Antibody-polypeptide conjugate and pharmaceutical use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

Antibody-peptide conjugates and their medical uses Technical Field

[0001] The present disclosure belongs to the field of biotechnology and relates to antibody-polypeptide conjugates and their medical uses. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] FGF21 is a multifunctional regulatory factor primarily produced by the liver, but also expressed in adipose tissue, pancreas, and skeletal muscle. It is secreted into the bloodstream and participates in regulating metabolism. By binding to its cell membrane receptor, KLB, FGF21 activates KLB and FGFR1c and their downstream signaling pathways, regulating glucose metabolism, lipid metabolism, and energy expenditure.

[0004] Endocrine peptides such as glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP) are secreted by the intestine or liver. They can widely regulate important physiological regulation related to metabolism, such as blood glucose levels, fat metabolism, and feeding behavior, by activating GLP-1R, GCGR, and GIPR, respectively.

[0005] In clinical practice, FGF21 fusion protein has achieved certain clinical effects in the treatment of non-alcoholic steatohepatitis (NASH). At the same time, GLP-1 / GIP / GCG single-activation or multi-activation peptides have also been shown to improve NASH patients.

[0006] From preclinical studies to clinical studies, it has been shown that FGF21 signal activation and GLP-1 are complementary in mechanism (Qi Pan et al., EBioMedicine, 63, 2021, 103202; Allegra Kaufman et al., Cell Reports Medicine, 2020, 1(4): 100057; Leiluo Geng et al., Nature Reviews Endocrinology, 2020, 16(11): 654-667; Kyle H. Flippo et al., Nature metabolism, 2021, 3(3): 309-317), and the combination of the two may have better therapeutic effects. At present, the industry mainly focuses on the development of bifunctional molecules in the form of fusion (for example, WO2022003169A1). These drug forms have a short half-life and can only be administered once a week. Therefore, there is an urgent need to develop multifunctional molecules with lower dosing frequency in clinical practice to avoid many problems such as the drugability of fusion proteins. Summary of the Invention

[0007] The present disclosure provides an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in the general formula (I): Ab-(LP) m (I)

[0008] in:

[0009] Ab is antibody;

[0010] P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof;

[0011] L is a linker connecting Ab and P;

[0012] wherein Ab binds directly to L via its amino acid residue at position 297, or Ab binds to L via its sugar chain or reconstructed sugar chain at position 297;

[0013] m represents an integer from 0 to 10.

[0014] The present disclosure provides an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in the general formula (I): Ab-(LP) m (I)

[0015] in:

[0016] Ab is antibody;

[0017] P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof;

[0018] L is a linker connecting Ab and P;

[0019] wherein Ab binds directly to L via its amino acid residue at position 297, or Ab binds to L via its sugar chain or reconstructed sugar chain at position 297;

[0020] m represents an integer of 1 to 10.

[0021] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described above, wherein the sugar chain remodeled at Asn297 of Ab is bound to L.

[0022] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the remodeled sugar chain structure is:

[0023] In the formula, the wavy line Indicates binding to Asn 297 of the Pc heavy chain;

[0024] P1 and P2 are the same or different and are each independently selected from hydroxyl, *-(CR p R q -CR sR t -O)s 1 - and *-(CR p R q -CR s R t -O)s 2 -(CR p R q -CR s R t -CR x R y -O)s 3 -(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano, amino, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano and amino; the asterisk * indicates binding to the linker L;

[0025] s 1 1-10, preferably 1-5; s 2 0-10, preferably 1-5; s 3 1-10, preferably 1-5; s 4 0-10, preferably 1-5;

[0026] The condition is that P1 and P2 are not hydroxyl or *-(CH2CH2O)s at the same time 1 -.

[0027] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P1 is a hydroxyl group, P2 is *-(CR p R q -CR s R t -O)s 2 -(CR p R q -CR s R t -CR x R y -O)s 3-(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl, s 2 1-5, s 3 1-5, s 4 1-5.

[0028] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P1 is a hydroxyl group, P2 is *-(CR p R q -CR s R t -O)s 2 -(CR p R q -CR s R t -CR x R y -O)s 3 -(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 Halogenated alkyl, s 2 is 1, s 3 is 1, s 4 is 1.

[0029] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P1 is a hydroxyl group, P2 is *-(CR p R q -CR s R t -O)s 2 -(CR p Rq -CR s R t -CR x R y -O)s 3 -(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently hydrogen or C 1-6 Alkyl, s 2 1-5, s 3 1-5, s 4 1-5.

[0030] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P1 is a hydroxyl group, P2 is *-(CR p R q -CR s R t -O)s 2 -(CR p R q -CR s R t -CR x R y -O)s 3 -(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently hydrogen or C 1-6 Alkyl, s 2 is 1, s 3 is 1, s 4 is 1.

[0031] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the remodeled sugar chain structure is:

[0032] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0033] Where R is -LP;

[0034] Ab, L, P and m are as defined in (I).

[0035] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof.

[0036] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR tripotonic peptide or an analog thereof.

[0037] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P is a GLP-1R monoagonist peptide or an analog thereof.

[0038] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P is a GLP-1R / GIPR dual agonist peptide or an analog thereof.

[0039] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P is a GLP-1R / GCGR dual agonist peptide or an analog thereof.

[0040] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P is a GLP-1R / GIPR / GCGR tripeptide or an analog thereof.

[0041] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein P comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.

[0042] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the amino acid sequence of P is as shown in SEQ ID NO: 20 or SEQ ID NO: 21.

[0043] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the amino acid sequence of P is as shown in SEQ ID NO: 22.

[0044] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is selected from IgG1, IgG2, IgG3 and IgG4 antibodies; preferably, Ab is an IgG1 antibody.

[0045] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody or an ActRIIA / B antibody.

[0046] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody.

[0047] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, HCDR1 of the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11.

[0048] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the amino acid sequence of HCDR1 of the heavy chain variable region of Ab is shown in SEQ ID NO: 6, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 8, and the amino acid sequence of LCDR1 of the light chain variable region is shown in SEQ ID NO: 9, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11.

[0049] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody.

[0050] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody; preferably, HCDR1 of the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 28.

[0051] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody; preferably, the amino acid sequence of HCDR1 of the heavy chain variable region of Ab is shown in SEQ ID NO: 23, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 24, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 25, and the amino acid sequence of LCDR1 of the light chain variable region is shown in SEQ ID NO: 26, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 27, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 28.

[0052] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, Ab is a chimeric antibody or a humanized antibody. In some embodiments, Ab is a humanized antibody.

[0053] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab comprises a framework region (FR) of a human antibody.

[0054] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13.

[0055] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the amino acid sequence of the heavy chain variable region of Ab is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 13.

[0056] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody; preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30.

[0057] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody; preferably, the amino acid sequence of the heavy chain variable region of Ab is as shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 30.

[0058] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the light chain constant region is a human kappa or lambda light chain constant region.

[0059] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 14 or 18, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 14, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 15.

[0060] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof according to any of the preceding items, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 18, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 15.

[0061] In some embodiments, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof according to any of the preceding items, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 31, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 31, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 32.

[0062] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO: 16 or 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 17.

[0063] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 16 or 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 17.

[0064] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 16, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 17.

[0065] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is a KLB antibody; preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 17.

[0066] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody; preferably, the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34.

[0067] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Ab is an ActRIIA / B antibody; preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 33, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 34.

[0068] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is -L a -L b -L c -L d -L e -,

[0069] L a Selected from: The asterisk * indicates that b Combined, wavy lines Indicates binding to the reconstructed sugar chain of Ab;

[0070] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-、-C(O)-(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR a R b -CR c R d -O)4-CR a R b -CR c R d -C(O)-、-CR f Rg -OC(O)- and -OC(O)-;

[0071] L c -NR h -CR i R j -CR m R n -;

[0072] L d is a PEG unit;

[0073] L e is -C(O)-;

[0074] R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, amino, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0075] or R a and R b Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R c and R d Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R f and R g Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R i and R j Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R m and R n Together with the carbon atom to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0076] or R a and R c Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, Ri and R m Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, which is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino.

[0077] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is -L a -L b -L c -L d -L e -,

[0078] L a Selected from: The asterisk * indicates that b Combined, wavy lines Indicates binding to the reconstructed sugar chain of Ab;

[0079] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-、-C(O)-(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR aR b -CR c R d -O)4-CR a R b -CR c R d -C(O)-、-CR f R g -OC(O)- and -OC(O)-;

[0080] L c -NR h -CR i R j -CR m R n -;

[0081] L d is a PEG unit;

[0082] L e is -C(O)-;

[0083] R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, hydroxy, cyano, amino, 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl, wherein the 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl are optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino groups;

[0084] or R a and R b Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R c and R dTogether with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R f and R g Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R i and R j Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R m and R n Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the 3 to 6-membered cycloalkyl or the 3 to 6-membered heterocyclic group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino groups;

[0085] or R a and R c Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R i and R m Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the 3 to 6-membered cycloalkyl or the 3 to 6-membered heterocyclic group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 The alkyl group is substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino.

[0086] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L b -C(O)-CR a R b -CR c R d -C(O)-, and R a 、R b 、R c and R d The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl; preferably, L b It is -C(O)-CH2-CH2-C(O)-.

[0087] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L c -NR h -CR i Rj -CR m R n -, and R h 、R i 、R j 、R m and R n The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl; preferably, L c It is -NH-CH2-CH2-.

[0088] In one embodiment, the antibody-polypeptide conjugate or pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the PEG unit comprises between 1 and 36 EG units, or between 4 and 24 EG units. In one embodiment, the PEG segment comprises 2 EG units, or 4 EG units, or 6 EG units, or 8 EG units, or 10 EG units, or 12 EG units, or 14 EG units, or 16 EG units, or 18 EG units, or 20 EG units, or 22 EG units, or 24 EG units.

[0089] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L d is 1 to 36 -O-CH2-CH2-; preferably, L d is 4 to 24 -O-CH2-CH2-; more preferably, L d is 4, 8, 12 or 24 -O-CH2-CH2-; most preferably, L d It is 8 -O-CH2-CH2-.

[0090] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is -L a -L b -L c -L d -L e -,

[0091] L a Selected from: The asterisk * indicates that b Combined, wavy lines Indicates binding to the reconstructed sugar chain of Ab;

[0092] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-;

[0093] L c -NR h -CR i R j -CR m R n -;

[0094] L d 4 to 24 -O-CH2-CH2-;

[0095] L e is -C(O)-;

[0096] R a 、R b 、R c 、R d 、R h 、R i 、R j 、R m and R n The same or different, and each independently a hydrogen atom or a C 1-6 alkyl.

[0097] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is

[0098] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein L is

[0099] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0100] in:

[0101] wherein Ab and m are as defined in the general formula (I).

[0102] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0103] wherein Ab and m are as defined in the general formula (I).

[0104] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0105] in:

[0106] wherein Ab and m are as defined in the general formula (I).

[0107] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0108] in:

[0109] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0110] Preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0111] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 16, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 17;

[0112] m is an integer from 1 to 4; preferably, m is 1 or 2; more preferably, m is 2.

[0113] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0114] in:

[0115] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0116] Preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0117] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 16 or 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 17;

[0118] m is an integer from 1 to 4; preferably, m is 1 or 2; more preferably, m is 2.

[0119] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0120] in:

[0121] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0122] Preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30;

[0123] More preferably, the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34;

[0124] m is an integer from 1 to 4; preferably, m is 1 or 2; more preferably, m is 2.

[0125] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0126] in:

[0127] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0128] Preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0129] More preferably, the amino acid sequence of the heavy chain of Ab is as shown in SEQ ID NO: 16, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 17;

[0130] m is an integer from 1 to 4; preferably, m is 1 or 2; more preferably, m is 2.

[0131] In some embodiments, the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items has a structure as shown in general formula (II):

[0132] in:

[0133] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0134] Preferably, the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30;

[0135] More preferably, the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34;

[0136] m is an integer from 1 to 4; preferably, m is 1 or 2; more preferably, m is 2.

[0137] In another aspect, the present disclosure provides a compound represented by formula (IIa) or a salt thereof:

[0138] wherein Ab and m are as defined in formula (I) or formula (II).

[0139] In some embodiments, the compound represented by the general formula (IIa) or a salt thereof as described above, wherein Ab is a KLB antibody or an ActRIIA / B antibody;

[0140] Preferably, (1) HCDR1 of the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11; or

[0141] (2) HCDR1 of the heavy chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0142] More preferably, (1) the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0143] (2) the heavy chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30;

[0144] Most preferably, (1) the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO: 16 or 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 17; or

[0145] (2) the heavy chain of the Ab comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34;

[0146] The definition of m is the same as that of the general formula (I) or (II).

[0147] In another aspect, the present disclosure provides a glucagon-like peptide-1 receptor agonist peptide or an analog thereof.

[0148] In some embodiments, the aforementioned glucagon-like peptide-1 receptor agonist peptide or analog thereof is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR tripotactic peptide or analog thereof.

[0149] In some embodiments, the glucagon-like peptide-1 receptor agonist peptide or analog thereof as described in any of the preceding items is a GLP-1R monoagonist peptide.

[0150] In some embodiments, the glucagon-like peptide-1 receptor agonist peptide or analog thereof as described in any of the preceding items is a GLP-1R / GIPR dual agonist peptide or analog thereof.

[0151] In some embodiments, the glucagon-like peptide-1 receptor agonist peptide or analog thereof as described in any of the preceding items is a GLP-1R / GCGR dual agonist peptide or analog thereof.

[0152] In some embodiments, the glucagon-like peptide-1 receptor agonist peptide or analog thereof as described in any of the preceding items is a GLP-1R / GIPR / GCGR tripotactic peptide or analog thereof.

[0153] In some embodiments, the glucagon-like peptide-1 receptor agonist peptide or its analogue as described in any of the preceding items comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; preferably, it comprises the amino acid sequence of SEQ ID NO: 21.

[0154] In some embodiments, the glucagon-like peptide-1 receptor agonist peptide or its analogue as described in any of the preceding items has an amino acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; preferably, its amino acid sequence is shown in SEQ ID NO: 21.

[0155] In another aspect, the present disclosure provides a compound represented by general formula (Ib) or a salt thereof: L'-P (Ib)

[0156] in:

[0157] P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR tripotactic peptide or an analog thereof; more preferably, P comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22;

[0158] L' is L aa -L b -L c -L d -L e -,

[0159] L aa selected from: where the asterisk * indicates combination with L b ;

[0160] L b selected from -C(O)-CR a R b -CR c R d -C(O)-, -C(O)-(CR a R b -CR c R d )2-C(O)-, -C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-, -C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c ​​​​​​​​​​​​​​​​​​​​​​​​​​

[0161] L c -NR h -CR i R j -CR m R n -;

[0162] L d is a PEG unit;

[0163] L e is -C(O)-;

[0164] R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, amino, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0165] or R a and R b Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R c and R d Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R f and R g Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R i and R j Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R m and R n Together with the carbon atom to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0166] or R a and R c Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R i and R mTogether with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, which is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino.

[0167] In another aspect, the present disclosure provides a compound represented by general formula (Ib) or a salt thereof: L'-P (Ib)

[0168] in:

[0169] P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R / GIPR / GCGR tripeptide or an analog thereof; more preferably, the amino acid sequence of P is as shown in SEQ ID NO: 20 or SEQ ID NO: 21;

[0170] L' is L aa -L b -L c -L d -L e -,

[0171] L aa Selected from: The asterisk * indicates that b Combine;

[0172] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-、-C(O)-(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f Rg -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR a R b -CR c R d -O)4-CR a R b -CR c R d -C(O)-、-CR f R g -OC(O)- and -OC(O)-;

[0173] L c -NR h -CR i R j -CR m R n -;

[0174] L d is a PEG unit;

[0175] L e is -C(O)-;

[0176] R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, amino, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0177] or R a and R b Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R c and R d Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R f and R gTogether with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R i and R j Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R m and R n Together with the carbon atom to which they are attached, they form a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0178] or R a and R c Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, R i and R m Together with the carbon atoms to which they are attached, they form a cycloalkyl or heterocyclic group, which is optionally substituted with one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino.

