Tyrosinase-mediated site-specific protein coupling and uses thereof

Abstract

The present disclosure relates to tyrosinase-mediated site-specific protein coupling and uses thereof. Specifically, the present disclosure provides tyrosinase-mediated site-specific protein coupling and uses thereof in the preparation of drugs (e.g., ADCs).

Description

[Technical Field]

[0001] This disclosure claims priority to a Chinese patent application (application number CN202310090079.0) filed on January 19, 2023.

[0002] The present disclosure relates to the biopharmaceutical field, and in particular to tyrosinase-mediated site-specific protein coupling and its use in the preparation of drugs (e.g., ADCs). [Background technology]

[0003] The design and development of antibody-drug conjugates (ADCs) involves antibody selectivity, the cytotoxicity of small molecule toxins, and the covalent bond between the antibody and the small molecule toxin. Coupling of antibodies to small molecule toxins is one of the core factors behind the clinical success of ADC drugs. Research on the coupling techniques used in approved ADCs has shown that Adcetris® and other ADCs are prepared by reducing interchain disulfide bonds to release cysteine ​​residues followed by linking to a linker, while Kadcyla® and other ADCs are prepared by random lysine coupling (Joubert N, et al. Pharmaceuticals (Basel). 2020 Sep 14;13(9):245). Both of these methods involve non-specific modification of lysine or cysteine, and the resulting ADCs are in fact highly heterogeneous mixtures that are difficult to characterize and purify, have heterogeneous pharmacokinetic properties, are poorly stable, and are prone to aggregation (Junutula JR, et al. Nat Biotechnol. 2008 Aug;26(8):925-32.).

[0004] Studies on site-specific modification of antibodies to generate homogeneous ADCs include Genentech's Thiomab technology, which inserts additional cysteines into antibody protein sequences to generate ADCs (Pillow TH, et al. J Med Chem. 2014 Oct 9;57(19):7890-9). Schultz et al. at the Scripps Research Institute introduced uniquely active non-natural amino acids (NNAAs) into antibodies and used them for selective modification of toxins (Hallam TJ, et al. Mol Pharm. 2015 Jun 1;12(6):1848-62). However, maleimide-based cysteine ​​coupling has been shown to be serum-instable, resulting in loss of ADC efficacy or off-target toxicity. The introduction of non-natural amino acids requires expression in recombinant cell systems or cell-free systems, which can result in low expression levels and raise concerns that non-natural amino acids may induce immunogenicity of ADCs in vivo.

[0005] Enzyme-mediated site-specific coupling technology is a novel technique for producing and preparing ADCs. Synaffix modified the specific glycan at position N297 of an antibody, introduced an azide group using endoglycosidases and glycosyltransferases, and then coupled a toxin to the antibody using click chemistry (Geel RV, et al. Bioconjug Chem. 2015 Nov 18;26(11):2233-42). However, the glycan at position N297 itself plays an important role in the solubility and stability of the antibody. Triphase, NBE Therapeutics, and Legochem have introduced specific amino acid sequences to the C-terminus of the antibody heavy or light chain, respectively, which are recognized by formylglycine synthase (FGE), transpeptidase (Sortase A), and isopropylacyltransferase (Prenyl transferase), thereby achieving site-specific modification of the toxin molecule. However, such C-terminal modification methods have certain limitations: they can only achieve site-specific coupling at a drug-antibody ratio (DAR) of 2, and require the additional introduction of a relatively long sequence (greater than five amino acids), which may pose a potential immunogenic risk (Walsh SJ, et al. Chem Soc Rev. 2021 Jan 21;50(2):1305-1353.).

[0006] Currently, achieving site-specific coupling by specifically modifying specific amino acids on antibodies using enzymes is one of the most advanced technologies in the field of site-specific coupling. Full-length IgG antibodies of the human isotype contain a conserved glutamine residue (Q295) at position 295 of their heavy chains. Therefore, site-specific modification of glutamine in antibodies can be performed using transglutaminase (mTG). However, Q295 is closely adjacent to the N-glycosylation site (N297), and it is generally believed that N-glycosylation prevents Q295 from being accessible to mTG. Therefore, to remove the N-glycosylation site, the Fc region of the antibody must be deglycosylated or mutated before the coupling reaction (see WO 2013 / 092998). At the same time, the overall reaction efficiency of mTG-mediated coupling reactions is relatively low. Jorick J. Bruins et al. used mushroom tyrosinase to site-specifically modify Y296 and Y300 near N297 (Bruins JJ, et al. Bioconjug Chem. 2021 Oct 20;32(10):2167-2172.). However, both transglutaminase and mushroom tyrosinase can only modify deglycosylated antibodies.

[0007] To date, there have been no reports of introducing additional mutations into antibody constant regions to achieve tyrosinase-mediated modification of specific amino acids in antibody constant regions, nor have there been any related uses of tyrosinase-mediated site-specific coupling in glycosylated intact antibodies. The present disclosure provides a tyrosinase-mediated site-specific modification technology for tyrosine in antibody constant regions or fragments thereof, which is characterized by a fast reaction rate, high specificity of site-specific modification, high uniformity of ADC products, and no need for antibody deglycosylation or removal of N-glycosylation site mutations. At the same time, by introducing multiple reactive tyrosines, multi-site site-specific coupling can be achieved, solving the problem that enzyme-mediated site-specific antibody coupling currently only has a DAR value of 2, providing options for higher DAR values ​​and enabling the development of more efficient and safer ADC drugs. Summary of the Invention

[0008] The present disclosure provides antibody constant regions or proteins comprising the constant regions (e.g., antibodies or antigen-binding fragments thereof), conjugates prepared therefrom, preparation methods, and uses for preparing medicaments.

[0009] Antibody or antigen-binding fragment thereof The present disclosure provides antibodies or antigen-binding fragments thereof that comprise an engineered tyrosine residue, which is a tyrosine residue (Tyr) resulting from mutation (e.g., substitution, replacement) of a natural amino acid residue.

[0010] In some embodiments, the naturally occurring amino acid residues or the resulting engineered tyrosine residues after mutation thereof may be one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16) located in the heavy chain constant region and / or the light chain constant region. For example, C H 1 region, hinge region, Fc region and / or CL region. H 1 region, hinge region, C H 2 areas, C HIn the present disclosure, the heavy chain constant region is located in the C3 region and / or the Cκ region and the Cλ region. H 1. Hinge region, C H 2 and C H Includes 3.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG type, such as an IgG1, IgG2, IgG3, or IgG4 type, such as a human or murine IgG1, IgG2, IgG3, or IgG4 type. In the present disclosure, human IgG1, IgG2, IgG3, and IgG4 are used interchangeably with IGHG1, IGHG2, IGHG3, and IGHG4.

[0012] In some embodiments, the naturally occurring amino acid residues are located at any one of positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof.

[0013] In some embodiments, the naturally occurring amino acid residues are located at any one of positions 135T, 137G, 192S, 223T, 298S, 329P of the heavy chain HC and position 202S of the light chain LC, or any combination thereof.

[0014] In some embodiments, the engineered tyrosine residue is located at any one of positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof.

[0015] In some embodiments, the engineered tyrosine residue is selected from any one of T135Y, G137Y, S192Y, T223Y, S298Y, P329Y of the heavy chain HC and S202Y of the light chain LC, or any combination thereof.

[0016] In some embodiments, the heavy chain HC is IGHG1. In some embodiments, the light chain LC is Cκ. Unless otherwise stated, all amino acid position numbers in this disclosure are defined according to the EU numbering system.

[0017] In some embodiments, the native amino acid residues at positions 135, 137, and 192 or the tyrosine residues to which they are mutated are C H 1 region, the natural amino acid residue at position 223 or its mutated tyrosine residue is located in the hinge region, and the natural amino acid residues at positions 298 and 329 or their mutated tyrosine residues are located in the C H The natural amino acid residue at position 202 or its mutated tyrosine residue is located in the Cκ region.

[0018] In some embodiments, the amino acid residues are located at any one of positions 135, 137, 192, 223, 298, and 329 of IGHG1 (or positions in IGHG2, IGHG3, and IGHG4 corresponding to positions 135, 137, 192, 223, 298, and 329 of IGHG1) and position 202 of Kappa LC (or positions in Lambda LC corresponding to position 202 of Kappa LC), or any combination thereof.

[0019] In some embodiments, the engineered tyrosine residue in the antibody or antigen-binding fragment thereof is HC's T135Y / T223Y, HC's T223Y / P329Y, HC's T135Y / P329Y, HC's T135Y / T223Y / P329Y, HC's T135Y / T223Y / P329Y / S192Y, HC T135Y / T223Y and LC S202Y, HC T135Y / T223Y and LC S202Y, HC's T223Y / P329Y and LC's S202Y, HC's T135Y / P329Y and LC's S202Y, G137Y for HC and S202Y for LC, HC T135Y / T223Y / P329Y and LC S202Y, HC T135Y / T223Y / P329Y / S192Y and LC S202Y, P329Y of HC and S202Y of LC, A combination selected from HC S192Y and LC S202Y, " / " represents "and".

[0020] In some embodiments, the antibody is an antibody that targets a tumor antigen, such as an anti-HER2 antibody, an anti-LIV-1 antibody, or an anti-TROP2 antibody.

[0021] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs), or the heavy chain variable region (VH) and light chain variable region (VL) of Trastuzumab.

[0022] The CDRs are as follows (defined according to the Kabat numbering system):

[0023] [C1] HCDR1:DTYIH (SEQ ID NO:51) [Case 2] HCDR2: RIYPTNGYTRYADSVKG (SEQ ID NO: 52) [C3] HCDR3: WGGDGFYAMDY (SEQ ID NO: 53) [C4] LCDR1:RASQDVNTAVA (SEQ ID NO: 54) [5] LCDR2:SASFLYS (SEQ ID NO: 55) [6] LCDR3:QQHYTTPPT (SEQ ID NO:56) [7] >Trastuzumab VH (SEQ ID NO: 57) [8] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS >Trastuzumab VL (SEQ ID NO: 58) [9] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

[0024] In some embodiments, the present disclosure provides an anti-HER2 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein: the heavy chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 3 to 10, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 2; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 11; The heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 24 to 27, 7, and 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2 or 11; for example, the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 26, 7, and 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 11, or the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 25 and 27, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2.

[0025] In some embodiments, the anti-LIV-1 antibody or antigen-binding fragment thereof comprises the following heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs), or heavy chain variable regions (VH) and light chain variable regions (VL):

[0026] The CDRs are as follows (defined according to the Kabat numbering system):

[0027] [C10] HCDR1:TYAMS (SEQ ID NO:59) [C11] HCDR2: TISDSDYYADNVKG (SEQ ID NO: 60) [C12] HCDR3:DDWDGDFDY (SEQ ID NO: 61) [C13] LCDR1:KSTQSLLYSDGETYLN (SEQ ID NO: 62) [C14] LCDR2:LVSKLDS (SEQ ID NO: 63) [C15] LCDR3:WQGSHFPQT (SEQ ID NO: 64) [C16] >0102 VH (SEQ ID NO: 65) [C17] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVVRDDWDGDFDYWGQGTLVTVSS >0102 VL (SEQ ID NO: 66) [C18] DVVMTQSPLSLPVTPGEPASISCKSTQSLLYSDGETYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCWQGSHFPQTFGGGTKVEIK.

[0028] In some embodiments, the present disclosure provides an anti-LIV-1 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence set forth in any one of SEQ ID NOs: 14-19 and the light chain comprises a sequence set forth in SEQ ID NO: 13, or wherein the heavy chain comprises a sequence set forth in SEQ ID NO: 12 and the light chain comprises a sequence set forth in SEQ ID NO: 20.

[0029] In some embodiments, the present disclosure provides an anti-LIV-1 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 42 and the light chain comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises the following heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs), or heavy chain variable regions (VH) and light chain variable regions (VL):

[0030] The CDRs are as follows (defined according to the Kabat numbering system):

[0031] [C19] HCDR1:DYAMH (SEQ ID NO:68)

[20] HCDR2: AITWNSGHIDYADSVEG (SEQ ID NO: 69)

[21] HCDR3: VSYLSTASSLDY (SEQ ID NO: 70) [C22] LCDR1:RASQGIRNYLA (SEQ ID NO: 71)

[23] LCDR2:AASTLQS (SEQ ID NO: 72)

[24] LCDR3:QRYNRAPYT (SEQ ID NO:73)

[25] >TROP2 VH (SEQ ID NO: 74)

[26] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASS >TROP2 VL (SEQ ID NO: 75)

[27] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC.

[0032] In some embodiments, the antigen-binding fragment includes, but is not limited to, any one of Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv).

[0033] protein The present disclosure provides proteins comprising antibody constant regions or fragments thereof containing one or more engineered tyrosine residues. The engineered tyrosines can be conjugated to a payload (e.g., a therapeutic agent, a diagnostic agent). The phenolic moiety of the engineered tyrosine residue is oxidized to an o-quinone moiety by tyrosinase, while the payload (e.g., a therapeutic agent, a diagnostic agent) contains an olefin or alkyne moiety, which is conjugated to the olefin or alkyne moiety via a [4+2] cycloaddition reaction.

[0034] The present disclosure provides proteins comprising antibody constant regions or fragments thereof containing one or more engineered tyrosine residues. The phenolic moieties of the engineered tyrosine residues can be oxidized to o-quinone moieties by monotyrosinase but not by polytyrosinase. In some embodiments, the monotyrosinase is derived from Bacillus megaterium or Verrucomicrobium spinosum, and the polytyrosinase is a tetrameric enzyme derived from a mushroom. In some embodiments, the term "derived from" refers to the amino acid sequence encoding the enzyme being derived from the bacterium, or the enzyme being secreted or prepared by the bacterium (or commercially available). In some specific embodiments, the mushroom-derived tetrameric tyrosinase is Sigma Aldrich T3824.

[0035] The present disclosure provides proteins comprising an antibody constant region or fragment thereof containing one or more engineered tyrosine residues. In some embodiments, the engineered tyrosine residues are not located near an N-glycosylation modification site, e.g., not within 10 amino acids of a glycosylation modification site, or the protein does not require removal / reduction of glycosylation modifications prior to reaction with tyrosinase. In some specific embodiments, the N-glycosylation modification is located at 297N of the heavy chain as defined according to the EU numbering system. In some embodiments, the engineered tyrosine residue is not located between amino acid residues 292 and 302, 293 and 303, 291 and 301, 293 and 302, or 292 and 301 of the heavy chain HC. In some embodiments, the protein is an N-glycosylation-modified protein, specifically a 297N-glycosylation-modified protein.

[0036] The present disclosure provides proteins comprising an antibody constant region or fragment thereof comprising one or more engineered tyrosine residues, wherein: I) the engineered tyrosine residue is located at any one of positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof; or II) The engineered tyrosine residues are located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof.

[0037] In some embodiments, if the engineered tyrosine residue is located at position 298 of the heavy chain HC, the protein (or antibody constant region or fragment thereof therein) must have glycosylation modifications removed / reduced and / or the native tyrosine at position 298 is mutated to a non-natural amino acid. In some embodiments, the native tyrosine at position 298 is selected from 296Y and / or 300Y. In some embodiments, the 296Y and / or 300Y are mutated to phenylalanine (Phe).

[0038] In some embodiments, the glycosylation modification is located at position 297N of the heavy chain. In some embodiments, the method for removing / reducing the glycosylation modification is selected from (A) contacting the protein with an amidase, (B) contacting the protein with an endoglycosidase, or (C) mutating the protein so that 297N is replaced with a non-glycosylated amino acid. Exemplary methods include (A) deglycosylating the protein (or antibody constant region or fragment thereof therein) by contacting it with an amidase (e.g., PNGase F) to obtain a protein (or antibody constant region or fragment thereof) from which the glycan has been removed, or (B) mutating the protein to obtain a protein (or antibody constant region or fragment thereof) from which the glycan has been removed by mutating the protein (or antibody constant region or fragment thereof) ... b(wherein b is 0 or 1), or (C) providing a mutated protein (or antibody constant region or fragment thereof) in which a glycosylated asparagine is substituted by a non-glycosylated amino acid.

[0039] The present disclosure provides proteins comprising an antibody constant region or fragment thereof comprising one or more engineered tyrosine residues, wherein the engineered tyrosine residues are located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof.

[0040] In some embodiments, the engineered tyrosine residue in the protein is located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof.

[0041] Illustratively, the combination is: 202Y for LC and 135Y for HC, 202Y for LC and 137Y for HC, LC 202Y and HC 192Y, 202Y of LC and 223Y of HC, 202Y of LC and 329Y of HC, HC 135Y / 137Y, HC 135Y / 192Y, HC 135Y / 223Y, HC 135Y / 329Y, HC 137Y / 192Y, HC 137Y / 223Y, HC 137Y / 329Y, HC 192Y / 223Y, HC 192Y / 329Y, HC 223Y / 329Y, 135Y / 137Y for HC and 202Y for LC, HC 135Y / 192Y and LC 202Y, 135Y / 223Y for HC and 202Y for LC, 135Y / 329Y for HC and 202Y for LC, 137Y / 192Y for HC and 202Y for LC, 137Y / 223Y for HC and 202Y for LC, 137Y / 329Y for HC and 202Y for LC, 192Y / 223Y for HC and 202Y for LC, HC 192Y / 329Y and LC 202Y, 223Y / 329Y for HC and 202Y for LC, HC 135Y / 137Y / 192Y, HC 135Y / 137Y / 223Y, HC 135Y / 137Y / 329Y, HC 135Y / 137Y / 192Y and LC 202Y, HC's 135Y / 137Y / 223Y and LC's 202Y, HC's 135Y / 137Y / 329Y and LC's 202Y, HC 135Y / 192Y / 223Y, HC 135Y / 192Y / 329Y, HC 135Y / 192Y / 223Y and LC 202Y, HC 135Y / 192Y / 329Y and LC 202Y, HC 137Y / 192Y / 223Y, HC 137Y / 192Y / 329Y, HC 137Y / 192Y / 223Y and LC 202Y, HC 137Y / 192Y / 329Y and LC 202Y, HC 137Y / 223Y / 329Y, 137Y / 223Y / 329Y for HC and 202Y for LC, HC 192Y / 223Y / 329Y, HC 192Y / 223Y / 329Y and LC 202Y, HC 135Y / 223Y / 329Y, Selected from HC 135Y / 223Y / 329Y and LC 202Y.

[0042] In some embodiments, the antibody constant region or fragment thereof in the protein is derived from an IgG, IgA, IgM, IgD, or IgE constant region or fragment thereof. In some specific embodiments, the IgG is preferably IgG1, IgG2, IgG3, or IgG4, such as IgG1, or IGHG1, IGHG2, IGHG3, or IGHG4.

[0043] In some embodiments, the antibody constant region or fragment thereof in the protein is a heavy chain constant region or fragment thereof and / or a light chain constant region or fragment thereof. In some embodiments, the heavy chain constant region is H 1 region, hinge region, C H In some embodiments, the light chain constant region is a Cκ region or a Cλ region. In some embodiments, the heavy chain constant region is a C H It further includes three areas.

[0044] In some embodiments, the antibody constant region or fragment thereof in the protein is glycosylated or non-glycosylated. In some embodiments, the glycosylation modification is a 297N glycosylation modification.

[0045] In some embodiments, the protein is selected from an antibody or antigen-binding fragment thereof, an Fc region or fragment thereof, or a fusion protein. In some embodiments, the antibody or antigen-binding fragment thereof is a monospecific antibody, a bispecific antibody, a multispecific antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-tumor antibody or antigen-binding fragment thereof, e.g., an anti-HER2 antibody or antigen-binding fragment thereof, an anti-LIV-1 antibody or antigen-binding fragment thereof, or an anti-TROP2 antibody or antigen-binding fragment thereof.

