoD-N322Q (Fan, SQ et al. (2012) J. Biol. Chem. 287:11272-11281) and EndoM-N175Q (Umekawa, M., Li, C. et al. (2010) J. Biol. Chem. 285:511-521) only transferred short complex-type N-glycans to the Fc domain. Endo-F3-D165Q (Giddens, JP et al. (2016) J. Biol. Chem. 291:9356-9370) only transferred glycans to the fucosylated Fc domain. EndoS-D233Q (Huang, W. et al. (2012) J. Am. Chem. Soc. 134:12308-12318) allows for the conjugation of various biantennary complex types, while EndoS2-D184M (Li, T., Tong et al. (2016) J. Biol. Chem. 291:16508-16518) has wild-type substrates including complex, high-mannose, and hybrid types.

[0009] While the use of antibody-drug conjugates (ADCs) for the local delivery of cytotoxic or cytostatic drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) allows for targeted delivery of the drug moiety to the tumor and its intracellular accumulation, systemic administration of unconjugated cytotoxic or cytostatic drugs can result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet (Mar. 15, 1986):603-05; Monoclonal Antibodies '84: Biological And Clinical (Thorpe, 1985, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Cancer Immunology, Cancer Applications, A. Pinchera et al. (eds.), pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, supra). Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 4,975,278 [Non-patent literature]

[0011] [Non-licensed Document 1] Bremer EGら(1984)J Biol Chem 259:14773~14777 pages [Non-licensed Document 2] WW Changら(2008)Proc Natl Acad Sci USA,105(33):11667~11672 pages [Non-licensed Document 3] Kannagiら(1983)EMBO J,2:2355~2361 pages [Non-licensed Document 4] Ishida(1992)EMBO J.11:3887~2895 pages [Non-licensed Document 5] Agataら(1996)Int.Immunol.8:765~772 pages [Non-licensed Document 6] Freemanら(2000)J Exp Med 192:1027~34 pages [Non-licensed Document 7] Latchmanら(2001)Nat Immunol 2:261~8 pages [Non-licensed Document 8] Sharmaら(2015)Science 348(6230): pages 56~61 [Non-licensed Document 9] Adams, GP and Weiner, LM (2005) Nat. Biotechnol. 23: 1147~1157 pages [Non-licensed Document 10] Aggarwal,SR(2012)Nat.Biotechnol.30:1191~1197 pages [Non-licensed Document 11] Aggarwal,SR(2014)Nat.Biotechnol.32:323~330 pages [Non-licensed Document 12] Hakomori,SI(2008)Biochim.Biophys.Acta.1780:325~346 pages [Non-licensed Document 13] Hakomori, S. and Zhang, Y. (1997) Chem. Biol. 4: 97~104 pages [Non-licensed Document 14] Zhang,S.ら(1997)Int.J.Cancer 73:42~49 pages [Non-licensed Document 15] Zhang,S.ら(1997)Int.J.Cancer 73:50~56 pages [Non-licensed Document 16] Jefferis,R.(2009)Nat.Rev.Drug Discoy.8:226~234 pages [Non-licensed Document 17] Ferrara, C. (2011) Proc.Natl.Acad.Sci.USA 108:12669~12674 pages [Non-licensed Document 18] Yamane-Ohnuki, N. and Satoh, M. (2009) MAbs, 1: 230-236 [Non-licensed Document 19] Yamane-Ohnuki,N.ら(2004)Biotechnol.Bioeng.87:614~622 pages [Non-licensed Document 20] Umana,P.ら(1999)Nat.Biotechnol.17:176~180 pages [Non-licensed Document 21] Kurogochi,M.ら(2015)PLoS One 10:e0132848 [Non-licensed Document 22] Huang,W.ら(2012)J.Am.Chem.Soc.134:12308~12318 pages [Non-licensed Document 23] Tai, T.ら(1975)J.Biol.Chem.250:8569~8575 pages [Non-licensed Document 24] Tarentino, ALら(1974)J.Biol.Chem.249:818~824 pages [Non-licensed Document 25] Kadowaki, S. (1990) Agric. Biol. Chem. 54: 97-106 [Non-licensed Document 26] Collin M and Olsen A. (2001) EMBO J. 20: 3046~3055 [Non-licensed Document 27] Shadnezhad,A.(2016)Future Microbiol 11:721~736 pages [Non-licensed Document 28] Trastoy, B. (2014) Proc.Natl.Acad.Sci.USA 111:6714~6719 pages [Non-licensed Document 29] Fan,SQら(2012)J.Biol.Chem.287:11272~11281 pages [Non-licensed Document 30] Umekawa,M.,Li,C.ら(2010)J.Biol.Chem.285:511~521 pages [Non-licensed Document 31] Giddens, JP (2016) J. Biol. Chem. 291: 9356~9370 pages [Non-licensed Document 32] Li.,T.,Tongら(2016)J.Biol.Chem.291:16508~16518 pages [Non-licensed Document 33] Syrigos and Epenetos (1999) Anticancer Research 19:605~614 pages [Non-licensed Document 34] Niculescu-Duvaz and びSpringer (1997) Adv.Drg.Del.Rev.26:151~172 pages [Non-licensed Document 35] Baldwin, 1986, Lancet (Mar.15, 1986): pages 603~05 [Non-licensed Document 36] Thorpe, 1985, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506. [Non-Patent Document 37] Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87 Summary of the Invention

[0012] In one aspect, the disclosure provides a method for preparing an engineered bioconjugate, comprising contacting a biomolecule with a glycosynthase and a modified glycan, thereby obtaining a first engineered bioconjugate, wherein the biomolecule further comprises an N-linked initial glycan; the glycosynthase comprises SEQ ID NO:1 or SEQ ID NO:2; the glycosynthase comprises a mutation located within residues 176-186, 225-237, or 273-289 of SEQ ID NO:1, or within residues 178-188, 227-239, or 275-291 of SEQ ID NO:2; the modified glycan comprises a substrate moiety and a first reactive moiety, wherein the substrate moiety is configured to react with the glycosynthase; and the biomolecule comprises an antibody or antigen-binding fragment thereof, a protein, or a peptide.

[0013] In another aspect, the present disclosure provides an engineered bioconjugate comprising a biomolecule and a modified glycan linked to the biomolecule, wherein the modified glycan comprises (i) a first polyethylene glycol (PEG) moiety and (ii) a first reactive moiety or a resultant moiety derived from a bioorthogonal reaction, wherein the resultant moiety comprises a triazole moiety, a DBCO-derived moiety, or a maleimide-derived moiety, and the biomolecule comprises an antibody or antigen-binding fragment thereof, a protein, or a peptide.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a plurality of engineered bioconjugates of the present disclosure and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides a method of treating cancer, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of the present disclosure.

[0016] In another aspect, the present disclosure provides: CLD a therapeutic conjugate comprising the formula: where C is a reactive moiety configured to react in a bioorthogonal reaction, L is a linker unit comprising a hydrophilic moiety, a cleavable moiety, and a spacer, and D is a therapeutic agent. [Brief explanation of the drawings]

[0017] [Figure 1-1] Figure 1 summarizes aspects of the overall process of the homogeneous platform. (A) The mAb was heterogeneous, containing a mixture of glycans. The glycan mixture was removed using wild-type EndoSz and α-fucosidase to generate mAb-GlcNAc. EndoSd-D232M and EndoSz-D234M were then used to conjugate the glycan-oxazoline, producing a homogeneous mAb. [Figure 1-2] (B) The glycan cleavage step uses only EndoSz enzyme to generate mAb-GlcNAc-F. After conjugation, the product is homogeneous mAb-glycan-F. [Figure 1-3] (C) An exemplary diagram of a biantennary glycan. [Figure 2]LC / MS / MS results of Herceptin-GlcNAc glycopeptide. Wild-type Herceptin was mixed with wild-type EndoSz and α-fucosidase to remove the glycans on the Fc region. The results showed that all of the glycans were removed, yielding over 99% Herceptin-GlcNAc. [Figure 3] An exemplary detection method for the homogeneous platform (shown by EndoSz-D234M). (A) HPLC analysis. Original Herceptin (green) had a retention time of 12.5 min, and Herceptin-GlcNAc (magenta) migrated to 11.4 min. During transglycosylation of NSCT-oxa, Herceptin-G2S2 (blue) could be clearly distinguished from 1N-G2S2 (semi-glycosylated) and 2N-G2S2 (fully glycosylated), which had retention times of 12.6 and 13.9 min, respectively. During the transglycosylation process, the peaks migrated sequentially from Herceptin-GlcNAc to Herceptin-1N-G2S2 and Herceptin-2N-G2S2, allowing the process to be monitored. (B) Comparative SDS-PAGE results. The semi-glycosylated and fully glycosylated Herceptin-G2S2 could not be readily identified. [Figure 4-1] Multiple sequence alignment of exemplary EndoS2, EndoS, EndoSz, and EndoSd, with the loop surrounding the active site labeled in light blue. [Figure 4-2] Multiple sequence alignment of exemplary EndoS2, EndoS, EndoSz, and EndoSd, with the loop surrounding the active site labeled in light blue. [Figure 5]Transglycosylation results over time. (A) Transglycosylation of NSCT-oxa and Herceptin-GlcNAc at a molar ratio of 20:1 with EndoSz-D234M. (B) EndoSd-D232M was used at a higher molar ratio of 150:1 (NSCT-oxa:Herceptin-GlcNAc). The reaction started with 100% Herceptin-GlcNAc. The percentages of Herceptin-1N-G2S2 and Herceptin-2N-G2S2 formed according to efficiency. With both enzymes, transglycosylation efficiencies of Herceptin-2N-G2S2 of over 90% could be reached. [Figure 6] Relative transglycosylation activity of various exemplary mutants. (A) EndoSz, (B) EndoSd. [Figure 7] Demonstration of homogeneous mAb efficacy. ADCC assay results. (A) ADCC results for Herceptin and Herceptin-G2S2. The data showed that Herceptin had higher ADCC activity. The EC50 values ​​for Herceptin and Herceptin-G2S2 were 15.29 (μg / mL) and 5.10 (μg / mL), respectively. (B) Summary of transglycosylation efficiency and ADCC for various mAbs. [Figure 8] Overall structure of apoEndoSz. (A) The glycosidase (red), leucine-rich repeat (yellow), hybrid lg (light blue), carbohydrate-binding motif (orange), and C-3HB (purple) domains are shown as cartoons. (B) Top and side views of the electrostatic surface are shown. (C) Diagram of the conserved surfaces of EndoSz. [Figure 9]Structure of the bound complex biantennary glycan in EndoSz-D234M. (A) The 2Fo-Fc electron density map contoured at 1.0σ is shown as a blue mesh. (B) Structure of the bound N-glycan (gray stick) within the β-barrel adjacent to the annotated loop. EndoSz-D234M is shown as transparent gray. (C) Cartoon representation of the substrate and EndoSz-D234M, complex N-linked glycan. The product is shown within the dashed rectangle. (D) Variable loops (green) in the conservation plot are labeled. Loop 1 (red ribbon), loop 2 (orange ribbon), loop 3 (yellow ribbon), loop 4 (green ribbon), loop 5 (blue ribbon), loop 6 (cyan ribbon), loop 7 (purple ribbon), and loop 8 (wheat ribbon) surrounding the active site are labeled. The α(1-6) antenna of the CT N-glycan is labeled. [Figure 10] Sugar substrate selectivity is dominated by loop 4 between EndoSz and EndoS2. (A) Bilayer structures of EndoSz / CT-N-glycan and EndoS2 / HM-N-glycan. The tilted helix 3 (labeled H3) of EndoSz is labeled, which structurally leads to a large deformation of loop 4 that interferes with HM-N-glycan binding. Loop 4 is indicated by a red arrow. EndoSz / CT-N-glycan (green) and EndoS2 / HM-N-glycan (blue) are colored, respectively. (B) Bilayer structures of EndoS2 / HM-N-glycan and unbound EndoS2. EndoS2 / HM-N-glycan (blue) and unbound EndoS2 (wheat) are colored, respectively. [Figure 11]The movement of loop 2 forms two binding grooves after N-glycan binding. (A) 3D view of the stacked structures of apo- and holo-EndoSzD234M. Apo-EndoSz-D234M (blue) and holo-EndoSz-D234M (gold) are shown as cartoons. The bound glycan in holo-EndoSz-D234M is shown as balls and sticks. The key residue W154 (stick) and the interacting Man(-2) and NAG(-8) are labeled. (B) Electrostatic surfaces of the unbound (left) and N-glycan-bound (right) EndoSz-D234M structures. W154 is circled in black. (C) Sequence alignment and conservation plot of loop 2 of EndoSz-D234M. [Figure 12-1] 2D diagram of EndoSz-D234M-sugar interactions. (A) Conformation A of GlcNAc(-1). (B) Conformation B of GlcNAc. The bound CT N-glycan is shown as a purple stick. Interacting residues of EndoSz through hydrogen bonds (orange) and hydrophobic interactions (red) are shown as orange sticks and red eyes, respectively. Hydrogen-bonded residues (blue) and residues with hydrophobic contacts (black) are labeled. [Figure 12-2] 2D diagram of EndoSz-D234M-sugar interactions. (A) Conformation A of GlcNAc(-1). (B) Conformation B of GlcNAc. The bound CT N-glycan is shown as a purple stick. Interacting residues of EndoSz through hydrogen bonds (orange) and hydrophobic interactions (red) are shown as orange sticks and red eyes, respectively. Hydrogen-bonded residues (blue) and residues with hydrophobic contacts (black) are labeled. [Figure 13-1] SDS-PAGE and CE-SDS results of the production of mAb-(NSCT-di-N3)2. (A) SDS-PAGE of R4702 (anti-TROP2 mAb). B) SDS-PAGE of TX05 (anti-H,ER2 mAb). [Figure 13-2] SDS-PAGE and CE-SDS results of the production of mAb-(NSCT-di-N3)2. (C) CE-SDS result analysis. [Figure 14]Results of MW analysis using unmodified MS. [Figure 15-1] LC and HC MS spectra of ADC-1 and ADC-2 incubated with HSA on days 0 and 6 for (A) R4702-MCCA-ADC (ADC-1) and (B) R4702-DBCO-ADC (ADC-2) at days 0 and 6. (C) Profiles of DAR changes. LC0, LC1, HC0, HC1, HC2, and HC3 represent the spectra of light and heavy chains carrying 0, 1, 2, and 3 payloads, respectively. [Figure 15-2] LC and HC MS spectra of ADC-1 and ADC-2 incubated with HSA on days 0 and 6 for (A) R4702-MCCA-ADC (ADC-1) and (B) R4702-DBCO-ADC (ADC-2) at days 0 and 6. (C) Profiles of DAR changes. LC0, LC1, HC0, HC1, HC2, and HC3 represent the spectra of light and heavy chains carrying 0, 1, 2, and 3 payloads, respectively. [Figure 15-3] LC and HC MS spectra of ADC-1 and ADC-2 incubated with HSA on days 0 and 6 for (A) R4702-MCCA-ADC (ADC-1) and (B) R4702-DBCO-ADC (ADC-2) at days 0 and 6. (C) Profiles of DAR changes. LC0, LC1, HC0, HC1, HC2, and HC3 represent the spectra of light and heavy chains carrying 0, 1, 2, and 3 payloads, respectively. [Figure 16] Payload release over time for R4702-MCCA-ADC (ADC-1) and R4702-DBCO-ADC (ADC-2) in human plasma. [Figure 17]Cytotoxicity assays of human tumor cell lines. Serial dilutions of the ADCs were used to evaluate the in vitro cytotoxic efficacy in several human tumor cell lines. Tumor cells were cultured with the ADCs for 6 days, and surviving cells were analyzed using CellTiter-Glo®. The IC50 of each ADC was calculated using Prism. (A) The efficacy of R4702-MCCA-ADC (ADC-1) and R4702-DBCO-ADC (ADC-2) was evaluated using NCI-H1975-C797S (human lung cancer) and DU-145 (human prostate cancer) tumor cells. (B) The efficacy of TX05-MCCA-ADC (ADC-3) and TX05-DBCO-ADC (ADC-4) was evaluated using NCI-N87 (human gastric cancer) and Capan-1 (human pancreatic cancer) tumor cells. [Figure 18-1] In vivo efficacy was evaluated in the NCI-H1975-C797S human lung cancer xenograft mouse model. NCI-H1975-C797S cells were cultured and implanted subcutaneously into the right flank of BALB / c nude mice. When the mean tumor volume reached 150-200 mm3, tumor-bearing mice were treated with a single dose of 10 or 3 mg / kg. Tumor volume (A) and body weight (B) were monitored twice weekly until day 22. Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1-(Ti-T1) / (Ci-C1)] × 100%. Ti and Ci represent the mean tumor volumes of the treated and vehicle groups at the end of the study (day 22), and T1 and C1 represent the mean tumor volumes of the treated and vehicle groups at the start of test administration. [Figure 18-2]In vivo efficacy was evaluated in the NCI-H1975-C797S human lung cancer xenograft mouse model. NCI-H1975-C797S cells were cultured and implanted subcutaneously into the right flank of BALB / c nude mice. When the mean tumor volume reached 150-200 mm3, tumor-bearing mice were treated with a single dose of 10 or 3 mg / kg. Tumor volume (A) and body weight (B) were monitored twice weekly until day 22. Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) = [1-(Ti-T1) / (Ci-C1)] × 100%. Ti and Ci represent the mean tumor volumes of the treated and vehicle groups at the end of the study (day 22), and T1 and C1 represent the mean tumor volumes of the treated and vehicle groups at the start of test administration. DETAILED DESCRIPTION OF THE INVENTION

[0018] Abbreviation ACN: acetonitrile, ADCC: antibody-dependent cellular cytotoxicity, DBCO: dibenzocyclooctyne, CDC: complement-dependent cytotoxicity, CDR: complementarity-determining region, FA: formic acid, FUT8: α-1,6-fucosyltransferase 8, GlcNAc: N-acetylglycosamine, HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol, IPTG: isopropyl-β-D-thiogalactopyranoside, TFA: trifluoroacetic acid, DMSO: dimethyl sulfoxide, NaOAc: sodium acetate, NaOH: sodium hydroxide sodium, HIC: hydrophilic interaction chromatography, ADC: antibody-drug conjugate, mAb: monoclonal antibody, ISTD: internal standard, HSA: human serum albumin, PBS: phosphate buffered saline, HC: heavy chain, LC: light chain, DAR: drug-antibody ratio, HRMS: high-resolution mass spectrometer, DL: drug linker, NSCT: sialylated complex N-glycan, ADC-1: R4702-MCCA-ADC, ADC-2: R4702-DBCO-ADC, ADC-3: TX05-MCCA-ADC, ADC-4: TX05-MCCA-ADC.

[0019] Antibody-drug conjugates (ADCs) are a promising approach for delivering therapeutic agents or imaging agents to target cells with reliable specificity and efficiency. Conjugation of payloads (e.g., therapeutic agents or imaging agents) to antibodies typically occurs at lysine or cysteine ​​residues on the antibody. Therefore, the drug-antibody ratio (DAR) of an ADC is determined according to the amount of lysine or cysteine ​​residues on the antibody. However, in practice, not all antibodies are conjugated identically in a conjugation reaction, resulting in ADC isomers with different DARs and conjugation sites. Furthermore, conjugation via lysine or cysteine ​​residues requires the use of reducing agents, which may affect the conformation of the antibody and thereby the efficacy of the ADC, which is another concern. With the above-mentioned technical challenges in mind, the present disclosure is directed to engineered antibody conjugates configured to conjugate payloads to antibodies via glycans. Preparation of the engineered antibody conjugates of the present disclosure includes glycosylation (eg, N-glycosylation) to link appropriate glycans to the antibody.

[0020] N-glycosylation is one of the most complex post-translational modifications, often resulting in significant heterogeneity in glycan structures, including high-mannose, hybrid, and complex types, depending on the recombinant expression system. Commercially available therapeutic antibodies typically exist as a mixture of glycoforms that are suboptimal for their respective therapeutic activity. Recently, glycoengineering to control Fc glycosylation to improve efficacy has attracted attention. Endoglycosidases, a family of at least 18 glycoside hydrolases (GHs) derived from Streptococcus pyogenes, have recently become the focus of attention for glycoengineering therapeutic antibodies. These enzymes can catalyze the hydrolysis of the β-1,4 linkage between two N-acetylglucosamines (GlcNAc) in the core of the N-linked glycan of human IgG. Furthermore, these enzymes remove complex glycans in the IgG Fc domain.

[0021] To conjugate a payload, the glycan linked to the antibody must be modified to provide a functional group for conjugation. The functional group of the modified glycan may affect the glycosylation efficiency of the endoglycosidase. As a result, the glycosylated antibody may have poor homogeneity. Although endoglycosidases are known in the art, their ability to glycosylate modified glycans cannot be predicted from their ability to glycosylate unmodified glycans. Therefore, it is important to identify capable endoglycosidases and suitable modified glycans for the purpose of solving the above-mentioned technical problems.

[0022] One aspect of the present disclosure is directed to a method for preparing an engineered bioconjugate. The bioconjugate may be a glycosylated antibody or antigen-binding fragment thereof, a glycoprotein, or a glycopeptide. Another aspect of the present disclosure is directed to an engineered bioconjugate or multiple engineered bioconjugates. Yet another aspect of the present disclosure is directed to a pharmaceutical composition comprising the engineered bioconjugate. Yet another aspect of the present disclosure is directed to a method of treating cancer using the pharmaceutical composition. Yet another aspect of the present disclosure is directed to a therapeutic conjugate.

[0023] Embodiments of the present disclosure relate to selected variants of glycosynthases that exhibit remarkable transglycosylation activity, transferring a wide range of high-mannose, hybrid, or complex N-glycans from activated oligosaccharide oxazolines to fucosylated or nonfucosylated GlcNAc peptides, proteins, or IgGs, with little or negligible product hydrolysis. The novel glycosynthase enzymes operate with surprisingly high efficiency, providing uniformly glycosylated glycopeptides, glycoproteins, and therapeutic antibodies and their Fc fragments with defined and diverse glycoforms. Further embodiments of the present disclosure may provide glycoengineered antibodies with enhanced effector functions, such as FcγIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC), as well as enhanced pharmacological properties. Embodiments of the present disclosure also enable rapid investigation of the effects of diverse Fc glycosylation of therapeutic antibodies on their effector functions.