[0179] In some embodiments, the compound represented by the general formula (Ib) or a salt thereof as described above, wherein P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR tripotactic peptide or an analog thereof; more preferably, P comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22;

[0180] L' is L aa -L b -L c -L d -L e -,

[0181] L aa Selected from: The asterisk * indicates that b Combine;

[0182] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-、-C(O)-(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NRe -(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR a R b -CR c R d -O)4-CR a R b -CR c R d -C(O)-、-CR f R g -OC(O)- and -OC(O)-;

[0183] L c -NR h -CR i R j -CR m R n -;

[0184] L d is a PEG unit;

[0185] L e is -C(O)-;

[0186] R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, hydroxy, cyano, amino, 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl, wherein the 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl are optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino groups;

[0187] or R a and R b Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R c and R d Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R f and R g Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R i and R j Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R m and R n Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino groups;

[0188] or R a and R c Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R i and R m Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the 3 to 6-membered cycloalkyl or the 3 to 6-membered heterocyclic group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 The alkyl group is substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino.

[0189] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items, wherein P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R / GIPR / GCGR tripeptide or an analog thereof; more preferably, the amino acid sequence of P is as shown in SEQ ID NO: 20 or SEQ ID NO: 21;

[0190] L' is L aa -L b -L c -L d -L e -,

[0191] L aa Selected from: The asterisk * indicates that b Combine;

[0192] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-、-C(O)-(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-、-C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR a R b -CR c R d-O)4-CR a R b -CR c R d -C(O)-、-CR f R g -OC(O)- and -OC(O)-;

[0193] L c -NR h -CR i R j -CR m R n -;

[0194] L d is a PEG unit;

[0195] L e is -C(O)-;

[0196] R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, hydroxy, cyano, amino, 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl, wherein the 3 to 6-membered cycloalkyl, 3 to 6-membered heterocyclyl, 6 to 10-membered aryl and 5 to 10-membered heteroaryl are optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino groups;

[0197] or R a and R b Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R c and R d Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R f and R gTogether with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R i and R j Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R m and R n Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino groups;

[0198] or R a and R c Together with the carbon atoms to which they are attached, they form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocyclic group, R i and R m Together with the carbon atoms to which they are attached, they form a 3 to 6-membered cycloalkyl or a 3 to 6-membered heterocyclic group, wherein the 3 to 6-membered cycloalkyl or the 3 to 6-membered heterocyclic group is optionally selected from oxo, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 The alkyl group is substituted by one or more substituents selected from hydroxyalkyl, hydroxy, cyano and amino.

[0199] In some embodiments, the compound represented by formula (Ib) or a salt thereof as described in any of the preceding items, wherein L aa Selected from: The asterisk * indicates that b combination.

[0200] In some embodiments, the compound represented by formula (Ib) or a salt thereof as described in any of the preceding items, wherein L b -C(O)-CR a R b -CR c R d -C(O)-, and R a 、R b 、R c and R d The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl; preferably, L b It is -C(O)-CH2-CH2-C(O)-.

[0201] In some embodiments, the compound represented by formula (Ib) or a salt thereof as described in any of the preceding items, wherein Lc -NR h -CR i R j -CR m R n -, and R h 、R i 、R j 、R m and R n The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl; preferably, L c It is -NH-CH2-CH2-.

[0202] In some embodiments, the compound represented by formula (Ib) or a salt thereof as described in any of the preceding items, wherein L d is 1 to 36 -O-CH2-CH2-; preferably, L d is 4 to 24 -O-CH2-CH2-; more preferably, L d is 4, 8, 12 or 24 -O-CH2-CH2-; most preferably, L d It is 8 -O-CH2-CH2-.

[0203] In some embodiments, the compound represented by formula (Ib) or a salt thereof as described in any of the preceding items, wherein L e It is -C(O)-.

[0204] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items, wherein P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR tripotactic peptide or an analog thereof; more preferably, P comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22;

[0205] L' is L aa -L b -L c -L d -L e -,

[0206] L aa Selected from: The asterisk * indicates that b Combine;

[0207] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-;

[0208] L c -NR h -CR i R j -CR m R n -;

[0209] L d 4 to 24 -O-CH2-CH2-;

[0210] L e is -C(O)-;

[0211] where R a 、R b 、R c 、R d 、R h 、R i 、R j 、R m and R n The same or different, and each independently a hydrogen atom or a C 1-6 alkyl.

[0212] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items, wherein P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R / GIPR / GCGR tripeptide or an analog thereof; more preferably, the amino acid sequence of P is as shown in SEQ ID NO: 20 or SEQ ID NO: 21;

[0213] L' is L aa -L b -L c -L d -L e -,

[0214] L aa Selected from: The asterisk * indicates that b Combine;

[0215] L b Selected from -C(O)-CR a R b -CR c R d -C(O)-;

[0216] L c -NR h -CR i R j -CR m R n -;

[0217] L d 4 to 24 -O-CH2-CH2-;

[0218] L e is -C(O)-;

[0219] where R a 、R b 、R c 、R d 、R h 、R i 、R j 、R m and R n The same or different, and each independently a hydrogen atom or a C 1-6 alkyl.

[0220] In some embodiments, the compound of formula (Ib) or a salt thereof as described in any of the preceding items has a structure as shown in formula (IIb): Wherein P' is the portion of P with the amino group removed from the end; preferably, P' is Among them L d The definition of is as defined in general formula (Ib).

[0221] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items is selected from the following structures:

[0222] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items is selected from the following structures:

[0223] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items is selected from the following structures:

[0224] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items is selected from the following structures:

[0225] In some embodiments, the compound represented by general formula (Ib) or a salt thereof as described in any of the preceding items is selected from the following structures:

[0226] In another aspect, the present disclosure provides a method for preparing an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as represented by formula (II), comprising the following steps:

[0227] The compound represented by general formula (IIa) or a salt thereof reacts with the compound represented by general formula (Ib) or a salt thereof to obtain the antibody-polypeptide conjugate represented by general formula (II) or a pharmaceutically acceptable salt thereof;

[0228] Where R is -LP;

[0229] Ab, L, P and m are as defined in the general formula (I) or (II), and L' is as defined in the general formula (Ib).

[0230] In another aspect, the present disclosure provides a compound represented by general formula (IIc) or a salt thereof: Among them L d The definition of is as defined in general formula (Ib) or general formula (IIb).

[0231] In some embodiments, the compound represented by the aforementioned general formula (IIc) or a salt thereof is selected from the following structures:

[0232] In some embodiments, the compound represented by the aforementioned general formula (IIc) or a salt thereof is selected from the following structures:

[0233] In another aspect, the present disclosure provides a method for preparing a compound represented by formula (IIb) or a salt thereof, comprising the following steps:

[0234] The compound represented by general formula (IIc) or a salt thereof is reacted with a carboxyl protecting agent (e.g., N-hydroxysuccinimide (HOSU)), and then reacted with P to obtain a compound represented by general formula (IIb) or a salt thereof;

[0235] P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR tripotactic peptide or an analog thereof; more preferably, P comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22;

[0236] P' is the part with the amino group removed from the end of P;

[0237] Among them L d The definition of is as defined in general formula (Ib) or general formula (IIb).

[0238] In another aspect, the present disclosure provides a method for preparing a compound represented by formula (IIb) or a salt thereof, comprising the following steps:

[0239] The compound represented by general formula (IIc) or a salt thereof is reacted with a carboxyl protecting agent (e.g., N-hydroxysuccinimide (HOSU)), and then reacted with P to obtain a compound represented by general formula (IIb) or a salt thereof;

[0240] P is a glucagon-like peptide-1 receptor agonist peptide or an analog thereof; preferably, P is a GLP-1R / GIPR / GCGR tripeptide or an analog thereof; more preferably, the amino acid sequence of P is as shown in SEQ ID NO: 20 or SEQ ID NO: 21;

[0241] P' is the part with the amino group removed from the end of P;

[0242] Among them L d The definition of is as defined in general formula (Ib) or general formula (IIb).

[0243] In some embodiments, the antibody-polypeptide conjugate represented by general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein m refers to the average number of polypeptides carried by each antibody-polypeptide conjugate molecule in the antibody-polypeptide conjugate population, and can also be expressed as a ratio of polypeptide to antibody.

[0244] In some embodiments, the antibody-polypeptide conjugate represented by general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the average of any two values; preferably, m is 1 to 10; further preferably, m is 1 to 4; more preferably, m is 1 or 2; most preferably, m is 2.

[0245] In some embodiments, the antibody-polypeptide conjugate represented by general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein m represents the average ratio of antibody-polypeptide, for example, m is an integer from 1 to 10; preferably, m is an integer from 1 to 4, that is, m is an integer from 1 to 4; further preferably, m is 1 or 2; more preferably, m is 2.

[0246] In some embodiments, the compound represented by the general formula (IIa) or a salt thereof as described in any of the preceding items, wherein m represents the average ratio of antibody-polypeptide, for example, m is an integer from 1 to 10; preferably, m is an integer from 1 to 4; further preferably, m is 1 or 2; more preferably, m is 2.

[0247] On the other hand, the present disclosure provides a pharmaceutical composition comprising an antibody-polypeptide conjugate represented by general formula (I) or general formula (II) as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0248] In another aspect, the present disclosure provides a pharmaceutical composition comprising the glucagon-like peptide-1 receptor agonist peptide or analog thereof as described in any one of the preceding items and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0249] In some embodiments, based on the total weight of the pharmaceutical composition, the pharmaceutical composition contains 0.01-99.99% of the antibody-polypeptide conjugate of general formula (I) or general formula (II) as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, or the glucagon-like peptide-1 receptor agonist peptide or its analogue as described in any of the preceding items. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the antibody-polypeptide conjugate of general formula (I) or general formula (II) as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, or the glucagon-like peptide-1 receptor agonist peptide or its analogue as described in any of the preceding items. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the antibody-polypeptide conjugate of general formula (I) or general formula (II) as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, or the glucagon-like peptide-1 receptor agonist peptide or its analogue as described in any of the preceding items. In some embodiments, the pharmaceutical composition contains 1%-99% of the antibody-polypeptide conjugate of general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof, or the glucagon-like peptide-1 receptor agonist peptide or an analog thereof as described in any of the preceding items. In some embodiments, based on the total weight of the pharmaceutical composition, the pharmaceutical composition contains 2%-98% of the antibody-polypeptide conjugate of general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof, or the glucagon-like peptide-1 receptor agonist peptide or an analog thereof as described in any of the preceding items.

[0250] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% of a pharmaceutically acceptable diluent or excipient, based on the total weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% of a pharmaceutically acceptable diluent or excipient. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 1% to 99% of a pharmaceutically acceptable diluent or excipient. In some embodiments, the pharmaceutical composition contains 2% to 98% of a pharmaceutically acceptable diluent or excipient.

[0251] On the other hand, the present disclosure provides a method for preventing or treating a disease, comprising administering to a subject an antibody-polypeptide conjugate represented by formula (I) or formula (II) as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0252] In another aspect, the present disclosure provides a method for preventing or treating a disease, comprising administering the glucagon-like peptide-1 receptor agonist peptide or an analog thereof as described in any one of the preceding items to a subject.

[0253] On the other hand, the present disclosure provides a use in preparing a medicament for preventing or treating a disease, comprising administering to a subject an antibody-polypeptide conjugate represented by general formula (I) or general formula (II) as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0254] In another aspect, the present disclosure provides a use in preparing a medicament for preventing or treating a disease, comprising administering the glucagon-like peptide-1 receptor agonist peptide or an analog thereof as described in any of the preceding items to a subject.

[0255] In another aspect, the present disclosure provides an antibody-polypeptide conjugate of formula (I) or formula (II) as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use as a medicament. In some embodiments, the medicament is used to prevent or treat a disease.

[0256] In another aspect, the present disclosure provides a glucagon-like peptide-1 receptor agonist peptide or an analog thereof as described in any of the preceding items for use as a medicament. In some embodiments, the medicament is used for preventing or treating a disease.

[0257] In some embodiments, the disease as described in any of the preceding items is a GLP-1R mediated disease.

[0258] In some embodiments, the disease as described in any of the preceding items is a GCGR-mediated disease.

[0259] In some embodiments, the disease as described in any of the preceding items is a GIPR-mediated disease.

[0260] In some embodiments, the disease as described in any of the preceding items is diabetes, obesity, liver disease, coronary artery disease or kidney disease; preferably, the disease is type II diabetes, obesity, metabolic dysfunction-associated fatty liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH, such as non-alcoholic steatohepatitis). BRIEF DESCRIPTION OF THE DRAWINGS

[0261] Figure 1-1 shows the curve of the activation of KLB & FGFR2c signaling pathway by the antibodies and conjugated molecules of the present application.

[0262] Figure 1-2 shows the curves of the antibodies and conjugated molecules of the present application for activating the KLB & FGFR3c signaling pathway.

[0263] Figures 1-3 show the curves of the antibodies and conjugated molecules of the present application on the activation of KLB & FGFR4 signaling pathways.

[0264] Figures 2-1, 2-2, 2-3 and 2-4 show the effects of the conjugated molecules of the present application on glucose tolerance in wild-type mice.

[0265] FIG3-1 shows the effects of the conjugated molecules APC-2 and APC-4 of the present application on the body weight of DIO mice.

[0266] FIG3-2 shows the effects of the conjugated molecules APC-2 and APC-4 of the present application on the food intake of DIO mice.

[0267] Figure 3-3 shows the effects of the conjugated molecules APC-2 and APC-4 of the present application on random blood glucose in DIO mice.

[0268] Figures 3-4 and 3-5 show the effects of the conjugated molecules APC-2 and APC-4 of the present application on glucose tolerance in DIO mice.

[0269] Figures 3-6 show the effects of the conjugated molecules APC-2 and APC-4 of the present application on serum cholesterol, serum low-density lipoprotein cholesterol, liver weight and serum alanine aminotransferase in DIO mice.

[0270] Figures 3-7 show the statistical results of Oil Red staining of the effects of the coupled molecules APC-2 and APC-4 of the present application on fat droplets in the liver tissue of DIO mice.

[0271] FIG4-1 shows the effect of the conjugated molecule APC-3 of the present application on the body weight of DIO mice.

[0272] FIG4-2 shows the effect of the conjugated molecule APC-3 of the present application on the food intake of DIO mice.

[0273] FIG4-3 shows the effect of the conjugated molecule APC-3 of the present application on random blood glucose in DIO mice.

[0274] Figures 4-4 and 4-5 show the effect of the coupled molecule APC-3 of the present application on glucose tolerance in DIO mice.

[0275] Figures 4-6 show the effects of the conjugated molecule APC-3 of the present application on liver weight, liver triglycerides, serum cholesterol, serum triglycerides, serum low-density lipoprotein cholesterol, serum alanine aminotransferase and serum aspartate aminotransferase in DIO mice.

[0276] FIG5-1 shows the effects of the antibody Ab1 and the conjugated molecule APC-4 of the present application on glucose tolerance in db / db mice.

[0277] FIG5-2 shows the effects of the antibody Ab1 and the coupled molecule APC-4 of the present application on random blood glucose in db / db mice.

[0278] FIG5-3 shows the effects of the antibody Ab1 and the conjugated molecule APC-4 of the present application on the fasting blood glucose levels of db / db mice on the 3rd and 31st days.

[0279] FIG5-4 shows the effects of the antibody Ab1 and the conjugated molecule APC-4 of the present application on HbA1c in db / db mice.

[0280] FIG5-5 shows the effects of the antibody Ab1 and the conjugated molecule APC-4 of the present application on serum triglycerides in db / db mice.

[0281] Figures 5-6 show the effects of the antibody Ab1 and the conjugated molecule APC-4 of the present application on the food intake of db / db mice. DETAILED DESCRIPTION

[0282] the term

[0283] In order to make the present disclosure more easily understood, certain technical and scientific terms are described below. Unless otherwise specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0284] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0285] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0286] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include mIL-2, IFNγ, TNFα, CCL-2, and IL-6.

[0287] The term "and / or" is intended to include both "and" and "or". For example, the phrase "A, B and / or C" is intended to include each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0288] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0289] The term "native GLP-1" refers to a peptide comprising the sequence of human GLP-1 (7-36 or 7-37).

[0290] The term "native GIP" refers to a peptide comprising the sequence of human GIP sequence (1-42).

[0291] The term "native GCG" refers to a peptide comprising the sequence of human GCG sequence (1-29).