[0046] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 51 to 53, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54 to 56. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 57, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 58, In some embodiments, the anti-LIV-1 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 59 to 61, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 62 to 64. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 66.

[0047] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 68 to 70, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 71 to 73. In some embodiments, the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 74, and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 75.

[0048] In some embodiments, the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0049] In some embodiments, the protein is a monomer or multimer, such as a dimer, trimer, or tetramer. For example, a tetramer is an IgG antibody with two heavy chains and two light chains. In some embodiments, there are two identical heavy chains and two identical light chains. In other embodiments, there are two different heavy chains and two different light chains.

[0050] In some embodiments, the protein comprises a polypeptide fragment at the N-terminus and / or C-terminus, wherein the fragment comprises one or more tyrosine residues. In some embodiments, the fragment is linked to the N-terminus and / or C-terminus of the protein directly or via a linker. In some embodiments, the linker is a GS or polyG linker. In some embodiments, the fragment is YGGGG, YGGGGS, which is linked to the N-terminus of the protein via a linker. In some embodiments, the fragment is GGGGY, SGGGGY, which is linked to the N-terminus of the protein via a linker. In some embodiments, the protein can comprise the N-terminal and C-terminal fragments simultaneously or separately.

[0051] In some embodiments, the term "engineered tyrosine residue" of the present disclosure refers to the presence of a non-wild-type amino acid residue in an amino acid sequence. For example, when aligning the wild-type and engineered sequences, an "engineered tyrosine residue" is a tyrosine residue that is not present in the corresponding wild-type polypeptide sequence and that is introduced into the polypeptide (by mutation (e.g., substitution, replacement) of an existing amino acid residue or by insertion of a tyrosine residue). In some embodiments, the polypeptide is any protein of the present disclosure, an antibody constant region or fragment thereof, or an antibody or antigen-binding fragment thereof.

[0052] In some embodiments, the protein has one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, fourteen, sixteen) engineered tyrosine residues.

[0053] In some embodiments, the engineered tyrosine residue is located at any one or any combination of positions 135, 137, 192, 223, 298, 329 of IHG1 (or at positions 135, 137, 192, 223, 298, 329 of IHG1 in IHG2, IHG3, or IHG4), and position 202 of Cκ (or at position 202 of Cκ in Cλ). In other embodiments, the engineered tyrosine residue is located at any one or any combination of positions 135, 137, 192, 223, 329 of IHG1 (or at positions 135, 137, 192, 223, 329 of IHG2, IHG3, or IHG4), and position 202 of Cκ (or at position 202 of Cκ in Cλ). In some embodiments, the numbering of the positions of IGHG1 above is according to the EU numbering system, and IGHG2, IGHG3, IGHG4 correspond to the corresponding positions of IGHG1, for example, with reference to FIG.

[0054] In some embodiments, an antibody constant region or fragment thereof is provided that comprises any one of the following: 1) C that contains (or has) the following: H 1 or a fragment thereof: 1-1) an amino acid substitution at position 135, such as 135Y or T135Y; 1-2) an amino acid substitution at position 137, such as 137Y or G137Y; 1-3) an amino acid substitution at position 192, such as 192Y or S192Y; 1-4) amino acid substitutions at positions 135 and 137, such as 135Y / 137Y or T135Y / G137Y; 1-5) amino acid substitutions at positions 135 and 192, such as 135Y / 192Y or T135Y / S192Y; 1-6) amino acid substitutions at positions 137 and 192, such as 137Y / 192Y or G137Y / S192Y; 1-7) amino acid substitutions at positions 135, 137, and 192, such as 135Y / 137Y / 192Y or T135Y / G137Y / S192Y; 2) a hinge region or fragment thereof comprising (or having) an amino acid substitution at position 223, e.g., 223Y or T223Y; 3) C that contains (or has) the following: H 2 or fragments thereof: 3-1) an amino acid substitution at position 298, such as 298Y or S298Y; 3-2) an amino acid substitution at position 329, such as 329Y or P329Y; 3-3) amino acid substitutions at positions 298 and 329, such as 298Y / 329Y or S298Y / P329Y; When an amino acid substitution at position 298 (e.g., 298Y or S298Y) is present, the CH2 or fragment thereof also contains an amino acid substitution at position 296X and / or 300X, which is any amino acid substitution except tyrosine (e.g., 296F and / or 300F, also e.g., 296F / 300F); 4) CL or a fragment thereof containing (or having) an amino acid substitution at position 202, such as 202Y or S202Y; 5) Any combination of 1) to 4) above, for example: 5-1) C is 1-1), 1-2), 1-3), 1-4), 1-5), 1-6) or 1-7) above. H 1) or a fragment thereof and the hinge region or a fragment thereof described above in 2), 5-2) A hinge region or a fragment thereof according to 2) above and C according to 3-1), 3-2) or 3-3) above H 2 or a fragment thereof, 5-3) C is 1-1), 1-2), 1-3), 1-4), 1-5), 1-6), or 1-7) above. H 1 or a fragment thereof and C which is 3-1), 3-2) or 3-3) above H 2 or a fragment thereof, 5-4) C above 1-1), 1-2), 1-3), 1-4), 1-5), 1-6) or 1-7). H 1 or a fragment thereof, the hinge region or a fragment thereof which is 2) above, and C which is 3-1), 3-2) or 3-3) above. H 2 or a fragment thereof, 5-5) C is 1-1), 1-2), 1-3), 1-4), 1-5), 1-6) or 1-7) above.H 1) or a fragment thereof and CL or a fragment thereof which is 4) above, 5-6) CL or a fragment thereof which is 4) above and C which is 3-1), 3-2) or 3-3) above H 2 or a fragment thereof, 5-7) a hinge region or a fragment thereof which is 2) above and a CL or a fragment thereof which is 4) above; 5-8) C above 1-1), 1-2), 1-3), 1-4), 1-5), 1-6) or 1-7). H 1 or a fragment thereof, C which is 3-1), 3-2) or 3-3) above H 2) or a fragment thereof, and CL or a fragment thereof which is 4) above, 5-9) C above 1-1), 1-2), 1-3), 1-4), 1-5), 1-6) or 1-7). H 1) or a fragment thereof, the hinge region or a fragment thereof which is 2) above, and the CL or a fragment thereof which is 4) above, 5-10) A hinge region or a fragment thereof that is 2) above, or C that is 3-1), 3-2) or 3-3) above H 2) or a fragment thereof, and CL or a fragment thereof which is 4) above, 5-11) C above 1-1), 1-2), 1-3), 1-4), 1-5), 1-6) or 1-7). H 1 or a fragment thereof, the hinge region or a fragment thereof which is 2) above, C which is 3-1), 3-2) or 3-3) above H 2 or a fragment thereof and CL or a fragment thereof which is 4) above.

[0055] In some embodiments, CL is Cκ or Cλ.

[0056] In some embodiments, the fragment comprises at least 10 amino acids around the amino acid position where the tyrosine substitution occurs, e.g., in the heavy chain, 10 amino acids around position 135 are positions 130-140, 10 amino acids around position 137 are positions 132-142, 10 amino acids around position 192 are positions 187-197, 10 amino acids around position 223 are positions 218-228, 10 amino acids around position 298 are positions 293-235, and 10 amino acids around position 329 are positions 324-334, and in the light chain, 10 amino acids around position 202 are positions 197-207.

[0057] In some embodiments, the C H In some embodiments, the hinge region or a fragment thereof has at least 90% sequence identity with SEQ ID NO: 46 based on the inclusion of tyrosine substitutions in 1-1) to 1-7) above. In some embodiments, the hinge region or a fragment thereof has at least 90% sequence identity with SEQ ID NO: 47 based on the inclusion of tyrosine substitutions in 2) above. In some embodiments, the hinge region or a fragment thereof has at least 90% sequence identity with SEQ ID NO: 47 based on the inclusion of tyrosine substitutions in 2) above. H 2 or a fragment thereof has at least 90% sequence identity with SEQ ID NO: 48 based on the inclusion of tyrosine substitutions 3-1) to 3-3) above. In some embodiments, the CL or a fragment thereof has at least 90% sequence identity with SEQ ID NO: 50 based on the inclusion of tyrosine substitutions 4) above.

[0058] In some embodiments, C H The amino acid sequence of the hinge region is set forth in any one of SEQ ID NOs: 84, 88 to 90. In some embodiments, the amino acid sequence of the hinge region is set forth in SEQ ID NO: 85. In some embodiments, H The amino acid sequence of the two regions is set forth in SEQ ID NO: 86. In some embodiments, the amino acid sequence of the Cκ region is set forth in SEQ ID NO:87.

[0059] In some embodiments, the antibody constant region or fragment thereof comprises C H 3, for example, a sequence set forth in SEQ ID NO: 49 or having at least 90% identity thereto.

[0060] In the present disclosure, "at least 90% sequence identity" includes at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0061] In some embodiments, the antibody constant region or fragment thereof is monomeric, eg, a polypeptide chain.

[0062] In some embodiments, the antibody constant region or fragment thereof is a multimer, such as a dimer, trimer, or tetramer.

[0063] In some embodiments, the antibody constant region or fragment thereof further comprises an antigen-binding domain, e.g., a heavy chain variable region (VH) and a light chain variable region (VL), an immunoglobulin single variable domain (or single domain antibody, VHH), or an scfv, that specifically binds to an antigen or an epitope thereof. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen, e.g., HER2, LIV-1, or TROP2.

[0064] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising the antibody constant region or a fragment thereof. The antibody or antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a recombinant antibody or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem di-scFv, or a tandem tri-scFv. In some embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody (e.g., a triabody). In some embodiments, the antibody or antigen-binding fragment thereof targets a tumor antigen such as HER2, LIV-1, or TROP2.

[0065] In some embodiments, proteins are provided that are fusion proteins, for example, fusion proteins of antibody constant regions or fragments thereof with other polypeptide therapeutic or diagnostic agents.

[0066] In some embodiments, an antibody constant region or fragment thereof is provided that comprises a polypeptide fragment containing one or more tyrosine residues at the N-terminus and / or C-terminus. In some embodiments, the fragment is linked to the N-terminus and / or C-terminus of the antibody constant region or fragment thereof, directly or via a linker. In some embodiments, the linker is a GS or polyG linker. In some embodiments, the fragment is YGGGG or YGGGGS, which is linked to the N-terminus of the antibody constant region or fragment thereof via a linker. In some embodiments, the fragment is GGGGY or SGGGGY, which is linked to the N-terminus of the antibody constant region or fragment thereof via a linker. In some embodiments, the antibody constant region or fragment thereof can comprise the N-terminal and C-terminal fragments, either simultaneously or separately.

[0067] In some embodiments, the protein does not need to have the 297N glycosylation modification removed / reduced.

[0068] In some embodiments, if amino acid 298 is not an engineered tyrosine residue, the protein does not need to remove / reduce the glycosylation modification at 297N.

[0069] In some embodiments, mutations may be present in other naturally occurring tyrosines in the protein in addition to the engineered tyrosine substitution sites provided by the present disclosure, and the amino acid mutations may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect function. In some specific embodiments, the amino acid mutations may occur in CDR regions and / or framework regions (FR regions) and / or constant regions.

[0070] In some embodiments, the protein comprises an antibody or antigen-binding fragment thereof provided above in this disclosure.

[0071] Complex The present disclosure provides use of the antibody or antigen-binding fragment thereof, or protein for preparing a conjugate. In some embodiments, the conjugate is a protein-drug conjugate, including an antibody-drug conjugate, an antibody-polypeptide conjugate, or a polypeptide-drug conjugate (e.g., an antibody constant region or fragment thereof-drug conjugate).

[0072] In some embodiments, the antibodies or antigen-binding fragments thereof, and proteins of the present disclosure do not require an amino acid mutation at 297N (e.g., N297G) to remove glycosylation, nor does it require removal / reduction of the glycosylation modification at 297N prior to preparation of the antibody-drug conjugate.

[0073] The present disclosure provides a conjugate having the structure shown in Formula I, or a pharmaceutically acceptable salt thereof: Pc-[L-(D) x ] yI wherein Pc is any of the antibodies or antigen-binding fragments thereof provided herein, or any of the proteins provided herein; L is a linker that covalently connects Pc to D, and L is linked to an amino acid in Pc, said amino acid being located in Pc; D is the payload, x is 1 to 20; y is 1 to 20.

[0074] In some embodiments, the amino acids are located in the heavy chain constant region and / or the light chain constant region of Pc. H 1 region, hinge region, Fc region and / or CL region. H 1 region, hinge region, C H 2 areas, C H It is located in the 3 region and / or the Cκ region and Cλ region.

[0075] In some embodiments, the amino acid is located at any one or combination of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC of the Pc antibody or antigen-binding fragment thereof, all of which are defined according to the EU numbering system.

[0076] In some embodiments, the heavy chain HC is IGHG1.

[0077] In some embodiments, the light chain LC is Cκ.

[0078] In some embodiments, the heavy chain HC is IGHG2, IGHG3, or IGHG4.

[0079] In some embodiments, the light chain LC is a Cλ.

[0080] In some embodiments, the amino acid is located at any one of positions 135, 137, 192, 223, 329 of IGHG1 (or at positions 135, 137, 192, 223, 329 of IGHG1 in IGHG2, IGHG3, IGHG4) and position 202 of Cκ (or at position 202 of Kappa LC in Cλ), or any combination thereof.

[0081] Illustratively, in some embodiments, the amino acid is HC 135th and 223rd place, HC 223rd and 329th, HC 135th and 329th, HC rankings 135th, 223rd and 329th, HC rankings 135th, 223rd, 329th and 192nd, HC 135th, 223rd and LC 202nd, HC 135th, 223rd and LC 202nd, 223rd and 329th in HC and 202nd in LC, 135th and 329th in HC and 202nd in LC, HC 135th, 223rd, 329th and LC 202nd, HC 135th, 223rd, 329th, 192nd and LC 202nd, Position 329 of HC and position 202 of LC, or It is ranked 192nd in HC and 202nd in LC.

[0082] In some embodiments, the amino acid is located in a non-glycosylation modified region of Pc, for example, not at position 297 of the heavy chain or within 10 amino acids around position 297 of the heavy chain.

[0083] In some embodiments, the Pc comprises a binding domain that specifically binds to a tumor antigen or is an anti-tumor antibody or antigen-binding fragment thereof, such as an anti-HER2 antibody or antigen-binding fragment thereof, an anti-LIV-1 antibody or antigen-binding fragment thereof, or an anti-TROP2 antibody or antigen-binding fragment thereof.

[0084] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises the heavy chain CDR (HCDR) and light chain CDR (LCDR), or the heavy chain variable region (VH) and light chain variable region (VL), of Trastuzumab. For example, the anti-HER2 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 51 to 53, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54 to 56, or the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 57, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 58.

[0085] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 3 to 10, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 2; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 11; The heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 24 to 27, 7, and 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2 or 11; for example, the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 26, 7, and 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 11, or the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 25 and 27, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2.

[0086] In some embodiments, the anti-LIV-1 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 59 to 61, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 62 to 64, or the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 66.

[0087] In some embodiments, the anti-LIV-1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 14 to 19 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 13, or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 12 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0088] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 68 to 70, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 71 to 73; or the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 74, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 75.

[0089] In some embodiments, the native tyrosine site of Pc can be mutated to reduce non-specific coupling or the effect of the native tyrosine on the tyrosinase reaction process. The amino acid mutations can be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function. In some specific embodiments, the amino acid mutations can occur in the CDR regions and / or framework regions (FR regions) and / or constant regions. In some specific embodiments, 296Y and / or 300Y in the antibody heavy chain constant region can be mutated, for example, to 296F and / or 300F.

[0090] In some embodiments, in the complex, L and Pc form a structure represented by Za or Zb, [ka] * represents the end of connection to Pc, ** represents the end of connection to the linker, and ---- represents a single bond or a double bond.

[0091] In some embodiments, in the complex, the structure represented by Za is a structure represented by formula Za1 or Za2, [ka] * represents the end connected to Pc, and ** represents the end connected to the linker.

[0092] In some embodiments, in the complex, the structure represented by Zb is a structure represented by formula Zb1 or Zb2, [ka] * represents the end connected to Pc, and ** represents the end connected to the linker.

[0093] In some embodiments, the amino acid linked to L is tyrosine and is oxidized to an o-quinone by tyrosinase. The o-quinone undergoes a [4+2] cycloaddition reaction with the linker to link Pc to L-(D)x. [4+2] cycloaddition reactions are well known in the art (see, for example, WO2014 / 065661, which is incorporated by reference). In some embodiments, the [4+2] cycloaddition reaction occurs under metal-free conditions.

[0094] In some other embodiments, the linker [ka] is decomposed under certain conditions. [ka] Form.

[0095] In some embodiments, the linker is stable extracellularly so that a conjugate comprising a binding domain that specifically binds to a tumor antigen remains intact when present in the extracellular environment but is cleavable upon internalization into a cell, such as a cancer cell. In some embodiments, D is cleaved from the antibody moiety when the conjugate enters a cell that expresses the particular tumor antigen.

[0096] In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, conjugates containing cleavable peptide moieties exhibit relatively lower aggregation levels, improved antibody-to-drug ratios, increased on-target killing of cancer cells, decreased off-target killing of non-cancer cells, and / or relatively higher drug loading (p) relative to conjugates containing other cleavable moieties.

[0097] In some embodiments, the addition of a cleavable moiety to a non-cleavable linker increases cytotoxicity and / or potency, hi some embodiments, the increased potency and / or cytotoxicity has increased potency and / or cytotoxicity in cancers that express moderate levels of a particular tumor antigen.

[0098] In some embodiments, the conjugate linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety can be cleaved by an enzyme, and the linker is an enzymatically cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In certain embodiments, compared to other cleavage mechanisms, an enzymatically cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above.

[0099] In some embodiments, the linker comprises a cleavable peptide moiety, which preferably comprises peptide residues consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0100] In some embodiments, the cleavable peptide moiety comprises valine-citrulline (Val-Cit). In some embodiments, ADCs comprising Val-Cit exhibit improved stability, reduced off-target cell killing, increased on-target cell killing, lower aggregation levels, and / or higher drug loading relative to ADCs comprising other amino acid units or other cleavable moieties.

[0101] In another aspect, the linkers provided in some embodiments comprise a cleavable sulfonamide moiety, said linkers being cleavable under reducing conditions.

[0102] In some embodiments, the linker comprises a cleavable disulfide moiety, wherein the linker is cleavable under reducing conditions.

[0103] In another embodiment, the linker in the conjugate of the present disclosure comprises at least one spacer unit connecting D to the cleavable moiety.

[0104] In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB), [ka] Includes:

[0105] In some embodiments, the spacer unit is p-aminobenzoyl [ka] Includes:

[0106] In some other embodiments, the spacer unit is -(CR 1 R 2 ) m1 -O(CR 1 R 2 ) m2 -CR 3 R 4 -C(O)-, -(CR 1 R 2 ) m1 NH-(CR 1 R 2 ) m2 -CR 3 R 4 -C(O)-, -(CR 1 R 2 ) m1 OCR 3 R 4 (CR 1 R 2 ) m2 -, -(CR 1 R 2 ) m1 OCR 3 R 4 -C(O)-, -(CR 1 R 2 ) m1 -O-(CR 1 R 2 ) m2 C(O)- or -(CR 1 R 2 ) m1 -S-(CR 1 R 2 ) m2 -CR 3 R 4 -C(O)-; Among them, R1 and R 2 are the same or different and are each independently selected from hydrogen, halogen, or alkyl groups; R 3 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, R 4 is hydrogen, a haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 3 and R 4 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, wherein m1 and m2 are each independently selected from 0, 1, 2, or 3;

[0107] In some embodiments, R 3 is selected from hydrogen, and R 4 is C 3-6 In some embodiments, R is selected from cycloalkyl groups, such as cyclopropyl or cyclobutyl groups. 3 is C 3-6 cycloalkyl groups, such as cyclopropyl or cyclobutyl groups, R 4 is selected from hydrogen.