[0024] According to embodiments of the present disclosure, novel glycosynthase enzymes comprise sequences selected from the sequences of SEQ ID NOs: 1-2. These variants exhibit unexpectedly improved transglycosylation activity and reduced hydrolysis activity. Thus, these variants can catalyze the efficient transfer of activated oligosaccharide donors to core GlcNAc acceptors, which may or may not be fucosylated.

[0025] According to certain embodiments, the glycosynthase enzyme can have at least about 80% sequence identity (e.g., 80%, 85%, 90%, 95%, or 98% (or a range of values ​​between any two numbers recited herein)) to the sequence of SEQ ID NOs: 1-2 and have the desired transglycosylation activity or a fragment thereof having transglycosylation activity. In some embodiments, the glycosynthase is as described in U.S. Patent No. 11,203,645, filed June 27, 2019, which is incorporated herein by reference.

[0026] definition It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The term "plural" means two or more. As used herein, the term "plural" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0027] As used herein, the terms "comprise," "include," or "have" are intended to describe the presence of certain features, integers, steps, operations, members, ingredients, and / or combinations thereof, but do not exclude the possible presence or addition of one or more other features, integers, steps, operations, members, ingredients, or combinations thereof. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed and semi-closed transitional phrases, respectively.

[0028] As used herein, the terms "can" and "may" are used interchangeably in this disclosure to indicate that a mentioned element, component, structure, feature, functionality, purpose, advantage, operation, step, process, apparatus, system, device, result, or description has the capability to be used for, included in, or present in, or otherwise represent, what is set forth in the description where the term is used (or referred to) for a particular embodiment.

[0029] As used herein, the term "about" refers to an amount, duration in time, or other measurable value and is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate.

[0030] As used herein, "substantially" means sufficient to function for its intended purpose. Thus, the term "substantially" allows for minor and insignificant variations from absolute or perfect conditions, dimensions, measurements, results, etc., that would be expected by one of ordinary skill in the art, but that do not appreciably affect overall performance. When used in reference to a numerical value or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within 10 percent.

[0031] As used herein, the term "linked" refers to two components that are connected directly to each other or indirectly to another component.

[0032] As used herein, μm means micrometer, μm 3 or um 3 means cubic micrometer, pL means picoliters, nL means nanoliters, and μL (or uL) means microliters.

[0033] As used herein, the term "glycan" refers to a polysaccharide, oligosaccharide, or monosaccharide. Glycans can be monomers or polymers of sugar residues, and can be linear or branched. Glycans can contain natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6' sulfo-N-acetylglucosamine, etc.).

[0034] As used herein, the terms "fucose," "core fucose," and "core fucose residue" are used interchangeably and refer to fucose linked to N-acetylglucosamine at the α-1,6-position.

[0035] As used herein, the terms "N-glycan," "N-linked glycan," "N-linked glycosylation," "Fc glycan," and "Fc glycosylation" are used interchangeably and refer to a glycan having an N-acetylglucosamine (GlcNAc) attached to the amide nitrogen of an asparagine residue in an Fc-containing polypeptide. The term "Fc-containing polypeptide" refers to a polypeptide comprising an Fc region, e.g., an antibody.

[0036] As used herein, the terms "glycosylation pattern" and "glycosylation profile" are used interchangeably and refer to the characteristic "fingerprint" of N-glycan species that are enzymatically or chemically released from a glycoprotein or antibody and analyzed for their carbohydrate structure using, for example, LC-HPLC, or MALDI-TOF MS. See, for example, the review in Current Analytical Chemistry, Vol. 1, No. 1 (2005), pp. 28-57, which is incorporated herein by reference in its entirety.

[0037] As used herein, the term "glycoengineered Fc" refers to N-glycans on the Fc region that have been enzymatically or chemically altered or engineered. As used herein, the term "Fc glycoengineering" refers to the enzymatic or chemical process used to generate a glycoengineered Fc.

[0038] As used herein, the term "initial N-linked glycan" refers to the N-linked glycan that is originally attached to a biomolecule before the biomolecule is treated in a reaction performed according to an embodiment of the present disclosure. The glycan may be a polysaccharide, oligosaccharide, or monosaccharide, or may be a monomer or polymer of sugar residues, and may be linear or branched as described herein.

[0039] As used herein, the term "bioconjugate" refers to a molecule formed by the combination of at least two entities, at least one or all of which are biological. All of the entities may be biological. For example, a bioconjugate may be a polypeptide conjugated to a glycan, a protein conjugated to a glycan, or an antibody or antigen-binding fragment thereof conjugated to a glycan. In some instances, a bioconjugate may further include a non-biological moiety, such as a chemical entity. The chemical entity may provide a therapeutic, diagnostic, or other function. As used herein, "bioconjugate" and "engineered bioconjugate" are interchangeable and refer to a conjugate created or obtained by laboratory techniques.

[0040] As used herein, the term "antibody drug conjugate" or "immunoconjugate" refers to the linkage between an antibody or antigen-binding fragment thereof and another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, or the like. The linkage may be a covalent bond or a non-covalent interaction such as electrostatic forces. Various linkers known in the art can be employed to form antibody drug conjugates. Furthermore, antibody drug conjugates can be provided in the form of a fusion protein expressed from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein created by joining two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein possessing functional properties derived from each of the original proteins.

[0041] The terms "homogeneous," "homogenous," "homogeneously," and "homogeneously," in relation to the glycosylation profile of an Fc region, are used interchangeably and are intended to mean a single glycosylation pattern represented by one desired N-glycan species with little or no trace of precursor N-glycans, e.g., comprising less than 95, 96, 97, 99% of the starting precursor material.

[0042] [Table 1]

[0043] As used herein, the terms "IgG," "IgG molecule," "monoclonal antibody," "immunoglobulin," and "immunoglobulin molecule" are used interchangeably.

[0044] As used herein, the term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Further, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγR1, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review by M. Daeron (1997) Annu. Rev. Immunol. 15:203-234). FcRs are reviewed in Ravetch and Kinet (1991) Annu. Rev. Immunol 9:457-92; Capel et al. (1994) Immunomethods 4:25-34; Haas et al. (1995) J. Lab. Clin. Med. 126:330-41). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al. (1976) J. Immunol. 117:587 and Kim et al. (1994) J. Immunol. 24:249).

[0045] As used herein, the term "effector function" refers to a biochemical event resulting from the interaction of the Fc region of an antibody with an Fc receptor of a ligand. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and others. Such effector functions can be assessed using a variety of assays known in the art.

[0046] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. Antibodies "arm" the cytotoxic cells and are necessary for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362 or US Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. (1998) PNAS (USA) 95:652-656.

[0047] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or a non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin (Fc). For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0048] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Antibodies generally contain six hypervariable regions: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). Several hypervariable region delineations are in use and are encompassed herein. The Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0049] "Framework" or "FW" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0050] The terms "Kabat variable domain residue numbering" or "Kabat amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain amino acid deletions or additions corresponding to shortenings of, or insertions into, FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody sequence in the regions of homology with the "standard" Kabat numbered sequence.

[0051] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fvs (scFvs), camelid antibodies (e.g., VHHs), disulfide-linked Fvs (sdFvs), Fab fragments, F(ab') fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; dAb fragments consisting of the VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of antibodies.

[0052] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0053] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404,097, WO 93 / 1161, and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0054] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0055] An "affinity matured" antibody is one with one or more alterations in one or more HVRs that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess the alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by shuffling VH and VL domains. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al. J. Immunol. 154(7):3310-9 (1995); and Hawkins et al. J. Mol. Biol. 226:889-896 (1992).

[0056] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of an antigen.

[0057] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0058] A "disorder" is any condition that would benefit from treatment with an antibody of the invention. This includes chronic or acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.

[0059] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders involving some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0060] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor," as referred to herein, are not mutually exclusive.

[0061] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include lung cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0062] As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing or reducing inflammation and / or tissue / organ damage, reducing the rate of disease progression, ameliorating or alleviating the disease state, and ameliorating or improving prognosis. In some embodiments, the antibodies of the invention are used to slow the progression of a disease or disorder.

[0063] An "individual" or "subject" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (e.g., cows), sport animals, pets (e.g., cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the vertebrate is a human.

[0064] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is a human.

[0065] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0066] A "therapeutically effective amount" of a substance / molecule of the present invention can vary according to factors such as the state of the disease, the age, sex, and weight of the individual, and the ability of the substance / molecule to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule are outweighed by the beneficial effects of treatment. A "prophylactically effective amount" refers to an amount that is effective, at a dosage and for a period of time necessary, to achieve the desired prophylactic effect. Typically, but not necessarily, since a prophylactic dose is used in subjects before or at an early stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0067] "Combination" refers to a combination therapy that is an amount of an ADC and / or an amount of another biological or chemical drug that is therapeutically effective and has a therapeutically additive or greater synergistic effect when administered together (as co-administration and / or co-formulation), sequentially or simultaneously, on the same or different days during a cycle of treatment.

[0068] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60C, and radioactive isotopes of ruthenium-177, strontium-89, and samarium (153Sm), chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, and is intended to include synthetic analogs and derivatives thereof.

[0069] The term "photodynamic therapy (PDT)," sometimes referred to as photochemotherapy, is a form of phototherapy involving light and photosensitizing chemicals used in combination with molecular oxygen to induce cell death (phototoxicity). It is used clinically to treat a wide range of medical conditions, including wet age-related macular degeneration, psoriasis, and atherosclerosis, and has shown some efficacy in antiviral treatment, including herpes. It also treats malignant cancers, including those of the head and neck, lung, bladder, skin, and prostate (Wang, SS et al. Cancer Journal. 8(2):154-63, 2002). The "photodynamic therapeutic agent" is selected from Photofrin, Laserphyrin, aminolevulinic acid (ALA), silicon phthalocyanine Pc-4, m-tetrahydroxyphenylcholine (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphiminex, Verteporfin, Purlytin, ATMPn, zinc phthalocyanine (ZnPc), protoporphin IX (PpIX), Pyropheophorbidea (PPa), or Pheophorbide a (PhA).

[0070] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine (also known as DMI), anthracyclines, pyrrolobenzodiazepines, α-amanitin, tubulysin, benzodiazepines, erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa, ethyleneimines and methylamersamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins;Duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards, such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega I1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein-enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevronic acid; eniluracil; amsacrine; Bestrab Sil; Bisantrene; Edatraxate; Defofamide; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;Platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatlaxate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine (XELODA® Roche); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0071] This definition of "chemotherapeutic agent" includes (i) antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON toremifene; (ii) chemotherapeutic agents that act to regulate the production of estrogen in the adrenal glands. aromatase inhibitors, which inhibit the enzyme aromatase, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestany, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and toloxa (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways thought to be involved in abnormal cell proliferation, such as PKC-alpha, Ralf, and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; (ix) vaccines, e.g., gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; (x) antiangiogenic agents, such as bevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0072] Protein kinase inhibitors include tyrosine kinase inhibitors that inhibit to some extent the tyrosine kinase activity of tyrosine kinases, such as ErbB receptors. Examples of tyrosine kinase inhibitors include EGFR-targeted drugs, such as (i) antibodies that bind to EGFR, including MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), and MAb 528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.), and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®, Imclone) and reshaped human 225 (H225) (WO 96 / 40210, Imclone Systems). Inc.); antibodies that bind to type II mutant EGFR (US Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR (US Pat. No. 5,891,996); and human antibodies that bind to EGFR, such as ABX-EGF (WO 98 / 50433); (ii) anti-EGFR antibodies conjugated to cytotoxic agents (EP 659439A2); and small molecules that bind to EGFR, such as ZD1839 or Gefitinib (IRESSA™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA™; Genentech / OSI), and AG1478, AG1571 (SU 5271; Sugen), quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazolines, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, e.g., CGP 59326, CGP 60261, and CGP 62706, as well as pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding ErbB); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396; ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); pan-ErbB inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); or US Pat. No. 5,804,396; WO 99 / 09016 (American Cyanamid); WO 98 / 43960 (American Cyanamid; WO 97 / 38983 (Warner Lambert); WO 99 / 06378 (Warner Lambert); WO 99 / 06396 (Warner Lambert); WO 96 / 30347 (Pfizer, Inc); WO 96 / 33978 (Zeneca); WO 96 / 3397 (Zeneca); and WO 96 / 33980 (Zeneca).

[0073] "Anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with the development of blood vessels. Anti-angiogenic factors can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis. An exemplary anti-angiogenic agent is an antibody that binds to vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®, Genentech).

[0074] The term "cytokine" is a general term for proteins released by one cell population and acting on another cell as intercellular mediators. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factors-α and -β; Mullerian inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-β; platelet growth factors; and transforming growth factors (TGF-β). Examples of cytokines include TGFs (TGF-α and TGF-β), insulin-like growth factor-I and -II, erythropoietin (EPO), osteogenic factor (BGF), interferons (e.g., interferon-α, -β, and -γ), colony-stimulating factors (CSFs), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors (TNF-α or TNF-β), and other polypeptide factors, including LIF and Kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native-sequence cytokines.

[0075] The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance that has reduced cytotoxicity to tumor cells compared to the parent drug and can be enzymatically activated or converted into a more active parent form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery, Directed Drug Delivery," Borchardt et al. (eds.), pp. 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, the chemotherapeutic agents listed above.

[0076] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an ADC. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylenebis(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may contain another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. Examples in which multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0077] A "pharmaceutically acceptable solvate" refers to an association of one or more solvent molecules with an ADC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0078] The complex N-linked oligosaccharides on each CH2 domain of IgG are important for the structure of the Fc region and therefore for the interaction with Fc receptors (Krapp et al. 2003; Woof and Burton 2004). The oligosaccharide chains of the IgG-Fc domain contain several N-acetylglucosamine (GlcNAc) and mannose (Man) residues, and finally galactose (Gal) and fucose (Fuc) residues, as well as sialic acid (Sia or N-acetylneuraminic acid, NANA). A GlcNAc with or without al-6 Fuc is attached to Asn297. GlcNAcpi-4 is attached to this first GlcNAc. Next, manβ1-4 is found, to which two Manα1-6 arms and a Manα1-3 arm are attached. Both arms contain an additional GlcNAcβ1-2, to which Galβ1-4 may or may not be attached. That is, the carbohydrate chain can contain zero, one, or two galactose residues, defined as G0, G1, and G2 glycoforms, respectively. Further modifications occur, including biantennary GlcNAcβ1-4 and capping of one or both terminal galactose residues with sialic acid or the presence of Galα1-3 residues. Enzymatic cleavage of Fc glycans by endoglycosidases causes modifications of the Fc region, thereby dramatically reducing IgG binding to Fcγ receptors (Allhorn et al. 2008). Despite 37% sequence identity between EndoS and EndoS2, they catalyze the hydrolysis of the β-1,4 linkage between two N-acetylglucosamines (GlcNAc) in the core of the N-linked glycan of human IgG. However, EndoS2 hydrolyzes hybrid and oligomannose structures, in addition to complex glycans, to a greater extent than EndoS (Sjögren et al. 2015).

[0079] Methods for preparing engineered bioconjugates Since the introduction of the first antibody therapy in the 1980s, over 240 therapeutic antibodies are in clinical trials, and the field is steadily expanding (Chan and Carter 2010). The role of IgG-Fc glycans in antibody function has attracted significant attention in the growing field of therapeutic monoclonal antibodies. Therefore, a major focus has been on engineering Fc-glycans that specifically interact with selected Fcγ receptors to improve the efficacy of therapeutic antibodies (Sondermann et al. 2013; Bournazos et al. 2014; Monnet et al. 2014; Quast and Lunemann 2014). Some key glycan modifications that dramatically affect effector function include: i) the absence of a core fucose residue attached to a reducing-end GlcNAc residue, resulting in increased affinity for FcγRIIIa and therefore increased antibody-dependent cellular cytotoxicity (Iidaet et al. 2006); ii) sialic acid-rich glycans on IgG, which have been proposed to enhance the anti-inflammatory response of IgG through enhanced interaction with the DC-SIGN receptor on dendritic cells and macrophages (Anthony et al. 2008; Anthony and Ravetch 2010; Pincetic et al. 2014); and iii) the presence of biantennary GlcNAc, which induces stronger ADCC compared to its parent counterpart. Recent improvements in biotechnology tools to control the Fc-glycosylation state of IgG have facilitated the development of therapeutic antibodies with predefined glycoforms. Thus, the glycosynthase enzymes of the present disclosure represent a major advance in the field of glycoengineering of peptides, proteins, and antibodies of interest, attaching a wide range of high-mannose, hybrid, and complex N-glycans for functional and structural studies.

[0080] One aspect of the present disclosure provides a method for preparing an engineered bioconjugate. The method includes contacting a biomolecule with a glycosynthase and a modified glycan, thereby obtaining a first engineered bioconjugate. The biomolecule includes an antibody or antigen-binding fragment thereof, a protein, or a peptide linked to an N-linked initial glycan. The modified glycan includes a substrate moiety and a first reactive moiety, the substrate moiety being configured to interact with the glycosynthase. In some embodiments, contacting the biomolecule with the glycosynthase and the modified glycan includes contacting a plurality of biomolecules with the glycosynthase and the modified glycan, thereby obtaining a plurality of first engineered bioconjugates. In certain embodiments, the homogeneity of the plurality of first engineered bioconjugates is at least 80%, 85%, 90%, 95%, or 99% or greater. High homogeneity indicates fewer isomers among the first engineered bioconjugates, suggesting good consistency and efficacy of the engineered bioconjugates prepared using the methods of the present disclosure. Without wishing to be bound by theory, the glycosynthases and / or modified glycans of the present disclosure are attributable to the preferred high homogeneity.

[0081] In some embodiments, the method further comprises contacting the first engineered bioconjugate with a payload conjugate or a salt thereof, thereby obtaining a second engineered bioconjugate. In some embodiments, the second engineered bioconjugate is an ADC having a therapeutic agent conjugated to the antibody via a modified glycan. The structures and characteristics of the payload conjugate and the second engineered bioconjugate are described in more detail below.

[0082] In some embodiments, contacting the first engineered bioconjugate with a payload conjugate or salt thereof comprises contacting the first engineered bioconjugate with a first payload conjugate and a second payload conjugate, wherein the first payload conjugate and the second payload conjugate are different. In certain embodiments, the first payload conjugate and the second payload conjugate are different in the payload of each of the two payload conjugates, the reactive moiety unit (C) of each of the two payload conjugates, and / or the linker unit (L) of each of the two payload conjugates.

[0083] Glycosylation (deglycosylation and transglycosylation): In some embodiments, contacting a biomolecule with a glycosynthase and a modified glycan comprises linking the modified glycan to an N-linked initial glycan. The modified glycan can be directly or indirectly linked to the N-linked initial glycan. In some embodiments, linking the modified glycan to an N-linked initial glycan comprises removing the N-linked initial glycan of the biomolecule, thereby obtaining a deglycosylated biomolecule, and contacting the deglycosylated biomolecule with a glycosynthase in the presence of the modified glycan.

[0084] The step of removing the N-linked initial glycans of the biomolecule can be a step of completely removing the N-linked initial glycans or a step of removing a portion of the N-linked initial glycans. In embodiments where only a portion of the N-linked initial glycans are removed, the modified glycan will be linked to the residue of the N-linked initial glycan. In certain embodiments, after the step of removing the N-linked initial glycans, the GlcNAc monosaccharide, which may or may not be fucosylated, remains on the biomolecule, and the modified glycan will be linked to the GlcNAc monosaccharide.

[0085] In some embodiments, the step of removing an N-linked initial glycan of a biomolecule can be performed using a glycosynthase of the present disclosure. The glycosynthase of the present disclosure comprises SEQ ID NO: 1 or SEQ ID NO: 2, and the glycosynthase comprises a mutation located at residues 176-186, 225-237, or 273-289 in SEQ ID NO: 1, or at residues 178-188, 227-239, or 275-291 in SEQ ID NO: 2. Glycosynthases of the present disclosure are described in more detail below.

[0086] Alternatively, the N-linked initial glycans of a biomolecule can be removed by using a glycosynthase different from the glycosynthases of the present disclosure. In certain embodiments, removing the N-linked initial glycans of a biomolecule, thereby obtaining a deglycosylated biomolecule, comprises mixing the glycosynthase with the biomolecule in a ratio of 1:500 to 1:1, 1:500 to 1:10, 1:500 to 1:20, 1:500 to 1:30, 1:500 to 1:50, 1:100 to 1:1, 1:100 to 1:10, 1:100 to 1:20, 1:100 to 1:30, 1:100 to 1:50, 1:50 to 1:1, 1:50 to 1:10, 1:50 to 1:20, or 1:50 to 1:30 in the absence of modified glycans. However, the methods of the present disclosure are not limited thereto. The ratio may vary depending on different circumstances.

[0087] In the present disclosure, exemplary glycosynthase enzymes are provided for transglycosylation at core fucosylated or non-fucosylated GlcNAc acceptors, including core fucosylated or non-fucosylated GlcNAc peptides, proteins, and IgG Fc domains or fragments thereof.

[0088] In some embodiments, two glycosynthase enzyme variants, EndoSd-D232M and EndoSz-D234M, are provided, which possess glycosynthase activity that enables the production of uniform mAb remodeling products. In addition to the previously reported EndoSd from Streptococcus dysgalactiae subsp. Dysgalactiae (NCBI GenBank accession number: ANI26082.1), novel enzymes were identified and isolated using protein BLAST database searches for EndoS and EndoSd sequences. Another candidate, a putative protein, EndoSz from Streptococcus equi subsp. Zooepidemicus Sz105 (NCBI GenBank accession number: KIS14581.1), was selected. Mutants of EndoSd and EndoSz were generated using multiple sequence alignment methodology. Results showed that both mutated enzymes possessed unexpectedly improved / enhanced glycosynthase activity for conjugating biantennary complex glycans to mAbs. mAbs suitable for conjugation were obtained / derived from a wide range of targets, including various biomarkers or various IgG types. Here, satisfactory conjugation results were demonstrated for OBI-888, Herceptin, Perjeta, Erbitux, Rituxan, OBI-898, Vectibix, Humira, Keytruda, and Bavencio. The efficiency of the enzymes in the conjugation reactions was also demonstrated, and the ADCC activity between heterogeneous and homogeneous mAbs in relevant cell lines was compared.