[0292] The terms "GLP-1", "GIP" or "GCG" unless further explained refer to native GLP-1, native GIP or native GCG, respectively.

[0293] The term "glucagon-like peptide-1 receptor agonist peptide" refers to GLP-1R agonist peptides, including GLP-1R single agonist peptides (i.e., peptides having single agonist activity against GLP-1R), as well as dual agonist peptides (including GLP-1R / GIPR dual agonist peptides (i.e., peptides having dual agonist activity against GLP-1R / GIPR), GLP-1R / GCGR dual agonist peptides (peptides having dual agonist activity against GLP-1R / GCGR)) and tripo-agonist peptides (GLP-1R / GIPR / GCGR tripo-agonist peptides (i.e., peptides having triple agonist activity against GLP-1R / GIPR / GCGR)).

[0294] The term "glucagon-like peptide-1 receptor agonist peptide analog" refers to a GLP-1R agonist peptide analog, including GLP-1R single agonist peptide analogs, as well as dual agonist peptide (GLP-1R / GIPR dual agonist peptide or GLP-1R / GCGR dual agonist peptide) analogs and tripotactin (GLP-1R / GIPR / GCGR tripotactin) analogs. For example, a peptide having an amino acid sequence containing 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof compared to the amino acid sequence of the GLP-1R agonist peptide. GLP-1R agonist peptide analogs include amidated forms, acid forms, pharmaceutically acceptable salt forms, and any other physiologically active forms of the molecule.

[0295] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0296] The term "amino acid mutation" includes amino acid substitutions (also known as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is expected that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 82aR means that the amino acid residue at position 82a is R. S82aR means that the amino acid residue at position 82a (also known as 82A) has mutated from S to R.

[0297] The term "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by a disulfide bond, and each heavy chain is linked to a light chain by a disulfide bond, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and full-length antibodies, as long as they exhibit the desired antigen-binding activity. For example, chimeric antibodies, humanized antibodies and fully human antibodies.

[0298] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, they can be divided into bispecific antibodies with different valencies, such as bispecific antibodies with different valencies. Based on whether the structure is symmetrical, they can be divided into bispecific antibodies with different symmetric structures and bispecific antibodies with different asymmetric structures. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, are formed by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue permeability. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragments) have a relatively large molecular weight. The Fc fragment helps purify the antibody and improve its solubility and stability. The Fc part may also bind to the receptor FcRn to increase the antibody serum half-life.

[0299] "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by a heavy chain constant region, and the natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL). The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody structure or having a heavy chain with an Fc region as defined herein. A natural complete antibody light chain comprises a light chain variable region VL and a constant region CL, wherein VL is located at the amino terminus of the light chain, and the light chain constant region comprises a kappa chain and a lambda chain; a heavy chain comprises a variable region VH and constant regions (CH1, CH2, and CH3), wherein VH is located at the amino terminus of the heavy chain, and the constant region is at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0300] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy or light chain involved in antigen binding. Herein, the heavy chain variable region (VH) and light chain variable region (VL) of an antibody each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs). The term "complementarity determining region" or "CDR" refers to the region within the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues excluding the CDR residues. The VH comprises three CDR regions: HCDR1, HCDR2, and HCDR3; the VL comprises three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus), in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0301] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art. Exemplary, as shown in Table 1.

[0302] Table 1. Relationships between CDR numbering systems

[0303] Unless otherwise specified, the variable regions and CDRs in this disclosure are numbered using the Kabat numbering convention.

[0304] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and reconstructed Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, such as by deleting the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) or by deleting the C-terminal glycine and lysine in the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise indicated, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0305] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0306] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant region and the framework region portion of the variable region).

[0307] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably to refer to antibodies whose variable and constant regions are human sequences. The term encompasses antibodies that are derived from human genes but have altered sequences, for example, to reduce potential immunogenicity, increase affinity, or eliminate cysteines or glycosylation sites that may cause undesirable folding. The term encompasses antibodies that are recombinantly produced in non-human cells (which may confer glycosylation that is not characteristic of human cells). The term also encompasses antibodies that have been bred in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0308] The term "affinity" refers to the overall strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0309] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured using methods well known in the art. For example, a biosensor system such as a system measuring surface plasmon resonance (e.g., Biacore) is used, or affinity in a solution is measured by solution equilibrium titration (SET).

[0310] The term "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0311] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence mutated Fc region) and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0312] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for the natural mutations that may be present in small amounts. In contrast, polyclonal antibody preparations are typically comprised of a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" represents the characteristic of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any ad hoc method. In some embodiments, the antibody provided by the present disclosure is a monoclonal antibody.

[0313] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody) and that can additionally be used in an animal to produce antibodies that can bind to the antigen. An antigen may have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0314] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope can be formed by a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity by folding of the antigen (i.e., by tertiary folding of a proteinaceous antigen). Conformational epitopes differ from linear epitopes in that antibody binding to a conformational epitope is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0315] The terms "capable of specific binding", "specific binding" or "binding" refer to an antibody that is able to bind to an antigen or an epitope of the antigen with a higher affinity than to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 In some embodiments, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0316] The term "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells with lytic activity (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fc gamma receptors (Fc gamma R) expressed on effector cells. For example, NK cells express Fc gamma RIIIa, while monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIIIa. The ADCC activity of the antibodies provided herein can be assessed using in vitro assays using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from the lysed cells (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins).

[0317] The term "antibody-dependent cellular phagocytosis (ADCP)" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes, such as macrophages or dendritic cells.

[0318] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death induction in which the Fc effector domain of a target-bound antibody binds to and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0319] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0320] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of the two sequences are identical at equivalent positions when two sequences are optimally aligned, introducing gaps when necessary, to obtain maximum sequence identity percentage, and not considering any conservative substitutions as part of sequence identity. For measuring sequence identity percentage, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters applicable to measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0321] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell in which they are introduced. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a vector that can transform a host cell and contains a nucleic acid sequence for the expression of one or more heterologous coding regions that are operably connected thereto, including guidance and / or control (together with the host cell). Expression constructs can include but are not limited to affecting or controlling transcription, translation and affecting the sequence of RNA splicing of the coding region operably connected thereto when there are introns.

[0322] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cells are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia truncatula, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha, polymorpha), Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa.

[0323] "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cell are included.

[0324] The term "alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., C 1-20 The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 alkyl), more preferably an alkyl group having 1 to 6 carbon atoms (i.e., C 1-6 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, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 ,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. The alkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0325] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups may be substituted or unsubstituted. When substituted, they may be substituted at any available point of attachment, with the substituent preferably being selected from one or more of a D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0326] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic all-carbon ring (i.e., a monocyclic cycloalkyl) or a polycyclic ring system (i.e., a polycyclic cycloalkyl) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3- to 20-membered cycloalkyl). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3- to 12-membered cycloalkyl group), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3- to 8-membered cycloalkyl group), and most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3- to 6-membered cycloalkyl group).

[0327] Non-limiting examples of the monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl and cyclooctyl.

[0328] The polycyclic cycloalkyl group includes: spirocycloalkyl group, fused cycloalkyl group and bridged cycloalkyl group.

[0329] The term "spiroalkyl" refers to a polycyclic ring system having a common carbon atom (called a spiro atom) between the rings, which may contain one or more double bonds within the ring, or one or more heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized to form nitrogen oxides; the sulfur may be optionally oxoed to form sulfoxides or sulfones, but does not include -OO-, -OS- or -SS-), provided that it contains at least one all-carbon ring and the point of attachment is on the all-carbon ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered spiroalkyl). The spiroalkyl preferably has 6 to 14 ring atoms (i.e., a 6- to 14-membered spiroalkyl), and more preferably has 7 to 10 ring atoms (i.e., a 7- to 10-membered spiroalkyl). The spirocycloalkyl group includes a monospirocycloalkyl group and a polyspirocycloalkyl group (such as a bispirocycloalkyl group, etc.), preferably a monospirocycloalkyl group or a bispirocycloalkyl group, more preferably a 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered monospirocycloalkyl group. Non-limiting examples include:

[0330] Its connection point can be at any position;

[0331] wait.

[0332] The term "fused cycloalkyl" refers to a polycyclic ring system in which two adjacent carbon atoms are shared between the rings, which is a monocyclic cycloalkyl fused to one or more monocyclic cycloalkyls, or a monocyclic cycloalkyl fused to one or more heterocyclyls, aryls, or heteroaryls, wherein the point of attachment is on the monocyclic cycloalkyl, which may contain one or more double bonds within the ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered fused cycloalkyl). The fused cycloalkyl is preferably a fused cycloalkyl having 6 to 14 ring atoms (i.e., a 6- to 14-membered fused cycloalkyl), more preferably a fused cycloalkyl having 7 to 10 ring atoms (i.e., a 7- to 10-membered fused cycloalkyl). The fused cycloalkyl group includes bicyclic fused cycloalkyl groups and polycyclic fused cycloalkyl groups (such as tricyclic fused cycloalkyl groups, tetracyclic fused cycloalkyl groups, etc.), preferably bicyclic fused cycloalkyl groups or tricyclic fused cycloalkyl groups, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered bicyclic fused cycloalkyl groups. Non-limiting examples include:

[0333] Its connection point can be at any position;

[0334]

[0335] wait.

[0336] The term "bridged cycloalkyl" refers to a full carbon polycyclic ring system that shares two carbon atoms that are not directly connected between the rings, which may contain one or more double bonds within the ring and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., a 5 to 20-membered bridged cycloalkyl). The bridged cycloalkyl preferably has a bridged cycloalkyl of 6 to 14 carbon atoms (i.e., a 6 to 14-membered bridged cycloalkyl), more preferably a bridged cycloalkyl of 7 to 10 carbon atoms (i.e., a 7 to 10-membered bridged cycloalkyl). The bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic bridged cycloalkyl or tricyclic bridged cycloalkyl. Non-limiting examples include:

[0337] Its connection point can be at any position.

[0338] The cycloalkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0339] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl) or a polycyclic heterocyclic ring system (i.e., a polycyclic heterocyclyl) containing at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxoed, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-) in the ring, and having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3- to 20-membered heterocyclyl). The heterocyclic group is preferably a heterocyclic group having 3 to 12 ring atoms (i.e., a 3- to 12-membered heterocyclic group), for example, a 4- to 12-membered heterocyclic group containing at least one nitrogen atom; further preferably, a heterocyclic group having 3 to 8 ring atoms (i.e., a 3- to 8-membered heterocyclic group); more preferably, a heterocyclic group having 3 to 6 ring atoms (i.e., a 3- to 6-membered heterocyclic group); and most preferably, a heterocyclic group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heterocyclic group).

[0340] Non-limiting examples of the monocyclic heterocyclic group include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and homopiperazinyl.

[0341] The polycyclic heterocyclic group includes a spiro heterocyclic group, a fused heterocyclic group and a bridged heterocyclic group.

[0342] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic ring system in which the rings share one atom (called a spiro atom), which may contain one or more double bonds in the ring and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS- or -SS-), provided that it contains at least one monocyclic heterocyclic group and the point of attachment is on the monocyclic heterocyclic group, which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered spiroheterocyclyl). The spiro heterocyclic radical preferably has a spiro heterocyclic radical (i.e., a 6 to 14 yuan spiro heterocyclic radical) of 6 to 14 ring atoms, more preferably a spiro heterocyclic radical (i.e., a 7 to 10 yuan spiro heterocyclic radical) with 7 to 10 ring atoms. The spiro heterocyclic radical includes monospiro heterocyclic radical and polyspiro heterocyclic radical (such as dispiro heterocyclic radical etc.), preferably monospiro heterocyclic radical or dispiro heterocyclic radical, more preferably 3 yuan / 4 yuan, 3 yuan / 5 yuan, 3 yuan / 6 yuan, 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 3 yuan, 5 yuan / 4 yuan, 5 yuan / 5 yuan, 5 yuan / 6 yuan, 5 yuan / 7 yuan, 6 yuan / 3 yuan, 6 yuan / 4 yuan, 6 yuan / 5 yuan, 6 yuan / 6 yuan, 6 yuan / 7 yuan, 7 yuan / 5 yuan or 7 yuan / 6 yuan monospiro heterocyclic radical. Non-limiting examples include:

[0343] wait.

[0344] The term "fused heterocyclyl" refers to a polycyclic heterocyclic ring system that shares two adjacent atoms between the rings, which may contain one or more double bonds within the ring and at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), which is a monocyclic heterocyclyl fused to one or more monocyclic heterocyclyls, or a monocyclic heterocyclyl fused to one or more cycloalkyl, aryl, or heteroaryl groups, wherein the point of attachment is on the monocyclic heterocyclyl, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered fused heterocyclyl). The fused heterocyclic radical preferably has a fused heterocyclic radical of 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic radical), more preferably a fused heterocyclic radical of 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic radical). The fused heterocyclic radical includes bicyclic and polycyclic fused heterocyclic radicals (such as tricyclic fused heterocyclic radicals, tetracyclic fused heterocyclic radicals, etc.), preferably bicyclic fused heterocyclic radicals or tricyclic fused heterocyclic radicals, more preferably 3 yuan / 4 yuan, 3 yuan / 5 yuan, 3 yuan / 6 yuan, 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 3 yuan, 5 yuan / 4 yuan, 5 yuan / 5 yuan, 5 yuan / 6 yuan, 5 yuan / 7 yuan, 6 yuan / 3 yuan, 6 yuan / 4 yuan, 6 yuan / 5 yuan, 6 yuan / 6 yuan, 6 yuan / 7 yuan, 7 yuan / 5 yuan or 7 yuan / 6 yuan bicyclic fused heterocyclic radicals. Non-limiting examples include:

[0345] wait.

[0346] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic ring system that shares two atoms that are not directly connected between the rings, which may contain one or more double bonds within the ring and at least one (e.g., 1, 2, 3, or 4) heteroatom selected from nitrogen, oxygen, and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS-, or -SS-), and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered bridged heterocyclic group). The bridged heterocyclic group is preferably a bridged heterocyclic group having 6 to 14 ring atoms (i.e., a 6- to 14-membered bridged heterocyclic group), and more preferably a bridged heterocyclic group having 7 to 10 ring atoms (i.e., a 7- to 10-membered bridged heterocyclic group). According to the number of constituent rings, heterocyclic groups can be divided into bicyclic bridged heterocyclic groups and polycyclic bridged heterocyclic groups (such as tricyclic bridged heterocyclic groups, tetracyclic bridged heterocyclic groups, etc.), preferably bicyclic bridged heterocyclic groups or tricyclic bridged heterocyclic groups. Non-limiting examples include:

[0347] wait.

[0348] The heterocyclic group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment. The substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclic group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group.

[0349] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aromatic group) or a polycyclic aromatic ring system (i.e., a polycyclic aromatic group) having a conjugated π electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 6- to 14-membered aromatic group). The aryl group is preferably an aromatic group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aromatic group). The monocyclic aromatic group is, for example, a phenyl group. Non-limiting examples of the polycyclic aromatic group include: naphthyl, anthracenyl, phenanthrenyl, etc. The polycyclic aromatic group also includes a phenyl group fused with one or more heterocyclic groups or cycloalkyl groups, or a naphthyl group fused with one or more heterocyclic groups or cycloalkyl groups, wherein the connection point is on the phenyl group or naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples include:

[0350] wait.

[0351] The aryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0352] The term "heteroaryl" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl) having a conjugated π electron system, which contains at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but excluding -OO-, -OS- or -SS-), and has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl group), more preferably a monocyclic heteroaryl group having 5 or 6 ring atoms (i.e., a 5- or 6-membered monocyclic heteroaryl group) or a bicyclic heteroaryl group having 8 to 10 ring atoms (i.e., an 8- to 10-membered bicyclic heteroaryl group), and most preferably a 5- or 6-membered monocyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur in the ring, or an 8- to 10-membered bicyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur in the ring.

[0353] The monocyclic heteroaryl groups include, but are not limited to, furyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazanyl, pyrrolyl, N-alkylpyrrolyl, pyridyl, pyrimidinyl, pyridonyl, N-alkylpyridone (e.g. etc.), pyrazinyl, pyridazinyl, etc.