[0108] In some embodiments, R 3 and R 4 are C together with the carbon atoms connected to them. 3-6 It forms a cycloalkyl group, for example a cyclopropyl group or a cyclobutyl group.

[0109] In some embodiments, the spacer unit is -(CH2)3-C(O)-, -CH2-O-CH2-C(O)-, -(CH2)2-O-CH2-C(O)-, [ka] The present invention includes a portion selected from the group consisting of:

[0110] In some embodiments, the linker may include at least one polyethylene glycol (PEG) moiety. The PEG moiety may be, for example, -(PEG) p1 -, [ka] is.

[0111] In some other embodiments, the linker is relatively short in length, but ADCs comprising a relatively short stretching unit (e.g., (PEG)2) exhibit relatively lower aggregation levels and / or relatively higher drug loading relative to ADCs comprising a relatively long stretching unit (e.g., (PEG)8).

[0112] In another aspect, the linker L in the conjugate of the present disclosure comprises a stretching unit (Str) that covalently bonds to P. In some embodiments, the stretching unit Str in the linker is a structural unit that connects to an o-quinone, or the stretching unit Str in the linker undergoes a [4+2] cycloaddition reaction with the o-quinone to connect P to L-(D). x [4+2] cycloaddition reactions are well known in the art (see, for example, WO2014 / 065661, which is incorporated by reference). In some embodiments, the [4+2] cycloaddition reaction occurs under metal-free conditions.

[0113] In some embodiments, the stretch unit Str in the linker comprises a cycloalkenyl group, a cycloalkynyl group, a heterocycloalkenyl group, or a heterocycloalkynyl group. As used herein, the cycloalkenyl group, the cycloalkynyl group, the cycloalkenyl group, or the heterocycloalkynyl group may be optionally substituted.

[0114] In some embodiments, the stretch unit Str in the linker comprises a cycloalkynyl group or a heterocycloalkynyl group.

[0115] In some embodiments, the stretch unit Str in the conjugate comprises a cycloheptynyl group, a cyclooctynyl group, a cyclononyl group, a cyclodecynyl group, a heterocycloheptynyl group, a heterocyclooctynyl group, a heterocyclononyl group, or a heterocyclodecynyl group.

[0116] In some embodiments, the stretch unit Str in the complex is [ka] The group includes a group selected from the group consisting of:

[0117] In some embodiments, the stretch unit Str in the complex is [ka] Among these, S (+) is anion B (-) is a cationic sulfur atom that is equilibrated to

[0118] In another aspect, in some embodiments, the stretch units Str and Pc in the linker are [ka] is.

[0119] In some other embodiments, the stretch units Str and Pc in the linker are [ka] and S (+) is anion B (-) is a cationic sulfur atom that is equilibrated to

[0120] In one embodiment, the stretch units Str and Pc in the linker are [ka] is.

[0121] In one embodiment, the stretch units Str and Pc in the linker are [ka] is.

[0122] In one embodiment, the stretch units Str and Pc in the linker are [ka] Forming Among them, R 8 are independently hydrogen, halogen, or C 1-6 Alkyl group, C 1-6 Alkoxy group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, aryl or heteroaryl groups optionally contain one or more halogens, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, R 9 is hydrogen, halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, aryl or heteroaryl groups optionally contain one or more halogens, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, R 10 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, t is an integer between 0 and 10, o is an integer between 0 and 10, * represents the end connected to Pc, and ** represents the end connected to the linker.

[0123] In one embodiment, the stretch units Str and Pc in the linker are [ka] Forming Among them, R 10 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, o is an integer between 0 and 10, * represents the end connected to Pc, and ** represents the end connected to the linker.

[0124] In one embodiment, the stretch units Str and Pc in the linker are [ka] Forming Among them, R 10 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C1-3 Alkyl group or C 1-3 substituted with an alkoxy group, o is an integer between 0 and 10, * represents the end connected to Pc, and ** represents the end connected to the linker.

[0125] In another aspect, the stretch unit Str of the linker in the conjugates provided by some embodiments comprises a cycloalkenyl group or a heterocycloalkenyl group.

[0126] In one embodiment, the stretch unit Str in the linker is [ka] wherein R a , R b are each independently hydrogen, C 1-6 selected from alkyl groups and aryl groups, and X 1 is C 1-3 Alkylene group, NR c or O, and R c is hydrogen or C 1-6 It is selected from alkyl groups.

[0127] In some other embodiments, the stretch units Str and Pc in the linker are [ka] is.

[0128] In one embodiment, the stretch units Str and Pc in the linker are [ka] is.

[0129] In one embodiment, the stretch units Str and Pc in the linker are [ka] is.

[0130] In one embodiment, the stretch units Str and Pc in the linker are [ka] Forming Among them, R 11 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, q is an integer between 0 and 10, * represents the end connected to Pc, and ** represents the end connected to the linker.

[0131] In some other embodiments, the stretch units Str and Pc in the linker are [ka] This structure is formed. Among them, R 11 are independently hydrogen, halogen, or C 1-6 Alkyl group, C 1-6 Alkoxy group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, aryl or heteroaryl groups optionally contain one or more halogens, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10and a heteroaryl group, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally contains one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, q is an integer between 0 and 10, r is an integer between 0 and 10, * represents the end connected to Pc, and ** represents the end connected to the linker.

[0132] In some embodiments, the linker in the conjugate is [ka] containing a chemical moiety represented by the formula: 12 are each independently hydrogen, C 1-3 Alkyl group or C 3-6 It is selected from cycloalkyl groups.

[0133] In some embodiments, the linker is [ka] It includes a chemical moiety represented by the formula:

[0134] The linker in the antibody conjugate provided by some embodiments comprises: [ka] a chemical moiety represented by the formula Among them, R 8 are independently hydrogen, halogen, or C 1-6 Alkyl group, C 1-6 Alkoxy group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, aryl or heteroaryl groups optionally contain one or more halogens, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, R9 is hydrogen, halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, aryl or heteroaryl groups optionally contain one or more halogens, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, R 10 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, t is an integer between 0 and 10, o is an integer between 0 and 10, ** indicates the end of the linker.

[0135] The linker in the conjugate provided by some embodiments is [ka] a chemical moiety represented by the formula Among them, R 11 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3substituted with an alkoxy group, R 12 are each independently hydrogen, C 1-3 Alkyl group or C 3-6 cycloalkyl groups, q is an integer between 0 and 10, ** indicates the end of the linker.

[0136] The linker in the conjugate provided by some embodiments is [ka] a chemical moiety represented by the formula Among them, R 11 are each independently a halogen, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, R 12 are each independently hydrogen, C 1-3 Alkyl group or C 3-6 cycloalkyl groups, q is an integer between 0 and 10, r is an integer between 0 and 10, ** indicates the end of the linker.

[0137] The linker in the conjugate provided by some embodiments is [ka] a chemical moiety represented by the formula Among them, R 11 are each independently a halogen, C 1-6 Alkyl group, C1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 3-6 Heterocycloalkyl group, C6 aryl group or C 5-10 and heteroaryl groups, wherein the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups optionally contain one or more halogen atoms, C 1-3 Alkyl group or C 1-3 substituted with an alkoxy group, R 12 are each independently hydrogen, C 1-3 Alkyl group or C 3-6 cycloalkyl groups, q is an integer between 0 and 10, r is an integer between 0 and 10, ** indicates the end of the linker.

[0138] Furthermore, the L-(D) in the antibody conjugate (ADC) provided by some embodiments x teeth, -Str-(Pep)-Sp-(D) x It is a chemical moiety represented by the formula Str is a stretching unit that covalently binds to Pc; Sp is a spacer unit, Pep is a cleavable peptide moiety.

[0139] L-(D) in the complexes provided by some embodiments x teeth, [ka] of which R 3 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 4 is hydrogen, a haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 3 and R 4are C together with the carbon atoms connected to them. 3-6 It forms a cycloalkyl group, and * indicates the end of the link to Pc.

[0140] L-(D) in the complexes provided by some embodiments x teeth, [ka] It is a chemical moiety represented by the formula 3 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 4 is hydrogen, a haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 3 and R 4 are C together with the carbon atoms connected to them. 3-6 p1 is selected from 2, 4, 6 or 8, and p2 is selected from 0, 1 or 2, forming a cycloalkyl group.

[0141] L-(D) in the complexes provided by some embodiments x teeth, [ka] wherein p1 is selected from 2, 4, 6, or 8, and p2 is selected from 0, 1, or 2.

[0142] L-(D) in the complexes provided by some embodiments x teeth, [ka] wherein p1 is selected from 2, 4, 6, or 8, and p2 is selected from 0, 1, or 2.

[0143] Additionally, some embodiments provide a conjugate comprising: [ka] [ka] It is expressed by the formula, where R 3 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 4 is hydrogen, a haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 3 and R 4 are C together with the carbon atoms connected to them. 3-6 forming a cycloalkyl group, y is selected from 1 to 20 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, p2 is selected from 0, 1, or 2, and Pc is an antibody or an antigen-binding fragment thereof, or an antibody constant region or a fragment thereof.

[0144] In another embodiment, the L-(D) in the complex x teeth, [ka] It is a chemical moiety represented by the formula:

[0145] Additionally, some embodiments provide a conjugate comprising: [ka] In this formula, y is selected from 1 to 20 and may be an integer or a decimal number, and Pc is an antibody or an antigen-binding fragment thereof.

[0146] In another embodiment, the payload D according to the present disclosure is selected from a therapeutic agent or a diagnostic agent.

[0147] In some embodiments, the therapeutic agent is a radioactive agent, a cytotoxic agent, a nucleic acid, or a polypeptide. In some embodiments, the diagnostic agent includes, but is not limited to, a fluorophore, a fluorescent dye, a radionuclide, or an enzyme.

[0148] In some embodiments, payload D is a topoisomerase I inhibitor or a microtubule inhibitor. In some embodiments, the topoisomerase I inhibitor includes, but is not limited to, exatecan or a derivative thereof. In other embodiments, the microtubule inhibitor includes, but is not limited to, eribulin or a derivative thereof.

[0149] In some embodiments, the payload D is a nucleic acid such as RNA, including siRNA, saRNA, or shRNA.

[0150] In some embodiments, the payload D is a radionuclide.

[0151] In some embodiments, the payload D is a polypeptide. In some embodiments, the polypeptide is covalently linked to the linker L at a cysteine ​​residue. In some embodiments, the polypeptide is covalently linked to the linker L at a terminal cysteine ​​residue. In some embodiments, the polypeptide does not contain a cysteine, and a cysteine ​​residue is introduced at the terminal or in a side chain and then covalently linked to the linker L, further forming an antibody-polypeptide coupling agent (APC), or the like.

[0152] In some embodiments, the polypeptide is covalently linked to the linker L at a tyrosine residue. In some embodiments, the polypeptide is covalently linked to the linker L at a terminal tyrosine residue. In some embodiments, the polypeptide does not contain tyrosine, and a tyrosine residue is introduced at the terminal or in a side chain and then covalently linked to the linker L, further forming an antibody-polypeptide coupling agent (APC), etc.

[0153] Conjugates provided by some embodiments include antibody-drug conjugates (ADCs) or antibody-polypeptide conjugates, such as conjugates of an antibody constant region or a fragment thereof (e.g., Fc) and a drug, antibody constant region or a fragment thereof (e.g., C). H 1, hinge, C H In some specific embodiments, the antibody constant region or a fragment thereof is any one of 1) to 5) above.

[0154] Method for preparing the complex The present disclosure provides a method for preparing a complex, the method comprising: a) providing a protein containing an engineered tyrosine residue; b) contacting the engineered tyrosine residue with tyrosinase to convert the phenol moiety of the engineered tyrosine residue to an o-quinone moiety; c) subjecting the o-quinone moiety to a [4+2] cycloaddition reaction with an olefin or alkyne compound containing a cycloalkenyl, heterocycloalkenyl, cycloalkynyl, or heterocycloalkynyl moiety; The protein is a protein comprising an antibody constant region or a fragment thereof, as provided in any of the above sections of the present disclosure. In some embodiments, the engineered tyrosine residue in the antibody constant region or fragment thereof is a mutation (eg, substitution, replacement) of a native amino acid residue to tyrosine.

[0155] In some embodiments, the engineered tyrosine residue is a C H 1 region, hinge region, Fc region and / or CL region. H 1 region, hinge region, C H It is located in the 2 region and / or the Cκ region and Cλ region.

[0156] In some embodiments, the engineered tyrosine residue is located at any one or combination of positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and position 202 of the light chain LC. In other embodiments, the engineered tyrosine is located at any one or combination of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC.

[0157] In some embodiments, the engineered tyrosine is selected from any one of 135Y, 137Y, 192Y, 223Y, 298Y, 329Y in the heavy chain HC and 202Y in the light chain LC, or any combination thereof, such as T135Y, G137Y, S192Y, T223Y, S298Y, P329Y, and S202Y in the light chain LC. In other embodiments, the engineered tyrosine is selected from any one of 135Y, 137Y, 192Y, 223Y, 329Y in the heavy chain HC and 202Y in the light chain LC, or any combination thereof, such as T135Y, G137Y, S192Y, T223Y, P329Y, and S202Y in the light chain LC, or any combination thereof.

[0158] In some embodiments, the engineered tyrosine is located at any one of positions 135, 137, 192, 223, 298, 329 of IGHG1 (or at positions 135, 137, 192, 223, 298, 329 of IGHG1 in IGHG2, IGHG3, IGHG4), or any combination thereof, and position 202 of Cκ. In other embodiments, the engineered tyrosine is located at positions 135, 137, 192, 223, 329 of IGHG1 (or at positions 135, 137, 192, 223, 329 of IGHG2, IGHG3, IGHG4), or any combination thereof, and position 202 of Cκ.

[0159] In some embodiments, the olefin or alkyne compound comprises a cycloalkenyl, heterocycloalkenyl, cycloalkynyl, or heterocycloalkynyl moiety, and optionally a payload D.

[0160] In some embodiments, the olefin or alkyne compound comprises a cycloalkenyl group, a heterocycloalkenyl group, a cycloalkynyl group, or a heterocycloalkynyl group moiety, and a payload, D.

[0161] In some embodiments, the o-quinone moiety in step b) undergoes a [4+2] cycloaddition reaction with an olefin or alkyne compound to form a structure represented by Za or Zb; [ka] * represents the end of connection to Pc, ** represents the end of connection to the linker, and ---- represents a single bond or a double bond.

[0162] In some embodiments, the structure represented by Za is a structure represented by formula Za1 or Za2, [ka] * represents the end connected to Pc, and ** represents the end connected to the linker.

[0163] In some embodiments, in the antibody conjugate, the structure represented by Zb is a structure represented by formula Zb1 or Zb2, [ka] * represents the end connected to Pc, and ** represents the end connected to the linker.

[0164] In some embodiments, the tyrosinase is a tyrosine oxidase.

[0165] In some embodiments, the tyrosinase is derived from Bacillus megaterium or Verrucomicrobium spinosum. In some embodiments, the tyrosinase is megaTYR from Bacillus megaterium, having the amino acid sequence set forth in SEQ ID NO:21 or at least 90% sequence identity thereto; VsTYR core from Verrucomicrobium spinosum, having the amino acid sequence set forth in SEQ ID NO:22 or at least 90% sequence identity thereto; or VsTYR sp from Verrucomicrobium spinosum, having the amino acid sequence set forth in SEQ ID NO:23 or at least 90% sequence identity thereto. The tyrosinase can be obtained by a preparation method commonly known in the art, for example, the method described in Example 6 of the present application.

[0166] The present disclosure further provides a protein comprising an antibody constant region or fragment thereof comprising an o-quinone chemical moiety. In some embodiments, the protein is any of the proteins provided above in this disclosure.

[0167] In some embodiments, the o-quinone chemical moiety is a C H 1 region, hinge region, Fc region and / or CL region. H 1 region, hinge region, C H 2 region and / or Cκ region.

[0168] In some embodiments, the o-quinone chemical moiety is located at any one of amino acid positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and amino acid position 202 of the light chain LC of the antibody constant region or fragment thereof, or any combination thereof. In other embodiments, the o-quinone chemical moiety is located at any one of amino acid positions 135, 137, 192, 223, 329 of the heavy chain HC and amino acid position 202 of the light chain LC of the antibody constant region or fragment thereof, or any combination thereof.

[0169] In some embodiments, the o-quinone chemical moiety is located in a non-glycosylated modified region of the antibody constant region or fragment thereof, e.g., is not located within a 10 amino acid range at or near position 297 (297N) of the heavy chain HC, e.g., is not located between amino acid residues 292 and 302, 293 and 303, 291 and 301, 293 and 302, or 292 and 301 of the heavy chain HC.

[0170] In some embodiments, the o-quinone chemical moiety is located at an engineered tyrosine residue selected from any one of 135Y, 137Y, 192Y, 223Y, 329Y in the heavy chain HC and 202Y in the light chain LC, or any combination thereof, e.g., any one of T135Y, G137Y, S192Y, T223Y, P329Y, and S202Y in the light chain LC, or any combination thereof.

[0171] In some embodiments, the o-quinone chemical moiety is located at an engineered tyrosine residue, the engineered tyrosine being located at any one of positions 135, 137, 192, 223, 329 of IGHG1 (or positions in IGHG2, IGHG3, IGHG4 corresponding to positions 135, 137, 192, 223, 329 of IGHG1) and position 202 of the Cκ region, or any combination thereof.

[0172] In some embodiments, the o-quinone chemical moiety is obtained by oxidation of a tyrosine residue by tyrosinase.

[0173] In some embodiments, the o-quinone chemical moiety is [ka] where * represents the end of the link to Pc.

[0174] The present disclosure further provides isotopic variations of the conjugate or its pharmaceutically acceptable salt. In some embodiments, the isotopic variations are deuterated.

[0175] In another aspect, the present disclosure further provides a pharmaceutical composition comprising an effective amount of the above-described complex, or a pharmaceutically acceptable salt thereof, or a deuterated version thereof, and a pharmaceutically acceptable excipient.

[0176] Pharmacable salts of the antibody drug conjugates described in the present disclosure may be selected from inorganic salts or organic salts.

[0177] The compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure includes cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and both racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures. All such compounds are intended to be within the scope of the present disclosure. Substituents such as alkyl groups may contain additional asymmetric carbon atoms. All such isomers and mixtures thereof are included within the scope of the present disclosure. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms may be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0178] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. Single enantiomers of certain compounds of the present disclosure can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, whereby the resulting diastereomeric mixture is isolated and the resulting diastereomeric mixture is cleaved to provide the desired enantiomer in pure form by cleavage of the corresponding groups. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxy) functional group, diastereomeric salts can be formed with an appropriate optically active acid or base, followed by diastereomeric separation and recovery using conventional methods well known in the art to obtain the enantiomers in pure form. Separation of enantiomers and diastereomers is typically accomplished using chromatography, employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., carbamates from amines).

[0179] JPEG2026503434000044.jpg40164

[0180] The compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also called protolytic tautomers) include interconversions via protolysis, such as keto-enol and imine-enamine, lactam-lactim isomerization. An example of a lactam-lactim equilibrium is between A and B shown below.

[0181] [ka]

[0182] All of the compounds in this disclosure can be depicted as Form A or Form B. All tautomeric forms are within the scope of the invention. The naming of a compound does not exclude any tautomeric forms.