[0089] In some embodiments, the present disclosure provides glycosynthase enzyme variants, which have at least about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence and / or structural homology thereto, and which exhibit improved transglycosylation activity toward fucosylated and nonfucosylated GlcNAc acceptors for a broad range of N-glycans, including high-mannose, hybrid, and complex types, and which enable efficient transfer of activated oligosaccharide donors to fucosylated and nonfucosylated GlcNAc acceptors to form homogeneous glycoforms of glycopeptides or glycoproteins or therapeutic antibodies. EndoSz and EndoSd variants are listed in the table below. In some embodiments, the glycosynthase is described in U.S. Patent No. 11,203,645, filed June 27, 2019, which is incorporated herein by reference.

[0090] [Table 2]

[0091] Modified glycan: As used herein, "modified glycan" refers to a glycan modified by a chemical entity. The chemical entity may include a polymer and at least one functional group that can serve as a functionalization or conjugation site for another moiety. Furthermore, the modified glycan includes a substrate moiety, which is configured to be catalyzed by a glycosynthase, as described herein, thereby linking the modified glycan to a biomolecule. In some embodiments, the structure of the substrate moiety can be selected based on the properties of the glycosynthase. In certain embodiments, the substrate moiety is an oxazoline moiety.

[0092] In one embodiment, the modified glycan has the formula:

[0093] [ka] is a synthetic glycan oxazoline containing a variety of high mannose, hybrid, and complex N-glycans having the formula: 1 is N-acetylglucosamine attached via -H or β-1,4 linkage, and R 2 and R 3 are the same or different,

[0094] [ka] are independently selected from the group consisting of:

[0095] In some embodiments, the first reactive moiety of the modified glycan of the present disclosure is configured to react with the unsaturated moiety in a bioorthogonal reaction, which may be copper-free click chemistry. In one example, the first reactive moiety comprises an azide group, but the modified glycan is not limited thereto.

[0096] In some embodiments, the modified glycan is a PEGylated glycan modified with a polyethylene glycol (PEG) moiety. Without wishing to be bound by any theory, the PEG moiety provides better spacing for click chemistry to occur, and is selected for its hydrophilicity, which facilitates biological reactions, and its favorable biocompatibility for clinical use. In other words, in some embodiments, the PEG moiety can be replaced with another hydrophilic polymer moiety that exhibits suitable biocompatibility. In certain embodiments, one end of the PEGylated glycan is covalently linked to, for example, a first reactive moiety, and the other end of the PEGylated glycan is linked to a substrate moiety. For example, the first reactive moiety can be linked to a PEG moiety, and the substrate moiety can be linked to the glycol moiety of the PEGylated glycan.

[0097] The length of the PEG moiety is not limited, but in some embodiments, the PEG moiety can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 72 ethylene oxide (OCHCH) subunits, or a range defined by the above endpoints, e.g., 2 to 72, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 4, 3 to 72, 3 to 65, 3 to 55, 3 to 45, 3 to 35, 3 to 25, 3 to 15, 3 to 10, 4 to 72, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, or 4 to 10 OCHCH subunits. In some embodiments, the PEG moiety may be linear, branched, or star-shaped. In certain embodiments, the PEG moiety may have 2, 3, 4, 5, 6, 7, or 8 arms. In certain embodiments, the PEG moiety may have a molecular weight of 4 KDa, 6 KDa, 8 KDa, 10 KDa, or 20 KDa.

[0098] Biomolecule: In some embodiments, the biomolecule comprises an antibody or antigen-binding fragment thereof, and the N-linked initial glycan is located in the constant region of the antibody or antigen-binding fragment. In certain embodiments, the N-linked initial glycan is located in the Fc region of the antibody or antigen-binding fragment, for example, at the N297 site of the Fc region. In some embodiments, the biomolecule is an antibody, and one or two N-linked initial glycans are linked to the N297 site of the Fc region of the antibody.

[0099] In some embodiments, the antibody is IgG, IgM, IgA, IgE, or IgD. A typical IgG consists of two antigen-binding fragments (Fab), which are connected to a constant region (Fc) via a flexible region. The Fab domain is involved in antigen recognition, and the N-glycan at Asn297 of the Fc domain interacts with the respective Fcγ receptors (e.g., FcγRIIIa and FcγRIIb) on effector cells and the C1q component of complement, activating effector functions including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Almost all therapeutic antibodies are N-glycosylated in each of the Fc domains of the homodimer at the conserved asparagine residue (N297). These N-linked glycans are typically biantennary complex with considerable structural heterogeneity, resulting in over 30 different glycoforms, in which the core heptasaccharide can be differentially modified with core fucose (Fuc), biantennary N-acetylglucosamine (GlcNAc), terminal galactose (Gal), and terminal sialic acid (Sia). The composition of N-glycans influences the conformation of the Fc domain and can therefore modulate antibody stability, pharmacokinetic profile, immunogenicity, effector function, antibody-mediated inflammation, and complement activation. For example, the absence of core fucose and the attachment of a biantennary GlcNAc moiety dramatically enhance antibody affinity for the FcγIIIa receptor (FcγRIIIa) on effector cells, resulting in more effective target elimination. Furthermore, terminal α-2,6-sialylated glycans, a minor component of antibodies and intravenous immunoglobulin (IVIG), are optimized structures that enhance anti-inflammatory properties.

[0100] In some embodiments, the biomolecule is an anti-Globo series antigen antibody or antigen-binding fragment thereof, an anti-HER2 antibody or antigen-binding fragment thereof, an anti-CD20 antibody or antigen-binding fragment thereof, an anti-TNF-alpha antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-TROP2 antibody, an anti-Nectin-4 antibody or antigen-binding fragment thereof, an anti-EGFR antibody or antigen-binding fragment thereof, an anti-HER3 antibody or antigen-binding fragment thereof, an anti-cMet antibody or antigen-binding fragment thereof, an anti-B7H3 antibody or antigen-binding fragment thereof, an anti-B7H4 antibody or antigen-binding fragment thereof, an anti-VEGF antibody or antigen-binding fragment thereof, an anti-Claudin 18.2 antibody or antigen-binding fragment thereof, an anti-Sirp-alpha antibody or antigen-binding fragment thereof, a TROP2xHER2 bispecific antibody, or a combination thereof. Globo series antigens may include Globo H, stage-specific embryonic antigen 4 (SSEA-4), or stage-specific embryonic antigen 3 (SSEA-3).

[0101] In certain embodiments, the antibody is OBI-888 (anti-Globo H monoclonal antibody). Exemplary OBI-888 is described in PCT patent publications (WO2015157629A2 and WO2017062792A1), the contents of which are incorporated by reference in their entireties.

[0102] In certain embodiments, the antibody is OBI-898 (anti-SSEA4 monoclonal antibody). An exemplary OBI-898 is described in PCT Patent Publication (WO2017172990A1) patent application, the contents of which are incorporated by reference in their entirety.

[0103] In certain embodiments, the antibody is R4702 (anti-TROP2 monoclonal antibody). An exemplary R4702 is described in PCT Patent Publication (WO2022222992A1) patent application, the contents of which are incorporated by reference in their entirety.

[0104] In some embodiments, the biomolecule is selected from, but is not limited to, Herceptin (trastuzumab), TX05 (trastuzumab biosimilar), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), Bavencio (avelumab), and R4702 (anti-TROP2 antibody).

[0105] In a further aspect, the present disclosure provides a composition of a fucosylated or non-fucosylated glycoengineered antibody or antigen-binding fragment comprising IgG molecules having the same N-glycan structures at each site in the Fc region, wherein the N-glycans are high mannose, hybrid, and complex;

[0106] [ka] where R 1 is N-acetylglucosamine attached via -H or β-1,4 linkage, and R 2 and R 3 are the same or different,

[0107] [ka] are independently selected from the group consisting of:

[0108] In another aspect, the present disclosure provides engineered bioconjugates that have unexpectedly improved effector functions, such as FcγIIIA binding and ADCC, compared to unmodified antibodies, to control immune responses.

[0109] Payload conjugates (linker-drug conjugates) Another aspect of the present disclosure provides a payload conjugate, which has the following formula: CLD wherein C is a second reactive moiety configured to react with the first reactive moiety of the modified glycan in a bioorthogonal reaction, L is a linker unit comprising a hydrophilic moiety, and D is a payload. The payload conjugate is configured to covalently link the payload to the biomolecule via the first reactive moiety of the modified glycan.

[0110] Second reactive moiety unit (C): For reaction with the first reactive moiety of the modified glycan, the second reactive moiety of the payload conjugate, in some embodiments, comprises an unsaturated moiety capable of reacting with an azide moiety to yield a triazole moiety. The unsaturated moiety may be, but is not limited to, an alkene moiety or an alkyne moiety. In some embodiments, the second reactive moiety is a bioorthogonal group, which is an unnatural and unperturbed chemical group. Some specific examples of second reactive moieties include, but are not limited to, a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN), a cyclic alkyne, a maleimide group, an α,β-unsaturated carbonyl group, or a sulfonylpyrimidine. Nevertheless, the methods of the present disclosure are not limited thereto. Other functional groups suitable for bioorthogonal reaction with the first reactive moiety of the modified glycan can also be selected for payload conjugation.

[0111] Linker unit (L): The hydrophilic portion of the linker (L) can be selected based on hydrophilicity and biocompatibility for clinical use. In certain embodiments, the hydrophilic portion comprises a second polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety can be replaced with another hydrophilic polymer moiety that exhibits suitable biocompatibility.

[0112] The length of the PEG moiety is not limited, but in some embodiments, the PEG moiety can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 72 ethylene oxide (OCHCH) subunits, or a range defined by the above endpoints, e.g., 2 to 72, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 4, 3 to 72, 3 to 65, 3 to 55, 3 to 45, 3 to 35, 3 to 25, 3 to 15, 3 to 10, 4 to 72, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, or 4 to 10 OCHCH subunits. In some embodiments, the PEG moiety may be linear, branched, or star-shaped. In certain embodiments, the PEG moiety may have 2, 3, 4, 5, 6, 7, or 8 arms. In certain embodiments, the PEG moiety may have a molecular weight of 4 KDa, 6 KDa, 8 KDa, 10 KDa, or 20 KDa.

[0113] In some embodiments, the linker may further comprise a cleavable moiety and a spacer. The cleavable moiety is configured to release the payload from the second engineered conjugate in vivo. Therefore, a portion of the function can be selected that can be catalyzed or digested by an enzyme normally present in the target environment in vivo. The enzyme may be a protease, such as matrix metalloproteinase 2 or matrix metalloproteinase 9, or a glycosidase. In certain embodiments, the cleavable moiety is a protease-sensitive peptide, including, but not limited to, Val-Cit, Val-Ala, Phe-Lys, Glu-Val-Cit, Glu-Val-Ala, Glu-Gly-Cit, Glu-Gly-Ala, Gly-Gly-Phe-Gly, Gly-Gly-Val-Cit, or Gly-Gly-Val-Ala. In other embodiments, the cleavable moiety is a glycosidase-sensitive sugar unit, including, but not limited to, glucuronic acid, iduronic acid, or galactose.

[0114] In some embodiments, the spacer may be an aromatic group, including but not limited to, a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, a 2,5-thienyl group, or an aminomethylene such as -NH-CH2-.

[0115] In some embodiments, the linker unit is:

[0116] [ka] where (PEG)m is a PEG moiety, m is an integer selected from 2 to 72, and Q SP is a spacer containing an aromatic group or an aminomethylene, and Q CL is a severable portion configured to be coupled to a payload, and L P is a connector unit configured to be linked to a second reactive moiety.

[0117] In some embodiments, the PEG moiety

[0118] [ka] where the wavy line is L P indicates the site of covalent attachment to R 20 is a PEG linking unit, and the PEG linking unit is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or

[0119] [ka] where R 21 is a PEG capping unit, and the PEG capping unit is H, SO3H, PO3H2, sugar derivatives, C1-C 10 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 Alkyl-NH2, C1-C10 Alkyl-COOH, C2-C 10 Alkyl-NH(C1-C3 alkyl), C2-C 10 alkyl-N(C1-C3 alkyl)2, where n is selected from 2-72, 4-72, or 8-72.

[0120] In certain instances, the linker unit (L) is

[0121] [ka] It has the following structure.

[0122] Payload (D): The payload can be a therapeutic agent or a diagnostic agent. In some embodiments, the diagnostic agent can be used for imaging.

[0123] In some embodiments, the therapeutic agent may be a toxin, a cytokine, a growth factor, a radionuclide, a hormone, an antiviral agent, an antibacterial agent, an immunomodulatory agent, an immunostimulatory agent, an anti-tumor agent, a chemotherapeutic agent, or a combination thereof.

[0124] In some embodiments, the toxin is at least one selected from the group consisting of pyrrolobenzodiazepines (e.g., PBDs); auristatins (e.g., MMAE, MMAF); maytansinoids (e.g., maytansine, DM1, DM4, DM21); duocamycins; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; tubulysins; enediynes (e.g., calicheamicin); anthracycline derivatives (PNUs) (e.g., doxorubicin); pyrrole-based kinesin spindle protein (KSP) inhibitors; cryptophycins; drug efflux pump inhibitors; sandramycin; amanitins (e.g., α-amanitin); and camptothecins (e.g., exatecan, deruxtecan).

[0125] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor, including topoisomerase I inhibitors and topoisomerase II inhibitors. In certain embodiments, the chemotherapeutic agent is a topoisomerase I inhibitor, which is a camptothecin (CPT) or non-camptothecin selected from irinotecan, topotecan, camptothecin, rubitecin, MLN576, exatecan, belotecan, seconeolithine, SN-38, Genz-644282, betulinic acid, β-lapachon, karenitekitin, gimatecan, namitecan, edotecan, SW044248, LMP744, T-2513, podocarpus flavone A, indimittecan, lurtotecan, TP3011, or 10-hydroxycamptothecin.

[0126] Exatecan may be selected as an example in the payload (D), and the linker-drug compound (i.e., payload conjugate) may have the following formula:

[0127] [ka] is further represented by

[0128] In one embodiment, the structure of the linker-drug compound (i.e., payload conjugate) has the following formula:

[0129] [ka] is expressed by

[0130] In one embodiment, the structure of the linker-drug compound (i.e., payload conjugate) has the following formula:

[0131] [ka] is expressed by

[0132] Engineered bioconjugates Another aspect of the present disclosure provides engineered bioconjugates. The engineered bioconjugates comprise a biomolecule and a modified glycan linked to the biomolecule, the modified glycan comprising (i) a first polyethylene glycol (PEG) moiety and (ii) a first reactive moiety or resulting moiety derived from a bioorthogonal reaction, and the biomolecule comprises an antibody or antigen-binding fragment thereof, a protein, or a peptide. In some embodiments, the first PEG moiety and the first reactive moiety are as described herein.

[0133] In some embodiments where the biomolecule comprises an antibody or antigen-binding fragment thereof, the modified glycan is linked to the antibody or antigen-binding fragment thereof at its Fc region. In certain embodiments, the modified glycan is located in the Fc region of the antibody or antigen-binding fragment, e.g., at the N297 position of the Fc region. In some embodiments where the biomolecule is an antibody, one or two N-linked initial glycans are linked to a GlcNAc monosaccharide at the N297 position of the Fc region of the antibody, e.g., at the N297 position. The GlcNAc monosaccharide may be fucosylated or non-fucosylated.

[0134] In some embodiments, the engineered bioconjugate further comprises a payload moiety, wherein the payload moiety is linked to the modified glycan via the resulting moiety. In such embodiments, the payload to biomolecule ratio (e.g., drug-to-antibody ratio) is 2:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0135] In certain embodiments, the payload moiety has the following formula: -LD wherein L is a linker unit comprising a hydrophilic moiety and is linked to the resulting moiety, and D is a payload. In some embodiments, the linker unit (L) and payload (D) are as described herein. In some embodiments, the engineered bioconjugate is an engineered bioconjugate prepared by a method for preparing an engineered bioconjugate according to an embodiment of the present disclosure.

[0136] In some embodiments, the resulting moiety may be a moiety resulting from a bioorthogonal or click reaction between a first reactive moiety and a second reactive moiety as described herein. In certain embodiments, the resulting moiety comprises a triazole moiety. In certain embodiments, the resulting moiety may comprise a DBCO-derived moiety or a maleimide-derived moiety.

[0137] In some embodiments, the present disclosure provides a plurality of engineered bioconjugates, each of which is an engineered bioconjugate according to an embodiment of the present disclosure, wherein the homogeneity of the plurality of engineered bioconjugates is at least 80%, 85%, 90%, 95%, or 99% or more.

[0138] In some embodiments, the engineered bioconjugate comprises a first payload moiety and a second payload moiety, wherein the first payload moiety and the second payload moiety are different. In certain embodiments, the first payload moiety and the second payload moiety are different in the payload of each of the two payload conjugates, the reactive moiety unit (C) of each of the two payload conjugates, and / or the linker unit (L) of each of the two payload conjugates.

[0139] In certain embodiments where the first payload moiety and the second payload moiety are different in each payload of the two payload conjugates, the payload of the first payload moiety can be a therapeutic agent and the payload of the second payload moiety can be an imaging agent (e.g., an imaging probe). In other embodiments, the payload of the first payload moiety can be a first therapeutic agent and the payload of the second payload moiety can be a second therapeutic agent.

[0140] The engineered bioconjugates of the present disclosure can include bioconjugates that have utility for anti-cancer activity. In certain embodiments, the engineered bioconjugates include antibodies conjugated to a therapeutic agent / payload via a linker, i.e., covalently linked, to form an ADC. The therapeutic agent has cytotoxic or cytostatic effects when not conjugated to the antibody. That is, the biological activity of the therapeutic agent / payload is modulated by conjugation to the antibody. In certain embodiments, the ADCs of the present disclosure can selectively deliver an effective dose of a cytotoxic agent to tumors, thereby achieving low, effective doses.

[0141] In some embodiments, the engineered bioconjugate is an ADC, the ADC having the following formula: Ab-(LD) n or a pharmaceutically acceptable salt or solvate thereof where Ab is an antibody that binds to TROP2, HER2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, claudin 18.2, Sirp-Alpha, or an antibody that binds to one or more tumor-associated antigens or cell surface receptors, D is a drug unit, L is a linker, and n is the drug-antibody ratio (DAR) in the range of 10 to 1.

[0142] Suitable exemplary linkers for ADCs are described, for example, in US Patent No. 7,595,292 (WO 2005 / 007197). The entire contents relating to linkers are incorporated herein by reference. The linker L covalently couples the antibody to the drug moiety / payload and does not contain a disulfide group. The linker is a bifunctional or polyfunctional moiety used to link one or more drug moieties / payloads (D) and antibody units (Ab) to form the ADC of Formula I. ADCs can be conveniently prepared using linkers with reactive functionalities that bind to the drug and antibody. Cysteine ​​thiols or amines, e.g., the N-terminus or amino acid side chains such as lysines, of the antibody (Ab) can form bonds with functional groups on the linker agent, drug moiety / payload, or drug-linker agent.

[0143] The linker is preferably stable extracellularly. Prior to transport or delivery into a cell, the ADC is preferably stable and unchanged, i.e., the antibody remains linked to the drug moiety / payload. The linker is stable outside the target cell and cleaved at an effective rate intracellularly. An effective linker (i) maintains the specific binding properties of the antibody, (ii) enables intracellular delivery of the conjugate or drug moiety / payload, (iii) is stable and unchanged, i.e., is not cleaved, until the conjugate is delivered or transported to its target site, and (iv) maintains the cytotoxic, cell-killing, or cytostatic effect of the maytansinoid drug moiety / payload. The stability of the ADC may be measured by standard analytical techniques, such as mass spectrometry, HPLC, and separation / analysis techniques such as LC / MS.

[0144] In another embodiment, the ADC specifically binds to TROP2, HER2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, claudin 18.2, and Sirp-Alpha and may inhibit the growth of tumor cells expressing TROP2, HER2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, claudin 18.2, and Sirp-Alpha.

[0145] Another aspect includes diagnostic and therapeutic uses of the compounds and compositions disclosed herein.

[0146] Another aspect is a method of killing or inhibiting the growth of tumor or cancer cells, comprising treating cells with an amount of an engineered conjugate or ADC according to an embodiment of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, that is effective to kill or inhibit the growth of the tumor or cancer cells.

[0147] Another aspect includes a method of treating a disease or disorder characterized by overexpression of TROP2, HER2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, Claudin 18.2, and Sirp-Alpha in a patient with the engineered conjugate or ADC.

[0148] Other aspects include methods of making, preparing, synthesizing, conjugating, and purifying engineered conjugates or ADCs, as well as intermediates for the preparation, synthesis, and conjugation of ADCs.

[0149] Pharmaceutical preparations Another aspect of the present disclosure provides a pharmaceutical composition comprising a plurality of engineered bioconjugates of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutical formulations comprising engineered conjugates of the present disclosure may be prepared for storage by combining an antibody having a desired purity with one or more optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed. (1980)) in the form of an aqueous solution, lyophilized, or other dry formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, histidine, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum These include proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0150] The formulations herein may contain more than one active compound as necessary for the particular indication being treated, including, but not limited to, those with complementary activities that do not have adverse effects on each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0151] Active ingredients can also be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th ed., Osol, A. (ed.) (1980).

[0152] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0153] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulins of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON These include DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods. If encapsulated immunoglobulins remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and altered immunogenicity. Rational strategies for stabilization can be devised depending on the mechanism involved. For example, if the mechanism of aggregation is found to be the formation of intermolecular S–S bonds via thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solution, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0154] The amount of antibody in the formulation before lyophilization is determined by considerations such as the desired dosage volume, mode of administration, and so forth. When the selected protein is an unmodified antibody (full-length antibody), an exemplary starting protein concentration is about 2 mg / mL to about 50 mg / mL, preferably about 5 mg / mL to about 40 mg / mL, and most preferably about 20 to 30 mg / mL. The protein is generally present in a solution. For example, the protein may be present in a pH buffer solution at a pH of about 4 to 8, preferably about 5 to 7. Exemplary buffering agents include histidine, phosphate, Tris, citrate, succinate, and other organic acids. The buffering agent concentration may be about 1 mM to about 20 mM, or about 3 mM to about 15 mM, depending, for example, on the buffer and the desired isotonicity of the formulation (e.g., the reconstituted formulation). A preferred buffering agent is histidine, since it can have cryoprotective properties, as demonstrated below. Succinate has been shown to be another useful buffering agent.