[0354] The polycyclic heteroaryl groups include, but are not limited to, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, phthalazinyl, benzimidazolyl, benzothiophenyl, quinazolinyl, benzothiazolyl, carbazolyl, and the like. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused to one or more aromatic groups, wherein the point of attachment is on the aromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused to one or more cycloalkyl or heterocyclic groups, wherein the point of attachment is on the monocyclic heteroaromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system. Non-limiting examples include:

[0355] wait.

[0356] The heteroaryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a D atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0357] The above-mentioned cycloalkyl, heterocyclyl, aryl and heteroaryl groups include residues derived from a parent ring atom by removing one hydrogen atom, or residues derived from the same parent ring atom or two different ring atoms by removing two hydrogen atoms, i.e., "divalent cycloalkyl", "divalent heterocyclyl", "arylene" and "heteroarylene".

[0358] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Two configurations.

[0359] The compounds of the present disclosure include all suitable isotopic derivatives of the compounds thereof. The term "isotopic derivative" refers to a compound in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass. Examples of isotopes that can be introduced into the compounds of the present disclosure include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, for example, 2 H (deuterium, D), 3 H (tritium, T), 11 C. 13 C. 14 C. 15 N. 17 O. 18 O. 32 p、 33 p、 33 S. 34 S. 35 S. 36 S. 18 F. 36 Cl, 82 Br, 123 I. 124 I. 125 I. 129 I and 131 I, etc., preferably deuterium.

[0360] Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations of the compounds disclosed herein, whether radioactive or not, are encompassed by the present disclosure. Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom, where the deuterium replacement can be partial or complete. Partial deuterium replacement refers to the replacement of at least one hydrogen atom with at least one deuterium atom.

[0361] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0362] The term "pharmaceutical composition" refers to a mixture containing one or more antibody-polypeptide conjugates described herein or pharmaceutically acceptable salts thereof and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0363] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0364] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. In certain embodiments, the individual or subject is a human.

[0365] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0366] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

[0367] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions that attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of a disease or slow the progression of a disease.

[0368] "Effective amount" is generally enough to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the damage (e.g., lung disease) caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is enough to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete treatment or prophylactic effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0369] Exemplary antibody-polypeptide conjugates or pharmaceutically acceptable salts thereof

[0370] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0371] in:

[0372] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0373] m is 2.

[0374] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0375] in:

[0376] the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0377] m is 2.

[0378] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0379] in:

[0380] The amino acid sequence of the heavy chain of Ab is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain is shown in SEQ ID NO: 17;

[0381] m is 2.

[0382] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0383] in:

[0384] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0385] m is 2.

[0386] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0387] in:

[0388] the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0389] m is 2.

[0390] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0391] in:

[0392] The amino acid sequence of the heavy chain of Ab is shown in SEQ ID NO: 16 or 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 17;

[0393] m is 2.

[0394] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0395] in:

[0396] the Ab's heavy chain variable region HCDR1 comprises the amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region LCDR1 comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0397] m is 2.

[0398] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0399] in:

[0400] the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30;

[0401] m is 2.

[0402] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0403] in:

[0404] the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34;

[0405] m is 2.

[0406] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0407] in:

[0408] (1) HCDR1 of the heavy chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 6, HCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 11; or

[0409] (2) HCDR1 of the heavy chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 28;

[0410] m is 2.

[0411] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0412] in:

[0413] (1) the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0414] (2) the heavy chain variable region of the Ab comprises the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30;

[0415] m is 2.

[0416] The embodiments disclosed herein disclose an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II):

[0417] in:

[0418] (1) the heavy chain of the Ab comprises the amino acid sequence of SEQ ID NO: 16, and the light chain comprises the amino acid sequence of SEQ ID NO: 17; or

[0419] (2) the heavy chain of the Ab comprises the amino acid sequence of SEQ ID NO: 33, and the light chain comprises the amino acid sequence of SEQ ID NO: 34;

[0420] m is 2.

[0421] DETAILED DESCRIPTION

[0422] The present disclosure is further described below with reference to the following examples and test cases, but these examples and test cases are not intended to limit the scope of the present disclosure. Experimental methods not specifying specific conditions in the examples or test cases of this disclosure generally followed conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents and materials not specifying their sources were purchased commercially.

[0423] Example

[0424] Example 1-1. Construction of recombinant cell lines

[0425] 1. Construction of human and cynomolgus macaque KLB & FGFR1c overexpressing cell lines

[0426] Cell lines expressing human KLB & FGFR1c complex (human KLB sequence see Uniprot ID: Q86Z14, human FGFR1c sequence see Uniprot ID: P11362) and cynomolgus monkey KLB & FGFR1c complex (cynomolgus monkey KLB sequence see Genebank ID: EHH53620.1, cynomolgus monkey FGFR1c sequence see SEQ ID NO: 1) were constructed.

[0427] Methods: The full-length genes of human KLB, monkey KLB, human FGFR1c, and monkey FGFR1c were cloned into the lentiviral expression vector pCDH (System bioscience, 01.SBI.CD514B-1). The viral expression vectors of KLB and FGFR1c carried different resistance genes. KLB and FGFR1c viruses of different species were packaged, respectively. HEK293T cells were co-transfected with the three plasmids pVSV-G, pCMV-dR8.91, pCDH-KLB, or pCDH-FGFR1c. CRL-11268) packaged viruses. To construct complex-overexpressing cell lines, CHO-K1 cells (ATCC, CCL-61) were co-infected with packaged viruses containing the KLB and FGFR1c genes at a specific ratio. 48 hours after infection, the virus-containing culture supernatant was removed, and after 5 days of pressure selection with the addition of the corresponding antibiotics, pools of CHO-K1 cells expressing the human or monkey KLB & FGFR1c complex were obtained. The cell pool expressing the human KLB & FGFR1c complex was flow cytometry-sorted to obtain monoclonal CHO-K1 cells stably expressing hKLB & hFGFR1c.

[0428] Amino acid sequence of cynomolgus monkey FGFR1c:

[0429] Note: In the above sequence,

[0430] 2. Construction of human KLB&FGFR2c, KLB&FGFR3c, and KLB&FGFR4 overexpressing cell lines

[0431] Cell lines expressing human KLB & FGFR2c complex (human KLB sequence see Uniprot ID: Q86Z14, human FGFR2c sequence see SEQ ID NO: 2), human KLB & FGFR3c complex (human FGFR3c sequence see SEQ ID NO: 3), and human KLB & FGFR4 complex (human FGFR4 sequence see SEQ ID NO: 4) were constructed.

[0432] A CHO-K1-hKLB monoclonal cell line stably expressing human KLB was constructed by lentiviral infection. Then, CHO-K1-hKLB&hFGFR2c, CHO-K1-hKLB&hFGFR3c, and CHO-K1-hKLB&hFGFR4 cell pools stably expressing human FGFR2c (human FGF receptor 2c), human FGFR3c (human FGF receptor 3c), and human FGFR4 (human FGF receptor 4) were constructed by lentiviral infection, respectively.

[0433] Amino acid sequence of human FGFR2c:

[0434] Amino acid sequence of human FGFR3c:

[0435] Amino acid sequence of human FGFR4:

[0436] 3. Construction of luciferase reporter gene overexpression cell lines expressing human, cynomolgus monkey, and mouse GLP1R and CRE and their regulation

[0437] Cell lines expressing human GLP1R (for human GLP1R sequence, see Uniprot ID: P43220), cynomolgus macaque GLP1R (for cynomolgus macaque GLP1R sequence, see Uniprot ID: A0A2K5WDY5), mouse GLP1R (for mouse GLP1R sequence, see Uniprot ID: O35659) and CRE and its regulated luciferase reporter gene stably expressed were constructed.

[0438] Expression plasmids encoding the full-length genes of human GLP1R, cynomolgus macaque GLP1R, and mouse GLP1R were obtained by molecular cloning. Viruses were then packaged and used to infect CHO-K1 cells. Puromycin selection was performed to obtain GLP1R / CHO-K1 cells that highly express human, cynomolgus macaque, and mouse GLP1R. The pGL4.29[CRE / hygro] plasmid was transfected into hGLP1R / CHO-K1, cynoGLP1R / CHO-K1, and mGLP1R / CHO-K1 cells using Lipofectamine 3000. Hygromycin B was added for selection to obtain monoclonal stable transfectant cell lines: hGLP1R CRE / CHO-K1, cynoGLP1R CRE / CHO-K1, and mGLP1R CRE / CHO-K1.

[0439] 4. Construction of luciferase reporter gene overexpression cell lines expressing human, cynomolgus monkey and mouse GIPR and CRE and their regulation

[0440] Cell lines expressing human GIPR (for human GIPR sequence, see Uniprot ID: P48546), cynomolgus macaque GIPR (for cynomolgus macaque GIPR sequence, see Uniprot ID: A0A2K5VD75), mouse GIPR (for mouse GIPR sequence, see Uniprot ID: Q0P543) and CRE and their regulated luciferase reporter genes stably were constructed.

[0441] Expression plasmids for the full-length genes of human GIPR, cynomolgus macaque GIPR, and mouse GIPR were obtained by molecular cloning. Viruses were then packaged and used to infect CHO-K1 cells. Puromycin selection was used to obtain GIPR / CHO-K1 cells that highly express human, cynomolgus macaque, and mouse GIPR. The pGL4.29[CRE / hygro] plasmid was transfected into hGIPR / CHO-K1, cynoGIPR / CHO-K1, and mGIPR / CHO-K1 cells using Lipofectamine 3000. Hygromycin B was added for selection to obtain stable cell pools of hGIPR CRE / CHO-K1, cyno GIPR CRE / CHO-K1, and mGIPR CRE / CHO-K1.

[0442] 5. Construction of luciferase reporter gene overexpression cell lines expressing human, cynomolgus monkey, mouse GCGR and CRE and their regulation

[0443] Cell lines expressing human GCGR (human GCGR sequence see Uniprot ID: P47871), cynomolgus macaque GCGR (cynomolgus macaque GCGR sequence see SEQ ID NO: 5), mouse GCGR (mouse GCGR sequence see Uniprot ID: Q61606) and CRE and their regulated luciferase reporter genes stably expressed were constructed.

[0444] Expression plasmids encoding the full-length genes of human GCGR, cynomolgus macaque GCGR, and mouse GCGR were obtained by molecular cloning. Viruses were then packaged and used to infect CHO-K1 cells. Selection with puromycin or G418 resulted in the generation of GCGR / CHO-K1 cells expressing high levels of human, cynomolgus macaque, and mouse GCGR. The pGL4.29[CRE / hygro] plasmid was transfected into hGCGR / CHO-K1, cynoGCGR / CHO-K1, and mGCGR / CHO-K1 cells using Lipofectamine 3000. Selection with hygromycin B resulted in the generation of stable monoclonal cell lines: hGCGR CRE / CHO-K1, cynoGCGR CRE / CHO-K1, and mGCGR CRE / CHO-K1.

[0445] Cynomolgus monkey GCGR amino acid sequence:

[0446] Example 1-2. Preparation of Antibodies

[0447] 1. Antibody Expression

[0448] The density of Lonza CHO-K1 cells stably overexpressing antibody Ab1 (whose amino acid sequence is derived from patent WO2023046071A1) was adjusted to 0.5×10 6 / mL, continue culturing the cells, and add feeds on days 4, 7, 9, and 11. Before each addition, check the sugar content in the culture medium with a blood glucose meter and replenish the sugar concentration to 8-12 g / L. Collect the expression supernatant on day 14 of culture and remove impurities by high-speed centrifugation.

[0449] Antibody Ab2 (obtained by Fc mutation of antibody Ab1) was expressed in HEK293 6e cells: on day 0, HEK293 6e cells were adjusted to 0.8×10 6 / mL, and continue culturing the cells. On day 1, transfection was performed using a transfection mixture of plasmids expressing the heavy and light chains of Ab2 antibody at a ratio of 2:3. On day 3, feed was added and the cells were cultured again. On day 7, the cell supernatant was collected and centrifuged at high speed to remove impurities.

[0450] 2. Antibody Purification

[0451] Purify using a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. Elute the target protein with 100 mM acetic acid, pH 3.0, and neutralize with 1 M Tris-HCl, pH 8.0. Concentrate the eluted sample appropriately and further purify using a Superdex 200 (GE) gel chromatography equilibrated with PBS to remove aggregates. Collect the monomer peak and aliquot for later use.

[0452] The sequence of antibody Ab1 is as follows:

[0453] Table 2. CDR sequences of antibody Ab1

[0454] The heavy chain variable region sequence of antibody Ab1:

[0455] The light chain variable region sequence of antibody Ab1:

[0456] Heavy chain constant region sequence 1:

[0457] Light chain constant region sequence 1:

[0458] Heavy chain sequence of antibody Ab1:

[0459] Light chain sequence of antibody Ab1:

[0460] Heavy chain constant region sequence 2:

[0461] Heavy chain sequence of antibody Ab2:

[0462] The light chain sequence of antibody Ab2 is the same as the light chain sequence of antibody Ab1 (SEQ ID NO: 17).

[0463] Note: In the above antibody sequences, the single underlined part is the CDR region sequence, the italic part is the variable region sequence, and the double underlined part is the antibody constant region sequence.

[0464] Example 1-3. Preparation of Antibodies

[0465] The amino acid sequence of antibody Ab3 was obtained from WHO Drug Information Vol. 27, No. 3, 2013. DNA encoding Ab3 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The Ab3 heavy and light chain genes were cloned into the expression vector pcDNA3.4, and the heavy and light chain expression vectors were simultaneously transfected into Expi293F using PEI (Polyethylenimine). TM 293F cells (Thermo Fisher Scientific, Catalog No. A14527) were cultured in serum-free medium for approximately 5 days, and the cell supernatant was harvested and the antibody was purified using Protein A affinity chromatography.

[0466] The purification steps are as follows: Celite is added to the cell culture and the cells and impurities are removed using a filter to obtain the cell supernatant. A MabSelect Sure (Cytiva, Cat. No. 17543801) affinity chromatography column is first washed with 0.2M NaOH, rinsed with pure water, and then equilibrated with PBS. The supernatant is passed through the affinity column and washed with PBS until the A280 value drops to baseline. The target protein is eluted with 0.1M acetate buffer (pH 3.5). The antibody solution is neutralized with 1M Tris-HCl (pH 8.0). The protein solution is dialyzed into PBS. The antibody concentration is determined by UV spectrophotometry. The antibody is sterilized by filtration and stored in a refrigerator (4°C).

[0467] The sequence of antibody Ab3 is as follows:

[0468] Table 3. CDR sequences of antibody Ab3

[0469] The heavy chain variable region sequence of antibody Ab3:

[0470] The light chain variable region sequence of antibody Ab3:

[0471] Heavy chain constant region sequence 3:

[0472] Light chain constant region sequence 2:

[0473] Heavy chain sequence of antibody Ab3:

[0474] Light chain sequence of antibody Ab3:

[0475] Note: In the above antibody full-length sequence, the single underlined part is the antibody CDR region sequence, the italic part is the variable region sequence, and the double underlined part is the antibody constant region sequence.

[0476] Example 2. Peptide Synthesis

[0477] The polypeptide compounds and derivatives provided in the present disclosure are obtained by solid-phase synthesis. The synthesis carrier is Rink-amide MBHA (Xi'an Lanxiao Technology) resin. The α-amino group of the amino acid derivatives used in the synthesis process is protected by an Fmoc group (fluorenylcarbonyl). The side chains of the amino acids are selected from the following protecting groups according to the different functional groups: the cysteine ​​side chain thiol, the glutamine side chain amide, and the histidine side chain imidazole group are protected by Trt (trityl), the arginine side chain guanidinium group is protected by Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), the tryptophan side chain indole group and the lysine side chain amino group are protected by Boc (tert-butyloxycarbonyl), the threonine side chain hydroxyl group, the tyrosine side chain phenol group, and the serine side chain hydroxyl group are protected by t-Bu (tert-butyl), and the glutamic acid and aspartic acid side chain carboxyl groups are protected by OtBu (tert-butyl ester), etc.

[0478] The chemical synthesis of the P1, P2 and P3 backbone peptide sequences was completed using the fluorenylmethoxycarbonyl (Fmoc) / tert-butyl (t-Bu) synthesis method on a Prelude-X fully automatic peptide synthesizer, where the resin used was Rink-amide MBHA resin with a degree of substitution of 0.54 mmol / g.