[0183] The present disclosure further includes some isotopically labeled compounds of the present disclosure that are the same as those described herein, except that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Illustrative isotopes that can be attached to compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0184] Unless otherwise specified, when a position is specifically designated as deuterium (D), it should be understood that the position is deuterium (i.e., at least 10% deuterium incorporated) with an abundance at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). For example, a compound having an abundance greater than the natural abundance of deuterium may be at least 1000 times more abundant, at least 2000 times more abundant, at least 3000 times more abundant, at least 4000 times more abundant, at least 5000 times more abundant, at least 6000 times more abundant, or even greater. The present disclosure further includes various deuterated forms of the compound of formula (I). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of formula (I) by referring to relevant literature. Deuterated forms of compounds of formula (I), when prepared, may use commercially available deuterated starting materials or may be synthesized by conventional techniques with deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuterated hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0185] Polynucleotides and Vectors The present disclosure provides polynucleotides and vectors encoding the proteins of the present disclosure (e.g., antibodies or antigen-binding fragments thereof). The polynucleotides of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the polynucleotides of the present disclosure are essentially isolated polynucleotides.

[0186] The polynucleotides of the present disclosure may be in the form of a vector, present within a vector, and / or part of a vector, which may be, for example, a plasmid, cosmid, YAC, or viral vector. The vector may, in particular, be an expression vector, i.e., a vector capable of expressing the antibodies or antigen-binding fragments thereof, or antibody constant regions or fragments thereof of the present disclosure in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors typically contain at least one polynucleotide of the present disclosure operably linked to one or more appropriate expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of these elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the antibodies or antigen-binding fragments thereof of the present disclosure include, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, and reporter genes.

[0187] The polynucleotides of the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence they encode, and / or may be isolated from suitable natural sources.

[0188] host cell The present disclosure provides recombinant host cells that express or are capable of expressing one or more proteins of the present disclosure (e.g., antibodies or antigen-binding fragments thereof) and / or that contain a polynucleotide or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0189] Bacterial cells include, for example, cells of Gram-negative strains (e.g., Escherichia coli, Proteus, and Pseudomonas) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0190] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0191] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0192] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0193] composition The present disclosure provides compositions comprising the conjugates (eg, antibody-drug conjugates) of the present disclosure.

[0194] In some embodiments, the unit dose of the conjugate in the pharmaceutical composition is 0.001 mg to 1000 mg.

[0195] In one embodiment, the pharmaceutical composition contains 0.01 to 99.99% of the complex, a medicinal salt thereof, or an isotopic derivative thereof, based on the total weight of the composition. In one embodiment, the pharmaceutical composition contains 0.1 to 99.9% of the complex, a medicinal salt thereof, or an isotopic derivative thereof. In one embodiment, the pharmaceutical composition contains 0.5 to 99.5% of the complex, a medicinal salt thereof, or an isotopic derivative thereof. In one embodiment, the pharmaceutical composition contains 1 to 99% of the complex, a medicinal salt thereof, or an isotopic derivative thereof. In one embodiment, the pharmaceutical composition contains 2 to 98% of the complex, a medicinal salt thereof, or an isotopic derivative thereof.

[0196] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0197] In some embodiments, a pharmaceutical composition is provided comprising an effective amount of an anti-HER2 antibody-drug conjugate, an anti-LIV-1 antibody-drug conjugate, an anti-TROP2 antibody-drug conjugate, and at least one pharmaceutically acceptable excipient for the treatment, alleviation, or prevention of cancer.

[0198] In some embodiments, an article of manufacture or product (e.g., a reagent kit) is provided that includes an antibody-drug conjugate of the present disclosure. Optionally, the article of manufacture includes a container and a label. The container may be, for example, a vial, syringe, or test tube. The container holds a composition that is effective for treating a medical condition. A label on or associated with the container indicates that the composition is used for treating a selected medical condition.

[0199] Therapeutic Methods and Pharmaceutical Uses The present disclosure provides methods of using the conjugates of the present disclosure to treat, alleviate, prevent, or diagnose a disease or condition.

[0200] In some embodiments, a method for ameliorating, alleviating, treating, or preventing a disease is provided, the method comprising administering a therapeutically effective amount of an anti-HER2 antibody-drug conjugate or a composition (including a pharmaceutical composition) comprising the same to a subject to treat cancer, such as breast cancer. For example, the method comprises administering a therapeutically effective amount of an anti-TROP2 antibody-drug conjugate or a composition (including a pharmaceutical composition) comprising the same to a subject to treat cancer. For example, the method comprises administering a therapeutically effective amount of an anti-LIV-1 antibody-drug conjugate or a composition (including a pharmaceutical composition) comprising the same to a subject to treat cancer.

[0201] Definition of Terms In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0202] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0203] "Engineered" refers to the manipulation of a nucleic acid or polypeptide molecule by synthetic methods (e.g., by recombinant techniques, by in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or by other methods commonly used in the art).

[0204] An "engineered tyrosine residue," in the case of an amino acid sequence, refers to the presence of a non-wild-type amino acid residue. For example, when aligning wild-type and engineered sequences, an "engineered tyrosine residue" is a tyrosine residue that is not present in the corresponding wild-type polypeptide sequence and that is introduced into the polypeptide (by mutation (e.g., substitution, replacement) of an existing amino acid residue or by insertion of a tyrosine residue).

[0205] The term "antibody" encompasses various antibody structures that exhibit the desired antigen-binding activity, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or antigen-binding portions). Antibodies may also refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant region, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as kappa chains or lambda chains depending on the difference in their constant regions. Each of the five types of Ig may have either a kappa chain or a lambda chain. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is highly variable and forms the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region (C region). The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

[0206] Antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly applicable in some embodiments. Generally, antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" broadly refers to a product, e.g., a cell or a polynucleotide, protein, or vector, that has been modified by introducing a heterologous polynucleotide or protein or by modifying a naturally occurring polynucleotide or protein, or that the cell is derived from a cell that has been so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form, or express naturally occurring genes that are aberrantly expressed, under-expressed, or not expressed at all.

[0207] "Antigen-binding fragment" covers single-chain antibodies (i.e., full-length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any one of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and preparing these antigen-binding fragments are known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0208] The determination or definition of CDRs can be achieved by elucidating the structure of an antibody and / or the structure of an antibody-ligand complex, thereby accurately delineating the CDRs and identifying the residues comprising the antibody's binding site. This can be achieved by any one of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Various analytical methods can be used to identify CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the location of several structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83.) The AbM numbering system uses an integrated suite of computer programs from the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd.).The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the base sequence (see, for example, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definition is based on analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, CDR positions can be identified as residues that contribute enthalpic contributions to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). It should be noted that the boundary definitions of other CDRs may not strictly follow one of the above methods, but may still overlap with at least a portion of the Kabat CDRs, although they may be shortened or extended based on predictions or experimental results that show that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is well known to those skilled in the art.

[0209] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When every position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, when each position in two DNA molecules is occupied by the same nucleotide, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, two sequences are compared when aligned to obtain the maximum percentage homology.

[0210] "Polynucleotide" refers to any DNA or RNA molecule, whether single- or double-stranded, and, if single-stranded, to the molecule of its complementary sequence, preferably double-stranded DNA. A polynucleotide is "operatively linked" when it is placed into a functional relationship with another polynucleotide sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the coding sequence.

[0211] A "host cell" includes an individual cell or cell culture that may be, or has been, a recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and due to natural, accidental, or deliberate mutations, the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide according to the present disclosure. "Cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. It should also be understood that due to deliberate or unintentional mutations, all progeny may not be precisely identical in DNA content. The scope of "host cell" encompasses mutant progeny that have the same function or biological activity as the cell screened for in the originally transformed cell.

[0212] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. Effective amount further refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as the condition being treated, the subject's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

[0213] "Optionally" or "optionally" means that the subsequently described event or circumstance may, but need not, occur; the description includes cases where the event or circumstance occurs and cases where it does not. "And / or" should be considered to specifically indicate that two specified features or components may or may not each have the other. Thus, the term "and / or," as used in the phrase "A and / or B" in this disclosure, includes "A and B," "A or B," "A" (alone) and "B" (alone). Unless the context clearly indicates otherwise, throughout the specification and claims, words such as "comprise," "have," "contain," and the like, should be understood to have an inclusive meaning, i.e., "including, but not limited to," rather than an exclusive or exhaustive meaning. In the context of mutations included in this disclosure, " / " denotes "and," e.g., "T223Y / P329Y" denotes "T223Y and P329Y."

[0214] As used herein, a "subject" or "patient" refers to mammals, particularly primates, and especially humans.

[0215] A "pharmaceutical composition" refers to a mixture containing one or more of the conjugates and other chemical components described herein, together with other components such as physiologically / pharmaceutically acceptable carriers and excipients, to facilitate administration to a living body and contribute to the absorption of the active ingredients, thereby further exerting biological activity.

[0216] "Excipient" includes, but is not limited to, any auxiliary agent, carrier, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier approved by a regulatory agency and acceptable for use in humans or domestic animals.

[0217] "Linker," "linking unit," "linker unit," "linker," or "linking fragment" refers to a chemical structural fragment or bond that is linked at one end to a protein of the present disclosure and at the other end to an agent, and may be linked to another linker before being attached to the agent.

[0218] The linker may comprise one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, also referred to herein as MCC), and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), which are derived from coupling with linker reagents. The linker may comprise a stretch unit, a spacer unit, an amino acid unit, and an elongation unit. The linker may be synthesized by methods known in the art, for example, by the method described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates drug release in cells, including, for example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020).

[0219] A "stretch unit" refers to a chemical fragment that is covalently attached to an antibody via a carbon atom at one end and to an amino acid unit, disulfide moiety, or polyethylene glycol moiety at the other end, and may be linked to the above moieties via another linker.

[0220] A "spacer unit" is a bifunctional compound structural fragment that can be used to couple an amino acid unit with a cytotoxic drug to ultimately form an antibody-drug conjugate, and this coupling method can selectively link the cytotoxic drug to the amino acid unit.

[0221] "Amino acid" refers to an organic compound whose molecular structure contains an amino group and a carboxyl group, both of which are directly linked to a -CH- structure. The general formula is H2NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Depending on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids can be classified as α-, β-, γ-, δ-, ε-, etc. In the biological world, amino acids that constitute natural proteins have a specific structural feature: their amino groups are directly linked to the α-carbon atom. α-amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc. An example of an unnatural amino acid is citrulline. As is well known to those skilled in the art, unnatural amino acids do not constitute natural proteins and are therefore not involved in the synthesis of antibodies in the present disclosure. The three-letter and one-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem., 243, p. 3558 (1968).

[0222] An "antibody-drug conjugate" refers to a ligand attached to a biologically active drug via a linking unit. In this disclosure, an "antibody drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment attached to a biologically active toxic drug, such as exatecan or a derivative thereof, via a linking unit.

[0223] "Therapeutic agent" means a large or small molecule that can be administered to a subject in need thereof to treat a medical condition. Therapeutic agents can be administered to treat or prevent the onset of, slow the progression of, or ameliorate one or more symptoms of a medical condition in a subject with that condition.

[0224] "Diagnostic agent" refers to a compound that can be used in in vivo and / or in hydroimaging studies such as CT, MRI, and X-ray. Non-limiting examples of diagnostic agents include fluorophores, fluorescent dyes, radionuclides, and enzymes.

[0225] The "drug loading" may be expressed as the ratio of the drug amount to the antibody amount. The drug loading range is such that each antibody (Pc) may be linked to 1 to 20 cytotoxic drugs (D), preferably 1 to 10. In an embodiment of the present disclosure, the drug loading is expressed as DAR or y. A low drug loading reduces efficacy, while a high drug loading adversely affects pharmacokinetics and toxicity. Exemplary values ​​may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an average value between any two values. Preferably, the DAR is 1 to 10, and more preferably, an average value of 1 to 8, 2 to 8, 2 to 7, 3 to 8, 3 to 7, 3 to 6, 4 to 7, 4 to 6, or 4 to 5. Conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA testing, monoclonal antibody molecular size variant assay (CE-SDS) and HPLC can specifically identify the average drug quantity in each ADC molecule after the coupling reaction.

[0226] The monoclonal antibody molecular size variant assay (CE-SDS) of the present disclosure can quantitatively determine the purity of recombinant monoclonal antibody products according to the capillary electrophoresis method (Chinese Pharmacopoeia) according to molecular size under reducing and non-reducing conditions using sodium dodecyl sulfate capillary electrophoresis (CE-SDS) with ultraviolet detection.

[0227] The amount of antibody-drug conjugate loaded is (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling the reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0228] "Alkyl group" refers to a saturated aliphatic hydrocarbon group, straight or branched, containing 1 to 12 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, and 3-methylbutyl. Alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and preferably are independently selected from halogen, C 1-3 Alkyl group or C 1-3 It is one or more groups selected from alkoxy groups.

[0229] An "alkenyl group" refers to an unsaturated aliphatic straight-chain or branched-chain hydrocarbon group containing one or more carbon-carbon double bonds. Exemplary alkenyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl groups. Examples include, but are not limited to, vinyl groups (i.e., vinyl), 1-propenyl groups, 2-propenyl groups (i.e., allyl groups), 2-methyl-1-propenyl groups, 1-butenyl groups, 2-butenyl groups (i.e., crotyl groups), and the like. Alkenyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0230] "Alkynyl group" refers to an unsaturated aliphatic straight-chain or branched-chain hydrocarbon group containing one or more carbon-carbon triple bonds. Exemplary alkynyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkynyl groups. Included are, but are not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl groups. As used in any context herein, alkynyl groups are optionally substituted in the same manner as alkyl groups.

[0231] The term "cycloalkyl group" refers to a saturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl group ring contains 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like, and polycyclic cycloalkyl groups include spirocyclic, fused, and bridged cycloalkyl groups. Cycloalkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment and are preferably independently selected from halogen, C 1-3 Alkyl group or C 1-3 It is one or more groups selected from alkoxy groups.

[0232] A "cycloalkenyl group" refers to an unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing one or more carbon-carbon double bonds. Cycloalkenyl groups contain 3 to 20 carbon atoms, including C2-C8, C4-C6, C8-C12, C14-C18, and C16-C20 cycloalkenyl groups. Cycloalkenyl groups may also be fused to an aryl or heteroaryl group. Exemplary cycloalkenyl groups include: [ka] Including, but not limited to:

[0233] Cycloalkenyl groups may also be substituted or unsubstituted, and when substituted, the substituents may be at any available point of attachment and are preferably independently selected from halogen, C 1-3 Alkyl group or C 1-3 It is one or more groups selected from alkoxy groups.

[0234] "Cycloalkynyl group" refers to an unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing one or more carbon-carbon triple bonds. Cycloalkynyl groups contain 3 to 20 carbon atoms, including C2-C8, C4-C6, C8-C12, C14-C18, and C16-C20 cycloalkynyl groups. Cycloalkynyl groups may also be fused to an aryl or heteroaryl group. Exemplary cycloalkynyl groups include: [ka] Including, but not limited to:

[0235] Cycloalkynyl groups may also be substituted or unsubstituted, and when substituted, the substituents may be at any available point of attachment and are preferably independently selected from halogen, C 1-3 Alkyl group or C 1-3 It is one or more groups selected from alkoxy groups.

[0236] "Heterocycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m(wherein m is an integer of 0 to 2), but does not include the ring moiety -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and more preferably 3 to 7 ring atoms. The heterocycloalkyl group may also be fused to an aryl group or a heteroaryl group. Non-limiting examples include a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a morpholinyl group, a thiomorpholinyl group, and the like.

[0237] "Heterocycloalkenyl group" refers to an unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m (wherein m is an integer of 0 to 2), but does not include the -OO-, -OS-, or -SS- ring moiety, and the remaining ring atoms are carbons containing one or more carbon-carbon double bonds. Heterocycloalkenyl groups contain 3 to 20 carbon atoms, and preferably include C2-C8, C4-C6, C8-C12, C14-C18, and C16-C20 heterocycloalkenyl groups. Heterocycloalkenyl groups may also be fused to an aryl or heteroaryl group. Exemplary heterocyclyl groups include: [ka] These include, but are not limited to, R a , R b are each independently hydrogen, C 1-6 R is selected from alkyl groups or aryl groups; c is hydrogen or C 1-6 The heterocycloalkenyl group may be substituted or unsubstituted, and if substituted, the substituents may be at any available point of attachment, and preferably are independently selected from halogen, C 1-3 Alkyl group or C 1-3 It is one or more groups selected from alkoxy groups.

[0238] "Heterocycloalkynyl group" refers to an unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m (wherein m is an integer of 0 to 2), but does not include the -OO-, -OS-, or -SS- ring moiety, and the remaining ring atoms are carbon, including one or more carbon-carbon triple bonds. Heterocycloalkynyl groups contain 3 to 20 carbon atoms, and include C2-C8, C4-C6, C8-C12, C14-C18, and C16-C20 heterocycloalkynyl groups. Heterocycloalkynyl groups may also be fused to an aryl or heteroaryl group. Exemplary heterocycloalkynyl groups include: [ka] Including, but not limited to:

[0239] Heterocycloalkynyl groups may also be substituted or unsubstituted, and when substituted, the substituents may be at any available point of attachment and are preferably independently selected from halogen, C 1-3 Alkyl group or C 1-3 It is one or more groups selected from alkoxy groups.

[0240] "Alkoxy group" refers to -O-(alkyl group), where alkyl is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy groups. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably independently halogen, C 1-3 Alkyl group or C 1-3 It may be one or more groups selected from alkoxy groups.

[0241] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl groups. The aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, in which the ring connected to the parent structure is an aryl ring, non-limiting examples of which are: [ka] Including, The aryl group may be substituted or unsubstituted, and when substituted, the substituents are preferably independently halogen, C 1-3 Alkyl group or C 1-3 It may be one or more groups selected from alkoxy groups.

[0242] The term "heteroaryl group" refers to a heteroaromatic group containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 6 to 12-membered, more preferably 5 or 6-membered. Non-limiting examples include imidazolyl, furanyl, thienyl, pyridyl, pyrimidine, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, and the like. [ka] Includes:

[0243] A "spirocycle" refers to a compound in which two rings share one atom. Non-limiting examples of spirocycloalkyl groups are: [ka] Includes:

[0244] "Fused ring" refers to a compound formed by the fusion of two or more rings by sharing two adjacent atoms. Non-limiting examples of fused cycloalkyl groups are: [ka] Includes:

[0245] "Bridged ring" refers to a structure formed by two or more ring structures sharing two non-adjacent ring atoms. Depending on the number of rings, bridged cycloalkyl groups can be classified as bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include: [ka]

[0246] "Hydroxy" refers to an -OH group.

[0247] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0248] A "cyano group" refers to -CN.

[0249] An "amino group" refers to -NH2.

[0250] A "nitro group" refers to -NO2.

[0251] "Oxo" refers to the =O substituent.

[0252] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, being independently replaced with the corresponding number of substituents. When the substituent is ketone or oxo (i.e., =O), two (2) hydrogens on the atom are replaced.

[0253] Technical proposal 1. An antibody-drug conjugate having the structure shown in formula I or a pharmaceutically acceptable salt thereof, Pc-[L-(D) x ] y I, wherein Pc is an antibody or an antigen-binding fragment thereof; L is a linker covalently linking Pc to D, and L is linked to an amino acid in Pc, said amino acid being located at any one of positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and position 202 of the light chain LC of the Pc antibody or antigen-binding fragment thereof, or any combination thereof, or said amino acid not being located within 10 amino acids of or near an N-glycosylation site; x is 1 to 20; y is 1 to 20; -D is the payload, Preferably, the N-glycosylation site is 297N.