[0155] A cryoprotectant is added to the formulation before lyophilization. In a preferred embodiment, the cryoprotectant is a non-reducing sugar, such as sucrose or trehalose. The amount of cryoprotectant in the formulation before lyophilization is generally an amount that renders the resulting formulation isotonic upon reconstitution. However, hypertonic reconstituted formulations may be preferred. Furthermore, the amount of cryoprotectant should not be too low to avoid unacceptable amounts of protein degradation / aggregation upon lyophilization. When the cryoprotectant is a sugar (e.g., sucrose or trehalose) and the protein is an antibody, an exemplary concentration of the cryoprotectant in the formulation before lyophilization is about 10 mM to about 400 mM, preferably about 30 mM to about 300 mM, and most preferably about 50 mM to about 100 mM.

[0156] The protein to cryoprotectant ratio is selected for each protein and cryoprotectant combination. In the case of an antibody as the selected protein and a sugar (e.g., sucrose or trehalose) as the cryoprotectant to produce an isotonic reconstituted formulation at a high protein concentration, the molar ratio of cryoprotectant to antibody may be about 100 to about 1,500 moles of cryoprotectant per mole of antibody, preferably about 200 to about 1,000 moles of cryoprotectant per mole of antibody, for example, about 200 to about 600 moles of cryoprotectant per mole of antibody.

[0157] In a preferred embodiment of the present invention, it has been found desirable to add a surfactant to the formulation before lyophilization. Alternatively, or in addition, a surfactant may be added to the lyophilized and / or reconstituted formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT™ series (Mona Surfactants include PEG-100 (Polyesterol-Based Proteins, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, and others). The amount of surfactant added is an amount that reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. For example, the surfactant may be present in the pre-lyophilized formulation in an amount of about 0.001-0.5%, preferably about 0.005-0.05%.

[0158] In certain embodiments of the invention, a mixture of a cryoprotectant (e.g., sucrose or trehalose) and a bulking agent (e.g., mannitol or glycine) is used in preparing the formulation before lyophilization. The bulking agent may allow for the production of a uniform lyophilized cake without excessive pockets therein.

[0159] Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences, 16th ed., edited by Osol, A. (1980), may be included in the pre-lyophilized formulation (and / or the lyophilized and / or reconstituted formulation), provided they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include additional buffering agents; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; and / or salt-forming counterions, such as sodium.

[0160] The pharmaceutical compositions and formulations described herein are preferably stable. A "stable" formulation / composition is one in which the antibody essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, pp. 247-301, Vincent Lee (ed.), Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time.

[0161] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes prior to or after lyophilization and reconstitution. Alternatively, sterility of the entire mixture may be achieved by autoclaving the components, except for protein, for example, at about 120°C for about 30 minutes.

[0162] After mixing the protein, cryoprotectant, and other optional ingredients together, the formulation is lyophilized. Many different lyophilizers are available for this purpose, such as the Hull 50® (Hull, USA) or GT20® (Leybold-Heraeus, Germany) lyophilizers. Lyophilization is achieved by freezing the formulation and then sublimating ice from the frozen contents at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or collapse temperature of the formulation. Typically, at a suitable pressure in the range of about 50 to 250 mTorr, shelf temperatures for primary drying will range from about −30 to 25°C (assuming the product remains frozen during primary drying). The formulation, size, and type of container holding the sample (e.g., glass vial) and the volume of liquid will primarily govern the time required for drying, which can range from a few hours to several days (e.g., 40 to 60 hours). The secondary drying stage may be carried out at about 0-40°C, depending primarily on the type and size of the container and the type of protein employed. However, it has been found herein that a secondary drying step is not always necessary. For example, the shelf temperature throughout the dehydration phase of lyophilization may be about 15-30°C (e.g., about 20°C). The time and pressure required for secondary drying will be those that produce a suitable lyophilized cake, depending, for example, on temperature and other parameters. The secondary drying time is governed by the desired residual moisture level in the product and typically requires at least about 5 hours (e.g., 10-15 hours). The pressure may be the same as that employed in the primary drying step. Lyophilization conditions may vary depending on the formulation and vial size.

[0163] In some cases, it may be desirable to lyophilize the protein formulation in the container in which the protein will be reconstituted to avoid a transfer step. The container in this example may be, for example, a 3, 5, 10, 20, 50, or 100 cc vial. As a general proposition, lyophilization will result in a lyophilized formulation with a moisture content of less than about 5%, preferably less than about 3%.

[0164] At the desired stage, typically when administering the protein to a patient, the lyophilized formulation may be reconstituted with a diluent to provide a protein concentration in the reconstituted formulation of at least 50 mg / mL, e.g., about 50 mg / mL to about 400 mg / mL, more preferably about 80 g / mL to about 300 mg / mL, and most preferably about 90 g / mL to about 150 mg / mL. Such high protein concentrations in the reconstituted formulation are considered particularly useful when the reconstituted formulation is intended for subcutaneous delivery. However, for other routes of administration, such as intravenous administration, a lower protein concentration in the reconstituted formulation (e.g., about 5 to 50 mg / mL or about 10 to 40 mg / mL of protein in the reconstituted formulation) may be desirable. In certain embodiments, the protein concentration in the reconstituted formulation is significantly higher than the concentration in the pre-lyophilized formulation. For example, the protein concentration in the reconstituted formulation may be about 2 to 40 times, preferably 3 to 10 times, and most preferably 3 to 6 times (e.g., at least 3 times or at least 4 times) the protein concentration in the pre-lyophilized formulation.

[0165] Reconstitution is generally performed at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, for example, on the type of diluent, excipients, and amount of protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives are described above, with aromatic alcohols such as benzyl or phenol alcohol being preferred preservatives. The amount of preservative employed is determined by evaluating various preservative concentrations for compatibility with the protein and testing for preservative effectiveness. For example, if the preservative is an aromatic alcohol (e.g., benzyl alcohol), it may be present in an amount of about 0.1-2.0%, preferably about 0.5-1.5%, and most preferably about 1.0-1.2%. Preferably, the reconstituted formulation has fewer than 6,000 particles greater than 10 μm in size per vial.

[0166] Therapeutic applications The engineered bioconjugates described herein may be used to treat patients with cancer. Methods of treatment include administering to a patient in need thereof an effective amount of the engineered bioconjugate or pharmaceutical composition described herein. Examples of cancers include, but are not limited to, cancers associated with and / or expressing the Globo series of antigens, including but not limited to, Globo H, SSEA-4, and SSEA-3; and cancers associated with and / or expressing HER2, TROP2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, Claudin 18.2, or Sirp-Alpha.

[0167] The subject treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment may be a human patient with, at risk of, or suspected of having cancer, including, but not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, cervical cancer, ovarian cancer, and prostate cancer. Subjects with cancer can be identified by routine medical testing. In particular, the cancer is a cancer that expresses a Globo series antigen.

[0168] In certain embodiments, the cancer is breast cancer.

[0169] Additionally, the engineered bioconjugates described herein may be used to treat patients with autoimmune or inflammatory diseases. Methods of treatment include administering to the patient an effective amount of a glycoengineered antibody or pharmaceutical composition described herein. Examples of autoimmune or inflammatory diseases include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, leukemia, and leukemia. No's syndrome, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranoproliferative glomerulonephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves' thyroid dysfunction, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss syndrome, asthma, psoriasis Arthritis, dermatitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, eczema, atherosclerosis, leukocyte adhesion deficiency, juvenile-onset diabetes, Reiter's disease, Behçet's disease, hemolytic anemia, atopic dermatitis, Wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, HIV and herpes-related diseases, systemic sclerosis, Sjögren's syndrome and glomerulonephritis, dermatomyositis, ANCA, aplastic anemia, autoimmune diseases These include infectious hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman syndrome, Goodpasture's syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), Guillain-Barré syndrome, large-vessel vasculitis, giant cell (Takayasu) arteritis, medium-vessel vasculitis, Kawasaki disease, and polyarteritis nodosa.

[0170] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present disclosure to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced therein.

[0171] Other Aspects of the Disclosure In another aspect, the present disclosure provides a method for remodeling core fucosylated or non-fucosylated GlcNAc-peptides, proteins, and IgG or IgG-Fc fragments, comprising the steps of providing a peptide / protein / antibody-GlcNAc acceptor or Fc fragment and reacting it with an activated oligosaccharide donor under the catalysis of Streptococcus dysgalactiae sp. dysgalactiae and Streptococcus equi sp. zooepidemicus Sz105 glycosynthase enzymes, thereby preparing substantially pure, essentially pure, and / or pure glycoforms of existing peptides, proteins, and monoclonal antibodies with heterogeneous glycosylation states.

[0172] In a further aspect, the present disclosure provides a method of using glycosynthase enzymes for glycan remodeling of therapeutic IgG or Fc fragments thereof, the method comprising: A. treating native or recombinant core-fucosylated or non-fucosylated therapeutic IgG or IgG-Fc fragment bearing heterogeneous N-glycans with an endoglycosidase (e.g., wild-type EndoS2) with or without bacterial alpha-fucosidase to hydrolyze the bond between two reducing-end GlcNAc residues and form a core-fucosylated or non-fucosylated GlcNAc-IgG acceptor; B. Transferring a wide range of predefined oligosaccharide building blocks in the form of activated oligosaccharide donors to core-fucosylated or non-fucosylated GlcNAc-IgG by transglycosylation using Streptococcus dysgalactiae sp. dysgalactiae and Streptococcus equi sp. zooepidemicus Sz105 glycosynthase enzymes to reconstitute the native beta-1,4 linkage, thereby remodeling core-fucosylated or non-fucosylated IgG or its Fc fragment by attaching predefined oligosaccharides. Includes. [Example]

[0173] Embodiments of the present invention are further illustrated by the following specific examples. Those skilled in the art will recognize that these specific examples are illustrative only and that other modifications and variations are possible without departing from the scope of the present invention. For example, the enzyme variants of the present invention can be used to glycoengineer any glycoprotein or glycopeptide, including antibodies. The specific examples described herein use an anti-CD20 antibody. However, those skilled in the art will recognize that other glycoproteins or antibodies can be used as well.

[0174] material Monoclonal anti-Globo H antibody OBI-888 was produced according to our previous procedures disclosed in PCT patent publications (WO2015157629A2 and WO2017062792A1). Monoclonal anti-SSEA4 antibody OBI-898 was produced according to our previous procedures disclosed in PCT patent publication (WO2017172990A1). Commercially available antibodies Herceptin (trastuzumab), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), and Bavencio (avelumab) are

[0175] [Table 3] Purchased from.

[0176] Biantennary glycans, sialylated complex N-glycans (NSCTs), were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan, D4065), and NSCT-oxazoline was synthesized as previously reported (Noguchi, M. et al. (2012) Helvetica chimica acta 95:1928-1936). Other glycans (M3, G0, and G2) and Bf-α-fucosidase were produced according to previous publications (Tsai, T. I. et al. (2017) ACS Chem. Biol. 12:63-72; Fairbanks, A. J. (2013) Pure Appl. Chem. 85:1847-1863).

[0177] Example 1 Cloning, overexpression, and purification of EndoSd-D232M and EndoSz-D234M and mutants The EndoSd and EndoSz genes from Streptococcus dysgalactiae sp. dysgalactiae (ANI26082.1) and Streptococcus equi sp. zooepidemicus Sz105 (KIS14581.1) were used for this study. The signal peptides at the N-terminus of both enzymes were deleted. To enhance transglycosylation activity, the protein sequences of EndoSd and EndoSz were aligned with EndoS-D233Q (Huang, W. et al. (2012) J. Am. Chem. Soc. 134:12308-12318) and found that the relative positions were D232 and D234 for EndoSd and EndoSz, respectively. We decided to mutate the relative positions D to M. Therefore, genes encoding amino acids 20–1067 of EndoSd-D232M and 20–1011 of EndoSz-D234M were synthesized and subcloned into pGEX-4T-1 with 5′-BamHI and 3′-XhoI restriction sites. For purification purposes, we further inserted six histidine residues at the C-terminus of EndoSd-D232M and EndoSz-D234M for affinity Ni-NTA column loading. Other mutants used in this study were generated by site-directed mutagenesis. Relevant primers were designed based on the mutated sites. EndoSd-D232M and EndoSz-D234M were used as template vectors, and the mutated vectors were amplified with Pfu DNA polymerase (Protech). The template vector (methylated DNA) was then digested with DpnI (Promega) for 2 h (37°C). The mutated vectors were transformed into DH5α competent cells for selection, and all mutants were confirmed by DNA sequencing.

[0178] All vectors were transformed into BL21(DE3) cells and grown at 37°C in TB medium containing ampicillin antibiotic (50 μg / mL). Protein production was induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) until the cell density (OD600) reached 0.6. After 5 h, cells were harvested by centrifugation (BACKMAN / JLA-8.1, 9000 g) for 15 min at 25°C. The cell pellet was resuspended in wash buffer containing 50 mM MOPS (pH 7.0), 300 mM NaCl, and 10 mM imidazole (100 mL of buffer per 1 L of cell pellet), and the cells were disrupted using a homogenizer (NanoLyzer N-10). After centrifugation at 12,000 g (BACKMAN / JA-10) for 60 min at 4°C, the pellet was discarded, and the supernatant was mixed with Ni-NTA resin (Roche) and gently rocked overnight at 4°C to ensure complete protein binding. The resin was loaded onto an open column, and unbound protein was washed with wash buffer until the unbound protein concentration was less than 1 mg / mL (as defined by Bradford assay, Thermo Scientific). Bound protein was eluted with elution buffer containing 50 mM MOPS (pH 7.0), 300 mM NaCl, and 250 mM imidazole. The eluted fraction was dialyzed against storage buffer containing 50 mM MOPS (pH 6.7) and concentrated using a 30 kDa cutoff cassette using a TFF (Millipore lab-scale). The final sample was assayed by SDS-PAGE and Bradford HPLC, respectively, to determine MW and concentration.

[0179] Example 2 Deglycosylation of OBI-888 with EndoS-WT and Bf-α-fucosidase to produce mAb-GlcNAc and mAb-GlcNAc(F) OBI-888 and Herceptin monoclonal antibodies (10 mg) were incubated with EndoS (10 μg) in 25 mM sodium citrate buffer (pH 6.5) and 100 mM NaCl for 4 h at 37°C. Complete cleavage of Fc N-glycans was analyzed by 4-12% gradient SDS-PAGE.

[0180] The N-glycans of OBI-888 (10 mg) were digested by incubation with EndoS-WT (10 μg) and Bf-α-fucosidase (10 mg) in Tris-HCl buffer (pH 7.4) at 37°C for 16 hours to generate OBI-888-GlcNAc. The commercially available antibodies Herceptin (trastuzumab), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), and Bavencio (avelumab) (10 mg) were digested using the same procedure as for OBI-888, except that the temperature was 30°C. Complete cleavage of the Fc N-glycans was analyzed by 4-12% gradient SDS-PAGE. Fucosylated mAb-GlcNAc (mAbs-GlcNAc-F) was produced only by EndoSz wild-type under similar conditions with an incubation time of 4 hours.

[0181] Example 3 Transglycosylation of glycans to mAb-GlcNAc and mAb-GlcNAc-F In a general procedure, 5 mg of mAb-GlcNAc / mAb-GlucNAc-F was incubated with EndoSz-D234M (167 μg) or EndoSd-D232M (1002 μg) at a molar ratio (mAb-GlcNAc:NSCT-oxa) of 1:20 and 1:150, respectively, in MOPS buffer (50 mM, pH 6.7) at 30°C for 20 min in a final volume of 500 μL. Some minor modifications were made depending on the experimental purpose and design (see Results). HPLC was employed to monitor transglycosylation efficiency.

[0182] Example 4: Purification of deglycosylated homogeneous mAb The reaction mixture was loaded onto a HiTrap Protein-A HP (5 mL, GE) prepacked column pre-equilibrated with PBS buffer. At each step, unbound contaminants were washed with a two-step pH gradient of 5 column volumes of PBS (pH 7.4) and glycine-HCl (pH 5.0). The bound antibody was eluted using sodium citrate (pH 3.0). The eluted fraction was immediately neutralized to pH 7.4 with Tris-HCl buffer (1 M, pH 9.0) and dialyzed overnight at 4°C using a 30 kDa cutoff dialysis cassette (Thermo) against a storage buffer containing 50 mM MOPS (pH 6.7) for mAb-GlcNAc(F) and 5 mM histidine and 150 mM NaCl for mAb-G2S2. All samples were concentrated using Amicon centrifugal membranes (cutoff 30 kDa, Millipore) and stored at 4°C [mAb-GlcNAc(F)] or −80°C [mAb-G2S2(F)].

[0183] [Example 5] LC / MS / MS of glycopeptide analysis Samples were first processed for buffer exchange into ddH2O using an Amicon Ultra-0.5 device with a 10 kDa cutoff. The samples were denatured in 0.1% RapiGest SF solution / 50 mM triethylammonium bicarbonate (TEABC), reduced with 5 mM dithiothreitol (DTT) at 60°C for 30 minutes, and then alkylated with 15 mM iodoacetamide in the dark at room temperature for 30 minutes. The resulting samples were subjected to in-solution trypsin digestion (trypsin:sample protein = 1:30) in 50 mM TEABC at 37°C overnight. After digestion, the samples were acidified with 0.5% trifluoroacetic acid (TFA) (v / v) and incubated at 37°C for 45 minutes. The acid-treated samples were centrifuged at 14,000 rpm for 30 minutes at 4°C to precipitate hydrolytic RapiGest SF by-products. Samples were analyzed using a Thermo Q-Exactive mass spectrometer (Thermo Scientific) coupled to an Ultimate 3000 RSLC system (Dionex). LC separation was performed using a C18 column (Acclaim PepMap RSLC, 75 μm × 150 mm, Thermo) with mobile phases A: 0.1% FA (formic acid) and B: 95% ACN (acetonitrile) / 0.1% FA. Table 3 lists the analytical solvent gradient.

[0184] [Table 4]

[0185] All MS scans were performed over the m / z range of 300 to 2000, and the 10 most intense ions from the MS scan were selected for the MS / MS scan.

[0186] Example 6: Enzymatic conjugation by HPLC Glycosynthase activity was analyzed by HPLC (Waters e2695) using a 2.1 x 150 mm UPLC glycoprotein amide column (Waters) with two different buffers (Buffer A: ddH2O / 0.3% v / v HFIP (1,1,1,3,3,3-hexafluoro-2-propanol), 0.1% v / v TFA; Buffer B: ACN, 0.3% v / v HFIP, 0.1% v / v TFA) and the gradient shown in Table 4.

[0187] [Table 5]

[0188] Prior to the experiment, the column was washed with 50% ACN and 50% ddH2O for 30 minutes and equilibrated with 15% Buffer A and 85% Buffer B until the system pressure stabilized. After ensuring baseline stability with a water-for-injection blank, the process began with a 2 μL sample injection. Over the course of the process (29 minutes), the flow rate was 0.2 mL / min, the column temperature was 65 °C, and the sample tray temperature was 5 °C.

[0189] Example 7: ADCC assay of engineered antibodies ADCC activity was analyzed using the ADCC Reporter Bioassay Complete Kit (Promega, G7015) with luciferase reporter cells. The relevant cell lines MCF7W (OBI-888), SKBR-3 (Herceptin, Perjeta), BxPC3 (Erbitux), and Raji (Rituxan) were selected for analysis. The cell lines shared the same procedure. Target cells were seeded into 96-well cell culture plates and incubated overnight at 37°C in a humidified 5% CO2 incubator. The culture medium was replaced with serially diluted homogenous antibodies and corresponding antibody standards in triplicate. ADCC bioassay effector cells were added to each well. The effector cell to target cell ratio was 3:1. A 6-hour induction was performed, followed by the addition of Bio-Glo Luciferase Assay Buffer. After 15 min, luminescence (RLU, relative luminescence units) was determined using a microplate reader (SpectraMax L, Molecular Devices, Sunnyvale, CA). The fold change in luminescence induction was calculated by the ratio of relative luminescence units (RLU) (induced) to RLU (no antibody control). EC 50 was determined by plotting x (concentration, μg / mL) versus y (fold change induced) and fitting the data with a 4PL nonlinear regression model using PRISM 6 software. Relative potency was estimated by parallel line analysis using a Gen5 microplate reader and Imager software (BioTek Instruments).

[0190] Example 8: Cleavage and conjugation of Herceptin Antibodies produced by CHO cells always contain a heterogeneous glycan at the N297 position of the Fc. To generate homogeneous mAbs and enhance ADCC, enzymatic modification of glycan cleavage and transglycosylation is an essential step in the homogeneous platform (Figure 1). Regarding glycan cleavage, EndoSz-WT has been reported to hydrolyze biantennary glycans, but it is not a general chitinase (Shadnezhad, A. et al. (2016) Future Microbiol 11:721-736). To demonstrate the capabilities of EndoSz-WT, we used it to hydrolyze native Herceptin and detected its N-glycan profile. The results showed that EndoSz-WT could hydrolyze biantennary hybrid and high-mannose glycans. EndoSz-WT and α-fucosidase enzymes produced over 99% Herceptin with a single N-acetylglycosamine at position N297 (Herceptin-GlcNAc) (Figure 2). However, for mAbs containing glycosylated Fab, additional enzymes, EndoH or EndoM (Shadnezhad, A. et al. (2016) Future Microbiol 11:721-736; Kadowaki, S. et al. (1990) Agric. Biol. Chem. 54:97-106), were combined in the cleavage step to completely remove the glycans.

