WO2022083720A1 - Glp1-gcgr antibody fusion protein variant and composition comprising same - Google Patents

Abstract

A GLP1-GCGR antibody fusion protein variant and a composition comprising same, specifically relating to a variant of a GLP1-GCGR antibody fusion protein, and in particular, to a glycosylated variant, a composition comprising a GLP1-GCGR antibody fusion protein and a variant thereof, preparation and characterization methods for the composition, and use of the composition.

Description

GLP1-GCGR antibody fusion protein variants and compositions comprising the same

This application claims the priority of the patent application filed on October 23, 2020 (application number CN202011143417.5) and the patent application filed on October 13, 2021 (application number CN202111192158.X).

technical field

The present disclosure relates to the field of biopharmaceuticals, and relates to glycosylation variants of GLP1-GCGR antibody fusion proteins, as well as pharmaceutical compositions comprising the glycosylation variants, and uses thereof.

Background technique

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

GLP1 is one of the most important hormones affecting insulin secretion. The biologically active GLP1 in the human body mainly includes two forms, GLP1(7-36) amide and GLP1(7-37). GLP1 is secreted by L cells in the small intestine, mainly promotes insulin secretion in a glucose concentration-dependent manner, protects islet β cells, and inhibits glucagon secretion to lower blood glucose levels. Glucagon is the opposite of insulin and mainly increases the body's blood sugar.

The glucagon receptor (GCGR) is a member of the B-type class of G protein-coupled receptors (GPCRs). Through G protein coupling, GCGR stimulation can activate adenylate cyclase and cAMP-dependent intracellular signaling pathways as well as phosphoinositide-mediated signaling. Subsequent increases in the expression of gluconeogenic enzymes including phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase promote gluconeogenesis. Furthermore, GCGR signaling can activate glycogen phosphorylase and inhibit hepatic glucose synthase, thereby promoting glycogenolysis. The study found that GCGR knockout mice showed a series of phenotypes such as increased GLP1, decreased hepatic glucose output, increased lipid metabolism, and decreased appetite.

Glycosylation of proteins is one of the most common post-translational modifications of proteins, which is the process of transferring carbohydrates to proteins (eg, amino acid residues on proteins) and forming glycosidic bonds under the action of glycosyltransferases.

There are two main types of glycosylation of proteins in mammals: N-linked glycosylation and O-linked glycosylation. In N-linked glycosylation, the oligosaccharide is covalently attached to asparagine; while in O-linked glycosylation, the attachment usually occurs at the hydroxyl group of serine or tyrosine. O-glycosylation can also use hydroxylysine as the attachment point, and then successively transfer sugar residues to form oligosaccharide chains. The process of Glucosyl-galactosyl hydroxylysine modification is as follows: Lysine is catalyzed by lysyl hydroxylase enzyme to form 5-hydroxylysine. Then further catalyzed by hydroxylysine-galactosyltransferase and galactosylhydroxy-lysine-glucosyltransferase, a galactose and glucose are connected, and finally a glucose-galactose-hydroxylysine modification is formed, The protein or peptide produces a mass gain of about 340Da. But glucose-galactose-hydroxylysine modifications are very rare in antibody products.

SUMMARY OF THE INVENTION

The present disclosure provides a glycosylation-modified variant of GLP1 comprising a hydroxylysine-based O-glycosylation modification; wherein the glycosylation-modified variant of GLP1 has higher stability. In some embodiments, wherein the glycosylation variant is not susceptible to peptide chain cleavage.

The present disclosure also provides a GLP1-GCGR antibody fusion protein with hydroxylysine-based O-glycosylation modification, wherein the glycosylation modification enables the fusion protein to have better stability, and the modification does not affect the activity of the fusion protein.

In some embodiments, the aforementioned glycosylation-modified variant of GLP1 comprises a galactose-hydroxylysine modification; in some embodiments, the aforementioned glycosylation-modified variant of GLP1 comprises a glucose-half Lactose-hydroxylysine modification.