[0254] Technical proposal 2.L and Pc form a structure represented by Za or Zb, [ka] The antibody-drug conjugate according to Technical Solution 1, wherein * represents the connection end with Pc, ** represents the connection end with the linker, and ---- represents a single bond or a double bond.

[0255] Technical Scheme 3: The antibody-drug conjugate according to Technical Scheme 1 or 2, wherein the amino acid linked to L is tyrosine and is oxidized to o-quinone by tyrosinase.

[0256] Technical proposal 4.L-(D) x teeth, [ka] It is a chemical moiety represented by the formula 3 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 4 is hydrogen, a haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 3 and R 4 are C together with the carbon atoms connected to them. 3-6The antibody-drug conjugate according to Technical Scheme 3, wherein p1 is selected from 2, 4, 6 or 8, and p2 is selected from 0, 1 or 2, forming a cycloalkyl group.

[0257] Technical proposal 5. [ka] [ka] It is expressed by the formula, where R 3 is hydrogen, C 3-6 Cycloalkylalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen; R 4 is hydrogen, a haloalkyl group or C 3-6 cycloalkyl groups, preferably hydrogen, or R 3 and R 4 are C together with the carbon atoms connected to them. 3-6 The antibody-conjugate according to Technical Scheme 3 or 4, wherein Pc forms a cycloalkyl group, y is selected from 1 to 20 and may be an integer or a decimal point, p1 is selected from 2, 4, 6 or 8, p2 is selected from 0, 1 or 2, and Pc is an antibody or an antigen-binding fragment thereof.

[0258] Technical proposal 6. A method for preparing an antibody-drug conjugate, comprising: a) providing an antibody or antigen-binding fragment thereof having an exposed tyrosine residue located at any one of positions 135, 137, 192, 223, 298, 329 of the heavy chain HC and position 202 of the light chain LC of the Pc antibody or antigen-binding fragment thereof, or any combination thereof, or which is not located within 10 amino acids of or near an N-glycosylation site; b) contacting the phenolic moieties of exposed tyrosine residues with tyrosinase to convert them to o-quinone moieties; c) subjecting the o-quinone moiety to a [4+2] cycloaddition reaction with an alkene or alkyne compound containing a cycloalkenyl, heterocycloalkenyl, cycloalkynyl, or heterocycloalkynyl moiety.

[0259] The method according to Technical Scheme 6, wherein the tyrosinase is derived from Bacillus megaterium or Verrucomicrobium spinosum.

[0260] Technical Scheme 7: The method according to Technical Scheme 6, wherein the alkene or alkyne compound comprises a cycloalkenyl group, a heterocycloalkenyl group, a cycloalkynyl group or a heterocycloalkynyl group moiety, and optionally comprises a payload D.

[0261] Technical proposal 8. In step b), the o-quinone moiety forms a structure represented by Za or Zb by a [4+2] cycloaddition reaction with an alkene or alkyne compound; [ka] The method according to any one of Technical Scheme 7, wherein * represents the connection end with Pc, ** represents the connection end with the linker, and ---- represents a single bond or a double bond. [Brief explanation of the drawings]

[0262] [Figure 1] Figure 1 shows the results of SDS-PAGE of purified recombinantly expressed tyrosinase: Figure 1A shows the results of SDS-PAGE of megaTYR, Figure 1B shows the results of SDS-PAGE of VsTYR core, and Figure 1C shows the results of SDS-PAGE of VsTYR sp. [Figure 2] Schematic of a one-pot coupling reaction mediated by tyrosinase catalysis. [Figure 3] Figure 3A shows the results of deconvolution mass spectrometry of a complex between a tyrosinase-mediated single-site mutant antibody and a small molecule. Figure 3A shows the results of deconvolution mass spectrometry of a complex between VsTYR core-mediated 0102 H30 and L-2, and Figure 3B shows the results of deconvolution mass spectrometry of a complex between VsTYR core-mediated 0102 L15 and L-2. [Figure 4]4A shows the results of deconvolution mass spectrometry of a complex between a tyrosinase-mediated multi-site mutant antibody and a small molecule, where FIG. 4A shows the results of deconvolution mass spectrometry of a complex between VsTYR core-mediated Her HH3 and L-2, FIG. 4B shows the results of deconvolution mass spectrometry of a complex between VsTYR core-mediated Her HH8 and L-2, and FIG. 4C shows the results of deconvolution mass spectrometry of a complex between VsTYR core-mediated Her HH9 and L-2. [Figure 5] This shows the results of purification of antibody-polypeptide complexes using HiLoad16 / 600 Superdex 200 pg. [Figure 6] 1 shows the results of ELISA for the binding of CBP-ADA-mut, ADA mut, and adalimumab to human TNFα. [Figure 7] Binding results of CBP-ADA-mut and ADA mut to chicken type II collagen. [Figure 8] Cell killing results of ADC in situ. [Figure 9] FIG. 1 shows an alignment of IGHG1, IGHG2, IGHG3, and IGHG4 sequences. DETAILED DESCRIPTION OF THE INVENTION

[0263] The present disclosure will be further explained in the following examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual, or conditions suggested by raw material or product manufacturers. Reagents for which specific sources are not specified are standard commercially available reagents.

[0264] The antibodies in the examples of this disclosure use the Kabat numbering system to define their CDRs unless otherwise specified.

[0265] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are 10 -6 The values ​​are shown in units of ppm. NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD) as the measurement solvents, and tetramethylsilane (TMS) as the internal standard.

[0266] For MS measurements, liquid chromatograph mass spectrometers Agilent 1200 / 1290DAD-6110 / 6120 Quadrupole MS (manufacturer: Agilent, MS model number: 6110 / 6120 Quadrupole MS), Waters ACQuity UPLC-QD / SQD (manufacturer: Waters, MS model number: Waters ACQuity Qda Detector / Waters SQ Detector), and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model number: THERMO Q Exactive) were used.

[0267] For high performance liquid chromatography (HPLC) analysis, high performance liquid chromatographs Agilent HPLC1200DAD, Agilent HPLC1200VWD or Waters HPLC e2695-2489 were used.

[0268] For chiral HPLC analysis, a high performance liquid chromatograph, Agilent 1260 DAD, was used.

[0269] For high-performance liquid preparative chromatography, preparative chromatographs Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 were used.

[0270] For chiral separation, a preparative chromatograph Shimadzu LC-20AP was used.

[0271] Combiflash Rf200 (TELEDYNE ISCO) was used as the CombiFlash high-speed preparative chromatograph.

[0272] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used as silica gel plates for thin layer chromatography. The specifications of the silica gel plates used for thin layer chromatography (TLC) are 0.15-0.2 mm, and the specifications for separating and purifying products by thin layer chromatography are 0.4-0.5 mm.

[0273] For silica gel column chromatography, 200-300 mesh silica gel manufactured by Yantai Huanghai Silica Gel was generally used as the carrier.

[0274] Known starting materials according to the present disclosure may be synthesized by adopting or following methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0275] In the examples, unless otherwise specified, all reactions can be carried out under an argon gas atmosphere or a nitrogen gas atmosphere.

[0276] The argon or nitrogen gas atmosphere refers to an argon or nitrogen gas balloon with a volume of about 1 L connected to the reaction flask.

[0277] The hydrogen gas atmosphere refers to a hydrogen gas balloon with a volume of approximately 1 L connected to the reaction flask.

[0278] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen gas generator or an HC2-SS hydrogenation apparatus were used.

[0279] The hydrogenation reaction was usually carried out by repeating the procedure of evacuating and filling with hydrogen gas three times.

[0280] A CEM Discover-S 908860 microwave reactor was used for the microwave reactions.

[0281] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0282] In the examples, unless otherwise specified, the reaction temperature is room temperature, 20°C to 30°C.

[0283] In the examples, thin layer chromatography (TLC) was used to monitor the progress of the reaction. The volume ratio of the developing solvent used in the reaction, the eluent system for column chromatography used to purify the compound, and the developing solvent system for thin layer chromatography was adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of a basic or acidic reagent such as triethylamine and acetic acid.

[0284] Example 1. Design of solvent-exposed sites on an IgG antibody mutated to tyrosine Using Trastuzumab Fab (PDB: 6MH2) and Trastuzumab Fc (PDB: 3D6G) as templates, bioinformatics methods were used to calculate the solvent accessible surface area (SASA (Å2)) of each amino acid in the antibody constant region and the solvent accessible surface area of ​​the mutant tyrosine after tyrosine substitution. Based on the secondary structure information of the mutation site, the possibility of site-specific modification of potential mutagenic sites was evaluated. The Trastuzumab heavy chain HC sequence is detailed in SEQ ID NO: 1, and the light chain LC sequence is detailed in SEQ ID NO: 2.

[0285] Jorick J. Bruins et al. used mushroom tyrosinase to site-specifically modify Y296 and Y300 near N297 (Bruins JJ, et al. Bioconjug Chem. 2021 Oct 20;32(10):2167-2172). SASA calculations revealed that Tyr296 was the most exposed of all Tyrs, with a value of 248, while Tyr300 had a value of only 41. Therefore, a SASA screening value of 40 was used. Based on the spatial conformations of Tyr296 and Tyr300, sites on or near the loop region (loop) with relatively high structural flexibility were screened as candidate sites.

[0286] The site selection for the IGHG1 antibody is shown in Table 1. For other types of IGHGs, similar calculation methods and screening were used, using PDB:4L4J, PDB:2QSC, PDB:5W5N, and PDB:3EO1 as templates. Selectable sites for the light chain are shown in Table 2. The hinge region connecting the Fab and Fc is also a potential site for site-specific modification, as it is relatively flexible. Position numbers in this disclosure are all defined according to the EU numbering system.

[0287] [Table 1-1] [Table 1-2]

[0288] [Table 2]

[0289] Example 2. Expression and identification of wild-type (wt) and single-site mutant antibodies Two molecular sequences of Trastuzumab and O102 were subjected to specific mutations at the mutation sites described in Example 1. Mutations were performed using Stratagene's QuikChange XL according to the manufacturer's protocol. Corresponding plasmid DNA was constructed in the pTT5 vector, and the desired mutations were confirmed by DNA sequencing. Antibody expression and purification were performed using ExpiCHO-S cells.

[0290] Specifically, ExpiCHO-S cells (Thermo, A29133) in logarithmic growth phase and in good condition were cultured at 6 × 10 6 The plasmid was diluted to 1000 cells / mL and inoculated into 50 mL of medium. 40 μL of plasmid was diluted with 2 mL of medium at a 3:2 ratio of LC:HC and mixed to homogeneity to obtain Solution 1. 160 μL of transfection reagent was diluted with 1.84 mL of medium and mixed to homogeneity to obtain Solution 2. Solution 2 was added to Solution 1 and gently mixed at room temperature for 1-5 minutes to obtain the mixed transfection solution. This was then added dropwise to the cell culture medium while shaking to ensure homogeneity. The shake flask was placed in an 8% CO2 atmosphere and cultured on a shaker at 37°C. After 18-22 hours, 8 mL of Feed (Thermo, A29133) and 0.3 mL of Enhancer (Thermo, A29133) were added, and the culture conditions were adjusted to 5% CO2 and 32°C. On days 12–14, the supernatant of the fermentation broth was collected by centrifugation and purified by one-step affinity chromatography (MabSelectSuRe column, GE, 175438) to purify the resulting antibody, and the liquid was exchanged into phosphate buffer at pH 5.5.

[0291] The expressed antibodies were identified using LC-MS. Specifically, the purified antibodies were diluted to 1 mg / mL and a final concentration of 0.1% formic acid was added to enhance sample protonation. The samples were analyzed using a ThermoFisher QE Plus mass spectrometer. The column was equilibrated with 90% mobile phase A (0.1% formic acid / water solution) and 10% mobile phase B (0.1% formic acid / acetonitrile) before sample injection, followed by gradient elution. After sampling, the mass spectrometry data of the target peaks was calculated using BioPharma Finder software.

[0292] The relevant information for the partially expressed antibodies is shown in Table 3, and the expressed molecular weights all agreed with the theoretical values.

[0293] [Table 3-1] [Table 3-2]

[0294] Example 3. Synthesis of Compound L-1 Step 1: Synthesis of compound 1b [ka] Compound 1a (prepared with reference to the method in J. Am. Chem. Soc. 2020, 142, 9285-9301) (100 mg, 0.317 mmol) was dissolved in DMF (5 mL), and EtN (96 mg, 0.951 mmol) and 3-[2-(2-aminoethoxy)ethoxy]-propionic acid (56 mg, 0.317 mmol) were added with stirring at room temperature. The reaction was allowed to proceed to essentially completion at room temperature. The mixture was concentrated under reduced pressure to dryness, and the crude product was purified by silica gel column chromatography (DCM / MeOH / HOAc = 15:1:0.01) to give compound 1b (63 mg) in 56% yield. Ms(ESI): m / z 376.1 [M+Na] + .

[0295] Step 2: Compound 1e [ka] Compound 1c (prepared according to the method of WO2022 / 161385, 73.6 mg, 0.119 mmol) and Fmoc-Gly-Gly-Phe-OH (65.6 mg, 0.131 mmol) were placed in a 50 mL reaction flask, THF (2 mL) and MeOH (3 mL) were added, and the mixture was cooled in an ice-water bath. DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 49.3 mg, 0.178 mmol) was added, the ice-water bath was removed, and the reaction was essentially complete at room temperature. The mixture was concentrated under reduced pressure to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 15:1) to obtain compound 1d (110 mg). Ms(ESI): m / z 1103.4 [M+H] + .

[0296] Compound 1d (150 mg, 0.136 mmol) was dissolved in anhydrous THF (3 mL), protected with Ar gas, cooled in an ice-salt bath, and the internal temperature was controlled at -10 °C. DBU (62 mg, 0.41 mmol) was added, and the mixture was stirred for 2 h while controlling the internal temperature at -10 °C. tert-Butyl dimethyl ether (MTBE, 10 mL) was added, filtered, and washed with MTBE (10 mL). After drying, crude product compound 1e (156 mg) was obtained. This was directly used in the next reaction. Ms(ESI): m / z 881.3 [M+H] + .

[0297] Step 3: Synthesis of compound L-1 Compound 1b (60 mg, 0.17 mmol) and compound 1e (HPLC purity 60%, 156 mg) were dissolved in a mixed solvent of DCM / MeOH (6 mL / 2 mL) and the temperature was controlled at -30 °C. DMTMM (75 mg, 0.254 mmol, 1.5 eq) was then added, and the reaction was stirred for 2 h while controlling the internal temperature at -17 °C. Water (15 mL) was added to quench the reaction, followed by extraction with DCM (8 mL × 2). The combined organic phases were washed once with saturated NaCl solution (20 mL), dried over NaSO, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The crude product was purified by preparative liquid chromatography (acetonitrile / water, 20 mM NHHCO) to obtain compound L-1 (41.9 mg, HPLC: 93.0%). Ms(ESI): m / z 1216.4 [M+H] + . 1 H NMR (400 MHz, CDCl3): δ 8.40-8.10 (m, 1H), 8.00-7.85 (m, 1H), 7.85-7.30 (m, 6H), 7.25-7.05 (m, 5H), 5.70-5.50 (m, 2H), 5.45-5.10 (m, 2H), 5.05-4.80 (m, 2H), 4.75-4.60 (m, 1H), 4.55-4.25 (m, 2H), 4.20-4.05 (m, 2H), 3.95-3.40 (m, 15H), 3.35-2.85 (m, 6H), 2.55-2.10 (m, 12H), 1.65-1.20 (m, 5H), 1.05-0.85 (m, 6H), 0.70-0.55 (m, 3H), 0.55-0.30 (m, 1H).

[0298] Example 4. Synthesis of Compound L-2 [ka] Compound 2b (prepared according to the method in Angew. Chem. Int. Ed. Eng. 2010, 49, 9422-9425) (83.7 mg, 0.258 mmol) and EtN (104 mg, 1.03 mmol) were dissolved in CHCl (5 mL), protected with Ar, and cooled in an ice-water bath. A solution of compound 2a (74.7 mg, 0.129 mmol) in CHCl (5 mL) was added and stirred overnight at room temperature. The mixture was concentrated under reduced pressure to dryness, and the crude product was purified by preparative liquid chromatography (acetonitrile / water, 20 mM NHHCO) to give compound L-2 (63 mg). Ms(ESI): m / z 865.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.86 (d, J = 1.6 Hz, 1H), 7.32 (dd, J = 8.0 2.0 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.31 (d, J = 9.6 Hz, 2H), 6.25-6.15 (m, 2H), 6.13 (d, J = 2.4 Hz, 1H), 3.30 (d, J = 8.4 Hz, 2H), 2.87 (q, J = 7.2 Hz, 8H), 2.83-2.69 (m, 8H), 2.48-2.39 (m, 5H), 1.48-1.28 (m, 6H), 0.85-0.68 (m, 2H), 0.49 (t, J = 7.2 Hz, 12H), 0.16-0.05 (m, 2H).

[0299] Example 5. Synthesis of Compound L-3 [ka] Step 1: Synthesis of compound 3b In a 100 mL single-neck flask, substrate 1e (240 mg, 0.272 mmol, 1.0 eq, prepared with reference to the method of EP3858386A1 Example 18) and compound 3a (141 mg, 0.356 mmol, 1.3 eq) were dissolved in DCM (18 mL) and MeOH (6 mL), purged with nitrogen gas three times, cooled to -30 °C, DMTMM (113 mg, 0.408 mmol, 1.5 eq) was added, and then the temperature was raised to -17 °C and stirred until the reaction was complete as monitored by LCMS. The reaction was purified by column chromatography (MeOH / DCM) to give a white solid (380 mg, used directly in the next step). MS (ESI): m / z 1262.6 [M+H] + .

[0300] Step 2: Synthesis of compound 3c In a 10 mL single-neck flask, substrate 3b (60 mg, 0.048 mmol, 1.0 eq) was dissolved in THF (1 mL) and cooled to -10 °C, followed by the addition of DBU (30 mg, 0.197 mmol, 4.1 eq). After the addition was complete and the mixture was maintained at -10 °C and stirred until the reaction was complete as monitored by LCMS, MTBE (5 mL) was added to the reaction, which was then warmed and stirred for 5 min, then directly filtered. The filter cake was washed twice with MTBE, dissolved in DCM (5 mL), and spun down to give the product (60 mg, crude, used directly in the next step) as a yellow solid. Ms (ESI): m / z 1040.8 [M+H] + .

[0301] Step 3: Synthesis of compound L-3 In a 10 mL single-neck flask, substrate 2c (60 mg crude, 0.048 mmol, 1.0 eq) and 1b (15 mg, 0.051 mmol, 1.1 eq, purchased from Kangfu Nuo Co., Ltd., lot number BCT-3-210428) were dissolved in DCM (1 mL), and DIEA (41 mg, 0.317 mmol, 6.6 eq) was added, followed by stirring at 20 °C overnight. The reaction was monitored for completion by LCMS, and the reaction solution was directly spun down and purified by reverse-phase preparative separation (acetonitrile / water, 20 mM NH4HCO3), twice, and lyophilized to give the product (5 mg, 78.5% purity) as a yellow solid. Ms(ESI): m / z 1192.6 [M+H] + . 1 H-NMR (400 MHz, CD3OD) δ 7.81 (s, 0.6H), 7.62-7.58 (m, 1H), 7.24-7.13 (m, 5H), 6.73-6.65 (m, 0.4H), 5.70-5.64 (m, 1H), 5.60-5.53 (m, 1H), 5.48-5.44 (m, 1H), 5.40-5.32 (m, 1H), 5.17-5.09 (m, 1H), 4.93-4.90 (m, 4H), 4.67 (d, J = 10.0 Hz, 1H), 4.59 (s, 1H), 4.42-4.35 (m, 1H), 4.28-4.23 (m, 1H), 3.83-3.77 (m, 4H), 3.72-3.67 (m, 3H), 3.64-3.60 (m, 1H), 3.54-3.44 (m, 6H), 3.26-3.13 (m, 4H), 3.07-2.96 (m, 1H), 2.90-2.78 (m, 1H), 2.50-2.46 (m, 2H), 2.39 (s, 3H), 2.36-2.17 (m, 6H), 2.02-1.86 (m, 4H), 1.73-1.64 (m, 2H), 1.58-1.50 (m, 1H), 1.36-1.25 (m, 3H), 1.07-0.88 (m, 3H), 0.71-0.48 (m, 4H).