[0479] The P1 sequence is: HAibHGTFTSDYSIαLLEERAAQEFVEWLLAGGGK-NH2

[0480] SEQ ID NO: 20;

[0481] The P2 sequence is: HAibHGTFTSDYSIαLLEEQAAQEFVEWLLAGGGK-NH2

[0482] SEQ ID NO: 21;

[0483] The P3 sequence is: HAibEGTFTSDVSSAibLEEEAAREFVAWLVEGGGK-NH2

[0484] SEQ DI NO:22.

[0485] Prior to the amino acid condensation step, the Fmoc group was removed using a 20% 4-methylpiperidine solution in DMF (two reactions, 8 minutes each). All standard amino acid condensations used an equimolar ratio of Fmoc amino acid, HATU (Suzhou Haofan Biotechnology), and two equivalents of 4-methylmorpholine at a 10-fold excess over the theoretical peptide loading for 25 minutes at room temperature. The exception was coupling to Cα-methylated amino acids such as Aib (2-aminoisobutyric acid) and the C-terminal amino acid of αL (2-methyl-L-leucine), where two or three condensations were performed, each 60 minutes long, to ensure complete condensation. Following peptide backbone synthesis, the resulting resin peptide was washed three times with DMF and then DCM, followed by vacuum drying. Then, 10 mL of freshly prepared cleavage buffer (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) was added and the reaction was shaken at room temperature for 3-4 hours. After the reaction, the mixture was filtered and the resin was washed twice with trifluoroacetic acid. The filtrates were combined and a large amount of methyl tert-butyl ether was added to precipitate the crude peptide solid. The crude peptides of P1, P2 and P3 backbone peptides were obtained by centrifugation and removal of the supernatant.

[0486] The crude peptide was dissolved in a mixed solvent containing 20% ​​acetic acid / water, filtered through a 0.22 μM membrane, and separated using a WATERS Prep150 LC reverse-phase high-performance liquid chromatography system with mobile phases A (0.1% trifluoroacetic acid, 10% acetonitrile, aqueous solution) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution). The chromatographic column was an X-SELECT OBD C-18 (WATERS) reverse-phase column. During the purification process, the chromatograph detection wavelength was set at 220 nm and the flow rate was 15 mL / min. The relevant fractions were collected and lyophilized to obtain pure P1, P2, and P3 backbone peptides with a yield of 20%. The pure backbone peptides were confirmed for purity and compound identity by analytical ultra-high performance liquid chromatography and liquid chromatography / mass spectrometry and used in the next reaction.

[0487] Example 3. Chemical Synthesis of C-Terminally DBCO-PEG8 Modified Peptides

[0488] 1. Synthesis of LP1

[0489] (1) Synthesis of activated intermediate 2

[0490] 177.7 mg (0.44 mmol) of dibenzocyclooctyne-N-hydroxysuccinimidyl ester (Compound A, Leyan) and 195 mg (0.44 mmol) of 1-amino-3,6,9,12,15,18,21,24-octaoxaheptaneacetic acid-27-oic acid (Compound B, Bidex Pharmaceuticals) were dissolved in N,N-dimethylformamide (2.5 mL). 285 mg (2.2051 mmol, 384 μL) of N,N-diisopropylethylamine was added, the atmosphere was purged with nitrogen three times, and the mixture was stirred at room temperature for 1 hour. The mixture was filtered and purified by HPLC. The corresponding sample was collected, dried at 30°C, and dried under vacuum to obtain Intermediate 1 (260 mg, 80.8% yield).

[0491] Dissolve 200.92 mg (0.28 mmol) of intermediate 1 and 34.9 mg (0.30 mmol) of N-hydroxysuccinimide (HOSU) in 2 mL of anhydrous THF / DMF (v:v = 3:1). Slowly add 47 μL (0.30 mmol) of N,N'-diisopropylcarbodiimide (DIC) to the reaction mixture with stirring. The mixture is allowed to react at room temperature for at least 12 hours. The reaction was monitored by analytical ultra-high performance liquid chromatography (UPLC). The conversion of the product, intermediate 2, was 77%. Intermediate 2 was used directly in the next reaction without purification.

[0492] (2) P1 peptide coupling modification

[0493] 90 mg (0.025 mmol) of P1 backbone peptide (see Example 2 for synthesis and purification methods) was weighed and dissolved in 4 mL of sodium carbonate solution (pH 10.8) and 2 mL of acetonitrile (final concentration not less than 15 mg / mL); after stirring for 10 minutes (the pH of the reaction system measured by a pH meter decreased), the pH of the reaction system was adjusted to 10.8 using sodium hydroxide solution, and stirring was continued for 10 minutes. After monitoring, the pH value remained unchanged; then, 358 μL of the intermediate 2 reaction system (actually containing 31.3 mg (0.038 mmol) of intermediate 2) was slowly added dropwise under stirring; after continuing the reaction for 20 minutes, ultra-performance liquid chromatography monitoring of the reaction indicated that the reaction was complete, and the reaction system was adjusted to pH 3-4 with trifluoroacetic acid and purified using preparative high-performance liquid chromatography. The product-related fractions were collected and lyophilized to obtain pure C-terminal modified polypeptide LP1. The purity and molecular weight of the LP1 pure product were determined by analytical ultra-high performance liquid chromatography and liquid chromatography / mass spectrometry, wherein the purity was 95.45% and the measured molecular weight was 4271.17.

[0494] 2. Synthesis of LP2

[0495] 90 mg (0.025 mmol) of P2 backbone peptide (see Example 2 for synthesis and purification methods) was weighed and dissolved in 4 mL of sodium carbonate solution (pH 10.8) and 2 mL of acetonitrile (final concentration not less than 15 mg / mL); after stirring for 10 minutes (the pH of the reaction system measured by the pH meter decreased), the pH of the reaction system was adjusted to 10.8 using sodium hydroxide solution, and the pH value was monitored to remain unchanged after stirring for 10 minutes; subsequently, 361 μL of the intermediate 2 reaction system (actually containing 31.6 mg (0.038 mmol) of intermediate 2) was slowly added dropwise under stirring; after continuing the reaction for 20 minutes, ultra-performance liquid chromatography monitoring of the reaction indicated that the reaction was complete, and the reaction system was adjusted to pH 3-4 with trifluoroacetic acid and purified using preparative high-performance liquid chromatography. The product-related fractions were collected and lyophilized to obtain a pure product of the C-terminally modified polypeptide LP2. The purity and molecular weight of the LP2 pure product were determined by analytical ultra-high performance liquid chromatography and liquid chromatography / mass spectrometry, wherein the purity was 97.56% and the measured molecular weight was 4243.13.

[0496] 3. Synthesis of LP3

[0497] 90 mg (0.026 mmol) of P3 backbone peptide (for synthesis and purification methods, see Example 2) was weighed and dissolved in 4 mL of sodium carbonate solution (pH 10.8) and 2 mL of acetonitrile (final concentration not less than 15 mg / mL); after stirring for 10 minutes (the pH of the reaction system measured by a pH meter decreased), the pH of the reaction system was adjusted to 10.8 using sodium hydroxide solution, and stirring was continued for 10 minutes. After monitoring, the pH value remained unchanged; then, under stirring, 324 μL of the intermediate 2 reaction system (actually containing 28.4 mg (0.034 mmol) of intermediate 2) was slowly added dropwise; after continuing the reaction for 20 minutes, ultra-performance liquid chromatography monitoring of the reaction indicated that the reaction was complete, and the reaction system was adjusted to pH 3-4 with trifluoroacetic acid and purified using preparative high-performance liquid chromatography. After that, the product-related fractions were collected and lyophilized to obtain a pure product of the C-terminally modified polypeptide LP3. The purity and molecular weight of the LP3 pure product were determined by analytical ultra-high performance liquid chromatography and liquid chromatography / mass spectrometry, wherein the purity was 96.00% and the measured molecular weight was 4117.99.

[0498] Example 4. Synthesis of sugar chain OLS-4

[0499] (2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-2-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-6-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,5-diol OLS-4

[0500] first step

[0501] 2-((4-methoxybenzyl)oxy)ethan-1-ol OLS-4b

[0502] Ethylene glycol OLS-4a (50.00 g, 805.57 mmol) was dissolved in anhydrous tetrahydrofuran (300 mL), and sodium hydroxide (4.93 g, 128.89 mmol, content 60%) and tetrabutylammonium iodide (5.37 g, 16.11 mmol) were added in sequence under nitrogen protection. The mixture was stirred at 0°C for 30 minutes, and then p-methoxybenzyl chloride (20.19 g, 128.89 mmol) was slowly added dropwise. The addition was completed in about 30 minutes, and the mixture was heated to 80°C and stirred for 1 minute. After 6 hours, the reaction solution was poured into ice water with stirring to quench, and ethyl acetate (500 mL) was added with stirring. The layers were separated, and the aqueous phase was extracted with ethyl acetate (150 mL×3). The organic phases were combined, washed with water (250 mL×3) and saturated sodium chloride solution (250 mL×2), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with eluent system C to give the title product OLS-4b (22.00 g, yield: 14.9%).

[0503] MS m / z(ESI):205.2[M+23].

[0504] Step 2

[0505] 2-((4-methoxybenzyl)oxy)ethyl 4-methylbenzenesulfonate OLS-4c

[0506] OLS-4b (22.00 g, 120.74 mmol) was dissolved in pyridine (98 mL). p-Toluenesulfonyl chloride (27.62 g, 144.88 mmol) was added at 0°C under nitrogen. The reaction was stirred at 0°C and slowly allowed to warm to room temperature for 16 hours. Ethyl acetate (300 mL) was added to dilute the reaction solution and washed with hydrochloric acid (10%, 150 mL × 3). The organic phase was washed sequentially with water (150 mL × 3) and saturated sodium chloride solution (250 mL × 2), dried over anhydrous sodium sulfate for 30 minutes, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to afford the title product, OLS-4c (36.40 g, 89.6% yield).

[0507] 1 H NMR(400MHz, CDCl3)δ7.81(d,2H),7.33(d,2H),7.21(d,2H),6.88(d,2H),4 .43(s,2H),4.23-4.17(m,2H),3.82(s,3H),3.68-3.61(m,2H),2.45(s,3H).

[0508] Step 3

[0509] 3-(2-((4-methoxybenzyl)oxy)ethoxy)propan-1-ol OLS-4d

[0510] OLS-4c (36.40 g, 108.20 mmol) and 1,3-propylene glycol (82.34 g, 1.08 mol) were dissolved in toluene (100 mL). Powdered potassium hydroxide (15.18 g, 270.51 mmol) was added at room temperature. The mixture was heated to 50°C under nitrogen and stirred for 72 hours. The reaction mixture was poured into ice water (200 mL). Concentrated hydrochloric acid was added dropwise until the pH reached 3-4. Ethyl acetate (300 mL) was added for extraction. The mixture was separated, and the aqueous phase was extracted with ethyl acetate (100 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate for 30 minutes. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to obtain the title product, OLS-4d (15.60 g, 60.0% yield).

[0511] 1 H NMR(400MHz, CDCl3)δ7.28(d,2H),6.89(d,2H),4.51(s,2H),3.81(s,3H),3.7 9(t,2H),3.68(t,2H),3.65-3.59(m,4H),2.5i1(br.s,1H),1.88-1.82(m,2H).

[0512] Step 4

[0513] 3-(2-((4-methoxybenzyl)oxy)ethoxy)propyl 4-methylbenzenesulfonate OLS-4e

[0514] OLS-4d (15.60 g, 64.92 mmol) was dissolved in a mixture of pyridine (52 mL) and dichloromethane (52 mL). p-Toluenesulfonyl chloride (18.56 g, 97.38 mmol) was added at 0°C. Under nitrogen, the reaction was stirred at 0°C and slowly returned to room temperature for 16 hours. Ethyl acetate (300 mL) was added to dilute the reaction solution, which was then washed sequentially with hydrochloric acid (10%, 150 mL × 3), water (150 mL × 3), and saturated sodium chloride solution (250 mL × 2). The solution was dried over anhydrous sodium sulfate for 30 minutes, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to afford the title product, OLS-4e (18.00 g, 70.3% yield).

[0515] MS m / z(ESI):417.3[M+23].

[0516] 1 H NMR(400MHz, CDCl3)δ7.80(d,2H),7.34(d,2H),7.27(d,2H),6.90(d,2H),4.48(s, 2H),4.16(t,2H),3.83(s,3H),3.53-3.49(m,6H),2.45(s,3H),1.96-1.91(m,2H).

[0517] Step 5

[0518] 14-Azido-1-(4-methoxyphenyl)-2,5,9,12-tetraoxatetradecane OLS-4f

[0519] OLS-4e (8.00 g, 20.28 mmol) and azide-diethylene glycol (3.72 g, 28.39 mol, AIMATE) were dissolved in toluene (33 mL). Powdered potassium hydroxide (2.84 g, 50.70 mmol) was added at room temperature. The mixture was heated to 60°C under nitrogen and stirred for 16 hours. The reaction mixture was poured into ice water (100 mL). Concentrated hydrochloric acid was added dropwise until the pH reached 3-4. Ethyl acetate (100 mL) was added for extraction. The mixture was separated and the aqueous phase was extracted with ethyl acetate (50 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate for 30 minutes. The desiccant was removed by filtration. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography using eluent System C to obtain the title product, OLS-4f (6.3 g, 87.9% yield).

[0520] MS m / z(ESI):376.3[M+23].

[0521] 1 H NMR (400MHz, CDCl3) δ7.29(d,2H),6.89(d,2H),4.52(s,2H),3.82(s,3H),3.70–3.56(m,14H),3.40(t,2H),1.93-1.87(m,2H).

[0522] Step 6

[0523] 2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethan-1-ol OLS-4g

[0524] OLS-4f (6.30 g, 17.83 mmol) was dissolved in dichloromethane (31 mL) and water (3.1 mL). Dichlorodicyanobenzoquinone (4.45 g, 19.61 mmol, Aladdin) was added at room temperature under nitrogen and stirred at 25°C for 1.5 hours. The reaction solution was filtered through celite, and the filtrate was diluted with dichloromethane (150 mL) and washed sequentially with saturated sodium bicarbonate solution (100 mL × 2), saturated sodium bisulfite solution (100 mL × 2), water (100 mL), and saturated sodium chloride solution (100 mL × 2). The product was dried over anhydrous sodium sulfate for 30 minutes, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to obtain the title product, OLS-4g (3.2 g, 76.9% yield).

[0525] Step 7

[0526] 2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethyl 4-methylbenzenesulfonate OLS-4h

[0527] OLS-4g (3.20 g, 13.72 mmol) was dissolved in pyridine (11 mL) and dichloromethane (11 mL), cooled to 0°C, and p-toluenesulfonyl chloride (3.14 g, 16.46 mmol) was added. The reaction was stirred at 0°C under nitrogen and slowly returned to room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (200 mL) and washed sequentially with hydrochloric acid (10%, 150 mL × 3), water (150 mL × 3), and saturated sodium chloride solution (250 mL × 2). The product was dried over anhydrous sodium sulfate for 30 minutes, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent System C to afford the title product, OLS-4h (4.80 g, 90.3% yield).

[0528] 1H NMR (400MHz, CDCl3) δ7.79(d,2H),7.34(d,2H),4.14(t,2H),3.67-3.57(m,8H),3.51-3.46(m,4H),3.38(t,2H),2.44(s,3H),1.81-1.75(m,2H).

[0529] Step 8

[0530] N-((2R,3R,4R,5S,6R)-5-(((2R,4aR,6S,7S,8S,8aR)-8-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-7-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxan-6-yl)oxy)-2,4-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide OLS-4i

[0531] OLS-3c (340 mg, 0.41 mmol), N,N-dimethylformamide (4.8 mL), and 2 mL of a solution of OLS-4h (398 mg, 1.03 mmol) in N,N-dimethylformamide were added sequentially to a reaction flask and cooled to 0°C. Sodium hydride (86 mg, 2.15 mmol, 60% content) was added and the mixture was allowed to react at 0°C for approximately 0.5 hour, then warmed to 20-30°C and reacted for approximately 0.5 hour. The reaction solution was poured into a mixture of saturated ammonium chloride solution (10 mL) and water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed sequentially with water (10 mL) and saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent System C to obtain the title product, OLS-4i (362 mg, 84.6% yield).