In some embodiments, the aforementioned glycosylation-modified variant of GLP1, wherein said GLP1 comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1-5.

In some embodiments, the aforementioned glycosylation modification variant of GLP1, wherein the glycosylation modification site is at K28 of GLP1. The amino acid positions described therein are numbered according to the natural sequence rules.

In some embodiments, the aforementioned glycosylation-modified variants of GLP1 comprise at least one glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned glycosylation-modified variants of GLP1 comprise at least 2 glucose-galactose-hydroxylysine modifications.

In some embodiments, the aforementioned glycosylation-modified variants of GLP1 comprise multiple glucose-galactose-hydroxylysine modifications.

The present disclosure provides a glycosylation-modified variant of a GLP1-GCGR antibody fusion protein, comprising the glycosylation-modified variant of GLP1 according to any one of the foregoing and an anti-GCGR antibody.

The present disclosure also provides a glycosylation-modified variant of a GLP1-GCGR antibody fusion protein, comprising a glycosylation-modified variant of GLP1 and an anti-GCGR antibody, wherein the glycosylation-modified variant of GLP1 comprises a glycosylation-modified variant based on O-glycosylation of hydroxylysine.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the anti-GCGR antibody comprises:

a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; and

A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the glycosylation-modified variant of GLP1 comprises a galactose-hydroxylysine modification.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the glycosylation-modified variant of GLP1 comprises a glucose-galactose-hydroxylysine modification.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the glycosylation-modified variant of GLP1 comprises a variant selected from the group consisting of SEQ ID NOs: 4, 1, 2, 3, and 5 any of the amino acid sequences shown.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification site is at K28 of GLP1.

In some embodiments, a glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein said GLP1 comprises the amino acid sequence of SEQ ID NO: 4, and wherein said hydroxylysine-based O-sugar The sylation modification site is at K28 of SEQ ID NO:4.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the anti-GCGR antibody comprises:

A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15; and

A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16, 17 or 18.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the anti-GCGR antibody comprises:

The heavy chain constant region is shown in SEQ ID NO:19, and the light chain constant region is shown in SEQ ID NO:20.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the anti-GCGR antibody comprises:

A heavy chain as set forth in SEQ ID NO:21, and a light chain as set forth in SEQ ID NO:22.

In some embodiments, a glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the C-terminus of said GLP1 is linked to an anti-GCGR antibody via a linker or directly.

In some embodiments, a glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the C-terminus of the GLP1 is linked to the N-terminus of the heavy chain variable region of the anti-GCGR antibody through a linker.

In some embodiments, a glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the C-terminus of the GLP1 is linked to the N-terminus of the light chain variable region of the anti-GCGR antibody through a linker.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the C-terminus of the GLP1 is linked to the heavy chain variable region of the anti-GCGR antibody through a linker (G ₄ S) ₃ N-terminal.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein comprises two first peptide chains with the same sequence and two second peptide chains with the same sequence, wherein:

a first peptide chain comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25 or 26; and

A second peptide chain comprising the amino acid sequence shown in SEQ ID NO:22.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein comprises two first peptide chains with the same sequence and two second peptide chains with the same sequence, wherein:

a first peptide chain comprising the amino acid sequence shown in SEQ ID NO: 23; and

A second peptide chain comprising the amino acid sequence shown in SEQ ID NO:22.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification is a galactose-hydroxylysine modification.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification is a glucose-galactose-hydroxylysine modification.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification site is in any of SEQ ID NOs: 23-26 A shown at K28 of the first peptide chain.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification comprises at least one glucose-galactose-hydroxylysine Acid modification.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification comprises at least 2 glucose-galactose-hydroxylysine amino acid modification.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the hydroxylysine-based O-glycosylation modification comprises a plurality of glucose-galactose-hydroxylysine Acid modification.