[0302] Example 6. Expression and purification of tyrosinase The gene sequences of three types of tyrosinase, megaTYR from Bacillus megaterium, VsTYR core from Verrucomicrobium spinosum, and VsTYR sp fused to the C-terminus or N-terminus with a His6 purification label, were each synthesized into the pet28a+ vector, and the vector was electroporated into E. coli BL21(DE3) to construct recombinant bacteria.

[0303] E. coli BL21(DE3) recombinant strains containing the constructed fusion protein gene were grown in 2*LB medium or other media commonly used for E. coli expression at 37°C to an OD of 0.8-1.0. 0.2 mM IPTG was added and the culture was incubated at 20°C and 220 RPM for 20 hours. After centrifugation at 10,000 g for 10 minutes, the cells were harvested and resuspended in wash buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris-HCl, pH 7.5). The cells were sonicated and centrifuged at 10,000 g for 30 minutes at 4°C. The supernatant was collected and purified using a Ni column. The elution buffer was 500 mM NaCl, 500 mM imidazole, 20 mM Tris-HCl, 0.02 mM CuSO, pH 7.4. The purified sample was exchanged with storage buffer (PBS, 15% glycerol, 0.02 mM CuSO4), and purity was detected by SDS-PAGE.

[0304] Table 4 provides specific information about the three tyrosinases. All three tyrosinases were correctly expressed in E. coli. Figures 1A to 1C are SDS-PAGE images.

[0305] [Table 4]

[0306] Example 7. Tyrosinase-mediated single-site mutagenesis and coupling of wild-type antibodies to small molecules The antibody expressed in Example 2 and the compounds synthesized in Examples 3 to 5 were subjected to a one-pot coupling reaction catalyzed by the tyrosinase expressed in Example 6. The specific reaction is shown in Figure 2. After an additional tyrosine was introduced into the antibody by mutation, tyrosinase was able to recognize and oxidize it to a quinone. The small molecule in the reaction system rapidly underwent a 4+2 Diels Alder reaction with the quinone via the linker, thereby forming an antibody conjugate.

[0307] The specific reaction conditions were as follows: the antibody concentration was controlled at approximately 2.5 mg / mL, the compound was added at a molar ratio of greater than 5:1, the small molecule was dissolved in DMSO, the final DMSO concentration was controlled to 10% or less, tyrosinase was added to a final concentration of 1 mg / mL after pre-cooling on ice, the reaction pH was controlled to around 5.5, and the reaction was carried out at 4°C for 16 hours, and the final reaction solution was stored at -40°C for detection.

[0308] To determine the reaction status of the corresponding HC / LC mutation sites, the coupling samples were analyzed by conventional RP-HPLC. The coupling samples were diluted to 1 mg / mL with PBS, pH 8.0, and reduced at 70°C for 10 minutes with 0.25 mol / L dithiothreitol (DTT). The samples were then analyzed by HPLC with a final concentration of 20% acetonitrile and 0.1% formic acid. The column was an Agilent PLRP-S 1000A, 8 μm column. The mobile phases were A: 0.1% TFA / HO, B: 0.1% TFA / ACN. The elution gradient is shown in Table 5. UV absorption was detected at 280 nm.

[0309] [Table 5-1] [Table 5-2] The coupled reaction mixture was diluted to 1 mg / mL and a final concentration of 0.1% formic acid was added to enhance sample protonation. The samples were analyzed using a ThermoFisher QE plus mass spectrometer. The column was equilibrated with 90% mobile phase A (0.1% formic acid / water solution) and 10% mobile phase B (0.1% formic acid / acetonitrile) prior to sample injection, followed by gradient elution. After sampling, the mass spectrometry data of the target peaks were calculated using BioPharma Finder software.

[0310] The DAR values ​​were determined based on the uncoupled and coupled mass spectrometry signal intensities, and the total DAR was calculated using the formula: Total DAR = (0 * Height D0 + 1 * Height D1 + ... + n * Height Dn) / (Height D0 + Height D1 + ... + Height Dn). The theoretical DAR value of a single-site mutant antibody was 2.

[0311] Table 6 shows the LC-MS and RP-HPLC results after coupling. After modification of wild-type Trastuzumab (Her H1) with VsTYR, the DAR was 0.20, while the DAR of the wild-type O102 molecule was 0.01. VsTYR demonstrated very low background modification in both Trastuzumab and O102 wild-type molecules. The introduction of single-site mutations, such as T135Y, G137Y, S192Y, T223Y, S298Y (the simultaneous introduction of N297G eliminates the influence of N-sugars on the enzymatic reaction, and the introduction of Y296F and Y300F eliminates the possibility of natural tyrosine at this position for enzyme catalysis), and P329Y, into the heavy chains of these two antibody molecules, or the introduction of a single-site mutation, S202Y, into the light chain, both achieved complete modification of the heavy and light chains of the corresponding antibodies. Furthermore, the heavy chain Q342Y mutation can improve the VsTYR modification ratio for Trastuzumab mutants to a DAR of 1.16, whereas the introduction of heavy chain T197Y cannot improve the modification ratio. Figures 3A and 3B show exemplary mass spectrometry results of tyrosinase-mediated complex deconvolution of some tyrosinase-mediated single-site mutant antibodies with small molecules. Furthermore, mushroom-derived tetrameric tyrosinase (Sigma Aldrich T3824) cannot modify either wild-type 0102 molecules or molecules with G137Y-introduced 0102 heavy chains.

[0312] [Table 6-1] [Table 6-2]

[0313] Example 8. Expression and identification of multi-tyrosine combinatorial mutant antibodies The additionally introduced highly reactive tyrosine demonstrated in Example 7 was introduced into Trastuzumab by a multi-site combination. At the same time, a highly reactive tyrosine was additionally introduced into Trastuzumab, and a GGGGY sequence was included at the C-terminus of the LC. The antibody was expressed according to the antibody expression and purification method of Example 2, and LC-MS identification was performed according to the method of Example 2. The relevant information for the partially expressed antibodies is shown in Table 7, and the expressed molecular weights were all consistent with the theoretical values.

[0314] [Table 7-1] [Table 7-2]

[0315] Example 9. Coupling of tyrosinase-mediated multi-site mutagenesis antibodies with compounds In this example, a one-pot coupling reaction was performed using the antibody expressed in Example 8 and the compounds synthesized in Examples 3 to 5 under the catalysis of the VsTYR core enzyme. The specific coupling conditions and LC-MS detection were the same as in Example 7, and the DAR values ​​were calculated simultaneously. Table 8 shows the LC-MS results after coupling. The measured DAR values ​​are consistent with theory, demonstrating that multiple highly reactive tyrosine mutations (Example 7) can be simultaneously introduced into the antibody constant region, achieving complete modification at multiple sites through the VsTYR tyrosinase reaction. Figures 4A to 4C show exemplary mass spectrometry results for the deconvolution of complexes between some tyrosinase-mediated multi-site mutant antibodies and small molecules.

[0316] [Table 8]

[0317] Example 10. Single-site mutant and wild-type antibody melting temperature (TM) analysis Differential scanning calorimetry (DSC) experiments were performed using a MicroCal PEAQ-DSC Automated microcalorimeter to measure the melting points of some of the single-site mutated antibodies described in Example 2 and to detect the stability of the antibodies after mutation. 350 μL of sample (controlled at 1.0 mg / mL) was placed in a 96-well plate, and the sample flask was placed in the sample cartridge. The sample position number was recorded. Each sample had two groups: blank and control. The blank was generally PBS. The run sequence included inserting one needle of wash solution every five samples, with the first three wells as blank and each subsequent well as sample. The blank was used as a baseline to verify the fitting model, and the corresponding melting point was calculated using the software. Table 9 shows the specific results, which indicate that the detected single-site mutation sites did not significantly affect the thermal stability of the protein.

[0318] [Table 9]

[0319] Example 11. Binding analysis of single-site mutant and wild-type antibodies to human FcRn FcRn affinity studies were performed on some of the Trastuzumab mutant antibodies and wild-type Trastuzumab (Her H1) in Example 2. Detection was performed using a Biacore 8K (GE Healthcare) instrument. The mobile phase used was PBS-P+ buffer solution (0.2 M PBS, 27 mM KCl, 1.37 M NaCl, 0.05% surfactant P20). Human FcRn (Acro Biosystems, FCN-H52W7) protein was prepared as a ligand using PBS-P+ buffer solution and captured using an anti-His antibody protein on a chip channel. Each antibody to be measured was prepared as an analyte using PBS-P+ buffer solution. The analyte was diluted two-fold starting from 500 nM for a total of seven concentration points. The flow rate through the experimental and reference channels was 30 μL / min, with an association time of 60 s and a dissociation time of 90 s. The regeneration buffer, 10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA), was run at a flow rate of 10 μL / min for 30 s. Data processing was performed using Biacore 8K Evaluation analysis software. The signal values ​​of the corresponding reference channel (Fc1) were subtracted from the signal values ​​of the detection channel (Fc2) to obtain a corrected signal curve. The affinity kinetic curve was fitted according to a 1:1 Langmuir binding model to calculate the association rate Ka, the dissociation rate Kd, and the dissociation constant (i.e., affinity KD value).

[0320] Table 10 shows that there is no apparent difference in FcRn affinity between the single-site mutations tested and the wild-type antibody.

[0321] [Table 10]

[0322] Example 12. Binding analysis of single-site mutant and wild-type antibodies to human CD16a In Example 2, CD16a affinity was examined for some Trastuzumab mutant antibodies and wild-type Trastuzumab (Her H1). In this experiment, detection was performed using a Biacore 8K (GE Healthcare) instrument. An anti-His sensor chip was selected, and for detection of human CD16a-F176 / human CD16a-V176 (Acro Biosystems, CDA-H5220, CD8-H52H4) receptors, the mobile phase was HBS-EP + A buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used. + Human CD16a-F176 and human CD16a-V176 receptors were prepared as ligands using buffer solutions, and then captured using anti-His antibody proteins on the chip channel. Each antibody to be measured was used as an analyte, and HBS-EP + The analyte was prepared using a buffer solution, and a two-fold gradient dilution was performed starting from 2000 nM, for a total of seven concentration points. The experimental and reference channels were run at a flow rate of 30 μL / min, with a 60 s binding time and a 60 s dissociation time. The regeneration buffer, 10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA), was run at a flow rate of 10 μL / min for 30 s. Data processing was performed using Biacore 8K Evaluation analysis software. The corresponding reference channel (Fc1) signal value was subtracted from the detection channel (Fc2) signal value to obtain a corrected signal curve. The affinity kinetic curve was fitted according to a 1:1 Langmuir binding model to calculate the binding rate Ka, the dissociation rate Kd, and the dissociation constant (i.e., affinity KD value).

[0323] Table 11 shows that two antibodies, Her H26 and Her H30, do not bind to CD16a, consistent with literature predictions. Among these, Her H26 removes the glycan that binds to CD16a, and the mutation P329 in Her H30 is a key amino acid for CD16a binding (Bogen JP, et al. Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2+1 Common Light Chain Antibody Architecture. Front Immunol. 2021 May 10;12:669496.). There was no significant difference in CD16a affinity between the remaining single-site mutations tested and the wild-type antibody.

[0324] [Table 11]

[0325] Example 13. Design, expression, and identification of other forms of IGHG single-site mutant antibodies Table 12 shows selectable sites in other forms of IGHG (IGHG2, IGHG3, IGHG4) calculated using a calculation method similar to that in Example 1. The hinge region connecting Fab and Fc is relatively flexible and is also a potential site for site-specific modification.

[0326] [Table 12-1] [Table 12-2] Note: SASA_ori(Å 2 ) is the original SASA (Å 2 ), SASA_mut(Å 2 ) is the SASA after mutation (Å 2[1]: IGHG2 uses template reference numbers PDB:4L4J, PDB:2QSC, IGHG4 uses template reference numbers PDB:5W5N, PDB:3EO1, and the Fc region of IGHG3 is defined according to the EU numbering system of IGHG1.

[0327] The designed mutation sites of the different forms of IGHG were created by single-site-directed mutagenesis on the sequence of the O102 molecule, and simultaneously expressed and identified using the non-mutated wild type (wt) as a control according to the method described in Example 2.

[0328] The relevant information for the partially expressed antibodies is shown in Table 13, and the expressed molecular weights all agreed with the theoretical values.

[0329] [Table 13]

[0330] Example 14. Tyrosinase-mediated alternative forms of IGHG single-site mutations and coupling of wild-type antibodies to small molecules In this example, the antibody expressed in Example 13 and the compounds synthesized in Examples 3 to 5 were subjected to a one-pot coupling reaction catalyzed by the VsTYR core enzyme. The specific coupling conditions and LC-MS detection were the same as in Example 7, and DAR values ​​were calculated simultaneously. Table 14 shows the DAR values ​​calculated by LC-MS after coupling. The 0102 H73 molecule with wild-type IGHG2 had a DAR of 0.08 after modification with VsTYR core; the 0102 H74 molecule with wild-type IGHG3 had a DAR of 0.00 after modification with VsTYR core; and the 0102 H40 molecule with wild-type IGHG4 had a DAR of 0.36 after modification with VsTYR core, indicating that VsTYR has a certain background modification relative to wild-type IGHG4. Introduction of single-site mutations such as T133Y and N197Y into IGHG2 and T138Y and P329Y into IGHG4 can increase the VsTYR modification ratio relative to the corresponding mutant molecules. Introduction of the P329Y single-site mutation into IGHG3 can achieve complete modification of the heavy and light chains of the corresponding antibody.

[0331] [Table 14]

[0332] Example 15. Expression and identification of other single (multiple) site mutant antibodies Other single- and multi-site mutant antibodies were expressed and identified according to the method described in Example 2. Among them, c-met H10 and c-met L15 are monoclonal antibodies targeting c-met, ADA mut is a site-specific mutation of HC P329Y and LC S202Y in the adalimumab sequence, and 1921 H10 L15 is a monoclonal antibody targeting TROP2. Table 15 shows that the molecular weights of the expressed antibodies all matched the theoretical values.

[0333] [Table 15-1] [Table 15-2]

[0334] Example 16. Synthesis of polypeptide P-1 P-1 peptide sequence: EQKLISEEDLGGSC(TCO-Mal) (SEQ ID NO: 80) [ka] Step 1: Resin peptide synthesis 1.1 Using 2-Cl resin with a substitution degree of 0.3 mmol / g, the Fmoc process was employed. Following the peptide sequence above, amino acids were sequentially condensed from the C-terminus to the N-terminus (right to left) until the condensation of the peptide chain was complete. 1 g of 2-Cl resin was placed in a clean reactor, 10 mL of DCM was added, and the mixture was immersed for 20 minutes to allow swelling. The reaction was then stopped, and the liquid was removed by suction filtration.

[0335] 1.2 Accurately weigh 0.3 mmol of Fmoc-Cys(Trt)-OH and 0.6 mmol of HoBt, dissolve them in 6 mL of DMF, add 0.6 mmol of DIC dropwise, activate for 10 minutes, add to a reaction column, react for 2 hours, stop the reaction, wash with an appropriate amount of DMF three times, add 2 mL of DIEA, 2 mL of methanol, and 6 mL of DCM, react for 35 minutes, cap the column, remove the reaction solution by suction, and wash the resin with DMF three times.

[0336] 1.3 FMOC Removal: 10 mL of 20% hexahydropyridine / DMF solution was added, and the mixture was stirred for 10 minutes to react. The liquid was then removed by suction. 10 mL of 20% hexahydropyridine / DMF solution was then added, and the mixture was stirred for 5 minutes to react. The liquid was then removed by suction. The mixture was washed five times with DMF, and a sample was taken for ninhydrin colorimetric detection, and the detected color (blue) was recorded.

[0337] 1.4 Condensation Reaction 0.9 mmol of Fmoc-Ser(tbu)-OH and 0.9 mmol of HoBt were weighed out in an amount three times the molar substitution of the resin, dissolved in 6 mL of DMF, and 0.9 mmol of DIC was added dropwise. After 10 minutes of activation, the column was added to the reaction column and reacted for 1.5 hours (no color development was detected by the ninhydrin colorimetric method), after which the column was removed by suction, and washed five times with DMF.

[0338] 1.5 Cycle Reaction Repeat steps 1.4 and 1.5 until the final glutamic acid condensation is complete. Then, wash with DMF three times. After sampling, the reaction mixture was colorless and detected by ninhydrin colorimetry, indicating completion of the reaction. After condensation is complete, remove Fmoc by following step 1.3. Then, wash with MeOH three times and suction filter the mixture until dry.

[0339] Step 2: Cleavage of the polypeptide Preparation of lysis reagent: Using 15 mL of lysis solution per 1 g of resin peptide, the amount of lysis reagent used was calculated: The necessary dissolving reagents, H2O and TFA, were added in order to the dissolving reaction flask in a ratio of TFA:TIS:EDT:H2O = 95:2:2:1. The temperature of the dissolving reagent was controlled at 0-10°C, and the dissolving reagent was added to the peptide resin with stirring. After the temperature of the system stabilized, the temperature was further controlled at 20-25°C, and the reaction was carried out with stirring for 2 hours.

[0340] The solution was filtered, precipitated with ice ether in an amount 10 times the volume of the filtrate, and washed four times by centrifugation. The precipitate was dried under reduced pressure at room temperature to obtain 0.7 g of a crude product.

[0341] Step 3: Purification of naked peptide A liquid chromatography system was prepared using 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile as mobile phase B. A 10 μm reversed-phase C18 column (50 × 250 mm) was used, with the UV detector set at 220 nm and the flow rate adjusted to 40 mL / min. The column was equilibrated with 10% acetonitrile for 10 minutes. The crude peptide was crushed and treated with a microwave in pure water until completely dissolved. The crude peptide was then filtered through a 0.45 μm filter membrane and the sample was injected.

[0342] Elution was performed at room temperature with an acetonitrile gradient from 10% for 0.01 min to 45% for 40 min. Changes in absorbance were noted, impurity peaks were removed, and the target product was collected. Analysis revealed a >95% purified solution. Lyophilization yielded 100 mg of the target product (white powder). After lyophilization, the polypeptide was analyzed for purity and MS detection. The purity was 95.684% and the MS molecular weight was 1507.62, calculated as a 22.11% yield of the naked peptide.

[0343] Step 4: Naked peptide modified TCO-mal 30 mg of the naked peptide was taken and dissolved in 10 mL of 10% aqueous acetonitrile, 10 mg of TCO-Mal was added, and the mixture was stirred at room temperature to react. The solution was removed every 30 minutes and subjected to MS detection until the molecular weight of the naked peptide, 1507.62, disappeared.

[0344] Step 5: Purification of the target peptide A liquid chromatography system was prepared using 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile as mobile phase B. A 10 μm reversed-phase C18 column (50 x 250 mm) was used. The UV detector was set at 220 nm, the flow rate was adjusted to 40 mL / min, and the column was equilibrated with 10% acetonitrile for 10 min. The reaction mixture was eluted with a 40-min gradient of 10% acetonitrile from 0.01 min to 45% acetonitrile. The target product was collected and analyzed to obtain a >95% purified solution. 15 mg of the target product (white powder) was collected and lyophilized. The polypeptide was lyophilized and analyzed for purity and MS detection. The product had a purity of 95.051% and a molecular weight of 1799.962, for a yield of 39.82%.