[0191] ENGase also possesses glycan conjugation function with appropriate mutations (Huang, W. et al. (2012) J. Am. Chem. Soc. 134:12308-12318; Li, T., Tong et al. (2016) J. Biol. Chem. 291:16508-16518). To accurately monitor the transglycosylation process, an HPLC method using an amide column was established. As shown in Figure 3, native Herceptin had a retention time of 12.5 min. After cleavage with EndoSz-WT and α-fucosidase, the retention time of Herceptin with one GlcNAc residue (Herceptin-GlcNAc) shifted to 11.4 min, with a molecular weight of 145,572 Da calculated by mass spectrometry. It is interesting to note that in the transglycosylation step of oxazoline sialylated complex N-glycans (NSCT-oxa) to Herceptin-GlcNAc (represented by EndoSz-D234M), the column clearly identified semi-glycosylated Herceptin (Herceptin-1N-G2S2) and fully glycosylated Herceptin (Herceptin-2N-G2S2), which were indistinguishable by SDS-PAGE. Herceptin-1N-G2S2 was found with a retention time of 12.6 min and a molecular weight of 147,574 Da, while Herceptin-2N-G2S2 had a retention time of 13.9 min and a molecular weight of 149,576 Da. The HPLC method can be applied to all mAbs. Therefore, the HPLC assay was used for further investigation.

[0192] [Example 9] Examination of transglycosylation of EndoSz-D234M and EndoSd-D232M Transglycosylation is the most critical step determining the quality of homogeneous mAbs in a homogeneous platform. According to a previous report (Li, T., Tong et al. (2016) J. Biol. Chem. 291:16508-16518), EndoS2 with the D184M mutation had high transglycosylation activity. To investigate transglycosylation through multiple sequence alignment, we first generated EndoSz-D234M and EndoSd-D232M (Figure 4). Generally, a higher sugar ratio results in higher conjugation efficiency. However, in practice, the goal of process optimization was to reduce the sugar amount and process within a reasonable time frame to save costs. Using Herceptin-GlcNAc and NSCT-oxa as study models, the transglycosylation activity of EndoSz-D234M and EndoSd-D232M was evaluated within 60 minutes, and more than 90% of Herceptin-2N-G2S2 was predicted to be formed.

[0193] Starting with a 40:1 NSCT-oxa / Herceptin molar ratio, EndoSz-D234M yielded over 90% Herceptin-2N-G2S2. Decreasing the NSCT-oxa / Herceptin molar ratio to 30:1 resulted in over 90% Herceptin-2N-G2S2 in 10 min. At a 20:1 NSCT-oxa / Herceptin ratio, Herceptin-2N-G2S2 reached 89.31% in 5 min and 91.85% in 10 min, remaining constant for up to 20 min before deglycosylation (Figure 5A). At a 10:1 NSCT-oxa / Herceptin molar ratio, Herceptin-2N-G2S2 yielded 62.61% Herceptin-2N-G2S2 in 5 min and 61.48% Herceptin-2N-G2S2 in 10 min, indicating the onset of deglycosylation. To determine the limits of NSCT-oxa use, the amount of NSCT-oxa / Herceptin was increased to 15:1 (molar ratio), yielding 80.8% Herceptin-2N-G2S2 in 5 minutes, with glycosylation initiated in 10 minutes. Therefore, the final conditions for transglycosylation with EndoSz-D234M enzyme were a 20:1 NSCT-oxa:antibody molar ratio and a 20-minute reaction time.

[0194] The time-dependent graph of EndoSz-D234M (Figure 5A) clearly illustrates the enzyme's behavior. Herceptin-GlcNAc decreased to almost 0% within 5 min, revealing the majority of fully glycosylated antibody. The enzyme rapidly bound to Fc and conjugated the glycan at the N297 position of Fc. However, the highly efficient transglycosylation enzyme also contributed to hydrolytic activity. In all studies, the reaction reached a peak efficiency of transglycosylation and remained constant for a period (20 min) while deglycosylation occurred. This demonstrates the importance of controlling the reaction time during the process. Notably, the data show that the decrease in the percentage of Herceptin-2N-G2S2 was accompanied by a major increase in the percentage of Herceptin-1N-G2S2 and a minor increase in the percentage of Herceptin-GlcNAc, implying that the enzyme has a target-selection preference in the hydrolysis reaction.

[0195] In contrast, applying the final transglycosylation conditions of EndoSz-D234M to the EndoSd-D232M enzyme showed inconsistent results. EndoSd-D232M produced only 46.06% Herceptin-2N-G2S2 at a molar ratio of 20:1 (NSCT-oxa / antibody), demonstrating the superior glycosynthase activity of EndoSz-D234M compared with EndoSd-D232M. To obtain more fully glycosylated antibody, the amounts of EndoSd-D232M and NSCT-oxa were increased to enhance transglycosylation efficiency. Using a fivefold excess of EndoSd-D232M enzyme, only 60% Herceptin-2N-G2S2 was produced. Increasing the amount of NSCT-oxa to 80:1 (molar ratio) resulted in unstable results for Herceptin-2N-G2S2, ranging from 80 to 90%. Finally, a 150:1 (molar ratio) of NSCT-oxa produced stable and reproducible data. The time-dependent graph (Figure 5B) showed that EndoSd-D232M slowly transferred NSCT-oxa to N297 (Fc region) despite the high amount of substrate used. At 5 min, Herceptin-1N-G2S2 (approximately 50%) had a higher amount than Herceptin-2N-G2S2 (approximately 40%). Herceptin-2N-G2S2 reached 80% by 10 min and 94% by 20 min. No deglycosylation was observed within 60 min.

[0196] In addition to using Herceptin-GlcNAc as an acceptor, we also investigated the transglycosylation activity of fucosylated Herceptin-GlcNAc (Herceptin-GlcNAc-F) for potential ADC applications. Using the best transglycosylation conditions described above, EndoSz-D234M and EndoSd-D232M yielded 94.29% and 94.75% yields of Herceptin-2N-G2S2F, respectively (Table 5). This demonstrated that the two enzymes possess transglycosylation activity on fucosylated substrates.

[0197] [Table 6]

[0198] In conclusion, EndoSz-D234M has better transglycosylation activity than EndoSd-D232M. EndoSz-D234M stably produced over 90% Herceptin-2N-G2S2 (Herceptin-2N-G2S2F) at a molar ratio of only 20:1 (NSCT-oxa:antibody), whereas EndoSd-D232M required 7.5 times more substrate.

[0199] [Example 10] Examination of transglycosylation of EndoSz and EndoSd mutants Previous reports have shown that some mutant sites, such as EndoS-D233Q and EndoS2-D184M, can increase transglycosylation activity toward glycans (Huang, W. et al. (2012) J. Am. Chem. Soc. 134:12308-12318; Li, T., Tong et al. (2016) J. Biol. Chem. 291:16508-16518). Recently, Shivatare et al. reported that the EndoS2-T138Q mutation increased activity better than EndoS2-D184M (Shivatare, SS et al. (2018) Chem. Commun. 54, 6161-6164). Based on multiple sequence alignment (Figure 4), the equivalent positions of T183, D232, D234, D280, S281, and T282 in EndoSz and T181, D230, D232, D278, S279, and T280 in EndoSd were selected as targets for site-directed mutagenesis. Among them, the D234 site in EndoSz and the D232 site in EndoSd were generated in several different forms, including positively / negatively charged and polar / nonpolar. Transglycosylation activity was assayed using the best conditions described above.

[0200] The activity assay results of the EndoSz mutants (Figure 6A) showed that EndoSz-D234M had the highest activity (defined as 100%), and EndoSz-D234Q (99.9%), EndoSz-D234S (98.8%), and D234F (98.6%) had competitively high activity. In contrast, EndoSz-D234R (4.9%) and EndoSz-D234H (4.5%) had low activity. The wild-type EndoSz also had a slight transglycosylation activity (24.3%). In addition to position D234, EndoSz-T183Q (89.1%), EndoSz-D232Q (31.4%), EndoSz-D280Q (34.0%), EndoSz-S281Q (12.9%), and EndoSz-T282Q (16.3%) did not have significantly increased transglycosylation activity compared to EndoSz-D234M.

[0201] Among the EndoSd mutants (Fig. 6B), EndoSd-D232M (defined as 100%), EndoSd-D232S (100.8%), and EndoSd-D278Q (108.0%) had equally high transglycosylation activity, whereas EndoSd-D232R (9.2%) and EndoSd-D232H (8.1%) had low activity. EndoSd wild-type had a relatively high transglycosylation activity (84%).

[0202] In conclusion, EndoSz-D234M and EndoSd-D278Q were shown to exhibit relatively good transglycosylation activity toward Herceptin-GlcNAc and to produce homogeneous Herceptin bearing NSCT-oxa in the Fc region.

[0203] [Example 11] Examination of conjugation of EndoSz-D234M to various sugars A homogeneous platform was designed to conjugate various sugars to the Fc region of Herceptin. Glycans M3, G0, and G2 were used for the EndoSz-D234M study. The results showed that all of the glycans were successfully conjugated to the Fc region at a molar ratio of 20:1 (NSCT-oxa:antibody). Except for M3, G0 and G2 reached over 90% fully glycosylated Herceptin (Table 6).

[0204] Table 6. Transglycosylation results of EndoSz-D234M with various glycans on various acceptors.

[0205] [Table 7]

[0206] M3 was able to yield 78% fully glycosylated Herceptin in 5 minutes, after which deglycosylation was initiated. To optimize the conjugation rate, increasing the M3 ratio to 30:1 (molar ratio) achieved 86.3% yield in 10 minutes before deglycosylation, and increasing the ratio to 40:1 (molar ratio) yielded 90% fully glycosylated Herceptin. The final conjugation conditions with M3 were 40:1 with a 10-minute reaction time, yielding 92.37% fully glycosylated Herceptin. This homogeneous platform was applicable not only to Herceptin-GlcNAc but also to Herceptin-GlcNAc-F, which conjugates various glycans to the Fc region, resulting in conjugation efficiencies exceeding 90%.

[0207] [Example 12] Transglycosylation of various antibodies The homogeneous platform is a powerful process for establishing homogeneous mAbs. Several other mAbs, including OBI-888, Perjeta, Erbitux, Rituxan, OBI-898, Vectibix, Humira, Keytruda, and Bavencio, were selected for conjugation with EndoSz-D234M. At a 20:1 molar ratio of NSCT-oxa to antibody, the results demonstrated the effectiveness of the homogeneous platform (Figure 7). The percentage of fully glycosylated mAb was OBI-888-G2S2: 87.57%, Perjeta-G2S2: 92.49%, Erbitux-G2S2: 87.92%, Rituxan-G2S2: 97.57%, OBI-898-G2S2: 89.73%, Vectibix-G2S2: 86.12%, Humira-G2S2: 93.68%, Keytruda-G2S2: 75.81%, and Bavercio-G2S2: 90.73%.

[0208] To evaluate the activity of the homogenous glycans against various mAbs, five antibodies were selected for ADCC bioassay (Figure 7B). The EC50 of all homogenous mAbs was increased compared to the original mAb. OBI-888 had the best ADCC improvement, increasing 26-fold.

[0209] [Example 13] Overall construction of complexes with EndoSz-D234M and glycans Our research identified EndoSz-D234M as having superior transglycosylation activity toward therapeutic IgG antibodies, which plays an important role in our homogeneous mAb platform. To understand the mechanism of transglycosylation, we performed crystallization and structural determination of the mutant EndoSz-D234M. For crystallization and structural analysis, we designed EndoSz-D234M, truncated from amino acids 99 to 974. As a result, we successfully obtained diffraction-quality crystals of EndoSz-D234M, which have two space groups, P212121 and P21. The P212121 crystal diffracted at a higher resolution (approximately 2.2 Å) than the P21 crystal (approximately 3.1 Å). The structures determined from the two crystal forms showed highly similar structural architecture (RMSD 0.75 Å calculated by SSM), and no significant conformational changes were observed between the two structures. Therefore, the high-resolution structure of EndoSz-D234M, which has the space group P212121, was referenced throughout the manuscript. The crystal structure of EndoSz-D234M revealed a monomeric V-shaped structure containing five major domains: a glycosidase hydrolase (GH) domain (aa 99–445), a leucine-rich repeat domain (aa 446–631), a hybrid Ig domain (aa 632–764), a carbohydrate-binding module (CBM) (aa 765–907), and a C-terminal three-helix bundle domain (aa 908–955) (Figure 8A). The CBM domain coordinates one calcium ion, the function of which will be discussed in the next section. The active site of the GH domain exhibited a highly negatively charged surface with conserved residues (Fig. 8B), providing a highly conserved catalytic residue and an environment for specific Endo-β-N-acetylglucosaminidase activity (Fig. 8C). However, several loops surrounding the active site of the GH and CBM domains exhibited more variable residues at the surface (Fig. 8C).In conjunction with the fact that the GH and CBM domains, located at opposite ends of the V-shaped architecture, face the same plane (Figure 8A), the CBM domain may facilitate or direct EndoSz to target the N-glycans of IgG by binding to a given position or one of the two N-linked glycans of the IgG protein and further cleaving or conjugating the N-glycans.

[0210] High-resolution data clearly identified extra density in the GH domain with sufficient quality to allow the construction of all 10 CT N-glycan moieties (Figure 9A). As previously mentioned, the structure of the EndoSz GH domain revealed a typical (α / β)8 TIM barrel fold, a cyclic eight-repeat β-strand / loop / α-helix configuration. The bound complex-type (CT) N-glycan consisted of two glycan antennae, α(1-3) and α(1-6), of the (α / β)8 barrel, surrounded by a Manβ1-4GlcNAc disaccharide and a loop on the upper barrel (Figure 9B). The nomenclature of the CT N-glycan used in the soaking experiments is shown in Figure 9C. The two sugar moieties, Manβ1-4GlcNAc, of the CT N-glycan resided in the cavity formed by the adjacent loop and the β-barrel core (Figure 9B). As shown in Figure 9B, the loops connecting the α-helix and β-strand were annotated as loop 1 (β1-β2; aa 121–146), loop 2 (β2-α1; aa 152–159), loop 3 (β3-α2; aa 186–207), loop 4 (β4-α3; aa 236–248), loop 5 (β5-α4; aa 282–291), loop 6 (β6-α5; aa 305–326), loop 7 (β9-α8; aa 348–380), and loop 8 (β10-α9; aa 402–429). In addition to the Manβ1-4GlcNAc disaccharide, two glycan antennae, α(1-3) and α(1-6), interacted with the adjacent loops, respectively. Compared with antenna α(1-3), antenna α(1-6) is located closer to the edge of the cavity formed by the GH domain and has more interactions with the GH domain (Figure 9B). Loops 3 and 4 of the GH domain interact with antenna α(1-6), while loops 1, 2, and 7 interact with antenna α(1-3). As shown in Figure 10, the altered H3 and loop 4 resulted in specific structural changes when comparing the structures of EndoSz-D234M and EndoS2. These specific structural changes indeed disrupted the binding of EndoSz-D234M to triantennary high-mannose triantennary N-glycans. Furthermore, conservation analysis of the neighboring loops (Figure 9D) indicated that loops 4, 6, 7, and 8 were more variable regions.Taken together, our results suggest that loop 4 plays an important role in substrate selectivity for biantennary glycans rather than triantennary glycans.

[0211] Structural comparison of the apo and holo EndoSz-D234M structures reveals that loop 2 (aa152–159) undergoes a dramatic conformational change (Figures 11A and 11B). In the apo EndoSz-D234M structure, Trp154 and Arg182 form typical cation-π interactions. However, upon the conformational change of loop 2, Trp154 flips its side chain and interacts with the core disaccharides Man(-2) and NAG(-8) via hydrogen bonds and weak π-stacking interactions, respectively, in the holo EndoSz-D234M structure. The dramatic movement of loop 2 reshapes the asymmetric groove in the GH domain to accommodate the two antennae of the CT N-glycan (Figure 11B). Both EndoSz-D234M and EndoS have the same tryptophan (Trp154 in EndoSz) in loop 2, whereas the analogous residue in EndoS2 is histidine (Fig. S11C), which may result in a different substrate-binding mechanism. Furthermore, the neighboring residues of Trp154 (His152, Asp153, and Thr155) are highly conserved (Fig. S11C), highlighting the importance of Trp154 in EndoSz-D234M.

[0212] The bound CT N-glycan utilized the reducing end GlcNAc and the trimannose core (-2, -3, and -7) to form the major contact points with EndoSz-D234M. Here, the first GlcNAc(-1) exhibited two conformations, A and B. Conformation A was the predominant conformation, as shown in Figure 12A. In conformation A, the O1 atom of GlcNAc(-1) interacted with Gln304, while the O6 and O7 atoms of GlcNAc(-1) did not interact with EndoSz. Furthermore, the N2 atom of the acetamide group of GlcNAc(-1) formed a hydrogen bond with the side chain of Met234. In conformation B (Figure 12B), the O1 atom of GlcNAc(-1) had no interaction, and the O6 and O7 atoms of GlcNAc(-1) interacted with Trp359 and Tyr306, respectively. The O2 and O4 atoms of Man(-2) formed hydrogen bonds with the side chain of Tyr401 and the indole nitrogen of Trp154, respectively. Furthermore, the sugar ring of Man(-2) formed a π-stacking interaction with Phe151. The O2 atom of Man(-3) formed a hydrogen bond with the side chain of Arg187. The O3, O4, and O6 atoms of Man(-7) formed hydrogen bonds with the indole nitrogen of Trp122, Arg120, and the side chain of Asn357, respectively. Because the substrate in the crystallization experiments did not contain a GlcNAc(+1) moiety, the major hydrogen-bonding interactions between the CT N-glycan and EndoSz were attributed to four moieties of the pentasaccharide core, namely, GlcNAc(-1), Man(-2), Man(-3), and Man(-7). Furthermore, GlcNAc(-8) on antenna α(1-3) had a π-π interaction with Trp154, whereas GlcNAc(-4) on antenna α(1-6) had a weak π-π interaction with His194 and a hydrogen bond with Arg187, which provided minor contacts between EndoSz-D234M and the bound CT N-glycan. In summary, the amino acid residues Arg120, Trp122, Phe151, Trp154, Arg187, His194, Met234, Gln304, Tyr306, Asn357, Trp359, and Tyr401 were the binding sites of EndoSz-D234M.

[0213] Example 14 Preparation of NSCT-2 (Oxazoline-NSCT-N3) NSCT-1 (235 mg, 0.097 mmol, purchased from Glytech, Inc., catalog no. GT-25261; HPLC purity >90%) and triethylamine (605 μL, 0.44 mmol) were dissolved in water (10 mL) and cooled to 0°C. An aqueous solution of 2-chloro-1,3-dimethyl-1H-benzimidazoline-3-ium chloride (1 M, 1.44 mL) was slowly added, and the resulting mixture was stirred at 0–5°C for 4 h. NaOH solution (0.01 M, 1 mL) was added, and the resulting mixture was concentrated under reduced pressure. After evaporation of most of the triethylamine, the remaining mixture was purified on a Sephadex® g-15 column. NSCT-2 was stabilized using 0.01 M NaOH as the eluent. Fractions containing the desired product were combined and lyophilized to give NSCT-2 (170 mg) as a white solid. 1H NMR (DO): δ 6.10 (d, J = 7.26 Hz, 1H, oxazoline H1), 5.24 (s, 1H, GluNAc H1), 4.97 (s, 1H, GluNAc H1), 4.76 (s, 1H, Man H1), 4.64-4.60 (m, 2H, two Gal H1), 4.46 (s, 1H, Neu5Ac H1), 4.45 (s, 1H, Neu5Ac H1), 4.40 (s, 1H, β-Man H3), 4.18 (d, J = 21 Hz, 4H, two Man and two GluNAc H2), 3.99-3.48 (m), 2.71 (dd, J1 = 12.9 Hz, J2 = 4.2 Hz, 2H, H3eq of two Neu5Ac), 2.15-2.02 (m, 15H), 1.58 (dd, J1 = J2 = 12.2 Hz, 2H, H3ax of two Neu5Ac).

[0214] [ka]

[0215] Example 15: Deglycosylation, transglycosylation, and purification of monoclonal antibodies 15-1. Deglycosylation of mAb with EndoSz-D234M to generate mAb-GlcNAc(Fuc) Seven monoclonal antibodies were used in this study. R4702 is an anti-TROP2 monoclonal antibody, and enfortumab was produced in-house; all other monoclonal antibodies were purchased: Herceptin (Roche), Perjeta (Roche), Erbitux (Meck), Rituxan (Roche), and TX05 (anti-HER2 mAb; Tanvex). Monoclonal antibodies were deglycosylated with EndoSz-D234M in 50 mM Tris (pH 7.2) at 37°C for 24–49 hours. Only antibodies with high-mannose N-glycan modifications were further treated with EndoH and incubated overnight at 25°C to completely remove glycans, yielding mAb-GlcNAc(Fuc). Complete cleavage of the Fc N-glycan was analyzed by SDS-PAGE and CE-SDS.

[0216] To investigate deglycosylation and transglycosylation, EndoSz-D234M for the glycan ADC platform was applied to other mAbs, including Herceptin, Perjeta, Rituxan, Erbitux, and enfortumab. For deglycosylation studies, mAbs were incubated with EndoSz-D234M at a weight ratio of 1:30 (EndoSz-D234M:mAb). EndoH was also added to Rituxan and enfortumab to completely cleave high-mannose glycans. The results below show the percentage of mAb-GlcNAc(Fuc) after the deglycosylation treatment. The cleavage rates of Herceptin-GlcNAc(Fuc): 94.91%, Perjeta-GlcNAc(Fuc): 95.04%, Rituxan-GlcNAc(Fuc): 97.25%, Erbitux-GlcNAc(Fuc): 100%, and enfortumab-GlcNAc(Fuc): 96.9% were all greater than 90% after EndoSz-D234M cleavage. To study transglycosylation by NSCT-2, 20 or 38 equivalents of NSCT-2 were added to mAb-GlcNAc(Fuc) along with EndoSz-D234M for 1.5–2 h at 37°C. The results showed that the percentages of mAb-(NSCT-di-N3)2 were Herceptin-(NSCT-di-N3)2: 89.27%, Perjeta-(NSCT-di-N3)2: 94.62%, Rituxan-(NSCT-di-N3)2: 94.88%, Erbitux-(NSCT-di-N3)2: 93.36%, and Enfortumab-(NSCT-di-N3)2: 95.8% by CE-SDS. These data demonstrate that EndoSz-D234M for the glycan ADC platform is applicable to various mAbs.