In some embodiments, the glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein has a molecular weight increase of about 340 Da or about 680 Da relative to the GLP1-GCGR antibody fusion protein without glycosylation. The theoretical average molecular weight of the modified glucose-galactose-hydroxylysine at the protein level is 340.2806Da. The molecular weight actually detected by mass spectrometry usually deviates from the theoretical molecular weight, but the deviation will not exceed 5Da. In this application, the molecular weight detected includes 340 Da.

In some embodiments, a glycosylation-modified variant of the aforementioned GLP1-GCGR antibody fusion protein, wherein the glycosylation-modified variant is fused to an antibody that does not contain a hydroxylysine-based O-glycosylation modification The protein undergoes a molecular weight increase of 340 Da ± 5 Da or 680 Da ± 10 Da; wherein said molecular weight is determined by deglycosylated reduced molecular weight LC-MS or deglycated intact molecular weight LC-MS analysis.

In some embodiments, glycosylation-modified variants of the aforementioned GLP1-GCGR antibody fusion proteins that are less susceptible to peptide chain degradation relative to GLP1-GCGR antibody fusion proteins that do not contain hydroxylysine-based O-glycosylation fracture, with higher stability.

In some embodiments, glycosylation-modified variants of the aforementioned GLP1-GCGR antibody fusion proteins, which relative to GLP1-GCGR antibody fusion proteins that do not contain hydroxylysine-based O-glycosylation, the glycosylation The first strand of the variant variant is not susceptible to breakage at positions K28, W25 and/or F22; the amino acid positions described therein are numbered according to the natural sequence rules.

In some embodiments, glycosylation-modified variants of the aforementioned GLP1-GCGR antibody fusion proteins, which relative to GLP1-GCGR antibody fusion proteins that do not contain hydroxylysine-based O-glycosylation, the glycosylation The first strand of the variant variant is not susceptible to breakage at positions K28, W25 and/or F22.

In some embodiments, glycosylation-modified variants of the aforementioned GLP1-GCGR antibody fusion proteins, which relative to GLP1-GCGR antibody fusion proteins that do not contain hydroxylysine-based O-glycosylation, the glycosylation The first strand of the variant variant is less prone to breakage at positions K28 and/or W25.

In some embodiments, glycosylation-modified variants of the aforementioned GLP1-GCGR antibody fusion proteins, which relative to GLP1-GCGR antibody fusion proteins that do not contain hydroxylysine-based O-glycosylation, the glycosylation The first strand of the variant variant is less prone to breakage at positions K28 and/or F22.

In some embodiments, glycosylation-modified variants of the aforementioned GLP1-GCGR antibody fusion proteins, which relative to GLP1-GCGR antibody fusion proteins that do not contain hydroxylysine-based O-glycosylation, the glycosylation The first strand of the variant variant is less prone to breakage at positions W25 and/or F22.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein is placed at 25°C for 4 days, wherein the first peptide chain of the glycosylation variant is not easily accessible at K28, W25 and /or a break at the F22 position, wherein said amino acid positions are numbered according to the rules of nature.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein is placed at 25°C for 4 days, wherein the first peptide chain of the glycosylation variant is at K28, W25 and/or The sum of the rates of fragmentation at the F22 site was less than 22%, less than 15%, or less than 5%.

In some embodiments, the glycosylation modification variant of the aforementioned GLP1-GCGR antibody fusion protein is placed at 25°C for 7 days, wherein the first peptide chain of the glycosylation variant is at K28, W25 and/or The sum of the rates of fragmentation at the F22 site was less than 33%, less than 20%, less than 15% or less than 5%.

The present disclosure provides a composition comprising a population of GLP1-GCGR antibody fusion proteins, wherein the population of GLP1-GCGR antibody fusion proteins comprises a glycosylation modified variant of the GLP1-GCGR antibody fusion protein of any one of the preceding .