[0345] Example 17. Synthesis of polypeptide P-2 P-2 peptide sequence: LRELHLNNNC(TCO-mal) (SEQ ID NO: 81) [ka] Step 1: Resin peptide synthesis 1.1 Using 2-CL resin with a substitution degree of 0.3 mmol / g, the Fmoc process was employed. Following the peptide sequence above, amino acids were sequentially condensed from the C-terminus to the N-terminus (right to left) until the condensation of the peptide chain was complete. 2 g of 2-CL resin was placed in a clean reactor, 20 mL of DCM was added, and the mixture was immersed for 20 minutes to allow swelling. The reaction was then stopped, and the liquid was removed by suction filtration.

[0346] 1.2 Accurately weigh 0.6 mmol of Fmoc-Cys(trt)-OH and 0.9 mmol of HoBt, dissolve them in 10 mL of DMF, add 0.9 mmol of DIC dropwise, activate for 10 minutes, add to a reaction column, react for 2 hours, stop the reaction, wash with an appropriate amount of DMF three times, add 3 mL of DIEA, 3 mL of methanol, and 10 mL of DCM, react for 35 minutes, cap the column, remove the reaction solution by suction, and wash the resin with DMF three times.

[0347] 1.3 Removal of FMOC 15 mL of 20% hexahydropyridine / DMF solution was added, and the mixture was stirred for 10 minutes to react. The liquid was then removed by suction. Another 15 mL of 20% hexahydropyridine / DMF solution was added, and the mixture was stirred for 5 minutes to react. The liquid was then removed by suction. The mixture was washed five times with DMF, and a sample was taken for ninhydrin color detection. The detected color (blue) was recorded.

[0348] 1.4 Condensation Reaction 1.8 mmol of Fmoc-Ser(tbu)-OH and 1.8 mmol of HoBt were weighed out in an amount three times the molar substitution of the resin, dissolved in 10 mL of DMF, and 2.7 mmol of DIC was added dropwise. After 10 minutes of activation, the column was added to the reaction column and reacted for 1.5 hours (no color development was detected by the ninhydrin colorimetric method), after which the column was removed by suction, and washed five times with DMF.

[0349] 1.5 Cycle Reaction Repeat steps 1.4 and 1.5 until the final leucine condensation is complete. Then, wash with DMF three times. After sampling, the reaction mixture was colorless and detected by ninhydrin colorimetry, indicating completion. After condensation is complete, remove Fmoc by following step 1.3. Then, wash with MeOH three times and suction filter until dry.

[0350] Step 2: Cleavage of the polypeptide Preparation of lysis reagent: The amount of lysis reagent required was calculated using 15 mL of lysis solution per 1 g of resin peptide: TFA:TIS:EDT:HO = 95:2:2:1. The lysis reagent, HO, and TFA were added sequentially to the reaction flask, and the temperature of the lysis reagent was controlled at 0-10°C. The lysis reagent was added to the peptide resin with stirring. After the system temperature stabilized, the temperature was further controlled at 20-25°C and the reaction was continued with stirring for 2 hours. The lysis solution was filtered and precipitated with 10 times the volume of the filtrate in ice ether. The precipitate was washed four times by centrifugation and dried under reduced pressure at room temperature to obtain 1.3 g of crude product.

[0351] Step 3: Purification of naked peptide A liquid chromatography system was prepared using 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile as mobile phase B. A 10 μm reversed-phase C18 column (50 × 250 mm) was used. The UV detector was set at 220 nm, the flow rate was adjusted to 40 mL / min, and the column was equilibrated with 10% acetonitrile for 10 min. The crude peptide was triturated and dissolved in 10% acetonitrile aqueous solution. The trituration was performed in a microwave oven until complete dissolution. The solution was filtered through a 0.45 μm filter membrane and the sample was injected. The elution was performed at room temperature with a gradient of 10% acetonitrile for 0.01 min to 45% acetonitrile for 40 min. The absorbance change was monitored, impurity peaks were removed, and the target product was collected. Analysis revealed a >95% purified solution. Lyophilization yielded 210 mg of the target product (white powder). After lyophilization of the polypeptide, the purity and MS detection were performed again, and the purity was 95.853% and the MS molecular weight was 1225.376, which was calculated as a yield of 27.38% of the naked peptide.

[0352] Step 4: Naked peptide modified TCO-mal 100 mg of the naked peptide was dissolved in 20 mL of 10% aqueous acetonitrile, 30 mg of TCO-Mal was added, and the mixture was stirred at room temperature to react. The solution was removed every 30 minutes and subjected to MS detection until the molecular weight of the naked peptide, 1225.376, disappeared.

[0353] Step 5: Purification of the target peptide A liquid chromatography system was prepared using 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile as mobile phase B. A 10 μm reversed-phase C18 column (50 x 250 mm) was used. The UV detector was set at 220 nm, the flow rate was adjusted to 40 mL / min, and the column was equilibrated with 10% acetonitrile for 10 min. The reaction mixture was eluted with a 40-min gradient of 10% acetonitrile from 0.01 min to 45% acetonitrile. The target product was collected and analyzed to obtain a solution with a purity of >95%. 50 mg of the target product (white powder) was collected and lyophilized. The polypeptide was lyophilized and analyzed for purity and MS detection. The product had a purity of 98.821% and a molecular weight of 1517.72 (MS). The yield was 40.36%.

[0354] Example 18. Coupling of tyrosinase-mediated single- (multiple-) site-mutated antibodies to polypeptides In this example, the antibody expressed in Example 15 and the polypeptides synthesized in Examples 16 and 17 were subjected to a one-pot coupling reaction catalyzed by the VsTYR core enzyme. The specific coupling conditions and LC-MS detection were the same as in Example 7, and the DAR values ​​were calculated simultaneously. Table 16 shows the calculated DAR values ​​after coupling, demonstrating that antibodies containing mutation sites can be coupled with the relevant polypeptides.

[0355] [Table 16]

[0356] Example 19. Preparation of antibody-polypeptide complexes using VsTYR core In this example, a conjugate (CBP-ADA-mut) between the ADA mut antibody of Example 18 and a P-2 polypeptide was prepared. The specific coupling conditions and LC-MS detection were the same as in Example 7, and the DAR was calculated simultaneously. The coupling reaction mixture was purified using a HiLoad 16 / 600 Superdex 200 pg (120 mL). The column was equilibrated and eluted with PBS 5.5 buffer. The peak of the target antibody conjugate was collected, and enzymes and small molecules were removed. Figure 5 shows a typical profile of the purified and collected antibody-polypeptide conjugate. The collected antibody-polypeptide conjugate was analyzed by SEC-HPLC and found to be >95% pure.

[0357] Example 20. Detection of in situ binding activity of antibody-polypeptide complexes An in vitro binding activity test was performed on the antibody-polypeptide complex CBP-ADA-mut prepared in Example 19. The antigen-binding sequence at the end of the CBP-ADA-mut antibody was adalimumab, which can bind to TNFα, and the polypeptide terminal sequence was CBP (collagen-binding peptide), which can bind to collagen protein (Katsumata K, et al. Targeting inflammatory sites through collagen affinity enhances the therapeutic efficacy of anti-inflammatory antibodies. Sci Adv. 2019 Nov 6;5(11):eaay1971.).

[0358] Detection was performed using the ELISA method. Human TNFα antigen (Sino Biological, 10602HNAE) and type II collagen (Chondrex, #20012) were coated onto a plate. After a wash-block-wash process, the sample to be measured was added and incubated. After washing again, secondary antibodies, anti-human IgG, HRP (BETHYL, A80 319P), and anti-human IgG (Fab specific) peroxidase antibody (Sigma, A0293), were added, respectively. After washing, TMB color development solution was added and incubated for 5 to 10 minutes. After that, stop solution was added and the OD450 value was measured.

[0359] Figure 6 shows that CBP-ADA-mut can bind to TNFα and has binding activity comparable to that of ADA-mut and adalimumab. Figure 7 shows that the antibody-polypeptide complex CBP-ADA-mut can bind to type II collagen via the CBP polypeptide coupled to it, but monoclonal ADA-mut without the CBP polypeptide cannot bind to type II collagen and therefore does not have the corresponding biological function.

[0360] Example 21. Detection of the killing activity of antibody-drug conjugates in vivo Using the coupling conditions of Example 7, the antibody TROP2 mut expressed in Example 15 was coupled to the L-1 small molecule synthesized in Example 3. The resulting ADC-1 had a DAR value of 4.00 as calculated by LC-MS and was purified and prepared using a procedure similar to that of Example 19. The resulting ADC-1 was subjected to in vitro cell experiments.

[0361] ADC-2 (interchain cys coupling) was prepared in parallel as a positive control, and isotype IgG1 DAR4 (interchain cys coupling) was prepared as a negative control.

[0362] ADC-2 has the structure shown below: [ka] The preparation method for ADC-2 was described in WO2021147993A1, n=4.0, and the heavy and light chain sequences of the PD3 antibody (TROP2 antibody) are shown in SEQ ID NOs: 44 and 45 of the present disclosure. The difference between hIgG1 DAR4 and ADC-2 was that the VH and VL at the antibody termini were those of an unrelated antibody.

[0363] The killing ability of ADC-1 was examined in cells with different levels of TROP2 expression by in vivo cell killing. A431 cells (highly TROP-expressing) were cultured in DMEM medium containing 10% FBS and passaged every 3 days at a passage ratio of 1:3 to 1:8. OVCAR-3 cells (moderately TROP-expressing) were cultured in RPMI-1640 medium containing 10% FBS and passaged every 3 days at a passage ratio of 1:2 to 1:4. HEK-293 cells were cultured in DMEM medium containing 10% FBS and passaged every 3 days at a passage ratio of 1:6 to 1:10. At the time of passage, the medium was aspirated and the cells were washed with PBS and digested with 2 mL of 0.25% pancreatin at 37°C for 5 minutes. Fresh medium was then added to stop the digestion and the cells were resuspended. 50 μL of the cell suspension was added to a 96-well cell culture plate at a concentration of 2 × 10 cells. 3 The cells were added at 1000 cells / well in DMEM containing 10% FBS, and PBS buffer was added around the periphery of the 96-well plate. The culture plate was then cultured overnight in an incubator (37°C, 5% CO2).

[0364] The next day, the ADC drug to be measured was prepared at a starting concentration of 200 nM and diluted 1:3 with complete medium to nine points, including a zero concentration point. 50 μL of the solution was added to the cell plate and incubated in an incubator (37°C, 5% CO2) for 6 days. Cell Titer-Glo reagent was added to a 96-well cell culture plate at 100 μL per well and incubated in the dark at room temperature for 10–15 min. Chemiluminescence signal readings were read using a PE Envision instrument and the data were processed using GraphPad software. ADC or toxin concentration was plotted on the x-axis against cell killing percentage on the y-axis.

[0365] The results show that ADC-1 has comparable killing ability to ADC-2 in high-TROP2 expressing A431 cells and moderate-TROP2 expressing OVCAR-3 cells, but is non-killing in HEK-293 TROP2-negative cells, and only at high concentrations exhibits non-specific killing (Figure 8). The data are shown in Table 17.

[0366] [Table 17]

[0367] Among them, ADC-1 and ADC-2 were both DAR4.

[0368] Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples and that various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Accordingly, the scope of the present disclosure is limited by the appended claims.

[0369] Sequence of the present disclosure: >Her wt HC (Sequence number 1)

[28] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her wt LC (Accession No. 2) [Chemical formula 29] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Her H9 HC (Accession No. 3) [Chemical formula 30] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H10 HC (Accession No. 4) [Chemical 31][[ID=**5**]] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H14 HC (Accession No. 5) Note: In the original text, the "化31" in line 4 is likely a chemical formula or some chemical-related notation. Since it's not clear what it exactly represents in English, I left it as "[Chemical 31]". If you can provide more context about this, a more accurate translation could be made. Also, I'm not sure if the "配列番号" in lines 2 and 8 should be translated as "Accession No." in a patent context. You may want to double-check this with a patent expert or based on specific requirements. [Chemical 32] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQYYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H19 HC (Accession No. 6) [Chemical 33] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKYHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H30 HC (SEQ ID NO: 7)

[34] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H33 HC (SEQ ID NO: 8)

[35] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGYPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H53 HC (Accession No. 9) [Chemical 36] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her H26 HC (Accession No. 10) [Chemical 37] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQFGYTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her L15 LC (SEQ ID NO: 11) [Chemical 38] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC >0102 wt HC (SEQ ID NO: 12) [Chemical 39] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 wt LC(SEQ ID NO: 13) [Chemical 40] DVVMTQSPLSLPVTPGEPASISCKSTQSLLYSDGETYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGSHFPQTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >0102 H9 HC(SEQ ID NO: 14) [Chemical 41] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H10 HC (SEQ ID No. 15) [Chemical 42] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H19 HC (SEQ ID No. 16) [Chemical formula 43] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKYHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H30 HC (Sequence number 17) [Chemical formula 44] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H53 HC (SEQ ID NO: 18)

[45] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H26 HC (SEQ ID NO: 19)

[46] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQFGYTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 L15 LC(SEQ ID No. 20) [Chemical 47] DVVMTQSPLSLPVTPGEPASISCKSTQSLLYSDGETYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGSHFPQTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC >megaTYR(SEQ ID No. 21)<0002&267>[Chemical 48] It should be noted that there might be a small error in your original text where " <0002&267>" is likely a typo and should probably be " ". I've translated it as best as possible with the given text.MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRN FGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSSLEHHHHHH >VsTYR core (SEQ ID NO: 22)

[49] MAKYHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIHTLDCPHGNWWFVVWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGVLDPNNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSPMFFPRGRARTLT RQNPGFDATTRRAVSIGTIRNALAPTDFITFGSGKTANHSESATQGILESQPHNNVHNNIGGFMQDLLSPTDPVFFAHHSNIDRLWDVWTRKQQRLGLPTLPTGANLPLWANEPFLFFIGPDGKPVAKNKAGDYATIGDFEYNYEPGSGEAVIPAASRPGEMNNKHHHHHH >VsTYR sp (SEQ ID NO: 23)

[50] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSAAGTMAKYHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIHTLDCPHGNWWFVVWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGVLDPNNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSPMFFPRGRARTLTRQNPGFDATTRRAVSIGTIRNALAPTDFITFGSGKTANHSESATQGILESQPHNNVHNNIGGFMQDLLSPTDPVFFAHHSNIDRLWDVWTRKQQRLGLPTLPTGANLPLWANEPFLFFIGPDGKPVAKNKAGDYATIGDFEYNYEPGSGEAV >Her HH3 HC (Accession No. 24) [Chemical 51] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKYHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her HH4 HC (Accession No. 25) [Chemical 52] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYLGTQTYICNVNHKPSNTKVDKKVEPKSCDKYHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her HH8 HC (Accession No. 26) [Chemical 53] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Her HH9 HC (Accession No. 27) [Chemical 54] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKYHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H73 HC (Sequence No. 28) [Chemical 55] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H74 HC (SEQ ID NO: 29)

[56] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDDWDGDFDYWGQGTLVTVSSASTKGPSV FPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE PKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK >0102 H40 HC (SEQ ID NO: 30)

[57] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >010۲ H75 HC (Sequence number 31) [Chemical 58] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSYSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >010۲ H76 HC (Sequence number 32) [Chemical 59] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >0102 H77 HC (SEQ ID NO: 33) [Chemical 60] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK >0102 H78 HC(SEQ ID NO: 34) [Chemical 61] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSYSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >0102 H79 HC (SEQ ID NO: 35)

[62] EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPEKRLEWVATISDSDYYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRDDWDGDFDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLYSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >c-met H10 HC (SEQ ID NO: 36)

[63] QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >c-met H10 LC (Accession No. 37)<000​​​​​​​​QVQLVESGGGVVQPGRSLRLSCAASGFSLSNYGVHWVRQAPGKGLEWLAVIWSGGSTNYAAAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYNYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >c-met L15 LC (Accession No. 39) [Chemical 66] DIVLTQSPDSLAVSLGERATINCRADKSVSTSTYNYLHWYQQKPGQPPKLLIYLASNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRDLPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC >ADA mut HC (Accession No. 40) [Chemical 67] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >ADA mut LC(SEQ ID NO: 41) [Chemical 68] DAVMTQTPLSLSVTPGQPASISCRSSQSLERSTGNTFLNWYLQKPGQSPQLLIYRVSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC >trop2 mut HC(SEQ ID NO: 42) [Chemical 69] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >trop2 mut LC (Accession No. 43) [Chemical Formula 70] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC >ADC-2 HC (Accession No. 44) [Chemical Formula 71] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ADC-2 LC (SEQ ID NO: 45)

[72] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >CH1 (SEQ ID NO: 46)

[73] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV > Hinge (SEQ ID NO: 47)

[74] EPKSCDKTHTCPPCP >CH2 (SEQ ID NO: 48)

[75] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK >CH3 (SEQ ID NO: 49)

[76] GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Cκ (SEQ ID NO: 50)

[77] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Her HH13 LC (SEQ ID NO: 67)

[78] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGY.

[0370]

[79] YGGGG (SEQ ID NO: 76),

[80] YGGGGS (SEQ ID NO: 77),

[81] GGGGY (SEQ ID NO: 78),

[82] SGGGGY (SEQ ID NO: 79),

[83] P-1: EQKLISEEDLGGSC (SEQ ID NO: 80),

[84] P-2: LRELHLNNNC (sequence number 81).

[0371] >CH1 T135Y (SEQ ID NO: 82)

[85] ASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV >CH1 G137Y (SEQ ID NO: 83)

[86] ASTKGPSVFPLAPSSKSTSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV >CH1 S192Y (SEQ ID NO: 84)

[87] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYLGTQTYICNVNHKPSNTKVDKKV > Hinge T223Y (SEQ ID NO: 85)

[88] EPKSCDKYHTCPPCP >CH2 P329Y (SEQ ID NO: 86)

[89] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALYAPIEKTISKAK >Cκ S202Y (SEQ ID NO: 87)

[90] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLYSPVTKSFNRGEC >CH1 T135Y / S192Y (SEQ ID NO: 88)

[91] ASTKGPSVFPLAPSSKSYSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYLGTQTYICNVNHKPSNTKVDKKV >CH1 G137Y / S192Y (SEQ ID NO: 89)

[92] ASTKGPSVFPLAPSSKSTSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSYLGTQTYICNVNHKPSNTKVDKKV >CH1 T135Y / G137Y (SEQ ID NO: 90)

[93] ASTKGPSVFPLAPSSKSYSYGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

Claims

1. A conjugate having the structure of Formula I or a pharmaceutically acceptable salt thereof: Pc-[L-(D) ｘ ] ｙ I、 Among them, Pc is a protein comprising an antibody constant region or a fragment thereof; L is a linker covalently linking Pc to D, and L is linked to an engineered tyrosine (Tyr) residue in said antibody constant region or fragment thereof, said engineered tyrosine residue is not located near an N-glycosylation site, and said Pc does not require removal / reduction of glycosylation modifications prior to reaction with tyrosinase; D is the payload, x is 1 to 20; y is 1 to 20; Preferably, said N-glycosylation site is 297N as defined according to the EU numbering system, Preferably, the phenolic moiety of the engineered tyrosine residue is capable of being oxidized to an o-quinone moiety by monotyrosinase but not by polytyrosinase. Complex.