[0532] MS m / z(ESI):1047.4[M+1].

[0533] 1H NMR(400MHz, CDCl3)δ7.40-7.16(m,25H),5.70(d,1H),5.44(s,1H),4.87(d,1H) ),4.84-4.78(m,3H),4.71-4.68(m,1H),4.57-4.50(m,4H),4.41-4.38(m,1H),4 .04-4.01(m,1H),3.97(t,1H),3.93-3.84(m,2H),3.76-3.68(m,3H),3.61-3.40 (m,16H),3.34-3.27(m,3H),3.11-3.05(m,2H),1.78-1.72(m,2H),1.51(s,3H).

[0534] Step 9

[0535] N-((2R,3R,4R,5S,6R)-5-(((2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-3-(benzyloxy)-5-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,4-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide OLS-4j

[0536] Compound OLS-4i (360 mg, 0.34 mmol) and dichloromethane (26 mL) were added sequentially to a reaction flask, cooled to -30 to -20°C under nitrogen, and trifluoroacetic acid (3.6 mL) was added dropwise. After the addition was complete, the temperature was raised to -5 to 5°C and stirred for approximately 1.5 hours. Methanol (5.1 mL) was added to quench the reaction, which was then diluted with dichloromethane (12.3 mL). The product was washed sequentially with saturated sodium bicarbonate solution (26 mL x 2) and saturated sodium chloride solution (13 mL), dried over anhydrous sodium sulfate, and filtered to remove the desiccant. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin-layer chromatography using developing solvent System C to afford the title product, OLS-4j (234 mg, 71.0% yield).

[0537] MS m / z(ESI):959.7[M+1].

[0538] 1H NMR (400MHz, CDCl3) δ7.31-7.17(m,20H),5.64(d,1H),4.87(d,1H),4.84-4.79(m,2H),4.70(s,2H),4.58-4.50(m,3H),4.43-4.39(m,2H),4.0 5(t,1H),3.82(t,1H),3.74-3.36(m,23H),3.30(t,2H),3.10-3.07(m,1 H),3.00-2.97(m,1H),2.21(br.s,1H),1.81-1.75(m,2H),1.66(s,3H).

[0539] Step 10

[0540] N-((3R,4R,5S,6R)-5-(((2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,4-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide OLS-4k (diastereoisomer mixture)

[0541] OLS-4j (150 mg, 158.72 μmol) was dissolved in tetrahydrofuran (8 mL) and water (2 mL), and palladium hydroxide (50.0 mg, 15% content), palladium on carbon (61 mg, 10% content), and hydrochloric acid (1 drop) were added. The atmosphere was replaced with hydrogen three times, and the reaction was stirred at room temperature for 12 hours. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in methanol (7 mL), and imidazole-1-sulfonyl azide hydrochloride (6 mg, 220.41 μmol), potassium carbonate (68 mg, 492.00 μmol), and copper sulfate (5.7 mg, 22.83 μmol) were added. The reaction was stirred at room temperature for 12 hours. Inorganic salts were removed by filtration, and the solid was rinsed with methanol (3 mL). The organic phases were combined and the filtrate was concentrated under reduced pressure to obtain the crude title product OLS-4k (161 mg). The product was directly used in the next reaction without purification.

[0542] MS m / z(ESI):599.3[M+1].

[0543] Step 11

[0544] (2S,3S,4S,5R,6R)-4-(2-(3-(2-(2-azidoethoxy)ethoxy)propoxy)ethoxy)-2-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-6-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,5-diol OLS-4

[0545] Crude compound OLS-4k (161 mg, 148.01 μmol) was dissolved in pure water (8 mL) under ice, and 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride (557 mg, 3.07 mmol) and cesium carbonate (3.00 g, 9.20 mmol) were added. The mixture was stirred at 0°C for 12 hours. The reaction mixture was purified by HPLC (Phenomenex C18 column, 150 x 25 mm, 10 μm; mobile phase: water (0.01% ammonia) and acetonitrile, gradient: acetonitrile 0% to 30%, flow rate: 25 mL / min) and lyophilized from 5 mol% sodium hydroxide solution to afford the title product, OLS-4 (35 mg, 38.2% total yield for the tenth and eleventh steps).

[0546] MS m / z(ESI):581.3[M+1].

[0547] 1 H NMR(400MHz, CDCl3)δ6.02(d,1H),4.65(s,2H),4.30(s,2H),4.09-3.93(m,5H),3.86 -3.77(m,4H),3.73-3.51(m,16H),3.40-3.28(m,5H),2.01(s,3H),1.88-1.81(m,2H).

[0548] Example 5-1. APC-1

[0549] (The triazole ring formed in this step has a geometric structure, and the resulting compound contains the two structures shown above as R.)

[0550] first step

[0551] Endo S enzyme (19.16 mg / mL, 0.104 mL) was added to a PBS buffer solution of antibody Ab1 (0.05 M PBS buffer solution at pH 6.5; 10 mg / mL, 20 mL) at 37°C. The solution was shaken in a water bath at 37°C for 12 hours, after which the reaction was stopped. The reaction solution was purified using a Hitrap Protein A HP protein purification column (elution phase: acetic acid buffer solution at pH 3.0) to obtain affinity eluate 1-a. The solution was exchanged to PBS buffer at pH 7.4 and stored refrigerated at 4°C.

[0552] Step 2

[0553] At 37°C, add Endo S enzyme (19.16 mg / mL, 0.104 mL) and OLS-4 (23.2 mg) to 1-a in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 20 mL). Place in a water bath shaker and shake at 37°C for 1 hour to stop the reaction. The reaction solution was purified using a Hitrap Protein AHP protein purification column (elution phase: acetic acid buffer, pH 3.0) to obtain the affinity eluate of 1-b. The solution was exchanged to PBS buffer, pH 7.4, and stored refrigerated at 4°C. 1 It represents the average number of sugar chains remodeled to the N297 position of the antibody heavy chain. Two N297 sites of the antibody heavy chain can be remodeled to two sugar chains, or one sugar chain.

[0554] Step 3

[0555] At 25°C, a PBS buffer solution of 1-b (pH = 7.4 PBS buffer; 8.0 mg / mL, 10.82 mL) was added, followed by the addition of 5.0 mL of 1,2-propylene glycol. The mixture was shaken thoroughly, followed by the addition of LP1 solution (6.83 mg, 1599 nmol, dissolved in a mixture of 625 μL of dimethyl sulfoxide and 625 μL of 1,2-propylene glycol). The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 50 mL of pH = 7.4 PBS buffer. The supernatant was purified using a Hitrap Protein A HP column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to a pH of approximately 5.0 with tris (hydroxymethylaminomethane hydrochloride) solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, APC-1, which was stored refrigerated at 4°C.

[0556] The average number of peptides bound to each antibody molecule was calculated by MS: y = 1.96.

[0557] Example 5-2. APC-2

[0558] (The triazole ring formed in this step has a geometric structure, and the resulting compound contains the two structures shown above as R.)

[0559] At 25°C, a PBS buffer solution of 1-b (pH = 7.4 PBS buffer; 8.0 mg / mL, 10.82 mL) was added, followed by the addition of 9.0 mL of 1,2-propylene glycol. The mixture was shaken thoroughly, followed by the addition of LP2 solution (12.4 mg, 2922 nmol, dissolved in a mixture of 1125 μL of dimethyl sulfoxide and 1125 μL of 1,2-propylene glycol). The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 50 mL of pH = 7.4 PBS buffer. The supernatant was then purified using a Hitrap Protein A HP column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to a pH of approximately 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, APC-2, which was stored refrigerated at 4°C.

[0560] The average number of peptides bound to each antibody molecule was calculated by MS: y = 1.72.

[0561] Example 5-3. APC-3

[0562] (The triazole ring formed in this step has a geometric structure, and the resulting compound contains the two structures shown above as R.)

[0563] first step

[0564] Endo S enzyme (19.16 mg / mL, 0.104 mL) was added to a PBS buffer solution of antibody Ab2 (0.05 M PBS buffer solution, pH 6.5; 10 mg / mL, 20 mL) at 37°C. The solution was shaken in a water bath at 37°C for 12 hours, after which the reaction was stopped. The reaction solution was purified using a Hitrap Protein A HP protein purification column (elution phase: acetic acid buffer solution, pH 3.0) to obtain affinity eluate 2-a. The solution was exchanged to PBS buffer solution, pH 7.4, and stored refrigerated at 4°C.

[0565] Step 2

[0566] At 37°C, add Endo S enzyme (19.16 mg / mL, 0.078 mL) and OLS-4 (17.4 mg) to a PBS buffer solution (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 15 mL) in 2-a. Place in a water bath shaker and shake at 37°C for 1 hour to stop the reaction. The reaction solution was purified using a Hitrap Protein AHP protein purification column (elution phase: acetic acid buffer, pH 3.0) to obtain the affinity eluate of 2-b. The solution was exchanged to a PBS buffer solution, pH 7.4, and stored refrigerated at 4°C. 1 It represents the average number of sugar chains remodeled to the N297 position of the antibody heavy chain. Two N297 sites of the antibody heavy chain can be remodeled to two sugar chains, or one sugar chain.

[0567] Step 3

[0568] At 25°C, a PBS buffer solution of 2-b (pH = 7.4 PBS buffer; 8.0 mg / mL, 18.25 mL) was added, followed by the addition of 14.6 mL of 1,2-propylene glycol. The mixture was shaken thoroughly, followed by the addition of LP2 solution (20.65 mg, 4866 nmol, dissolved in a mixture of 1825 μL of dimethyl sulfoxide and 1825 μL of 1,2-propylene glycol). The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 50 mL of pH = 7.4 PBS buffer. The supernatant was then purified using a Hitrap Protein A HP column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to a pH of approximately 5.0 with tris (hydroxymethylaminomethane) hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, APC-3, which was stored refrigerated at 4°C.

[0569] The average number of peptides bound to each antibody molecule was calculated by MS: y = 1.89.

[0570] Example 5-4. APC-4

[0571] (The triazole ring formed in the third step has a geometric structure, and the resulting compound contains the two structures shown above as R.)

[0572] To a 5 mL solution of crude product 1-b, 4 mL of 1,2-propylene glycol was added. The mixture was shaken thoroughly, followed by the addition of LP-3 solution (5.56 mg, 1350 nmol, dissolved in a mixture of 500 μL of dimethyl sulfoxide and 500 μL of 1,2-propylene glycol). The mixture was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 50 mL of PBS 7.4 buffer. The supernatant was purified using a MabSelect protein purification column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to approximately pH 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, APC-4, which was stored refrigerated at 4°C.

[0573] The average drug binding number per antibody molecule was calculated by MS: y = 1.94.

[0574] Example 5-5. APC-5

[0575] (The triazole ring formed in this step has a geometric structure, and the resulting compound contains the two structures shown above as R.)

[0576] first step

[0577] Endo S enzyme (5.06 mg / mL, 0.061 mL) was added to a PBS buffer solution of antibody Ab3 (0.05 M PBS buffer solution at pH 6.5; 10 mg / mL, 3.1 mL) at 37°C, and the mixture was shaken in a water bath at 37°C for 12 hours to stop the reaction. The reaction solution was purified using a MabSlect protein purification column (elution phase: acetic acid buffer solution at pH 3.0) to obtain an affinity eluate of 3-a, which was then exchanged to a PBS buffer solution at pH 6.5 and stored refrigerated at 4°C.

[0578] Step 2

[0579] At 37°C, Endo S enzyme (5.06 mg / mL, 0.061 mL) and OLS-4 (3.72 mg) were added to a PBS buffer solution (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 3.1 mL) in 3-a. The mixture was shaken in a water bath at 37°C for 1.5 hours, after which the reaction was stopped. The reaction solution was purified using a MabSlect protein purification column (elution phase: acetic acid buffer, pH 3.0) to obtain the affinity eluate in 3-b. The solution was then exchanged to a PBS buffer solution, pH 7.4, and stored refrigerated at 4°C. 1 It represents the average number of sugar chains remodeled to the N297 position of the antibody heavy chain. Two N297 sites of the antibody heavy chain can be remodeled to two sugar chains, or one sugar chain.

[0580] Step 3

[0581] At 25°C, a solution of 3-b in PBS buffer (pH = 7.4; 8.0 mg / mL, 0.875 mL) was added to 700 μL of 1,2-propylene glycol. After shaking, LP3 solution (0.93 mg, 226 nmol, dissolved in 87.5 μL of dimethyl sulfoxide and 87.5 μL of 1,2-propylene glycol) was added. The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 10 mL of pH = 7.4 PBS buffer. The supernatant was purified using a MabSlect protein purification column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to approximately pH 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, APC-5, which was stored refrigerated at 4°C.

[0582] The average number of peptides bound to each antibody molecule was calculated by MS: y = 1.93.

[0583] Example 5-6. APC-6

[0584] (The triazole ring formed in this step has a geometric structure, and the resulting compound contains the two structures shown above as R.)

[0585] At 25°C, a solution of 3-b in PBS buffer (pH = 7.4; 8.0 mg / mL, 0.875 mL) was added to 700 μL of 1,2-propylene glycol. After shaking, LP2 solution (0.96 mg, 226 nmol, dissolved in a mixture of 87.5 μL of dimethyl sulfoxide and 87.5 μL of 1,2-propylene glycol) was added. The reaction was allowed to react on a shaker at room temperature for 12 hours. The reaction solution was centrifuged, and the supernatant was removed and diluted with 10 mL of pH = 7.4 PBS buffer. The supernatant was purified using a MabSlect protein purification column (elution phase: acetate buffer, pH 3.0). The resulting solution was adjusted to approximately pH 5.0 with tris hydrochloride solution (1 M, pH = 8.0) to obtain the affinity eluate of the title product, APC-6, which was stored refrigerated at 4°C.

[0586] The average number of peptides bound to each antibody molecule was calculated by MS: y = 1.89.

[0587] Test Case

[0588] Test Example 1. Affinity Experiment

[0589] 1-1. BIAcore detection of affinity between conjugated molecule KLB and human KLB antigen

[0590] Antibody molecules were affinity-captured using a Protein A biosensor chip (Cat.#29127556, Cytiva). Antigen molecules were passed across the chip surface, and the reaction signals were monitored in real time using a Biacore 8K instrument to generate binding and dissociation curves. After each experimental cycle, the biosensor chip was washed and regenerated with 10mM Glycine-HCl (pH 1.5). A 1:1 model was used for data fitting to obtain antibody affinity data. Specific experimental results are shown in Table 4-1 below.

[0591] Table 4-1. Affinity test results of samples and hKLB antigen

[0592] The experimental results show that the disclosed antibodies or conjugated molecules have good binding function with human KLB antigen, and the binding ability of the conjugated molecules is comparable to that of antibodies Ab1 and Ab2.

[0593] 1-2. BIAcore Detection of the Affinity of the Conjugated Molecule to Human ActRIIA / B

[0594] The affinity of the disclosed antibodies and APC molecules for human ActRIIA and ActRIIB was tested using Biacore. Antibodies were affinity-captured using a Protein A biosensor chip (Cytiva, 29127556). The antigens ActRIIA (Sino Biological) and ActRIIB (Acro Biosystems) were then passed over the chip surface. The reaction signals were detected in real time using an instrument to obtain binding and dissociation curves. After each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl, pH 1.5. A 1:1 model was used for data fitting. Specific experimental results are shown in Tables 4-2 and 4-3 below.

[0595] Table 4-2. Affinity test results of samples and ActRIIA antigen

[0596] Table 4-3. Affinity test results of samples and ActRIIB antigen

[0597] The experimental results show that the affinity of the disclosed conjugate molecules APC-5 and APC-6 for the receptors ActRIIA and ActRIIB is comparable to that of the antibody Ab3.