The disclosure provides a population of GLP1-GCGR antibody fusion proteins, wherein the population of GLP1-GCGR antibody fusion proteins comprises glycosylation-modified variants of any of the preceding GLP1-GCGR antibody fusion proteins.

In some embodiments, the population of GLP1-GCGR antibody fusion proteins is homogeneous.

In other embodiments, the population of GLP1-GCGR antibody fusion proteins is heterogeneous. Glycosylation-modified variants of GLP1-GCGR antibody fusion proteins comprising hydroxylysine-based O-glycan modifications in the population, and/or GLP1-GCGR antibody fusion proteins not comprising hydroxylysine-based O-glycan modifications .

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins is a mixture comprising: GLP1-GCGR antibodies containing other modifications (ie, modifications other than glucose-galactose-hydroxylysine) Fusion protein and GLP1-GCGR antibody fusion protein containing glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins is a mixture comprising: a GLP1-GCGR antibody fusion protein glycosylation modification variant containing a glucose-galactose-hydroxylysine modification and GLP1-GCGR antibody fusion protein without glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the glycosylation-modified variant of the GLP1-GCGR antibody fusion protein comprises:

a first peptide chain comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25 or 26; and

A second peptide chain comprising the amino acid sequence shown in SEQ ID NO:22.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the glycosylation-modified variant of the GLP1-GCGR antibody fusion protein comprises a glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the glycosylation-modified variant of the GLP1-GCGR antibody fusion protein comprises the first peptide chain shown in SEQ ID NO: 23 and the first peptide chain shown in SEQ ID NO: 23 The second peptide chain shown in ID NO: 22 and comprising one or more glucose-galactose-hydroxylysine modifications.

In some embodiments, the aforementioned GLP1-GCGR antibody fusion protein population, wherein the glycosylation modification variant of the GLP1-GCGR antibody fusion protein is:

i) homodimeric variants, wherein both first peptide chains comprise hydroxylysine-based O-glycosylation modifications; or

ii) Heterodimeric variants in which only one of the first peptide chains contains a hydroxylysine-based O-glycosylation modification.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the hydroxylysine-based O-glycosylation modification of the glycosylation-modified variant of the GLP1-GCGR antibody fusion protein occurs at the first at K28 of the peptide chain. The amino acid positions described therein are numbered according to the natural sequence rules.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the glycosylation-modified variant of the GLP1-GCGR antibody fusion protein has a hydroxylysine-based O-glycosylation modification comprising at least 1 A glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the glycosylation modification variant of the GLP1-GCGR antibody fusion protein comprises at least 2 hydroxylysine-based O-glycosylation modifications Glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned population of GLP1-GCGR antibody fusion proteins, wherein the hydroxylysine-based O-glycosylation modification of the glycosylation modification variant of the GLP1-GCGR antibody fusion protein comprises a plurality of glucoses -Galactose-hydroxylysine modification.

In some embodiments, the aforementioned GLP1-GCGR antibody fusion protein population, wherein the glycosylation modification variant of the GLP1-GCGR antibody fusion protein is:

i) homodimeric variants, wherein both first peptide chains comprise hydroxylysine-based O-glycosylation modifications; or

ii) Heterodimeric variants in which only one of the first peptide chains contains a hydroxylysine-based O-glycosylation modification.

In some embodiments, the aforementioned GLP1-GCGR antibody fusion protein population, wherein the glycosylation modification variant of the GLP1-GCGR antibody fusion protein is:

i) homodimeric variants, wherein both first peptide chains comprise glucose-galactose-hydroxylysine modifications; or

ii) Heterodimeric variants in which only one of the first peptide chains contains a glucose-galactose-hydroxylysine modification.