[0217] Table 7. Deglycosylation and transglycosylation of various mAbs

[0218] [Table 8]

[0219] 15-2. Transglycosylation of mAb-GlcNAc(Fuc) with oxazoline-NSCT-N3 (NSCT-2) to generate mAb-(NSCT-di-N3)2 Briefly, mAb-GlcNAc(Fuc) was incubated with 20–38 equivalents of NSCT-2 for 1.5–2 h at 37°C to generate mAb-(NSCT-di-N3)2. The efficiency of transglycosylation was monitored by SDS-PAGE and CE-SDS.

[0220] 15-3. Purification of mAb-(NSCT-di-N3)2 The transglycosylation mixture was adjusted to a final concentration of 3M sodium chloride and loaded onto a HiTrap Phenyl HP (Cytiva) column pre-equilibrated with PBS and 3M NaCl. Unbound contaminants were washed away with 5 CV of equilibration buffer (PBS and 3M NaCl). mAb-(NSCT-di-N3)2 was eluted with a 20 CV linear gradient of 30 to 100% elution buffer (20 mM sodium phosphate and 20% IPA, pH 7.2). The eluted fraction was loaded onto a pre-packed HiTrap Protein A HP column (Cytiva). Impurities were washed away with a two-step pH gradient: 100 mM sodium citrate, pH 6.0, and pH 5.5, each 5 CV. Bound antibody was eluted using 50 mM sodium citrate (pH 3.5). The eluted fraction was immediately neutralized to its native pH with 1 M Tris-HCl (pH 9.0) and the buffer was changed to 20 mM sodium acetate (pH 5.0) using an Amicon centrifuge membrane (30 kDa cutoff, Millipore). The purified mAb-(NSCT-di-N3)2 was stored at -80°C.

[0221] Example 16 Preparation of ADC 16-1.MCCA-PEG 24 Preparation of -VA-PAB-Exatecan (Linker-Drug Compound 1)

[0222] Step 1: N-PM-0015 and DIPEA were added to a suspension of exatecan mesylate in DMF at room temperature. The suspension became a clear brown solution within 5 minutes. The mixture was stirred at room temperature for 20 hours. After the reaction was complete, the reaction mixture was added to stirred TBME over 30 minutes to obtain a precipitate. After stirring for 30 minutes, the solid was collected by filtration, followed by high vacuum drying to obtain crude N-PM-0016. This crude product was used in the next step without further purification.

[0223] Step 2 A stirred suspension of N-PM-0016 in DCM was cooled to -20°C. TFA liquid pre-cooled to -10°C was added to the N-PM-0016 solution over 60 minutes. The mixture was stirred at -20°C for 10 hours. After the reaction was complete, the reaction mixture was added to stirred TBME over 30 minutes to obtain a precipitate. After stirring for 30 minutes, the solid was collected by filtration, followed by high vacuum drying to obtain crude N-PM-0018. This crude product was used in the next step without further purification.

[0224] Step 3 To a solution of N-DT-0013 in DMF, HATU and NMM were added. The mixture was stirred at room temperature for 2 hours. A solution of N-PM-0018 and NMM in DMF was added to the solution of N-DT-0013 at room temperature over 30 minutes. The mixture was stirred at room temperature for another 2 hours. After the reaction was complete, the reaction mixture was added to TBME with stirring over 30 minutes to obtain a precipitate. After stirring for 30 minutes, the solid was collected by filtration and purified by reverse-phase chromatography (eluent: ACN / water). The pure fractions were combined and extracted with 10% MeOH / DCM to obtain N-PM-0017.

[0225] [ka]

[0226] 16-2.DBCO-PEG 24 Preparation of -VA-PAB-exatecan (Linker-Drug Compound 2) Step 1: N-(9-Fmoc)-L-glutamic acid γ-tert-butyl ester monohydrate (152.2 mg, 0.35 mmol), m-PEG24-amine (380.9 mg, 0.35 mmol), and HATU (159.7 mg, 0.42 mmol) were dissolved in DMF / CHCl (3.5 mL) at room temperature. NMM (115.8 μL, 1.05 mmol) was added. After the addition, the resulting mixture was stirred at room temperature for 18 h. The reaction solution was concentrated under vacuum at 30-35 °C, and the residue was purified by flash silica gel column chromatography (CHCl / MeOH = 15 / 1 to 12 / 1) to give 502.5 mg of Glu-1 in 96.0% yield.

[0227] [ka]

[0228] Step 2: To a solution of Glu-1 (413.8 mg, 0.27 mmol) in CHCl / MeOH (13.8 mL) (1 / 1) was added EtNH (1.38 mL). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was concentrated under vacuum at 30–35 °C and azeotroped with toluene (5 mL × 3) to remove excess EtNH. The resulting material was dried under high vacuum to give crude Glu-2. This crude product was used in the next step without further purification.

[0229] [ka]

[0230] Step 3: To a solution of crude Glu-2 (352.3 mg, 0.27 mmol) and DBCO acid (101.4 mg, 0.33 mmol) in DMF / CHCl (5.5 mL) (1 / 1), HATU (157.8 mg, 0.45 mmol) and NMM (91.6 μL, 0.83 mmol) were added separately. The reaction mixture was stirred at room temperature for 16 h. After completion of the reaction, the reaction solution was concentrated under vacuum at 30-35 °C, and the residue was purified on a flash silica gel column (CHCl / MeOH (15 / 1)) to give 367.9 mg of DBCO-1 in 85.2% yield from Glu-1.

[0231] [ka]

[0232] Step 4: A solution of DBCO-1 (350.0 mg, 0.22 mmol) in CHCl (6.9 mL) was cooled to 0 °C. TFA (1.8 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 4–6 h. After the reaction was completed, the reaction mixture was concentrated under vacuum at 30–35 °C, and the residue was purified by flash silica gel column (CHCl / MeOH = 12 / 1) to give 175.4 mg of DBCO-2 in 52.0% yield.

[0233] [ka]

[0234] Step 5: N-PM-0018 (26.5 mg, 0.031 mmol), DBCO-2 (45.9 mg, 0.031 mmol), and HATU (13.9 mg, 0.037 mmol) were dissolved in DMF (0.61 mL). NMM (10.1 μL, 0.092 mmol) was added. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the resulting mixture was purified by preparative HPLC to give 48.9 mg of DL-2 in 71.5% yield. 11H NMR (600 MHz, d-MeOH) δ 7.70 - 7.50 (m, 6H), 7.46 - 7.36 (m, 5H), 7.33 - 7.10 (m, 3H), 5.56 (d, 1H, J = 16.0 Hz), 5.36 (dd, 1H, J = 16.0, 6.2 Hz), 5.33 - 5.29 (m, 1H), 5.27 (d, 1H, J = 20.2 Hz), 5.21 - 5.13 (m, 2H), 5.13 - 5.05 (m, 2H), 4.47 - 4.41 (m, 1H), 4.23 - 4.12 (m, 2H), 3.73 - 3.52 (m, 102H), 3.50 - 3.46 (m, 1H), 3.43 - 3.38 (m, 1H), 3.35 (s, 3H), 3.27 - 3.17 (m, 1H), 3.15 - 3.07 (m, 1H), 2.81 - 2.70 (m, 1H), 2.37 (s, 3H), 2.36 - 2.24 (m, 4H), 2.45 - 2.40 (m, 1H), 2.24 - 2.00 (m, 5H), 2.00 - 1.78 (m, 4H), 1.43 (d, 1H, J = 7.1 Hz), 1.03 - 0.94 (m, 9H); HRMS (ESI) m / z found [(M+2H) / 2] + , 1121.0605 C 113 H 164 FN9O 36 2+ , calculated value 1121.0553.

[0235]

Table 9

[0236]

Chem.

[0237] 16 - 3.MCCA - PEG 24 Preparation of ADC Using 16 - 3.MCCA - PEG - VA - PAB - Exatecan (Linker - Drug Compound 1) R4702 and TX05 mAb (10 mg / mL, 50 mL total) in reaction buffer (50 mM histidine, 20 mM EDTA, pH 7.0) were cooled to 12–16 °C. R4702 and TX05 were treated with TCEP-HCl (2.29 mg; 0.00799 mmol) in reaction buffer (0.46 mL) for 2–6 h at 12–16 °C. To reduce the antibody solution, payload-linker N-PM-0017 (39.94 mg; 0.0183 mmol) in DMSO was added and conjugated for 1 h at 12–16 °C. After conjugation was complete, the buffer was exchanged into storage buffer (20 mM sodium acetate, pH 5.0, containing 0.1% (w / w) polysorbate 80) through a UF / DF dialysis membrane to a final concentration of 10.14 mg / mL and a total volume of 41.9 mL. The final mean drug-antibody ratio (DAR) values ​​for R4702-MCCA-ADC (ADC-1) and TX05-MCCA-ADC (ADC-3) were 4.5 and 4.7, respectively, as determined by hydrophilic interaction chromatography (HIC).

[0238] 16-4.DBCO-PEG 24 Preparation of ADCs using -VA-PAB-exatecan (Linker-Drug Compound 2) DL-2 (16.35 mg) was dissolved in 1635 μL of DMSO to form a DL-2 solution. DL-2 solution (1614 μL) was slowly added to R4702-(NSCT-diN3)2 solution (18 mL, antibody concentration 5 mg / mL in 20 mM NaOAc, pH 5.0) and shaken at 25°C for 6.5 hours. After conjugation was complete, residual DL-2 was partially removed by buffer exchange (20 mM NaOAc, pH 5.0) using PES Amicons. The crude ADC was further purified using an HIC column to obtain ADC-2. After buffer exchange with storage buffer (20 mM NaOAc, pH 5.0), the concentration of R4702-DBCO-ADC (ADC-2) was adjusted to 5.02 mg / mL and sterilized by passing through a ProMax™ Syringe Filter (PVDF, 0.22 μm). Ultimately, 9.9 mL of ADC-2 was produced with a drug-antibody ratio (DAR) value of 3.8 (determined by HIC).

[0239] Additionally, DL-2 (34.58 mg) was dissolved in 1729 μL of DMSO to form a DL-2 solution. DL-2 solution (1108 μL) was slowly added to TX05-(NSCT-diN3)2 solution (25.33 mL, antibody concentration 4.88 mg / mL in 20 mM NaOAc, pH 5.0) and stirred at 25°C for 6 hours. After conjugation was complete, the crude ADC was further purified using Spectrum® Hollow Fiber Filter Modules (buffer: 20 mM NaOAc, pH 5.0) to obtain TX05-DBCO-ADC (ADC-4). ADC-4 was adjusted to approximately 5 mg / mL and sterilized by passing through a ProMax™ Syringe Filter (PVDF, 0.22 μm). Finally, 17.7 mL of ADC-4 (concentration 4.53 mg / mL) was produced with a drug-antibody ratio (DAR) value of 3.9 (determined by HIC).

[0240] Example 17: Analysis of glycan-engineered ADCs 17-1. CE-SDS analysis for determination of deglycosylation and glycosylation CE-SDS analysis was performed under reducing conditions. A Beckman Coulter PA800Plus system equipped with a UV photodiode array detector (using a wavelength of 220 nm) was used in this study. A bare fused-silica capillary (ID 50 m × total length 30 cm) with an effective capillary separation length of 20 cm was rinsed with 0.1 M NaOH, 0.1 M HCl, and SDS gel buffer before injection. For capillary separation, electrokinetic injection mode was applied at -5 kV for 20 s with reverse polarity, followed by a voltage of -15 kV. The total separation time was 35 min. 60 μg of test material was sampled and diluted in 120 μL of sample buffer (1% SDS in dilute PBS (pH 7.0)). The diluted sample was then mixed with 5 μL of 2ME and 2 μL of a 10 kDa internal standard, followed by incubation at 65°C for 10 min. Finally, the sample was cooled to room temperature for CE-SDS analysis.

[0241] EndoSz-D234M demonstrated high deglycosylation and transglycosylation activity for the glycan ADC platform. As a model for glycan ADC production, we produced R4702-DBCO-ADC (ADC-2) and TX05-DBCO-ADC (ADC-4) using EndoSz-D234M with R4702 and TX05 mAbs. For ADC-2, R4702 mAb was deglycosylated with EndoSz-D234M along with the additional enzyme EndoH to cleave high-mannose glycans. The yield of deglycosylated R4702 (R4702-GlcNAc(Fuc)) bearing one GlcNAc or possibly fucose residue was approximately 96.42%. Next, R4702-GlcNAc(Fuc) was mixed with 20 equivalents of modified complex N-glycan (NSCT-2) at 37°C for 1.5 hours to generate R4702-(NSCT-di-N3)2. CE-SDS results showed that EndoSz-D234M produced 96.51% of R4702-(NSCT-di-N3)2. For ADC-4, EndoSz-D234M was used to hydrolyze the biantennary hybrid glycan on the TX05 mAb, producing 94.5% of TX05-GlcNAc(Fuc). In the transglycosylation step, NSCT-2 was added to the mixture of TX05-GlcNAc(Fuc) and EndoSz-D234M. Using 15 equivalents of NSCT-2 relative to TX05-GlcNAc(Fuc), 96.7% TX05-(NSCT-di-N3)2 was obtained after 4.5 h of incubation at 15 °C with EndoSz-D234M catalyst (Figure S13C). These data demonstrate that EndoSz-D234M effectively generates mAb-(NSCT-di-N3)2 for glycan ADC production.

[0242] 17-2. Analysis of unmodified MW by LC-MS Analysis of the unmodified MW was performed using a Q-Exactive mass spectrometer (Thermo Scientific) coupled to a vanquish HPLC system (Thermo). LC separation was performed using an Agilent PLRP-S column with a gradient program. Test samples were diluted to 0.5 mg / mL with HO and no further deglycosylation or reduction processes were performed before LC-MS analysis. A full MS scan was performed over the m / z range of 1500 to 5000 for molecular weight analysis. Raw data were processed using Protein Deconvolution 4.0 to obtain molecular weights.

[0243] The results of the native molecular weight analysis and reduced MS analysis of each species are shown in Figure 14, demonstrating deglycosylation and transglycosylation by EndoSz-D234M. The difference in mass between R4702-GlcNAc(Fuc) and R4702 indicated that the structure of the N-glycan on R4702-GlcNAc(Fuc) was GlcNAc(Fuc). The N-glycan of R4702 could be cleaved to form R4702-GlcNAc(Fuc), as assessed by native MW analysis. The MW of R4702-(NSCT-di-N3)2 confirmed that NSCT-2 was successfully transferred to R4702-GlcNAc(Fuc) by EndoSz-D234M. The MW of R4702-DBCO-ADC (ADC-2) demonstrated that there were four payloads conjugated to R4702. Furthermore, reduced MW analysis also demonstrated that the modifications were located in the heavy chain.

[0244] 17-3. Analysis of Drug-to-Antibody Ratio (DAR) in the Presence of Human Serum Albumin (HSA) Maleimide linkers have been widely used for conjugating antibodies to payloads. Nevertheless, thioether linkages are susceptible to deconjugation via a reverse Michael reaction, resulting in payload loss and reduced efficacy. Therefore, maleimide-linked payloads can bind to plasma thiols (e.g., human serum albumin, HSA) and cause off-target toxicity. 1,2 To compare DAR changes in the presence of HSA, R4702-MCCA-ADC (ADC-1) and R4702-DBCO-ADC (ADC-2) were added to 3% HSA in PBS to a final concentration of 500 μg / mL and incubated at 37°C in a shaking water bath for 0, 24, and 144 hours. The incubated samples were purified with streptavidin magnetic beads coated with anti-idiotypic antibodies. The mixtures were gently shaken at room temperature for 1.5 hours, then washed three times with HEPES-buffered saline and twice with deionized water, and eluted with 2% formic acid. To calculate the DAR change, the eluted ADC was reduced with TCEP (final concentration 20 mM) for 30 min at room temperature to generate light chain (LC) and heavy chain (HC) fragments, followed by LC-HRMS analysis. The following formula was used to calculate the average DAR: (LC1 / (LC0+LC1))×2+(HC1 / (HC0+HC1+HC2+HC3))×2+(HC2 / (HC0+HC1+HC2+HC3))×4+HC3 / (HC0+HC1+HC2+HC3))×6 where LC0 and LC1 are the reconstituted areas of the light chain signal with 0 and 1 payload, respectively, and HC0, HC1, HC2, and HC3 are the reconstituted areas of the heavy chain signal with 0, 1, 2, and 3 payloads, respectively.

[0245] The restored mass spectra of ADC-1 and ADC-2 incubated in HSA for 0 and 144 hours are shown in Figure 15. After 144 hours (6 days) of incubation, loss of the maleimide linker-payload was observed for ADC-1 (Figure 15A). However, no extra peaks corresponding to HC2 degradation products were detected for ADC-2 (Figure 15B). According to the DAR formula, a time-dependent decrease in the average DAR was observed for ADC-1 (Figure 15C). The results indicated that ADC-2 did not undergo deconjugation via a reverse Michael reaction.

[0246] 17-4. In vitro stability in human plasma Pooled human plasma (prepared with heparin) was spiked with R4702-MCCA-ADC (ADC-1) and R4702-DBCO-ADC (ADC-2) to a final concentration of 200 μg / mL and incubated in a shaking water bath at 37°C for 0, 24, 96, 168, and 336 hours. To determine the released payload, the incubated samples were deproteinized using acetonitrile (ACN) with an internal standard (IS) followed by LC-MS / MS analysis. The percentage of the theoretical maximum was calculated as the amount of released payload divided by the theoretical amount of ADC DAR4 payload × 100%.

[0247] The stability of ADC-1 and ADC-2 in human plasma was monitored over 14 days at 37°C. Based on the ratio of released payload to the theoretical amount of payload (DAR4), the percentage of released payload for ADC-1 and ADC-2 increased over time, as shown in Figure 16. After 14 days of incubation in human plasma, the percentage of released payload from ADC-1 and ADC-2 was approximately 2.2% and 0.6%, respectively. This result indicated that ADC-2 released less payload than ADC-1 in human plasma.

[0248] Example 18: In vitro cytotoxicity assay of glycan-engineered ADCs Tumor cells (2 × 10 3Cells (cells / well) were seeded into 96-well plates and treated with ADCs for 6 days. CellTiter-Glo® Reagent (Cat. G7572, Promega) was prepared by adding CellTiter-Glo® Buffer to lyophilized CellTiter-Glo® Substrate. After 6 days of treatment, the reconstituted CellTiter-Glo® Reagent was added to the culture medium containing the cells at a 1:1 ratio. The plate was placed on an orbital shaker for 2 minutes to induce cell lysis, then incubated at room temperature for 10 minutes, and the luminescence signal was recorded by a luminometer. The viability of each treated sample was compared to the untreated control. The IC of each ADC was calculated. 50 was calculated by Prism.

[0249] The in vitro efficacy of the ADCs in several tumor cell lines was evaluated by cytotoxicity assay (Figure 17). IC of R4702-MCCA-ADC (ADC-1) and R4702-DBCO-ADC (ADC-2) in the NCI-H1975-C797S lung cancer cell line 50 The IC values ​​of ADC-1 were similar (77.64 and 98.42 nM) in DU145 prostate cancer cells. 50 The IC50 of ADC-3 was slightly lower than that of ADC-2 (140.3 and 261.1 nM). Similar results were observed for TX05-MCCA-ADC (ADC-3) and TX05-DBCO-ADC (ADC-4). In NCI-N87 gastric cells, the IC50 of ADC-3 was slightly lower than that of ADC-4 (2.904 and 5.515 nM), while in Capan-1 pancreatic cancer cells, ADC-3 and ADC-4 had similar IC50s. 50 These results suggest that the site-specific glycan-conjugated ADCs (ADC-2 and ADC-4) exhibit similar or slightly lower cytotoxicity than the cysteine-conjugated ADCs (ADC-1 and ADC-3).

[0250] Example 19: In vivo efficacy study of glycan-engineered ADCs in NCI-H1975-C797S lung cancer cell-derived xenografts The in vivo efficacy of R4702-MCCA-ADC (ADC-1) and R4702-DBCO-ADC (ADC-2) was evaluated using NCI-H1975-C797 human lung cancer cells. Female BALB / c nude mice were housed under specific pathogen-free conditions. Mice were acclimated for at least 3 days before the start of the study. Food (LabDiet 5010, PMI, USA) and water (sterile RO water) were provided ad libitum throughout the study period. All animal experiments were approved by the Institutional Animal Care and Use Committee at the National Laboratory Animal Center in Taiwan.

[0251] Tumor cells were washed and resuspended in PBS. Viable cells (1 × 10 7 The cells (1000 cells / mouse) were mixed with the same volume of Matrigel (Cat. 356234, BD) and injected subcutaneously into the right flank of female BALB / c nude mice (200 μL / mouse). The average tumor volume was 150–200 mm. 3 When tumor-bearing mice reached 100 mg / kg / day, they were divided into distinct groups. ADC or vehicle control was administered as a single dose via tail vein injection. The day of administration was designated as day 1. Tumor growth and mouse body weight were monitored twice weekly until day 22. The efficacy of the ADC was evaluated as tumor growth inhibition (TGI). TGI was calculated using the following formula: TGI (%) = [1-(Ti-T1) / (Ci-C1)] × 100%. Ti and Ci represent the mean tumor volumes of the treatment and vehicle groups at the end of the study (day 22), while T1 and C1 represent the mean tumor volumes of the treatment and vehicle groups at the start of test administration. The experimental design, test items, dose concentrations, dosing frequency, administration route, and animal numbers are listed in Table 9.

[0252] [Table 10]

[0253] The in vivo efficacy of ADC-1 and ADC-2 was evaluated in an NCI-H1975-C797S lung cancer xenograft mouse model. NCI-H1975-C797S cancer cells were implanted into BALB / c nude mice. Tumor growth (Figure 18A) and mouse body weight (Figure 18B) were recorded throughout the study. Antitumor efficacy was assessed using tumor growth inhibition (TGI). TGI was calculated by comparing treatment groups with vehicle control based on tumor size on days 1 and 22. Both groups treated with 10 mg / kg ADC-1 and ADC-2 exhibited similarly excellent antitumor efficacy (TGI > 100%, P = 0.24). At 3 mg / kg, ADC-1 and ADC-2 similarly exhibited partial inhibition of tumor growth (TGI: 42.7% and 66.2%, respectively, P = 0.37). The results suggest that site-specific glycan-conjugated ADC-2 exhibits similar antitumor efficacy as cysteine-conjugated ADC-1.