[0598] Test Example 2. Combined Experiment

[0599] 2-1. Flow cytometry detection of the binding ability of conjugated molecules to KLB-overexpressing cell lines

[0600] This test example uses flow cytometry to detect the binding ability of the conjugate molecule to CHO-K1 cell lines overexpressing human KLB & FGFR1c and monkey KLB & FGFR1c (for detailed preparation, see Example 1). Specifically, the cells were cultured in 10% FBS medium in a 37°C, 5% CO2 incubator for 2 days. The number of cells per well was 0.8×10 5 Cells were added to the cell plate, centrifuged at 300g for 5 minutes, and washed once with PBS. A final concentration of the conjugated molecule or antibody was prepared by serially diluting the conjugate molecule or antibody from 50 nM to 11 concentrations, with 100 μL added to each well. The plate was incubated at 4°C for 1 hour, washed twice with PBS, and 100 μL of Alexa488-Goat Anti-human IgG (H+L) fluorescent secondary antibody diluted 1:1000 was added to each well. The plate was incubated at 4°C for 30 minutes. The plate was washed three times with PBS, and 100 μL of PBS was added to each well for reading. The experimental results are shown in Table 5-1.

[0601] Table 5-1. Binding ability of samples to KLB overexpressing cell lines

[0602] The experimental results showed that the conjugate molecules disclosed herein maintained the binding ability of antibodies Ab1 and Ab2 to human and cynomolgus monkey KLB antigens.

[0603] 2-2. Flow cytometry detection of the ability of conjugated molecules to bind to ActRIIA / B

[0604] Human ActRIIA (Sino Biological) and ActRIIB (Acro Biosystems) proteins were coated onto 96-well plates (Thermo, 442404) at 50 ng / well with PBS and incubated overnight at 4°C. After washing, the plates were blocked with 1% BSA at room temperature for 1 hour. After washing, serially diluted antibodies were added and incubated at room temperature for 1 hour. After washing, HRP-goat anti-human Fc secondary antibody (Goat Anti-Human IgG Antibody, Fc-specific, HRP-conjugated, Sigma) was added and incubated at room temperature for 0.5 hour. After washing, TMB chromogenic substrate (KPL, 52-00-03) was added and incubated at room temperature for 5-10 minutes. The reaction was terminated by adding 1 M H2SO4, and the OD450 was read using a VERSAmax plate reader (Molecular Devices). The experimental results are shown in Table 5-2.

[0605] Table 5-2. Binding ability of samples to ActRIIA / B antigen

[0606] The experimental results show that the binding ability of the disclosed conjugate molecules APC-5 and APC-6 to the receptors ActRIIA and ActRIIB is slightly weaker than that of the antibody Ab3.

[0607] Test Example 3: Detection of the activation activity of conjugated molecules on KLB & FGFR1c

[0608] The activation effect of the coupled molecules on the recombinant cell lines CHO-K1-hKLB&hFGFR1c and CHO-K1-cynoKLB&cynoFGFR1c was tested by in vitro cell experiments. The recombinant cell lines were stably transformed with the luciferase reporter gene regulated by GAL4 and the GAL4-Elk1 fusion protein gene. The activation activity was measured by EC 50 Value representation.

[0609] The experimental method is as follows:

[0610] On the first day of the experiment, CHO-K1-hKLB&hFGFR1c or CHO-K1-cynoKLB&cynoFGFR1c cells were seeded at a density of 15,000 cells / well in a 96-well plate (Corning, #3903) using DMEM / F12 medium supplemented with 10% FBS, 10 μg / mL puromycin, and 800 μg / mL G418. 100 μL of cell suspension was added to each well, and only 100 μL of PBS was added to the periphery of the 96-well plate. The cells were incubated overnight at 37°C in a 5% CO2 incubator. On the second day, the medium was discarded, and 50 μL of starvation medium (DMEM / F12 without FBS) was added to each well. The cells were transfected with pFA2-Elk1 and pFR-Luc at a 1:6 ratio using Lipofectamine 3000. 10 μL of the plasmid and Lipofectamine 3000 mixture was added to each well. After transfection, the well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. On the third day, 60 μL of the coupled molecule or antibody to be tested was added to each well in a gradient dilution with starvation medium. The final concentration of the coupled molecule or antibody was 4-fold gradient dilution starting from 100 nM. The starvation medium was set as the blank control well, and the well plate was placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. On the fourth day, the 96-well cell culture plate was removed, 60 μL of signal-activated fluorescent detection reagent (One-glo Luciferase Assay System, Promega, E6120) was added to each well, and the luminescent signal value was read using a multi-function microplate reader (EnVision2015, PerkinElmer). GraphPad Prism was used to fit the curve and calculate the EC based on the logarithmic concentration and signal value of the antibody. 50 The experimental results are shown in Table 6.

[0611] Table 6. Results of the activation activity experiments of the samples on KLB and FGFR1c

[0612] The experimental results show that the activation activity of the disclosed conjugate molecule on human KLB&FGFR1c and cynomolgus monkey KLB&FGFR1c is comparable to that of antibodies Ab1 and Ab2.

[0613] Test Example 4: Detection of the activation activity of conjugated molecules on CHO-K1 / hKLB cells expressing FGFR2c / FGFR3c / FGFR4

[0614] The selectivity of the conjugate molecule for FGFR2c, FGFR3c or FGFR4 was determined by in vitro cell assays to determine the activation effect of the tested conjugate molecule on CHO-K1 / hKLB cell lines expressing human FGFR2c, FGFR3c or FGFR4. The specific method is as follows:

[0615] On the first day of the experiment, CHO-K1-hKLB&hFGFR2c, CHO-K1-hKLB&hFGFR3c, and CHO-K1-hKLB&hFGFR4 cells were seeded at a density of 15,000 cells per well in a 96-well plate (Corning, #3903) using DMEM / F12 medium supplemented with 10% FBS, 10 μg / mL puromycin, and 800 μg / mL G418. 100 μL of cell suspension was added to each well, and only 100 μL of PBS was added to the periphery of the 96-well plate. The cells were incubated overnight at 37°C in a 5% CO2 incubator. On the second day, the medium was discarded, and 50 μL of starvation medium (DMEM / F12 without FBS) was added to each well. The cells were transfected with pFA2-Elk1 and pFR-Luc at a 1:6 ratio using Lipofectamine 3000, with 10 μL of the plasmid and Lipofectamine 3000 mixture added to each well. After transfection, the well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. On the third day, 60 μL of the coupled molecule or antibody to be tested was added to each well, which was diluted in a gradient with starvation medium. The final concentration of the coupled molecule or antibody was 9 concentration points of 4-fold gradient dilution starting from 200 nM. The starvation medium was set as the blank control well, and the well plate was placed in a 37°C, 5% CO2 cell incubator for 24 hours. On the fourth day, the 96-well cell culture plate was removed, and 60 μL of signal-activated fluorescent detection reagent (One-glo Luciferase Assay System, Promega, E6120) was added to each well. The luminescent signal value was read using a multi-function microplate reader (EnVision2015, PerkinElmer). GraphPad Prism was used to fit the curve and calculate the EC based on the logarithmic concentration and signal value of the antibody. 50The experimental results are shown in Figures 1-1 to 1-3.

[0616] The experimental results showed that the conjugate molecules disclosed herein had no activation effect on hKLB&FGFR2c, hKLB&FGFR3c and hKLB&FGFR4, which was consistent with antibodies Ab1 and Ab2.

[0617] Test Example 5: Detection of the agonist activity of conjugated molecules on GLP1R

[0618] The activation effect of the coupled molecules on the recombinant cell lines CHO-K1-hGLP1R / CRE, CHO-K1-cynoGLP1R / CRE and CHO-K1-mouseGLP1R / CRE was tested by in vitro cell experiments. The recombinant cell lines were stably transformed with CRE and its regulated luciferase reporter gene, and the activation activity was measured by EC 50 The experimental method is as follows:

[0619] On the first day of the experiment, CHO-K1-hGLP1R / CRE, CHO-K1-cynoGLP1R / CRE, or CHO-K1-mouseGLP1R / CRE cells were seeded at a density of 18,000 cells / well in a 96-well plate (Corning, #3903) using DMEM / F12 medium supplemented with 10% FBS, 10 μg / mL puromycin, and 200 μg / mL hygromycin B. 90 μL of cell suspension was added to each well, and only 100 μL of PBS was added to the periphery of the 96-well plate. The plates were incubated overnight at 37°C, 5% CO2. On the second day, 10 μL of the test conjugate molecule serially diluted in PBS was added to each well. The final concentration of the conjugate molecule was achieved by a four-fold serial dilution starting from 400 nM. A blank control well was prepared with starvation medium. The plates were incubated at 37°C, 5% CO2 for 5 hours. Remove the 96-well cell culture plate, add 50 μL of signal-activated fluorescent detection reagent (One-glo Luciferase Assay System, Promega, E6120) to each well, and read the luminescent signal value using a multi-function microplate reader (EnVision2015, PerkinElmer). GraphPad Prism was used to fit the curve based on the logarithmic concentration of the antibody and the signal value, and the EC 50 The experimental results are shown in Table 7-1 and Table 7-2.

[0620] Table 7-1. Experimental results of activation activity of coupled molecules APC-1 to APC-3 on GLP1R

[0621] Table 7-2. Experimental results of activation activity of coupled molecules APC-4 to APC-6 on GLP1R

[0622] Test Example 6: Detection of GIPR agonist activity by conjugated molecules

[0623] The activation effect of the coupled molecules on the recombinant cell lines CHO-K1-hGIPR / CRE, CHO-K1-mouseGIPR / CRE, and CHO-K1-cynoGIPR / CRE was tested by in vitro cell experiments (Cisbio, 62AM4PEB). 50 The value indicates its activation activity. The experimental method is as follows:

[0624] On the first day of the experiment, cells were cultured in DMEM / F12 medium containing 10% FBS, puromycin resistance, and hygromycin B resistance. When the cells grew to 70-80%, they were trypsinized, digested and resuspended in complete medium, centrifuged and discarded, and resuspended in Buffer A (HBSS + 20 mM HEPES + 0.1% casein). The cells were counted and the cell density was adjusted to 2x10 5 1000 cells / mL. Prepare a 2x concentration of antibody using Buffer B (Buffer A + 2mM IBMX). Add 5μL of the 2x concentration of antibody to each well of a 384-well plate, centrifuge at low speed, and incubate at 25°C for 30 min. Prepare cAMP-d2 and anti-cAMP-Eu-Cryptate in the dark, mix at a 1:4 or 1:19 ratio with cAMP lysis buffer, add 5μL / well of the prepared cAMP-d2 solution, and then add 5μL / well of the anti-cAMP-Eu-Cryptate solution. Incubate at 25°C in the dark for 1 h. Luminescence signals were read using a multi-function microplate reader (EnVision 2015, PerkinElmer). The experimental results are shown in Tables 8-1 and 8-2.

[0625] Table 8-1. Experimental results of the activation activity of coupled molecules APC-1 to APC-3 on GIPR

[0626] Table 8-2. Experimental results of the activation activity of the coupled molecule APC-6 on GIPR

[0627] Test Example 7: Detection of GCGR agonist activity by conjugated molecules

[0628] The activation effect of the coupled molecules on the recombinant cell lines CHO-K1-hGCGR / CRE, CHO-K1-cynoGCGR / CRE and CHO-K1-mouseGCGR / CRE was tested by in vitro cell experiments. The recombinant cell lines were stably transformed with CRE and its regulated luciferase reporter gene, and the activation activity was measured by EC 50 The experimental method is as follows:

[0629] On the first day of the experiment, CHO-K1-hGCGR / CRE cells were cultured in DMEM / F12 medium supplemented with 10% FBS, 1 mg / mL G418, and 200 μg / mL hygromycin B (CHO-K1-cynoGCGR / CRE and CHO-K1-mouseGCGR / CRE cells were cultured in DMEM / F12 medium supplemented with 10 μg / mL puromycin and 200 μg / mL hygromycin B). Cells were seeded at a density of 18,000 cells / well in a 96-well plate (Corning, #3903), with 90 μL of cell suspension per well. Only 100 μL of PBS was added to the periphery of the 96-well plate. The plates were incubated at 37°C, 5% CO2, and incubated overnight. On the second day, 10 μL of the conjugate molecule to be tested was added to each well, and the final concentration of the conjugate molecule was diluted 4 times starting from 1600 nM. The starvation medium was set as the blank control well, and the well plate was placed in a 37°C, 5% CO2 cell culture incubator for 5 hours. The 96-well cell culture plate was removed, and 50 μL of signal-activated fluorescent detection reagent (One-glo Luciferase Assay System, Promega, E6120) was added to each well. The luminescent signal value was read using a multi-function microplate reader (EnVision2015, PerkinElmer). GraphPad Prism was used to fit the curve based on the logarithmic concentration of the antibody and the signal value and calculate the EC 50 The experimental results are shown in Table 9-1 and Table 9-2.

[0630] Table 9-1. Experimental results of activation activity of coupled molecules APC-1 to APC-3 on GCGR

[0631] Table 9-2. Experimental results of the activation activity of the coupled molecule APC-6 on GCGR

[0632] Test Example 8: Detection of the agonistic activity of conjugated molecules on MIN-6 cells

[0633] The activation activity of the coupled molecules on mouse GLP1R, GIPR, and GCGR positive cells MIN-6 was tested by in vitro cell experiments. 50 The value indicates its activation activity. The experimental method is as follows:

[0634] On the first day of the experiment, MIN-6 cells were cultured in DMEM (high glucose) medium containing 15% FBS and 0.05 mM β-mercaptoethanol. When the cells grew to 70%-80%, they were trypsinized and resuspended in HBSS + 20 mM HEPES + 0.1% BSA (buffer A). The cells were centrifuged, the old solution was discarded, and the cells were resuspended in buffer A. The cells were counted and the cell density was adjusted to 2x10 5 Cells / mL. Antibody was prepared at a 2x concentration (3200 nM) using Buffer B (Buffer A + 2 mM IBMX). 1000 cells were plated in a 384-well plate. 5 μL of the 2x concentration antibody was added and centrifuged briefly at low speed. The cells were incubated at 25°C for 30 min. cAMP-d2 and anti-cAMP-Eu-Cryptate were prepared in a 1:4 ratio with cAMP lysis buffer. 5 μL of the prepared cAMP-d2 solution was added to each well, followed by 5 μL of anti-cAMP-Eu-Cryptate solution per well. The cells were incubated at 25°C for 1 h in the dark. Luminescence signals were read using a multi-function microplate reader (EnVision 2015, PerkinElmer). The results are shown in Table 10.

[0635] Table 10. Results of the experimental test on the agonistic activity of the coupled molecules on MIN-6 cells

[0636] Test Example 9: Determination of peptide stability in human serum

[0637] 4 μL of P2, P3, and a control peptide (HGEGTFTSDVSSYLEEEAAKEFVAWLVKGGG (SEQ ID NO: 35), sequence derived from patent WO2020125744A1) dissolved solution was added to 396 μL of pre-incubated serum to a final concentration of 10 μM. 30 μL of the peptide-serum mixture was placed in a new sterile centrifuge tube and incubated in a 37°C metal bath with shaking for 24 hours. Immediately after incubation, 120 μL of room temperature quenching solution (methanol containing an internal standard (verapamil, 10 ng / mL)) was added to the peptide-serum mixture sample to stop the reaction. Vortex for 5 minutes. The samples in the plate were centrifuged at 4000 rpm for 15 minutes or 12000 rpm for 10 minutes at 4°C to precipitate the protein. 80 μL or 100 μL of the supernatant was transferred to a new 96-well plate, and 80 μL or 100 μL of water was added for LC-MS / MS analysis. The specific experimental results are shown in Table 11.

[0638] Table 11. Stability test results of P2 and P3 in human serum

[0639] The experimental results showed that the stability of P2 and P3 in human serum was better than that of the control peptide.

[0640] Test Example 10: Stability determination of conjugated molecules in human, rhesus monkey, and rat serum

[0641] Dissolve 300 μg of the conjugated molecule in 1.5 mL of human, rhesus monkey, and rat serum, respectively, and store in a 37°C incubator. Samples were collected on days 1, 2, 7, and 14, and the concentrations of the KLB and peptide ends of the conjugated molecule were determined by ELISA or HTRF. Detailed experimental results are shown in Table 12.

[0642] Table 12. Results of stability test of coupled molecules in serum

[0643] The results showed that APC-1, APC-2 and APC-3 were stable in serum.