In some embodiments, the aforementioned glycosylation-modified variants are present in at least 0.1% of the GLP1-GCGR antibody fusion protein population. In some embodiments, the aforementioned GLP1-GCGR antibody fusion protein population, wherein the glycosylation modification variant comprises at least 1% of the GLP1-GCGR antibody fusion protein population. In some embodiments, the aforementioned glycosylation-modified variants are present in at least 10% of the population of GLP1-GCGR antibody fusion proteins. The content of the glycosylation-modified variants described therein is measured by LC-MS (liquid chromatography-mass spectrometry).

The present disclosure also provides a pharmaceutical composition comprising the glycosylation-modified variant of GLP1 as described in any one of the preceding items, or the glycosylated-modified variant of GLP1-GCGR antibody fusion protein as described in any of the preceding items, or A population of GLP1-GCGR antibody fusion proteins as previously described, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

The present disclosure also provides a method of reducing blood glucose concentration in a subject, the method comprising administering to the subject a therapeutically effective amount of a glycosylation-modified variant of GLP1 as described in any of the preceding items, or as described in any of the preceding items The glycosylation modification variant of the GLP1-GCGR antibody fusion protein, or the aforementioned GLP1-GCGR antibody fusion protein population, or the aforementioned pharmaceutical composition.

In some embodiments, the therapeutically effective amount is a unit dose of the composition comprising 0.1-3000 mg of a glycosylation-modified variant of GLP1 as described in any preceding item, or a GLP1-GCGR antibody as described in any preceding item A glycosylation-modified variant of the fusion protein, or a population of GLP1-GCGR antibody fusion proteins as described above, or a pharmaceutical composition as described above.

The present disclosure also provides glycosylation-modified variants of GLP1 as described in any preceding item, or glycosylation-modified variants of GLP1-GCGR antibody fusion proteins as described in any preceding item, or GLP1-GCGR as previously described Use of the antibody fusion protein population or the aforementioned pharmaceutical composition in the preparation of a medicament for treating metabolic disorders.

A glycosylation-modified variant of GLP1 as described in any one of the preceding items, or a glycosylation-modified variant of a GLP1-GCGR antibody fusion protein as described in any preceding item, or a GLP1-GCGR antibody as described above A population of fusion proteins, or a pharmaceutical composition as previously described, can be used as a medicament, preferably as a blood sugar lowering medicament.

The present disclosure also provides a method of treating a metabolic disorder, the method comprising administering to a subject a therapeutically effective amount of a glycosylation-modified variant of GLP1 as described in any preceding item, or a GLP1- A glycosylation-modified variant of a GCGR antibody fusion protein, or a population of GLP1-GCGR antibody fusion proteins as described above, or a pharmaceutical composition as described above.

The present disclosure also provides glycosylation-modified variants of GLP1 as described in any preceding item, or glycosylation-modified variants of GLP1-GCGR antibody fusion proteins as described in any preceding item, or GLP1-GCGR as previously described Use of the antibody fusion protein population or the aforementioned pharmaceutical composition in the preparation of a medicament for treating metabolic disorders.

A glycosylation-modified variant of GLP1 as described in any one of the preceding items, or a glycosylation-modified variant of a GLP1-GCGR antibody fusion protein as described in any preceding item, or a GLP1-GCGR antibody as described above The fusion protein population, or the pharmaceutical composition as described above can be used as a drug for the treatment of metabolic disorders.

In some embodiments, wherein the metabolic disorder is selected from the group consisting of: metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia Glycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerosis, and prediabetic states.

The sequences and related properties of the anti-GLP1 and GLP1-GCGR antibody fusion proteins involved in the present disclosure have been described in the International Application Publication No. WO2020125744, the entire contents of which are incorporated herein by reference.