[0254] The present disclosure discloses selected glycosynthase variants that exhibit excellent transglycosylation activity with a wide range of N-glycans, including high-mannose, hybrid, and complex types.

[0255] In a preferred embodiment, the high mannose, hybrid, and complex N-glycans are in the active oxazoline form.

[0256] In some embodiments, the high mannose N-glycan described herein is selected from the group consisting of Man3GlcNAc, Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, Man8GlcNAc, and Man9GlcNAc. In a preferred embodiment, the high mannose N-glycan is Man5GlcNAc.

[0257] In some embodiments, the hybrid N-glycans described herein comprise at least one alpha-2,6- or alpha-2,3-terminal sialic acid on the alpha-1,3 arm, and the alpha-1,6 arm comprises a trimannose residue.

[0258] In some embodiments, the hybrid N-glycans described herein comprise at least one terminal galactose on the alpha-1,3 arm and the alpha-1,6 arm comprises a trimannose residue.

[0259] In some embodiments, the hybrid N-glycans described herein comprise at least one terminal GlcNAc on the alpha-1,3 arm and the alpha-1,6 arm comprises a trimannose residue.

[0260] In some embodiments, the complex glycans are biantennary, triantennary, and tetraantennary complex glycans.

[0261] In some embodiments, the biantennary complex N-glycans described herein comprise at least one α-2,6 or α-2,3 terminal sialic acid. In preferred embodiments, the N-glycans comprise two α-2,6 and / or α-2,3 terminal sialic acids.

[0262] In some embodiments, the biantennary complex N-glycans described herein comprise at least one terminal galactose or GlcNAc. In preferred embodiments, the N-glycans comprise two terminal galactoses and / or GlcNAcs.

[0263] In some embodiments, the biantennary complex N-glycans described herein comprise at least one alpha-1,2-fucose. In preferred embodiments, the N-glycans comprise two alpha-1,2-fucoses.

[0264] In some embodiments, the biantennary complex N-glycans described herein comprise at least one alpha-1,3-fucose. In preferred embodiments, the N-glycans comprise two alpha-1,3-fucoses.

[0265] In some embodiments, the biantennary complex N-glycans described herein comprise a bisecting GlcNAc.

[0266] In some embodiments, the biantennary complex N-glycans described herein comprise at least one LacNAc repeat unit. In a preferred embodiment, the N-glycan comprises two LacNAc repeat units.

[0267] In some embodiments, the triantennary complex N-glycans described herein comprise at least one α-2,6 or α-2,3 terminal sialic acid. In preferred embodiments, the N-glycans comprise three α-2,6 and / or α-2,3 terminal sialic acids.

[0268] In some embodiments, the triantennary complex N-glycans described herein comprise at least one terminal galactose or GlcNAc. In preferred embodiments, the N-glycans comprise three terminal galactoses and / or GlcNAcs.

[0269] In some embodiments, complex glycans are biantennary and triantennary complex glycans containing asymmetric antennae in the alpha-1,3 or alpha-1,6 arms.

[0270] In some embodiments, the hybrid, biantennary, and triantennary complex N-glycans described herein comprise α-2,6 or α-2,3 terminal sialic acids, while in other embodiments, the hybrid, biantennary, and triantennary complex N-glycans comprise α-2,6 terminal sialic acids.

[0271] While the present invention has been described in connection with a limited number of embodiments, it will be recognized that those skilled in the art, having the benefit of this disclosure, may devise other embodiments that do not depart from the scope of the invention as described herein. Accordingly, the scope of the present invention is limited only by the appended claims.

[0272] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described herein. All publications and patents specifically mentioned herein are incorporated by reference for all purposes, including describing and disclosing chemicals, cell lines, vectors, animals, devices, statistical analyses, and methodologies reported in the publications used in connection with the present invention. All references cited herein should be construed as an indication of the level of skill in the art. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0273] Before the present materials and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, materials, and agents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which will be limited only by the appended claims.

[0274] Additional Embodiments Embodiment 1: A method of preparing an engineered bioconjugate comprising contacting a biomolecule with a glycosynthase and a modified glycan, thereby obtaining a first engineered bioconjugate, wherein the biomolecule further comprises an N-linked initial glycan; the glycosynthase comprises SEQ ID NO:1 or SEQ ID NO:2; and the glycosynthase comprises a mutation located within residues 176-186, 225-237, or 273-289 of SEQ ID NO:1, or within residues 178-188, 227-239, or 275-291 of SEQ ID NO:2; the modified glycan comprises a substrate moiety and a first reactive moiety, wherein the substrate moiety is configured to react with the glycosynthase; and the biomolecule comprises an antibody or antigen-binding fragment thereof, a protein, or a peptide.

[0275] Embodiment 2: The method of embodiment 1, wherein the biomolecule comprises an antibody or an antigen-binding fragment thereof, and the N-linked initial glycan is located in the constant region of the antibody or antigen-binding fragment.

[0276] Embodiment 3: The method of embodiment 1 or embodiment 2, wherein the biomolecule comprises an antibody or antigen-binding fragment thereof, and the N-linked initial glycan is located in the Fc region of the antibody or antigen-binding fragment.

[0277] Embodiment 4: The method of embodiment 3, wherein the N-linked initial glycan is located at the N297 position of the Fc region.

[0278] Embodiment 5: The method of any one of embodiments 1 to 4, wherein contacting the biomolecule with the glycosynthase and the modified glycan comprises linking the modified glycan to an N-linked initial glycan.

[0279] Embodiment 6: The method of any one of embodiments 1 to 5, wherein the substrate moiety of the modified glycan is an oxazoline moiety.

[0280] Embodiment 7: The method of any one of embodiments 1-6, wherein the first reactive moiety is configured to react with the unsaturated moiety in a bioorthogonal reaction.

[0281] Embodiment 8: The method of embodiment 7, wherein the bioorthogonal reaction is copper-free click chemistry.

[0282] Embodiment 9: The method of any one of embodiments 1-8, wherein the first reactive moiety comprises an azide group.

[0283] Embodiment 10: The method of any one of embodiments 1 to 9, wherein the modified glycan is a PEGylated glycan modified with a first polyethylene glycol (PEG) moiety.

[0284] Embodiment 11: The method of embodiment 10, wherein the first polyethylene glycol (PEG) moiety comprises 2 to 72 OCH2CH2 subunits.

[0285] Embodiment 12: The method of embodiment 10 or embodiment 11, wherein the first PEG moiety is a linear PEG, a branched PEG, or a star PEG.

[0286] Embodiment 13: The method of any one of embodiments 10 to 12, wherein the first end of the PEGylated glycan is covalently linked to a first reactive moiety.

[0287] Embodiment 14: The method of embodiment 13, wherein the first reactive moiety is covalently linked to the PEG moiety.

[0288] Embodiment 15: The method of any one of embodiments 10 to 14, wherein the second end of the PEGylated glycan is covalently linked to a substrate moiety.

[0289] Embodiment 16: The method of embodiment 15, wherein the substrate moiety is covalently linked to the glycol moiety of the PEGylated glycan.

[0290] Embodiment 17: The method of any one of embodiments 1 to 16, wherein the modified glycan is a glycan oxazoline.

[0291] Embodiment 18: The glycan oxazoline is

[0292] [ka] Contains the formula for R 1 is N-acetylglucosamine attached via -H or β-1,4 linkage, and R 2 and R 3 are the same or different,

[0293] [ka] 18. The method of embodiment 17, wherein said IL-16 is independently selected from the group consisting of:

[0294] Embodiment 19: The method of any one of embodiments 1 to 18, wherein contacting the biomolecule with the glycosynthase and the modified glycan comprises removing an N-linked initial glycan of the biomolecule, thereby obtaining a deglycosylated biomolecule, and contacting the deglycosylated biomolecule with the glycosynthase in the presence of the modified glycan.

[0295] Embodiment 20: The method of embodiment 19, wherein removing N-linked initial glycans of the biomolecule, thereby obtaining a deglycosylated biomolecule, comprises mixing the glycosynthase and the biomolecule in a ratio of 1:500 to 1:1, 1:500 to 1:10, 1:500 to 1:20, 1:500 to 1:30, 1:500 to 1:50, 1:100 to 1:1, 1:100 to 1:10, 1:100 to 1:20, 1:100 to 1:30, 1:100 to 1:50, 1:50 to 1:1, 1:50 to 1:10, 1:50 to 1:20, or 1:50 to 1:30 in the absence of modified glycans.

[0296] Embodiment 21: The method of embodiment 19 or embodiment 20, wherein the deglycosylated biomolecule comprises a GlcNAc monosaccharide.

[0297] Embodiment 22: The method of embodiment 21, wherein the GlcNAc monosaccharide is fucosylated.

[0298] Embodiment 23: The method of embodiment 21, wherein the GlcNAc monosaccharide is non-fucosylated.

[0299] Embodiment 24: The method of any one of embodiments 1 to 23, wherein the step of contacting the biomolecule with the glycosynthase and the modified glycan comprises contacting a plurality of biomolecules with the glycosynthase and the modified glycan, thereby obtaining a plurality of first engineered bioconjugates, wherein the homogeneity of the plurality of first engineered bioconjugates is at least 80%, 85%, 90%, 95%, or 99% or more.

[0300] Embodiment 25: The method of claim 2, further comprising contacting the first engineered bioconjugate with a payload conjugate or a salt thereof, thereby obtaining a second bioconjugate, wherein the payload conjugate has the formula: CLD wherein C is a second reactive moiety configured to react with a first reactive moiety of the modified glycan in a bioorthogonal reaction; L is a linker unit comprising a hydrophilic moiety; and D is a payload.

[0301] Embodiment 26: The method of embodiment 25, wherein the second reactive moiety comprises an unsaturated moiety (e.g., an alkene moiety or an alkyne moiety).

[0302] Embodiment 27: The method of embodiment 25 or embodiment 26, wherein the second reactive moiety is a non-natural and non-perturbing chemical group.

[0303] Embodiment 28: The method of any one of embodiments 25-27, wherein the second reactive moiety is a benzocyclooctyne group (DBCO), a bicyclononyne (BCN), a cyclic alkyne, a maleimide group, an α,β-unsaturated carbonyl group, or a sulfonylpyrimidine.

[0304] Embodiment 29: The method of any one of embodiments 25-28, wherein the hydrophilic moiety comprises a second polyethylene glycol (PEG) moiety.

[0305] Embodiment 30: The method of embodiment 29, wherein the second PEG moiety comprises 2 to 72 OCH2CH2 subunits.

[0306] Embodiment 31: The method of embodiment 29 or embodiment 30, wherein the second PEG moiety is a linear PEG, branched PEG, or star PEG.

[0307] Embodiment 32: The method of any one of embodiments 25 to 31, wherein the linker unit further comprises a cleavable moiety.

[0308] Embodiment 33: The method of embodiment 32, wherein the cleavable moiety is a protease-sensitive peptide or a glycosidase-sensitive sugar unit.

[0309] Embodiment 34: The method of any one of embodiments 25 to 33, wherein the linker unit further comprises a spacer comprising an aromatic group or an aminomethylene.

[0310] Embodiment 35: The linker unit is

[0311] [ka] Contains the formula (PEG)m is a PEG moiety, m is an integer selected from 2 to 72, and Q SP is a spacer containing an aromatic group or an aminomethylene, and Q CL is a severable portion configured to be coupled to a payload, and L P 35. The method of any one of embodiments 25-34, wherein is a connecting unit configured to be linked to a second reactive moiety.

[0312] Embodiment 36: The PEG moiety

[0313] [ka] The wavy line is L P indicates the site of covalent attachment to R 20is a PEG linking unit, and the PEG linking unit is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or

[0314] [ka] and R 21 is a PEG capping unit, and the PEG capping unit is H, SO3H, PO3H2, sugar derivatives, C1-C 10 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 Alkyl-NH2, C1-C 10 Alkyl-COOH, C2-C 10 Alkyl-NH(C1-C3 alkyl), C2-C 10 36. The method of embodiment 35, wherein n is selected from alkyl-N(C1-C3 alkyl)2 and n is selected from 8 to 72.

[0315] Embodiment 37: The linker unit (L) is

[0316] [ka] 37. The method of any one of embodiments 25 to 36, having the structure:

[0317] Embodiment 38: The method of any one of embodiments 25 to 37, wherein the payload is a therapeutic agent.

[0318] Embodiment 39: The method of embodiment 38, wherein the therapeutic agent comprises an antiviral agent, an antibacterial agent, an immunomodulatory agent, an immunostimulatory agent, an anti-tumor agent, or a combination thereof.

[0319] Embodiment 40: The method of any one of embodiments 25 to 39, wherein the payload comprises a toxin, a cytokine, a growth factor, a radionuclide, a hormone, or a combination thereof.

[0320] Embodiment 41: The method of any one of embodiments 25 to 40, wherein the payload is selected from pyrrolobenzodiazepines (e.g., PBDs), auristatins (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, DM1, DM4, DM21), duocamycins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, tubulysins, enediynes (e.g., calicheamicin), anthracycline derivatives (PNU) (e.g., doxorubicin), pyrrole kinesin spindle protein (KSP) inhibitors, cryptophycins, drug efflux pump inhibitors, sandramycin, amanitins (e.g., alpha-amanitin), and camptothecins (e.g., exatecan, deruxtecan).

[0321] Embodiment 42: The payload conjugate comprises:

[0322] [ka] 42. The method of any one of embodiments 25 to 41, comprising the structure:

[0323] Embodiment 43: The method of any one of embodiments 1 to 42, wherein the mutation comprises an alteration at residue 183, 232, 234, 280, 281, or 282 of SEQ ID NO: 1, or at residue 181, 230, 232, 278, 279, or 280 of SEQ ID NO: 2.

[0324] Embodiment 44: The method of embodiment 43, wherein the mutations comprise D234E, D234R, D234H, D234M, D234V, D234L, D234F, D234T, D234Q, T183Q, D232Q, D280Q, S281Q, T282Q in SEQ ID NO: 1, or D232E, D232R, D232H, D232M, D232V, D232L, D232F, D232T, D232Q, T181Q, D230Q, D278Q, S279Q, T280Q in SEQ ID NO: 2.

[0325] Embodiment 45: The method of any one of embodiments 1 to 44, wherein the biological molecule is an anti-Globo series antigen antibody or an antigen-binding fragment thereof, an anti-HER2 antibody or an antigen-binding fragment thereof, an anti-CD20 antibody or an antigen-binding fragment thereof, an anti-TNF-alpha antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-TROP2 antibody or an antigen-binding fragment thereof, an anti-EGFR antibody or an antigen-binding fragment thereof, an anti-Nectin-4 antibody or an antigen-binding fragment thereof, an anti-HER3 antibody or an antigen-binding fragment thereof, an anti-cMet antibody or an antigen-binding fragment thereof, an anti-B7H3 antibody or an antigen-binding fragment thereof, an anti-B7H4 antibody or an antigen-binding fragment thereof, an anti-VEGF antibody or an antigen-binding fragment thereof, an anti-claudin 18.2 antibody or an antigen-binding fragment thereof, an anti-Sirp-alpha antibody or an antigen-binding fragment thereof, a TROP2xHER2 bispecific antibody, or a combination thereof.

[0326] Embodiment 46: The method of embodiment 45, wherein the Globo series antigens comprise Globo H, stage-specific embryonic antigen 4 (SSEA-4), or stage-specific embryonic antigen 3 (SSEA-3).

[0327] Embodiment 47: The method of any one of embodiments 1 to 46, wherein the biomolecule is selected from Herceptin (trastuzumab), TX05 (trastuzumab biosimilar), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), Bavencio (avelumab), and BSI04702 (anti-TROP2 antibody).

[0328] Embodiment 48: An engineered bioconjugate comprising a biomolecule and a modified glycan linked to the biomolecule, wherein the modified glycan comprises (i) a first polyethylene glycol (PEG) moiety and (ii) a first reactive moiety or a resultant moiety derived from a bioorthogonal reaction, and the biomolecule comprises an antibody or antigen-binding fragment thereof, a protein, or a peptide.

[0329] Embodiment 49: The engineered bioconjugate of embodiment 48, wherein the biomolecule comprises an antibody or an antigen-binding fragment thereof, and the engineered glycan is linked to the antibody or antigen-binding fragment thereof at its Fc region.

[0330] Embodiment 50: The engineered bioconjugate of embodiment 49, wherein the modified glycan is linked to the antibody or antigen-binding fragment at the N297 site of the Fc region.

[0331] Embodiment 51: The engineered bioconjugate of embodiment 50, wherein the modified glycan is linked to a GlcNAc monosaccharide at the N297 site of the Fc region.

[0332] Embodiment 52: The engineered bioconjugate of embodiment 51, wherein the GlcNAc monosaccharide is fucosylated.

[0333] Embodiment 53: The engineered bioconjugate of embodiment 51, wherein the GlcNAc monosaccharide is non-fucosylated.

[0334] Embodiment 54: The engineered bioconjugate of any one of embodiments 48 to 53, wherein the first reactive moiety is configured to interact with an alkyne moiety in a bioorthogonal reaction.

[0335] Embodiment 55: The engineered bioconjugate of any one of embodiments 48 to 54, wherein the bioorthogonal reaction is copper-free click chemistry.

[0336] Embodiment 56: The engineered bioconjugate of any one of embodiments 48-55, wherein the first reactive moiety comprises an azide group.

[0337] Embodiment 57: The engineered bioconjugate of any one of embodiments 48 to 56, wherein the first PEG moiety comprises 2 to 72 (OCH2CH2) subunits.

[0338] Embodiment 58: The engineered bioconjugate of embodiment 57, wherein the first PEG moiety is a linear PEG, branched PEG, or star PEG.

[0339] Embodiment 59: The engineered bioconjugate of any one of embodiments 48 to 58, wherein the modified glycan is a first modified glycan, and the engineered bioconjugate further comprises a first payload moiety, wherein the first payload moiety is linked to the first modified glycan via the resulting moiety.

[0340] Embodiment 60: The first payload portion -LD wherein L is a linker unit comprising a hydrophilic moiety and is linked to the resulting moiety; and D is a payload.

[0341] Embodiment 61: The engineered bioconjugate of embodiment 59 or embodiment 60, wherein the resulting moiety comprises a triazole moiety.

[0342] Embodiment 62: The engineered bioconjugate of embodiment 61, wherein the resulting moiety comprises a DBCO-derived moiety or a maleimide-derived moiety.

[0343] Embodiment 63: The engineered bioconjugate of any one of embodiments 60-62, wherein the hydrophilic moiety comprises a second polyethylene glycol (PEG) moiety.

[0344] Embodiment 64: The engineered bioconjugate of embodiment 63, wherein the second PEG moiety comprises 2 to 72 OCH2CH2 subunits.

[0345] Embodiment 65: The engineered bioconjugate of embodiment 10 or embodiment 63 or embodiment 64, wherein the second PEG moiety is a linear PEG, a branched PEG, or a star PEG.

[0346] Embodiment 66: The engineered bioconjugate of any one of embodiments 60 to 65, wherein the linker unit further comprises a cleavable moiety.

[0347] Embodiment 67: The engineered bioconjugate of embodiment 66, wherein the cleavable moiety is a protease-sensitive peptide or a glycosidase-sensitive sugar unit.

[0348] Embodiment 68: The engineered bioconjugate of any one of embodiments 60 to 67, wherein the linker unit further comprises a spacer comprising an aromatic group or an aminomethylene.

[0349] Embodiment 69: The linker unit comprises:

[0350] [ka] Contains the formula (PEG)m is a PEG moiety, m is an integer selected from 2 to 72, and Q SP is a spacer containing an aromatic group or an aminomethylene, and Q CL is the cleavable moiety linked to the payload, and L P 69. The engineered bioconjugate of any one of embodiments 60-68, wherein is a connecting unit linked to the resulting moiety.

[0351] Embodiment 70: The PEG moiety

[0352] [ka] The wavy line is L P indicates the site of covalent attachment to R 20 is a PEG linking unit, and the PEG linking unit is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or

[0353] [ka] and R 21 is a PEG capping unit, and the PEG capping unit is H, SO3H, PO3H2, sugar derivatives, C1-C 10 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 Alkyl-NH2, C1-C 10 Alkyl-COOH, C2-C 10 Alkyl-NH(C1-C3 alkyl), C2-C 10 70. The engineered bioconjugate of embodiment 69, wherein n is selected from alkyl-N(C1-C3 alkyl)2 and n is selected from 8-72.

[0354] Embodiment 71: A linker unit

[0355] [ka] 71. The engineered bioconjugate of any one of embodiments 60-70, having the structure:

[0356] Embodiment 72: The engineered bioconjugate of any one of embodiments 59 to 71, wherein the payload is a therapeutic agent.

[0357] Embodiment 73: The engineered bioconjugate of embodiment 72, wherein the therapeutic agent comprises an antiviral agent, an antibacterial agent, an immunomodulatory agent, an immunostimulatory agent, an antitumor agent, a chemotherapeutic agent, or a combination thereof.

[0358] Embodiment 74: The engineered bioconjugate of any one of embodiments 59 to 73, wherein the payload comprises a toxin, cytokine, growth factor, radionuclide, hormone, or combination thereof.

[0359] Embodiment 75: The engineered bioconjugate of any one of embodiments 59 to 74, wherein the payload is selected from pyrrolobenzodiazepines (e.g., PBDs), auristatins (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, DM1, DM4, DM21), duocamycins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, tubulysins, enediynes (e.g., calicheamicin), anthracycline derivatives (PNUs) (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors, cryptophycins, drug efflux pump inhibitors, sandramycin, amanitins (e.g., alpha-amanitin), and camptothecins (e.g., exatecan, deruxtecan).

[0360] Embodiment 76: The engineered bioconjugate of any one of embodiments 59 to 75, further comprising a second payload moiety, wherein the second payload is linked to the engineered bioconjugate via a second modified glycan.