[0644] Test Example 11.1: Simultaneous PK testing of the efficacy of a single dose of a conjugated molecule on acute glucose tolerance in C57BL / 6J mice

[0645] This experiment used male C57BL / 6J mice, administered a single dose, and used the IPGTT method to examine the effects of acute glucose tolerance in mice. The specific experimental plan is as follows:

[0646] All experimental animals received a single intraperitoneal injection of the conjugate molecule. The dose was 1 mpk for APC-1, 4 mpk and 1 mpk for APC-3, and 4 mpk and 1 mpk for APC-4. The day before the experiment, animals were randomly divided into groups (n=6) according to body weight. After a 16-hour fast, fasting blood glucose was measured. The drug was administered intraperitoneally 60 minutes before (-60 minutes) the intraperitoneal injection of glucose. Blood glucose was measured at 0 minutes in each group, followed immediately by intraperitoneal injection of glucose solution (2 g / kg). Blood glucose was measured 15 minutes, 30 minutes, 60 minutes, and 120 minutes later. On the seventh day, each group underwent a second IPGTT assay. PK sampling: 100 μL of serum was collected from the same batch of animals for analysis of blood drug concentrations at 3, 48, 168, 336, 504, and 672 hours after administration of the conjugate molecule. The experimental results are shown in Table 13 and Figures 2-1 to 2-4.

[0647] Table 13. Pharmacokinetic results in mice

[0648] The experimental results showed that when the conjugate molecules APC-1, APC-3 and APC-4 of the present disclosure were administered as a single dose to wild-type mice, they had extremely significant acute hypoglycemic ability on day 0 compared with the vehicle group (Figures 2-1 and 2-2); on day 7 after administration, APC-1, APC-3 and APC-4 still had significant acute hypoglycemic ability compared with the vehicle group (Figures 2-3 and 2-4).

[0649] PK test results showed that APC-1, APC-3 and APC-4 all had good stability in mice. Test Example 11.2: Stability test of conjugated molecules in C57BL / 6J mice after single administration

[0650] This study used male C57BL / 6J mice, who were administered a single intraperitoneal injection of 20 mpk of the conjugate molecules APC-1 and APC-3, and 40 mpk of APC-4. The animals were randomly divided into groups (n = 4 or 6) based on body weight the day before the experiment. 100 μL of serum was collected at 3, 48, 168, 336, 504, and 672 hours after dosing for conjugate stability analysis. The results are shown in Table 14.

[0651] Table 14. PAR value analysis of coupled molecules

[0652] The experimental results showed that the coupled molecules APC-1, APC-3 and APC-4 have good stability in mice.

[0653] Test Example 12.1: In vivo efficacy testing of conjugated molecules in DIO mice

[0654] Male high-fat diet-induced DIO mice were ordered from Jicui Yaokang, with a 12 / 12-hour light-dark cycle, free access to food and water, and adaptive feeding. According to body weight and random blood glucose, the mice were divided into 3 groups, with 6 mice in each group. Blood glucose and body weight of the mice were tested one day before administration (day-1). The experimental animals were intraperitoneally injected with APC-2 at a dose of 8 mpk and 2 mpk (once a week), and APC-4 at a dose of 8 mpk and 2 mpk (once a week). The test results are shown in Figures 3-1 to 3-7. Statistical analysis was performed using one-way ANOVA (****: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05, relative to the vehicle control group).

[0655] The experimental results showed that after multiple administration of the disclosed conjugate molecules APC-2 and APC-4, the body weight (Figure 3-1), food intake (Figure 3-2) and random blood glucose (Figure 3-3) of DIO mice were dose-dependently reduced, the glucose tolerance of mice was improved (Figures 3-4 and 3-5), and serum cholesterol, serum low-density lipoprotein cholesterol, liver weight or serum alanine aminotransferase (Figure 3-6) and liver fat (Figure 3-7) were reduced.

[0656] Test Example 12.2: Multiple-dose drug administration of conjugated molecules in DIO mice

[0657] Male high-fat feeding-induced DIO mice were ordered from Jicui Yaokang, with a 12 / 12 hour light-dark cycle, free access to food and water, and adaptive feeding. After the DIO mice were adaptively fed until their weight stabilized, on Day-1, they were weighed and random blood glucose was tested. They were randomly divided into groups (n=6) based on their body weight and food intake (and reference to random blood glucose). On Day 0, each group of mice began to be dosed with APC-3. The doses of APC-3 were 10 mpk, 3 mpk, and 1 mpk (once a week). The test results are shown in Figures 4-1 to 4-6. The data are expressed as the mean and the standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA (****: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05, relative to the vehicle control group).

[0658] The experimental results showed that after multiple administrations, the disclosed conjugate molecule APC-3 was able to dose-dependently reduce the body weight (Figure 4-1), food intake (Figure 4-2) and random blood glucose (Figure 4-3) of DIO mice, and improve the glucose tolerance of mice (Figures 4-4 and 4-5); reduce the liver mass and liver triglycerides as well as serum cholesterol, serum triglycerides, serum low-density lipoprotein cholesterol, serum alanine aminotransferase, and serum aspartate aminotransferase of mice (Figure 4-6).

[0659] Test Example 13: In vivo efficacy testing of conjugated molecules in db / db mice

[0660] Male db / db (BKS) experimental animals were ordered from Jicui Yaokang and placed on a 12 / 12 hour light / dark cycle with free access to food and water. They were acclimated for 17 days before dosing began. The day before dosing (day-1), the HbA1c, blood glucose, and body weight of the mice were measured. The mice were randomly divided into groups according to blood glucose (or HbA1c) and body weight. The vehicle-administered group had 6 mice per group, and the other dosing groups had 9 mice per group, with 3 mice per cage. The APC-4 administration doses were 8 mpk, 2 mpk, 0.5 mpk (once a week, respectively), and 4 mpk (once every 2 weeks). The test results are shown in Figures 5-1 to 5-6. The data are expressed as the mean and standard error of the mean (SEM). Statistical analysis was performed using t-test (****: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05, relative to the vehicle control group).

[0661] Experimental results showed that the disclosed conjugate molecule APC-4, after the first administration, was able to improve glucose tolerance in db / db mice in a dose-dependent manner (Figure 5-1). Repeated administration of APC-4 significantly reduced random blood glucose (Figure 5-2), fasting blood glucose (Figure 5-3), HbA1c (Figure 5-4), serum triglycerides (Figure 5-5), and food intake (Figure 5-6) in db / db mice. The 4mpk (Q2W) and 2mpk (QW) administration groups of APC-4 had comparable effects on improving glucose tolerance and reducing random blood glucose, fasting blood glucose, HbA1c, serum triglycerides, and food intake, demonstrating that APC-4 has good PK in db / db mice.

Claims

1. An antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, having a structure represented by the general formula (I): Ab-(L-P) m (I) Wherein: Ab is an antibody; P is a glucagon-like peptide-1 receptor agonist peptide or an analogue thereof; L is a linker connecting Ab and P; wherein the sugar chain reconstructed from Ab at its Asn297 binds to L; m represents an integer from 1 to 10.

2. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the reconstructed sugar chain structure is: In the formula, the wavy line It means binding to the Asn at position 297 of the heavy chain of Pc; P1 and P2 are the same or different and are each independently selected from hydroxy, *-(CR p R q -CR s R t -O)s 1 -, and *-(CR p R q -CR s R t -O)s 2 -(CR p R q -CR s R t -CR x R y -O)s 3 -(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxy group, a cyano group, an amino group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, and the cycloalkyl group, heterocyclic group, aryl group, and heteroaryl group are optionally substituted with one or more substituents selected from an oxo group, a halogen, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxy group, a cyano group, and an amino group; the asterisk * represents binding to the linker L; s 1 is from 1 to 10, preferably from 1 to 5; s 2 is from 0 to 10, preferably from 1 to 5; s 3 is from 1 to 10, preferably from 1 to 5; s 4 is from 0 to 10, preferably from 1 to 5; Provided that P1 and P2 are not hydroxyl or *-(CH2CH2O)s at the same time 1 -; Preferably, P1 is a hydroxyl group, and P2 is *-(CR p R q -CR s R t -O)s 2 -(CR p R q -CR s R t -CR x R y -O)s 3 -(CR p R q -CR s R t -O)s 4 -, where R p 、R q 、R s 、R t 、R x and R y are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, and a haloalkyl group, and s 2 is 1, s 3 is 1, s 4 is 1; More preferably, the reconstructed sugar chain structure is as follows:

3. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, having a structure as shown in general formula (II): wherein R is -L-P; The definitions of Ab, L, P and m are as defined in claim 1.

4. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR triple agonist peptide or an analogue thereof; preferably, P comprises the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:

22.

5. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein Ab is selected from IgG1, IgG2, IgG3 and IgG4 antibodies; preferably, Ab is an IgG1 antibody.

6. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein Ab is a KLB antibody or an ActRIIA / B antibody; preferably, (1) HCDR1 of the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO:6, HCDR2 comprises the amino acid sequence of SEQ ID NO:7, and HCDR3 comprises the amino acid sequence of SEQ ID NO:8, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:9, LCDR2 comprises the amino acid sequence of SEQ ID NO:10, and LCDR3 comprises the amino acid sequence of SEQ ID NO:11; or (2) HCDR1 of the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO:23, HCDR2 comprises the amino acid sequence of SEQ ID NO:24, and HCDR3 comprises the amino acid sequence of SEQ ID NO:25, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:26, LCDR2 comprises the amino acid sequence of SEQ ID NO:27, and LCDR3 comprises the amino acid sequence of SEQ ID NO:28; more preferably, (1) the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO:12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:13; or (2) the heavy chain variable region of Ab comprises the amino acid sequence of SEQ ID NO:29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:30; most preferably, (1) the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO:16 or 19, and the light chain comprises the amino acid sequence of SEQ ID NO:17; or (2) the heavy chain of Ab comprises the amino acid sequence of SEQ ID NO:33, and the light chain comprises the amino acid sequence of SEQ ID NO:

34.

7. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein L is -L a -L b -L c -L d -L e -, L a Selected from: where the asterisk * indicates combination with L b combination, wavy line It represents binding to the reconstructed sugar chain of Ab; L b Selected from -C(O)-CR a R b -CR c R d -C(O)-, -C(O)-(CR a R b -CR c R d )2-C(O)-, -C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-, -C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR a R b -CR c R d -O)4-CR a R b -CR c R d -C(O)-, -CR f R g -O-C(O)- and -O-C(O)-; L c is -NR h -CR i R j -CR m R n -; L d is a PEG unit; L e is -C(O)-; R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxyl group, a cyano group, an amino group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, and the cycloalkyl group, heterocyclic group, aryl group and heteroaryl group are optionally substituted with one or more substituents selected from an oxo group, a halogen, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxyl group, a cyano group and an amino group; or R a and R b together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R c and R d together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R f and R g together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R i and R j together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R m and R n together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, said cycloalkyl or heterocyclic group being optionally substituted by one or more substituents selected from oxo group, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino; or R a and R c together with the carbon atoms to which they are respectively attached form a cycloalkyl or heterocyclic group, R i and R m together with the carbon atoms to which they are respectively attached form a cycloalkyl or heterocyclic group, and the cycloalkyl or heterocyclic group is optionally substituted by one or more substituents selected from oxo group, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino.

8. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 7, wherein L b is -C(O)-CR a R b -CR c R d -C(O)-, and R a 、R b 、R c and R d are the same or different and each independently is a hydrogen atom or C 1-6 alkyl; preferably, L b is -C(O)-CH2-CH2-C(O)-.

9. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 7, wherein L c is -NR h -CR i R j -CR m R n -, and R h 、R i 、R j 、R m and R n are the same or different and each independently is a hydrogen atom or a C 1-6 alkyl; preferably, L c is -NH-CH2-CH2-.

10. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 7, wherein L d is 1 to 36 -O-CH2-CH2-; preferably, L d is 4 to 24 -O-CH2-CH2-; more preferably, L d is 4, 8, 12 or 24 -O-CH2-CH2-; most preferably, L d is 8 -O-CH2-CH2-.

11. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein L is 12. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, having a structure as shown in general formula (II): Wherein: Or The definitions of Ab and m are as defined in claim 1.

13. A glucagon-like peptide-1 receptor agonist peptide or an analogue thereof, which comprises the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:

22.

14. A compound represented by the general formula (Ib) or a salt thereof: L'-P (Ib) Wherein: P is a glucagon-like peptide-1 receptor agonist peptide or an analogue thereof; preferably, P is a GLP-1R single agonist peptide or a GLP-1R / GIPR / GCGR triple agonist peptide or an analogue thereof; more preferably, P comprises the amino acid sequence of SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22; L' is L aa -L b -L c -L d -L e - L aa Selected from: where the asterisk * represents the combination with L b Combination; L b Selected from -C(O)-CR a R b -CR c R d -C(O)-, -C(O)-(CR a R b -CR c R d )2-C(O)-, -C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d )2-C(O)-, -C(O)-CR a R b -CR c R d -C(O)-NR e -(CR a R b -CR c R d -O)2-CR f R g -C(O)-, -C(O)-CR a R b -CR c R d -NR e -C(O)-(CR a R b -CR c R d -O)4-CR a R b -CR c R d -C(O)-, -CR f R g -O-C(O)- and -O-C(O)-; L c is -NR h -CR i R j -CR m R n -; L d is a PEG unit; L e is -C(O)-; R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 、R j 、R m and R n are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxy group, a cyano group, an amino group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group, and the cycloalkyl group, heterocyclic group, aryl group and heteroaryl group are optionally substituted by one or more substituents selected from an oxo group, a halogen, an alkyl group, a haloalkyl group, an alkoxy group, a hydroxyalkyl group, a hydroxy group, a cyano group and an amino group; or R a and R b together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R c and R d together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R f and R g together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R i and R j together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, R m and R n together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclic group, said cycloalkyl or heterocyclic group being optionally substituted by one or more substituents selected from oxo group, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino; or R a and R c together with the carbon atoms to which they are respectively attached form a cycloalkyl or heterocyclic group, R i and R m together with the carbon atoms to which they are respectively attached form a cycloalkyl or heterocyclic group, and the cycloalkyl or heterocyclic group is optionally substituted by one or more substituents selected from oxo group, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino.

15. The compound of the general formula (Ib) or a salt thereof according to claim 14, wherein L aa is selected from: where the asterisk * represents binding to L b ; and / or L b is -C(O)-CR a R b -CR c R d -C(O)-, and R a 、R b 、R c and R d are the same or different and each independently is a hydrogen atom or C 1-6 alkyl; preferably, L b is -C(O)-CH2-CH2-C(O)-; and / or L c is -NR h -CR i R j -CR m R n -, and R h 、R i 、R j 、R m and R n are the same or different and each independently is a hydrogen atom or C 1-6 alkyl; preferably, L c is -NH-CH2-CH2-; and / or L d is 1 to 36 -O-CH2-CH2-; preferably, L d is 4 to 24 -O-CH2-CH2-; more preferably, L d is 4, 8, 12 or 24 -O-CH2-CH2-; most preferably, L d is 8 -O-CH2-CH2-; and / or L e is -C(O)-.

16. A compound of the general formula (Ib) or a salt thereof according to claim 14 or 15, which is selected from the following structures:

17. A method for preparing an antibody-polypeptide conjugate represented by the general formula (II) or a pharmaceutically acceptable salt thereof, comprising the following steps: The compound represented by the general formula (IIa) or a salt thereof reacts with the compound represented by the general formula (Ib) or a salt thereof to obtain an antibody-polypeptide conjugate represented by the general formula (II) or a pharmaceutically acceptable salt thereof; Wherein R is -L-P; The definitions of Ab, L, P and m are as defined in claim 1, and the definition of L' is as defined in claim 14.

18. A pharmaceutical composition, which comprises the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, the glucagon-like peptide-1 receptor agonist peptide or an analogue thereof according to claim 13, and one or more pharmaceutically acceptable carriers, diluents or excipients.

19. A method for preventing or treating a disease, the method comprising administering to a subject the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, the glucagon-like peptide-1 receptor agonist peptide or an analogue thereof according to claim 13 or the pharmaceutical composition according to claim 18; preferably, the disease is diabetes, obesity, liver disease, coronary artery disease or kidney disease; more preferably, the disease is type II diabetes, obesity, metabolic dysfunction-related fatty liver disease and metabolic dysfunction-related steatohepatitis (non-alcoholic steatohepatitis).