The GLP1 sequences referred to in this disclosure are as follows:

Table 1. GLP-1 sequences

The anti-GCGR antibody related sequences involved in the present disclosure are as follows:

Table 2. hu1803 antibody CDR sequences

The variable regions of anti-GCGR antibodies are as follows:

>hu1803_VH.1:

>hu1803_VH.1A:

>hu1803_VH.1B:

>hu1803_VH.1C:

>hu1803_VL.1

>hu1803_VL.1A:

>hu1803_VL.1B:

Table 3. Combinations of light and heavy chain variable regions of different humanized antibodies

  hu1803_VL.1 hu1803_VL.1A hu1803_VL.1B hu1803_VH.1 hu1803-1 hu1803-5 hu1803-9 hu1803_VH.1A hu1803-2 hu1803-6 hu1803-10 hu1803_VH.1B hu1803-3 hu1803-7 hu1803-11 hu1803_VH.1C hu1803-4 hu1803-8 hu1803-12

The variable regions of the antibody light and heavy chains indicated by the antibody names in the table above can be linked with the constant regions of the antibody light and heavy chains respectively to form a full-length antibody. Unless otherwise specified in the present disclosure, when forming a full-length antibody, the light chain variable region is linked with the Kappa chain constant region shown in SEQ ID NO: 20 to form an antibody light chain, and the heavy chain variable region is linked with SEQ ID NO: 19 The IgG4-AAs shown are linked to form antibody heavy chains.

The heavy chain constant region sequence of IgG4-AA is as follows:

The light chain (Kappa chain) constant region sequence of the antibody is as follows:

Exemplary anti-GCGR antibody sequences:

hu1803-9 heavy chain sequence:

hu1803-9 light chain sequence:

The GLP1 polypeptide is linked to the N-terminus of the anti-GCGR antibody heavy chain directly or through a linker (G ₄ S) ₃ , and expressed together with the anti-GCGR antibody light chain through the CHO expression system to obtain the GLP1-GCGR antibody fusion protein in the present disclosure (which The structure is shown in Figure 1). The sequences of exemplary GLP1-GCGR antibody fusion proteins are shown below:

1. Fusion protein hu1803-9D:

hu1803-9D first chain

hu1803-9D second chain

2. Fusion protein hu1803-9A:

hu1803-9A first chain

hu1803-9A second chain:

3. Fusion protein hu1803-9B:

hu1803-9B first chain

hu1803-9B second chain:

4. Fusion protein hu1803-9C:

hu1803-9C first chain

hu1803-9C second chain:

Description of drawings

Figure 1: Schematic diagram of the structure of the GLP1-GCGR antibody fusion protein of the present disclosure.

Figure 2: Spectral identification of GLP1-GCGR antibody fusion protein.

Figures 3A to 3B: Mass spectrometry identification after Ides digestion; Figure 3A shows the mass spectrum of the Fd part; Figure 3B shows the mass spectrum of the Fc/2 monomer. The abscissa is the mass (Da); the ordinate is the mass spectrum signal intensity.

Figure 4A to Figure 4B: MS secondary spectrum of the O-glycosylated peptide at position K28; Figure 4A is the mass spectrum of the identification of the O-glycosylated modification site at position K28; Figure 4B is the O-glycosylated peptide at position K28 The glycosylation modification characteristic fragment mass spectrum of , in which 1944.825Da is the parent ion, 1782.7725Da is the parent ion-162Da, and 1620.7184Da is the parent ion-324Da. The abscissa is the mass (Da); the ordinate is the mass spectrum signal intensity.

Figure 5A to Figure 5C: The first-strand mass spectrometry detection results of GLP1-GCGR antibody fusion protein; Figure 5A is the first-strand mass spectrometry detection results of normal GLP1-GCGR; Figure 5B is the first-strand mass spectrometry detection of GLP1-GCGR mutant 1 Results; Figure 5C is the first-strand mass spectrometry detection result of GLP1-GCGR mutant 2. The abscissa is the mass (Da); the ordinate is the mass spectrum signal intensity.

Figure 6: HIC analysis profile of GLP1-GCGR antibody fusion protein.