[0361] Embodiment 77: The engineered bioconjugate of embodiment 76, wherein the first payload moiety and the second payload moiety are different.

[0362] Embodiment 78: The engineered bioconjugate of embodiment 76 or embodiment 77, wherein the first payload portion comprises a first payload (D1) and the second payload portion comprises a second payload (D2), and the first payload (D1) is different from the second payload (D2).

[0363] Embodiment 79: The engineered bioconjugate of any one of embodiments 76 to 78, wherein the first modified glycan is different from the second modified glycan.

[0364] Embodiment 80: The engineered bioconjugate of any one of embodiments 48 to 79, wherein the biomolecule is an anti-Globo series antigen antibody or antigen-binding fragment thereof, an anti-HER2 antibody or antigen-binding fragment thereof, an anti-CD20 antibody or antigen-binding fragment thereof, an anti-TNF-alpha antibody or antigen-binding fragment thereof, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-TROP2 antibody or antigen-binding fragment thereof, an anti-EGFR antibody or antigen-binding fragment thereof, an anti-Nectin-4 antibody or antigen-binding fragment thereof, an anti-HER3 antibody or antigen-binding fragment thereof, an anti-cMet antibody or antigen-binding fragment thereof, an anti-B7H3 antibody or antigen-binding fragment thereof, an anti-B7H4 antibody or antigen-binding fragment thereof, an anti-VEGF antibody or antigen-binding fragment thereof, an anti-claudin 18.2 antibody or antigen-binding fragment thereof, an anti-Sirp-alpha antibody or antigen-binding fragment thereof, a TROP2xHER2 bispecific antibody, or a combination thereof.

[0365] Embodiment 81: The engineered bioconjugate of embodiment 80, wherein the Globo series antigen comprises Globo H, stage-specific embryonic antigen 4 (SSEA-4), or stage-specific embryonic antigen 3 (SSEA-3).

[0366] Embodiment 82: The engineered bioconjugate of any one of embodiments 48 to 81, wherein the biomolecule is selected from Herceptin (trastuzumab), TX05 (trastuzumab biosimilar), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), Bavencio (avelumab), and BSI04702 (anti-TROP2 antibody).

[0367] Embodiment 83: An engineered bioconjugate according to any one of embodiments 48 to 82, prepared by a method according to any one of embodiments 1 to 46.

[0368] Embodiment 84: A plurality of engineered bioconjugates, each of which is an engineered bioconjugate of any one of embodiments 48 to 83, wherein the homogeneity of the plurality of engineered bioconjugates is at least 80%, 85%, 90%, 95%, or 99% or greater.

[0369] Embodiment 85: A pharmaceutical composition comprising a plurality of engineered bioconjugates according to embodiment 84 and a pharmaceutically acceptable carrier.

[0370] Embodiment 86: A method of treating cancer, comprising administering an effective amount of the pharmaceutical composition of embodiment 85 to a patient in need thereof.

[0371] Embodiment 87: The method of embodiment 86, wherein the cancer is a cancer that expresses an antigen of the Globo series, HER2, TROP2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, Claudin 18.2, or Sirp-Alpha.

[0372] Embodiment 88: The method of embodiment 86 or embodiment 87, wherein the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, renal cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0373] Embodiment 89: CLD 1. A therapeutic conjugate comprising the formula: A therapeutic conjugate, wherein C is a reactive moiety configured to react in a bioorthogonal reaction, L is a linker unit comprising a hydrophilic moiety, a cleavable moiety, and a spacer, and D is a therapeutic agent.

[0374] Embodiment 90: The therapeutic conjugate of embodiment 89, wherein the reactive moiety comprises an unsaturated moiety.

[0375] Embodiment 91: The therapeutic conjugate of embodiment 90, wherein the unsaturated moiety is an alkene or alkyne moiety.

[0376] Embodiment 92: The therapeutic conjugate of any one of embodiments 89 to 91, wherein the reactive moiety is a non-natural and non-perturbing chemical group.

[0377] Embodiment 93: The therapeutic conjugate of any one of embodiments 89 to 92, wherein the reactive moiety is a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN), a cyclic alkyne, a maleimide group, an α,β-unsaturated carbonyl group, or a sulfonylpyrimidine.

[0378] Embodiment 94: The therapeutic conjugate of any one of embodiments 89 to 93, wherein the hydrophilic moiety comprises a polyethylene glycol (PEG) moiety.

[0379] Embodiment 95: The therapeutic conjugate of embodiment 94, wherein the PEG moiety comprises 2 to 72 OCH2CH2 subunits.

[0380] Embodiment 96: The therapeutic conjugate of embodiment 94 or embodiment 95, wherein the first PEG moiety is a linear PEG, a branched PEG, or a star PEG.

[0381] Embodiment 97: The therapeutic conjugate of any one of embodiments 89 to 96, wherein the cleavable moiety is a protease-sensitive peptide comprising Val-Cit, Val-Ala, Phe-Lys, Glu-Val-Cit, Glu-Val-Ala, Glu-Gly-Cit, Glu-Gly-Ala, Gly-Gly-Phe-Gly, Gly-Gly-Val-Cit, Gly-Gly-Val-Ala, or a glycosidase-sensitive sugar unit comprising glucuronic acid, iduronic acid, or galactose.

[0382] Embodiment 98: The therapeutic conjugate of any one of embodiments 89 to 97, wherein the spacer comprises an aromatic group including a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, a 2,5-thienyl group, or an aminomethylene (e.g., —NH—CH—).

[0383] Embodiment 99: The linker unit comprises:

[0384] [ka] Contains the formula (PEG)m is a PEG moiety, m is an integer selected from 2 to 72, and Q SP is a spacer containing an aromatic group or an aminomethylene, and Q CL is a severable portion configured to be coupled to a payload, and L P is a connecting unit configured to be linked to a second reactive moiety.

[0385] Embodiment 100: The PEG moiety

[0386] [ka] The wavy line is L P indicates the site of covalent attachment to R 20 is a PEG linking unit, and the PEG linking unit is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or

[0387] [ka] and R 21 is a PEG capping unit, and the PEG capping unit is H, SO3H, PO3H2, sugar derivatives, C1-C 10 (Hetero)alkyl groups, C3-C 10(Hetero)cycloalkyl groups, C2-C 10 Alkyl-NH2, C1-C 10 Alkyl-COOH, C2-C 10 Alkyl-NH(C1-C3 alkyl), C2-C 10 The therapeutic conjugate of embodiment 99, wherein n is selected from alkyl-N(C1-C3 alkyl)2 and n is selected from 8-72.

[0388] Embodiment 101: A linker unit

[0389] [ka] The therapeutic conjugate of any one of embodiments 89 to 100, having the structure:

[0390] Embodiment 102: The therapeutic conjugate of any one of embodiments 89 to 111, wherein the therapeutic agent comprises a toxin, a cytokine, a growth factor, a radionuclide, a hormone, an antiviral agent, an antibacterial agent, an immunomodulatory agent, an immunostimulatory agent, an antitumor agent, or a combination thereof.

[0391] Embodiment 103: The therapeutic agent is selected from pyrrolobenzodiazepines (e.g., PBDs), auristatins (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, DM1, DM4, DM21), duocamycins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, tubulysins, enediynes (e.g., calicheamicin), anthracycline derivatives (PNU) (e.g., doxorubicin), pyrrole kinesin spindle protein (KSP) inhibitors, cryptophycins, drug efflux pump inhibitors, sandramycin, amanitins (e.g., alpha-amanitin), and camptothecins (e.g., exatecan, deruxtecan). The therapeutic conjugate of any one of embodiments 89 to 112.

[0392] Embodiment 104:

[0393] [ka] The therapeutic conjugate of any one of embodiments 89 to 103, having the structure:

[0394] Sequence Listing

[0395] [ka]

[0396] [ka]

[0397] [ka]

[0398] [ka]

Claims

1. 1. A method of preparing an engineered bioconjugate, comprising contacting a biomolecule with a glycosynthase and a modified glycan, thereby obtaining a first engineered bioconjugate, the biomolecule further comprises an N-linked initial glycan; the glycosynthase comprises SEQ ID NO:1 or SEQ ID NO:2, and the glycosynthase comprises a mutation located within residues 176-186, 225-237, or 273-289 of SEQ ID NO:1, or within residues 178-188, 227-239, or 275-291 of SEQ ID NO:2; the modified glycan comprises a substrate moiety and a first reactive moiety; a substrate portion configured to react with a glycosynthase; and The method, wherein the biomolecule comprises an antibody or an antigen-binding fragment thereof, a protein, or a peptide.

2. 2. The method of claim 1, wherein the biomolecule comprises an antibody or an antigen-binding fragment thereof, and the N-linked initial glycan is located in the Fc constant region of the antibody or antigen-binding fragment.

3. The method of claim 2, wherein the N-linked initial glycan is located at the N297 position of the Fc region.

4. 10. The method of claim 1, wherein contacting the biomolecule with a glycosynthase and a modified glycan comprises linking the modified glycan to an N-linked initial glycan.

5. The method of claim 1 , wherein the substrate moiety of the modified glycan is an oxazoline moiety.

6. 10. The method of claim 1, wherein the first reactive moiety is configured to react with the unsaturated moiety in a bioorthogonal reaction.

7. 7. The method of claim 6, wherein the bioorthogonal reaction is copper-free click chemistry.

8. The method of claim 1 , wherein the first reactive moiety comprises an azide group.

9. The method of claim 1 , wherein the modified glycan is a PEGylated glycan modified with a first polyethylene glycol (PEG) moiety.

10. The first polyethylene glycol (PEG) moiety is 2 to 72 OCH ２ CH ２ The method of claim 9, comprising a subunit.

11. 10. The method of claim 9, wherein the first PEG moiety is a linear PEG, a branched PEG, or a star PEG.

12. 10. The method of claim 9, wherein the first end of the PEGylated glycan is covalently linked to a first reactive moiety.

13. The method of claim 9 , wherein the second end of the PEGylated glycan is covalently linked to a substrate moiety.

14. The method of claim 1 , wherein the modified glycan is a glycan oxazoline.

15. Glycan oxazoline 【Chemistry 1】 and R １ is —H or N-acetylglucosamine attached via a β-1,4 linkage, and R ２ and R ３ are the same or different, 【Chemistry 2】 15. The method of claim 14, wherein the hydroxyl group is independently selected from the group consisting of:

16. contacting the biomolecule with the glycosynthase and the modified glycan removes the N-linked initial glycan of the biomolecule, thereby obtaining a deglycosylated biomolecule; and contacting the deglycosylated biomolecule with a glycosynthase in the presence of the modified glycan; The method of claim 1 , comprising:

17. 17. The method of claim 16, wherein removing N-linked initial glycans of the biomolecule, thereby obtaining a deglycosylated biomolecule, comprises mixing the glycosynthase and the biomolecule in a ratio of 1:500 to 1:1, 1:500 to 1:10, 1:500 to 1:20, 1:500 to 1:30, 1:500 to 1:50, 1:100 to 1:1, 1:100 to 1:10, 1:100 to 1:20, 1:100 to 1:30, 1:100 to 1:50, 1:50 to 1:1, 1:50 to 1:10, 1:50 to 1:20, or 1:50 to 1:30 in the absence of modified glycans.

18. 17. The method of claim 16, wherein the deglycosylated biomolecule comprises a GlcNAc monosaccharide.

19. 19. The method of claim 18, wherein the GlcNAc monosaccharide is fucosylated or non-fucosylated.

20. 2. The method of claim 1, wherein the step of contacting a biomolecule with a glycosynthase and a modified glycan comprises contacting a plurality of biomolecules with the glycosynthase and the modified glycan, thereby obtaining a plurality of first engineered bioconjugates, wherein the homogeneity of the plurality of first engineered bioconjugates is greater than 80%.

21. 2. The method of claim 1, wherein the mutation comprises an alteration at residue 183, 232, 234, 280, 281, or 282 of SEQ ID NO:1, or residue 181, 230, 232, 278, 279, or 280 of SEQ ID NO:

2.

22. The mutation, D234E, D234R, D234H, D234M, D234V, D234L, D234F, D234T, D234Q, T183Q, D232Q, D280Q, S281Q, T282Q in SEQ ID NO: 1, or D232E, D232R, D232H, D232M, D232V, D232L, D232F, D232T, D232Q, T181Q, D230Q, D278Q, S279Q, and T280Q in SEQ ID NO: 2 22. The method of claim 21, further comprising:

23. The method of claim 1, wherein the biological molecule is an anti-Globo series antigen antibody or an antigen-binding fragment thereof, an anti-HER2 antibody or an antigen-binding fragment thereof, an anti-CD20 antibody or an antigen-binding fragment thereof, an anti-TNF-alpha antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-TROP2 antibody or an antigen-binding fragment thereof, an anti-EGFR antibody or an antigen-binding fragment thereof, an anti-Nectin-4 antibody or an antigen-binding fragment thereof, an anti-HER3 antibody or an antigen-binding fragment thereof, an anti-cMet antibody or an antigen-binding fragment thereof, an anti-B7H3 antibody or an antigen-binding fragment thereof, an anti-B7H4 antibody or an antigen-binding fragment thereof, an anti-VEGF antibody or an antigen-binding fragment thereof, an anti-claudin 18.2 antibody or an antigen-binding fragment thereof, an anti-Sirp-alpha antibody or an antigen-binding fragment thereof, a TROP2 x HER2 bispecific antibody, or a combination thereof.

24. 24. The method of claim 23, wherein the Globo series antigens include Globo H, stage-specific embryonic antigen 4 (SSEA-4), or stage-specific embryonic antigen 3 (SSEA-3).

25. 2. The method of claim 1, wherein the biomolecule is selected from Herceptin (trastuzumab), TX05 (trastuzumab biosimilar), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), Bavencio (avelumab), and R4702 (anti-TROP2 antibody).

26. Biomolecules and Modified glycans linked to biomolecules 1. An engineered bioconjugate comprising: The modified glycan is (i) a first polyethylene glycol (PEG) moiety, and (ii) a first reactive moiety or resulting moiety derived from a bioorthogonal reaction Including, the resulting moiety comprises a triazole moiety, a DBCO-derived moiety, or a maleimide-derived moiety; An engineered bioconjugate, wherein the biomolecule comprises an antibody or antigen-binding fragment thereof, a protein, or a peptide.

27. 27. The engineered bioconjugate of claim 26, wherein the biomolecule comprises an antibody or an antigen-binding fragment thereof, and the modified glycan is linked to the antibody or antigen-binding fragment thereof at the N297 site of its Fc region.

28. 27. The engineered bioconjugate of claim 26, wherein the modified glycan is linked to a fucosylated or non-fucosylated GlcNAc monosaccharide at the N297 site of the Fc region.

29. 27. The engineered bioconjugate of claim 26, wherein the first reactive moiety comprises an azide group and is configured to interact with the alkyne moiety in a bioorthogonal reaction.

30. 30. The engineered bioconjugate of claim 29, wherein the bioorthogonal reaction is copper-free click chemistry.

31. The first PEG moiety is 2 to 72 (OCH ２ CH ２ 27. The engineered bioconjugate of claim 26, comprising a .sup.-(A) ...

32. 27. The engineered bioconjugate of claim 26, wherein the first PEG moiety is a linear PEG, a branched PEG, or a star PEG.

33. 33. The engineered bioconjugate of any one of claims 26-32, further comprising a payload moiety, wherein the payload moiety is linked to the modified glycan via the resulting moiety.

34. 34. The engineered bioconjugate of claim 33, wherein the payload moiety is a first payload moiety and the engineered bioconjugate further comprises a second payload moiety, and wherein the first payload moiety and the second payload moiety are different.

35. 34. The engineered bioconjugate of claim 33, wherein the payload moiety is a therapeutic agent.

36. 36. The engineered bioconjugate of claim 35, wherein the therapeutic agent comprises an antiviral agent, an antibacterial agent, an immunomodulatory agent, an immunostimulatory agent, an antitumor agent, a chemotherapeutic agent, a toxin, a cytokine, a growth factor, a radionuclide, a hormone, or a combination thereof.

37. 34. The engineered bioconjugate of claim 33, wherein the payload moiety is selected from pyrrolobenzodiazepines (e.g., PBD), auristatins (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, DM1, DM4, DM21), duocamycins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, tubulysins, enediynes (e.g., calicheamicin), anthracycline derivatives (PNU) (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors, cryptophycins, drug efflux pump inhibitors, sandramycin, amanitins (e.g., alpha-amanitin), and camptothecins (e.g., exatecan, deruxtecan).

38. 27. The engineered bioconjugate of claim 26, wherein the biomolecule is an anti-Globo series antigen antibody, an anti-HER2 antibody, an anti-CD20 antibody, an anti-TNF-alpha antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TROP2 antibody, an anti-EGFR antibody, an anti-Nectin-4 antibody, an anti-HER3 antibody, an anti-cMet antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-VEGF antibody, an anti-Claudin 18.2 antibody, an anti-Sirp-alpha antibody, a TROP2xHER2 bispecific antibody, or a combination thereof.

39. 39. The engineered bioconjugate of claim 38, wherein the Globo series antigen comprises Globo H, stage-specific embryonic antigen 4 (SSEA-4), or stage-specific embryonic antigen 3 (SSEA-3).

40. 27. The engineered bioconjugate of claim 26, wherein the biomolecule is selected from Herceptin (trastuzumab), TX05 (trastuzumab biosimilar), Perjeta (pertuzumab), Erbitux (cetuximab), Rituxan (rituximab), Vectibix (panitumumab), Humira (adalimumab), Keytruda (pembrolizumab), Bavencio (avelumab), and R4702 (anti-TROP2 antibody).

41. 27. The engineered bioconjugate of claim 26, prepared by the method of any one of claims 1 to 25.

42. 41. A pharmaceutical composition comprising a plurality of the engineered bioconjugates of claim 40 and a pharmaceutically acceptable carrier.

43. 42. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 41.

44. The method of claim 42, wherein the cancer expresses a Globo series antigen, HER2, TROP2, Nectin-4, HER3, cMet, B7H3, B7H4, VEGF, claudin 18.2, or Sirp-Alpha.

45. 43. The method of claim 42, wherein the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, renal cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

46. C-L-D 1. A therapeutic conjugate comprising the formula: C is a reactive moiety configured to react in a bioorthogonal reaction; L is a linker unit comprising a hydrophilic moiety, a cleavable moiety, and a spacer; and A therapeutic conjugate wherein D is a therapeutic agent.

47. 46. ​​The therapeutic conjugate of claim 45, wherein the reactive moiety comprises an unsaturated moiety.

48. 47. The therapeutic conjugate of claim 46, wherein the unsaturated moiety is an alkene moiety or an alkyne moiety.

49. 46. ​​The therapeutic conjugate of claim 45, wherein the reactive moiety is a non-natural and non-perturbing chemical group.

50. 48. The therapeutic conjugate of any one of claims 45 to 47, wherein the reactive moiety is a dibenzocyclooctyne group (DBCO), a bicyclononyne (BCN), a cyclic alkyne, a maleimide group, an α,β-unsaturated carbonyl group, or a sulfonylpyrimidine.

51. The hydrophilic portion has 2 to 72 (OCH ２ CH ２ 46. ​​The therapeutic conjugate of claim 45, comprising a polyethylene glycol (PEG) moiety comprising a .sup.(C) subunit.

52. 52. The therapeutic conjugate of claim 51, wherein the first PEG moiety is a linear PEG, a branched PEG, or a star PEG.

53. 47. The therapeutic conjugate of claim 46, wherein the cleavable moiety is a protease-sensitive peptide comprising Val-Cit, Val-Ala, Phe-Lys, Glu-Val-Cit, Glu-Val-Ala, Glu-Gly-Cit, Glu-Gly-Ala, Gly-Gly-Phe-Gly, Gly-Gly-Val-Cit, or Gly-Gly-Val-Ala, or a glycosidase-sensitive sugar unit comprising glucuronic acid, iduronic acid, or galactose.

54. The spacer is an aromatic group including a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, a 2,5-thienyl group, or an aminomethylene (—NH—CH ２ 47. The therapeutic conjugate of claim 46, comprising:

55. The linker unit is 【Transformation 3】 Contains the formula (PEG)m is a PEG moiety, where m is an integer selected from 2 to 72; Q ＳＰ is a spacer containing an aromatic group or an aminomethylene; Q ＣＬ is a severable portion configured to be coupled to a payload; L Ｐ is a connecting unit configured to be linked to a second reactive moiety.

56. The PEG part 【Chemistry 4】 Contains the formula The wavy line is L Ｐ indicates the site of covalent attachment to R ２０ is a PEG binding unit, and the PEG binding unit is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO ２ -, alkyl-S-, or 【Transformation 5】 and R ２１ is a PEG capping unit, and the PEG capping unit is H, SO ３ H, P.O. ３ H ２ , sugar derivative, C １ ~C １０ (hetero)alkyl group, C ３ ~C １０ (hetero)cycloalkyl group, C ２ ~C １０ Alkyl-NH ２ , C １ ~C １０ Alkyl-COOH, C ２ ~C １０ Alkyl-NH(C １ ~C ３ alkyl), C ２ ~C １０ Alkyl-N(C １ ~C ３ alkyl) ２ and n is selected from 2 to 72.

57. The linker unit is 【Transformation 6】 56. The therapeutic conjugate of claim 55, having the structure:

58. 47. The therapeutic conjugate of claim 46, wherein the therapeutic agent comprises a toxin, a cytokine, a growth factor, a radionuclide, a hormone, an antiviral agent, an antibacterial agent, an immunomodulatory agent, an immunostimulatory agent, an antitumor agent, or a combination thereof.

59. 47. The therapeutic conjugate of claim 46, wherein the therapeutic agent is selected from pyrrolobenzodiazepines (e.g., PBD), auristatins (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, DM1, DM4, DM21), duocamycins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, tubulysins, enediynes (e.g., calicheamicin), anthracycline derivatives (PNU) (e.g., doxorubicin), pyrrole-based kinesin spindle protein (KSP) inhibitors, cryptophycins, drug efflux pump inhibitors, sandramycin, amanitins (e.g., alpha-amanitin), and camptothecins (e.g., exatecan, deruxtecan). 【Request Item 60】 【Chemistry 7】 60. The therapeutic conjugate of any one of claims 46 to 59, having the structure:

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