2. The monotyrosinase is derived from Bacillus megaterium or Verrucomicrobium spinosum, and the polytyrosinase is a tetrameric enzyme preferably derived from a mushroom. The composite of claim 1.

3. the engineered tyrosine residue is not located between amino acid residues 292 and 302 of the heavy chain HC; The complex according to claim 1 or 2.

4. A conjugate having the structure of Formula I or a pharmaceutically acceptable salt thereof: Pc-[L-(D) ｘ ] ｙ I、 Among them, Pc is a protein comprising an antibody constant region or a fragment thereof; L is a linker covalently linking Pc to D, and L is linked to an engineered tyrosine residue in the antibody constant region or fragment thereof, wherein the engineered tyrosine residue is located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC of Pc, or any combination thereof, wherein the amino acid residue positions are as defined according to the EU numbering system; D is the payload, x is 1 to 20; y is 1 to 20; Complex.

5. The engineered tyrosine residue in the Pc is located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC of the Pc, or any combination thereof, and preferably 202Y in LC and 135Y in HC, 202Y in LC and 137Y in HC, 202Y in LC and 192Y in HC, 202Y in LC and 223Y in HC, 202Y in LC and 329Y in HC, HC 135Y / 137Y, HC 135Y / 192Y, HC 135Y / 223Y, HC 135Y / 329Y, HC 137Y / 192Y, HC 137Y / 223Y, HC 137Y / 329Y, HC 192Y / 223Y, HC 192Y / 329Y, HC 223Y / 329Y, 135Y / 137Y in HC and 202Y in LC; 135Y / 192Y in HC and 202Y in LC; 135Y / 223Y in HC and 202Y in LC; 135Y / 329Y in HC and 202Y in LC; 137Y / 192Y in HC and 202Y in LC; 137Y / 223Y in HC and 202Y in LC; 137Y / 329Y in HC and 202Y in LC; 192Y / 223Y in HC and 202Y in LC; 192Y / 329Y in HC and 202Y in LC; 223Y / 329Y in HC and 202Y in LC; HC 135Y / 137Y / 192Y, HC 135Y / 137Y / 223Y, HC 135Y / 137Y / 329Y, 135Y / 137Y / 192Y in HC and 202Y in LC; 135Y / 137Y / 223Y in HC and 202Y in LC; 135Y / 137Y / 329Y in HC and 202Y in LC; HC 135Y / 192Y / 223Y, HC 135Y / 192Y / 329Y, 135Y / 192Y / 223Y in HC and 202Y in LC; 135Y / 192Y / 329Y in HC and 202Y in LC; HC 137Y / 192Y / 223Y, HC 137Y / 192Y / 329Y, 137Y / 192Y / 223Y in HC and 202Y in LC; 137Y / 192Y / 329Y in HC and 202Y in LC; HC 137Y / 223Y / 329Y, 137Y / 223Y / 329Y in HC and 202Y in LC; HC 192Y / 223Y / 329Y, 192Y / 223Y / 329Y in HC and 202Y in LC; HC 135Y / 223Y / 329Y, A combination selected from HC 135Y / 223Y / 329Y and LC 202Y, The composite according to any one of claims 1 to 4.

6. L and Pc together form a structure represented by Za or Zb, 【Chemistry 1】 * represents the connection end with Pc, ** represents the connecting end with L, --- is a single or double bond, A composite according to any one of the preceding claims.

7. the amino acid linked to L is the engineered tyrosine residue, and the phenol moiety of the engineered tyrosine residue is oxidized to an o-quinone moiety by tyrosinase; A composite according to any one of the preceding claims.

8. said L comprising a cleavable peptide moiety; A composite according to any one of the preceding claims.

9. L comprises an amino acid unit, and the amino acid unit preferably comprises a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and is more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), A composite according to any one of the preceding claims.

10. The L comprises a spacer unit connecting to D, the spacer unit preferably being p-aminobenzyloxycarbonyl (PAB), 【Chemistry 2】 Including, A composite according to any one of the preceding claims.

11. L comprises a spacer unit linked to D, the spacer unit being -(CR １ R ２ ) ｍ１ -O(CR １ R ２ ) ｍ２ -CR ３ R ４ -C(O)-、 -(CR １ R ２ ) ｍ１ NH-(CR １ R ２ ) ｍ２ -CR ３ R ４ -C(O)-、 -(CR １ R ２ ) ｍ１ O-CR ３ R ４ (CR １ R ２ ) ｍ２ -、 -(CR １ R ２ ) ｍ１ OCR ３ R ４ -C(O)-、 - (CR １ R ２ ) ｍ１ -O-(CR １ R ２ ) ｍ２ C(O)- or -(CR １ R ２ ) ｍ１ -S-(CR １ R ２ ) ｍ２ -CR ３ R ４ -C(O)-, Among them, R １ and R ２ are the same or different and are each independently selected from hydrogen, halogen, or alkyl groups; R ３ is hydrogen, C ３－６ cycloalkylalkyl group or C ３－６ cycloalkyl groups, R ４ is hydrogen, a haloalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; Or, R ３ and R ４ are C together with the carbon atoms connected to them. ３－６ forming a cycloalkyl group, m1 and m2 are each independently selected from 0, 1, 2, or 3; A composite according to any one of the preceding claims.

12. The spacer unit is -(CH ２ ) ３ -C(O)-、-CH ２ -O-CH ２ -C(O)-、-(CH ２ ) ２ -O-CH ２ -C(O)-、 【Transformation 3】 including a structure selected from The composite of claim 11.

13. L includes a stretch unit (Str) that is covalently bound to Pc, and Str undergoes a [4+2] cycloaddition reaction with an o-quinone obtained by oxidation of a tyrosine residue by tyrosinase. A composite according to any one of the preceding claims.

14. The Str includes a cycloalkenyl group, a cycloalkynyl group, a heterocycloalkenyl group, or a heterocycloalkynyl group. The complex of claim 13.

15. The Str is 【Chemistry 4】 containing a group selected from The complex of claim 13 or 14.

16. The Str and Pc are 【Transformation 5】 That is, The composite of claim 15.

17. The Str is 【Transformation 6】 and Among them, R ａ , R ｂ are each independently hydrogen, C １－６ selected from alkyl groups or aryl groups, X １ is C １－３ Alkylene group, NR ｃ or O, R ｃ is hydrogen or C １－６ selected from alkyl groups, The complex of claim 13 or 14.

18. The Str and Pc are 【Transformation 7】 That is, The composite of claim 17.

19. L-(D) ｘ teeth, -Str-(Pep)-Sp-D, Str is a stretching unit that is covalently bound to Pc, Sp is a spacer unit, Pep is a cleavable peptide moiety. A composite according to any one of the preceding claims.

20. L-(D) ｘ teeth, 【Transformation 8】 It contains the chemical structure Among them, R ３ is hydrogen, C ３－６ cycloalkylalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; R ４ is hydrogen, a haloalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; Or, R ３ and R ４ are C together with the carbon atoms connected to them. ３－６ forming a cycloalkyl group, * indicates the connection end with Pc. A composite according to any one of the preceding claims.

21. L-(D) ｘ teeth, 【Chemistry 9】 It is a chemical structure represented by the formula: Among them, R ３ is hydrogen, C ３－６ cycloalkylalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; R ４ is hydrogen, a haloalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; Or, R ３ and R ４ are C together with the carbon atoms connected to them. ３－６ forming a cycloalkyl group, p1 is selected from 2, 4, 6 or 8; p2 is selected from 0, 1 or 2; A composite according to any one of the preceding claims. 【Request Item 22】 【Chemistry 10-1】 【Chemistry 10-2】 It is expressed by the formula: Among them, R ３ is hydrogen, C ３－６ cycloalkylalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; R ４ is hydrogen, a haloalkyl group or C ３－６ cycloalkyl groups, preferably hydrogen; Or, R ３ and R ４ are C together with the carbon atoms connected to them. ３－６ forming a cycloalkyl group, y is selected from 1 to 20 and may be an integer or a decimal number; p1 is selected from 2, 4, 6 or 8; p2 is selected from 0, 1 or 2; A composite according to any one of the preceding claims.

23. The antibody constant region or a fragment thereof in the Pc is derived from an IgG, IgA, IgM, IgD, or IgE constant region or a fragment thereof, and the IgG is preferably IgG1, IgG2, IgG3, or IgG4, and more preferably IGHG1, IGHG2, IGHG3, or IGHG4. A composite according to any one of the preceding claims.

24. the antibody constant region or fragment thereof in the Pc is a heavy chain constant region or fragment thereof and / or a light chain constant region or fragment thereof; Preferably, the heavy chain constant region is C Ｈ 1 region, hinge region, C Ｈ the light chain constant region is selected from any one of two regions or any combination thereof, and the light chain constant region is a Cκ region; Preferably, the heavy chain constant region is C Ｈ It includes three areas: A composite according to any one of the preceding claims.

25. The antibody constant region or fragment thereof in the Pc is glycosylated or non-glycosylated, preferably, the glycosylation is 297N glycosylation; A composite according to any one of the preceding claims.

26. the Pc is selected from an antibody or an antigen-binding fragment thereof, an Fc region or a fragment thereof, and a fusion protein, and the antibody or antigen-binding fragment thereof is preferably a monospecific antibody, a bispecific antibody, a multispecific antibody, or an antigen-binding fragment thereof; A complex according to any one of claims 1 to 4.

27. The Pc is an anti-tumor antibody or an antigen-binding fragment thereof, and the antibody is preferably an anti-HER2 antibody, an anti-LIV-1 antibody, or an anti-TROP2 antibody.

27. The complex of claim 26.

28. The Pc is selected from a mouse antibody, a chimeric antibody, a humanized antibody, and a fully human antibody or an antigen-binding fragment thereof; 28. The complex of claim 26 or 27.

29. The Pc is a monomer or a polymer, and the polymer is preferably a dimer, trimer, or tetramer; A composite according to any one of the preceding claims.

30. the Pc comprises a polypeptide fragment at the N-terminus and / or C-terminus, the fragment comprising one or more tyrosine residues; Preferably, the fragment is linked to the N-terminus and / or C-terminus of Pc, either directly or via a linker; More preferably, the linker is a GS or polyG linker; Most preferably, the N-terminus of Pc comprises YGGGG, YGGGGS, and / or the C-terminus comprises GGGGY, SGGGGY. A composite according to any one of the preceding claims.

31. D is a therapeutic agent or a diagnostic agent, Preferably, D is a radioactive agent, a cytotoxic agent, a nucleic acid, a polypeptide, a fluorophore, a fluorescent dye, a radionuclide, an enzyme, an antibiotic, a chelating agent, a lipid, an antibody or an antigen-binding fragment thereof; More preferably, D is exatecan or a derivative thereof, eribulin or a derivative thereof. A composite according to any one of the preceding claims.

32. wherein x is 1 and y is 1 to 8; A composite according to any one of the preceding claims.

33. A protein comprising an antibody constant region or a fragment thereof, the antibody constant region or fragment thereof comprises one or more engineered tyrosine residues, the phenolic moieties of which are capable of being oxidized to o-quinone moieties by monotyrosinase but not by polytyrosinase; Preferably, the monotyrosinase is derived from Bacillus megaterium or Verrucomicrobium spinosum, and the polytyrosinase is a tetrameric enzyme, preferably derived from a mushroom. protein.

34. the engineered tyrosine residue is not located near an N-glycosylation site, and / or the Pc does not require removal / reduction of glycosylation modifications prior to reaction with tyrosinase; Preferably, said N-glycosylation site is 297N as defined according to the EU numbering system, More preferably, the engineered tyrosine residue is not located between amino acid residues 292 and 302 of the heavy chain HC. The protein of claim 33.

35. Removing / reducing said glycosylation modifications comprises: 1) contacting the protein with an amidase, preferably PNGase F, to obtain a protein from which glycans have been removed; 2) modifying the protein by contacting it with an endoglycosidase to form an N-glycoprotein of glycans having the structure -GlcNAc(Fuc)b, where b is 0 or 1; or 3) mutating the protein so that 297N is replaced by a non-glycosylated amino acid; 35. The protein of claim 34.

36. A protein comprising an antibody constant region or a fragment thereof, the antibody constant region or fragment thereof comprises one or more engineered tyrosine residues, the engineered tyrosine residues being located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof; protein.

37. The engineered tyrosine residues in the Pc are located at any one of positions 135, 137, 192, 223, 329 of the heavy chain HC and position 202 of the light chain LC, or any combination thereof, and preferably 202Y in LC and 135Y in HC, 202Y in LC and 137Y in HC, 202Y in LC and 192Y in HC, 202Y in LC and 223Y in HC, 202Y in LC and 329Y in HC, HC 135Y / 137Y, HC 135Y / 192Y, HC 135Y / 223Y, HC 135Y / 329Y, HC 137Y / 192Y, HC 137Y / 223Y, HC 137Y / 329Y, HC 192Y / 223Y, HC 192Y / 329Y, HC 223Y / 329Y, 135Y / 137Y in HC and 202Y in LC; 135Y / 192Y in HC and 202Y in LC; 135Y / 223Y in HC and 202Y in LC; 135Y / 329Y in HC and 202Y in LC; 137Y / 192Y in HC and 202Y in LC; 137Y / 223Y in HC and 202Y in LC; 137Y / 329Y in HC and 202Y in LC; 192Y / 223Y in HC and 202Y in LC; 192Y / 329Y in HC and 202Y in LC; 223Y / 329Y in HC and 202Y in LC; HC 135Y / 137Y / 192Y, HC 135Y / 137Y / 223Y, HC 135Y / 137Y / 329Y, 135Y / 137Y / 192Y in HC and 202Y in LC; 135Y / 137Y / 223Y in HC and 202Y in LC; 135Y / 137Y / 329Y in HC and 202Y in LC; HC 135Y / 192Y / 223Y, HC 135Y / 192Y / 329Y, 135Y / 192Y / 223Y in HC and 202Y in LC; 135Y / 192Y / 329Y in HC and 202Y in LC; HC 137Y / 192Y / 223Y, HC 137Y / 192Y / 329Y, 137Y / 192Y / 223Y in HC and 202Y in LC; 137Y / 192Y / 329Y in HC and 202Y in LC; HC 137Y / 223Y / 329Y, 137Y / 223Y / 329Y in HC and 202Y in LC; HC 192Y / 223Y / 329Y, 192Y / 223Y / 329Y in HC and 202Y in LC; HC 135Y / 223Y / 329Y, A combination selected from HC 135Y / 223Y / 329Y and LC 202Y, A protein according to any one of claims 33 to 36.

38. The antibody constant region or a fragment thereof is derived from an IgG, IgA, IgM, IgD, or IgE constant region or a fragment thereof, and the IgG is preferably IgG1, IgG2, IgG3, or IgG4, and more preferably IGHG1, IGHG2, IGHG3, or IGHG4. A protein according to any one of claims 32 to 37.

39. the antibody constant region or fragment thereof is a heavy chain constant region or fragment thereof and / or a light chain constant region or fragment thereof; Preferably, the heavy chain constant region is C Ｈ 1 region, hinge region, C Ｈ the light chain constant region is selected from any one of two regions or any combination thereof, and the light chain constant region is a Cκ region; Preferably, the heavy chain constant region is C Ｈ It includes three areas: A protein according to any one of claims 32 to 38.

40. Said C Ｈ The amino acid sequence of the hinge region is represented by SEQ ID NO: 85, and the amino acid sequence of the C Ｈ The amino acid sequence of the two regions is represented by SEQ ID NO: 86, and the amino acid sequence of the Cκ region is represented by SEQ ID NO:

87.

40. The protein of claim 39.

41. The antibody constant region or fragment thereof in the Pc is glycosylated or non-glycosylated, preferably, the glycosylation is 297N glycosylation; A protein according to any one of claims 32 to 40.

42. an antibody or antigen-binding fragment thereof, an Fc region or fragment thereof, or a fusion protein, wherein the antibody or antigen-binding fragment thereof is preferably a monospecific antibody, a bispecific antibody, a multispecific antibody, or an antigen-binding fragment thereof; A protein described in any one of claims 32 to 41.

43. an anti-tumor antibody or an antigen-binding fragment thereof, the anti-tumor antibody being preferably an anti-HER2 antibody, an anti-LIV-1 antibody, or an anti-TROP2 antibody; More preferably, the anti-HER2 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 51 to 53, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54 to 56; or the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 57, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 58; More preferably, the anti-LIV-1 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 59 to 61, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 62 to 64; or the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 66; More preferably, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 68 to 70, and the VL comprises LCDR1, LCDR, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 71 to 73; or the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 74, and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

75.

43. The protein of claim 42.

44. The antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

44. A protein according to claim 42 or 43.

45. Monomeric or polymeric, preferably dimeric, trimeric or tetrameric; A protein according to any one of claims 32 to 44.

46. the Pc comprises a polypeptide fragment at the N-terminus and / or C-terminus, the fragment comprising one or more tyrosine residues; Preferably, the fragment is linked to the N-terminus and / or C-terminus of the protein, either directly or via a linker; More preferably, the linker is a GS or polyG linker; Most preferably, the N-terminus of the protein comprises YGGGG, YGGGGS and / or the C-terminus comprises GGGGY, SGGGGY. A protein described in any one of claims 32 to 45.

47. 1. A method for preparing a complex, comprising: a) providing a protein comprising an antibody constant region or a fragment thereof comprising an engineered tyrosine residue; b) contacting the protein of a) with tyrosinase to convert the phenol moiety of the engineered tyrosine residue to an o-quinone moiety; c) subjecting the o-quinone moiety to a [4+2] cycloaddition reaction with an olefin or alkyne compound containing a cycloalkenyl, heterocycloalkenyl, cycloalkynyl, or heterocycloalkynyl moiety; Preferably, the protein in a) is a protein according to any one of claims 32 to 46. method.

48. The tyrosinase is preferably a monotyrosinase derived from Bacillus megaterium or Verrucomicrobium spinosum, 48. The method of claim 47.

49. The protein does not require removal / reduction of glycosylation modifications prior to reaction with tyrosinase; 49. The method of claim 47 or 48.

50. the olefin or alkyne compound comprises a cycloalkenyl group, a heterocycloalkenyl group, a cycloalkynyl group, or a heterocycloalkynyl group moiety, and optionally a payload (D); 50. The method according to any one of claims 47 to 49.

51. D is a therapeutic agent or a diagnostic agent, Preferably, D is a radioactive agent, a cytotoxic agent, a nucleic acid, a polypeptide, a fluorophore, a fluorescent dye, a radionuclide, an enzyme, an antibiotic, a chelating agent, a lipid, an antibody or an antigen-binding fragment thereof; More preferably, D is exatecan or a derivative thereof, eribulin or a derivative thereof.

51. The method of claim 50.

52. In step b), the o-quinone moiety undergoes a [4+2] cycloaddition reaction with an olefin or alkyne compound to form a structure represented by Za or Zb; 【Chemistry 11】 * indicates the end of the linkage to the protein. ** represents the end of the linker, --- is a single or double bond, 52. The method according to any one of claims 47 to 51.

53. An isotope-substituted complex of any one of claims 1 to 32, Preferably, the isotope substitution product is a deuterated product. Isotopically substituted compounds.

54. Encoding the protein of any one of claims 33 to 46. Polynucleotide.

55. comprising or expressing the polynucleotide of claim 54. vector.

56. 1. A pharmaceutical composition comprising: a therapeutically effective amount of the conjugate of any one of claims 1 to 32; a pharmaceutically acceptable carrier, diluent or excipient; Pharmaceutical compositions.

57. A method for preparing a complex of a protein according to any one of claims 33 to 46, use.

58. antibody drug conjugates, polypeptide drug conjugates, antibody polypeptide conjugates, An isotope-substituted product of the complex according to any one of claims 1 to 32 and the complex according to claim 53.

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