Figure 7A to Figure 7B: HIC collection sample peaks 3 (Figure 7A) and 4 (Figure 7B) deglycosylated intact molecular weight mass spectrometry results; "Intact" is the peak of normal GLP1-GCGR deglycosylated intact antibody fusion protein; "Intact+ 340 Da" represents a peak with 1 glucose-galactose-hydroxylysine modification on the basis of normal GLP1-GCGR. The abscissa is the mass (Da); the ordinate is the mass spectrum signal intensity.

Detailed ways

the term

To make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

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

Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," and the like, should be construed in an inclusive rather than an exclusive or exhaustive sense; that is, " including but not limited to".

The terms "and/or" such as "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide explicit support for either or both meanings.

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

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 the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been modified later, such as hydroxyproline, gamma-carboxyglutamic acid, and O-orthophosphoserine. Amino acid analogs are those that have the same basic chemical structure as a naturally occurring amino acid (i.e., a hydrogen-bonded alpha carbon, carboxyl, amino, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine amino acid methylsulfonium). Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a compound having a structure that differs from the general chemical structure of amino acids but functions in a similar manner to naturally occurring amino acids.

The term "antibody fusion protein" refers to a biologically active fusion protein formed by linking a protein (polypeptide) of interest with an antibody. The fusion protein has the biological activity of the linked protein as well as immunoglobulin activity. In some embodiments, the antibody fusion protein described in the present disclosure refers to a fusion protein of a GLP1 polypeptide and an anti-GCGR antibody (GLP1-GCGR antibody fusion protein), wherein the GLP1 is fused to the heavy chain variable region of an anti-GCGR antibody or the N-terminus of the light chain variable region. In some embodiments, the GLP1 polypeptide is linked to the N-terminus of the variable region of the heavy chain of the anti-GCGR antibody through a linker to form a GLP1-GCGR antibody fusion protein with a tetrapeptide structure. In some embodiments, the GLP1-GCGR antibody fusion protein has two first chains with the same sequence and two second chains with the same sequence, wherein the first peptide chain comprises SEQ ID NOs: 23, 24 The amino acid sequence shown in , 25 or 26; and a second peptide chain comprising the amino acid sequence shown in SEQ ID NO: 22.

As used herein, a "glycosylated variant" of a GLP1-GCGR antibody fusion protein is a fusion protein that has one or more attachments to a GLP1-GCGR antibody compared to an unglycosylated GLP1-GCGR antibody fusion protein Carbohydrate moieties of fusion proteins. In one embodiment, the glycosylation variant has an oligosaccharide structure attached to the first peptide chain of the GLP1-GCGR antibody fusion protein. In one embodiment, the glycosylation variant has a hydroxylysine-based O-glycosylation modification attached to the first peptide chain of the GLP1-GCGR antibody fusion protein. In certain embodiments, the glycosylation variant has a glucose-galactose-hydroxylysine modification attached to GLP1 of one or both first chains of a GLP1-GCGR antibody fusion protein. In certain embodiments, the glycosylation site is at the K28 amino group of the first chain. In certain embodiments, both first chains are glycosylated (homodimeric variants). In certain other embodiments, only one first strand is glycosylated (heterodimeric variant). In certain embodiments, the oligosaccharides covalently attached to K28 in the first strand may be heterogeneous among glycosylation variants.

N-linked by "N-linked glycosylation" refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X is any amino acid except proline, are the carbohydrate moieties bound to the asparagine side chain enzyme Linked recognition sequence. It is known to those skilled in the art that, for example, each of mouse IgG1, IgG2a, IgG2b and IgG3, as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 regions have a single N-linked sugar at amino acid residue 297 basement site.

"O-linked glycosylation" or "O-glycosylation" refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose sugars to a hydroxyl-containing amino acid. O-linked glycosylation sites are typically the hydroxyl side chains of natural amino acids (eg, serine, threonine) or unnatural amino acids (eg, 5-hydroxyproline or 5-hydroxylysine). Exemplary O-linked sugar residues include, but are not limited to, N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose, or xylose. In the present disclosure, the glucose-galactose-hydroxylysine modification is an O-glycosylation modification, in which the lysine in the GLP1 sequence is first hydroxylated, and then galactose and glucose are sequentially linked.

"GLP1 polypeptide", "GLP-1 polypeptide", "GLP1 peptide" or "GLP1" refers to a peptide capable of binding to and activating the GLP1 receptor. Including GLP-1, GLP-1 analogs and GLP-1 receptor peptide agonists, some specific GLP-1 peptides such as: Lixisenatide/AVE0010/ZP10/Lyxumia, Exenatide /Exendin-4/Byetta/Bydureon/ITCA 650/AC-2993, Liraglutide/Victoza, Semaglutide, Taspoglutide, Syncria/Albiglutide ( Albiglutide), Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide/HM-11260C, CM-3, GLP-1Eligen, ORMD-0901, NN-9924 , NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022 , TT-401, BHM-034, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, Exenatide-XTEN and Glucagon-Xten, exemplary GLP1 in this disclosure Contains the sequences shown in SEQ ID NOs: 1-5.

"GCGR" is a glucagon receptor, which is a member of the GPCR family. After glucagon binds to GCGR, it mainly activates downstream pathways to accelerate glycogenolysis, lipolysis and/or gluconeogenesis. Raise blood sugar.

The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments so long as they exhibit the desired Antigen binding activity.

"Native antibody" refers to naturally-occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has a variable domain (VH), also known as a variable heavy domain or heavy chain variable domain, followed by 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 light chain variable domain, followed by a constant light (CL) domain. Antibody light chains include two types, kappa (κ) and lambda (λ), according to their constant domain amino acid sequences. According to the different composition and sequence of amino acids in the constant region of the antibody heavy chain, antibodies can be divided into five categories, or antibody isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, delta chain, gamma chain, alpha chain, and epsilon chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Each of the five classes of Ig can have a kappa chain or a lambda chain.

The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly and are variable regions; the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). Each light chain contains 3 CDR regions: LCDR1, LCDR2, and LCDR3; each heavy chain contains 3 CDR regions: HCDR1, HCDR2, and HCDR3. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The terms "complementarity determining regions", "CDRs" or "hypervariable regions" refer to regions within a variable domain that primarily contribute to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering scheme, "ABM" numbering scheme, "contact" numbering scheme (see Martin, ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImMunoGenTics (IMGT) numbering scheme (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278) et al. The relationship between these numbering systems is well known to those skilled in the art.

For example, for the classical format, following Kabat's rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light The CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).

Following Chothia's rule, CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).

Following the IMGT rules, CDR amino acid residue numbers in VH are approximately 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3), and CDR amino acid residues in VL are approximately 27-38 (CDR1 ), 56-65 (CDR2) and 105-117 (CDR3).

Following AbM rules, CDR amino acids in VH are numbered 26-35 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).

Unless otherwise stated, the "Kabat" numbering convention applies to the variable regions and CDR sequences in the examples of the present disclosure.

The term "antibody framework" or "FR region" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

"Antibody constant region domain" refers to domains derived from the constant regions of the light and heavy chains of antibodies, including CL and CH1, CH2, CH3 domains derived from different classes of antibodies. The constant regions of the present disclosure also include "conventional variants" of the human antibody heavy chain constant regions and human antibody light chain constant regions, which refer to the human-derived variable regions disclosed in the prior art that do not alter the structure and function of the antibody variable regions Variants of the heavy chain constant region or light chain constant region of Technically known YTE mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations that obtain a knob-into-hole structure (giving the antibody heavy chain a combination of knob-Fc and hole-Fc) that have been confirmed The antibody has new properties, but does not change the function of the variable region of the antibody.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd, dAb; camelid VHH domains; diabodies; linear antibodies; ); and multispecific antibodies formed from antibody fragments.

"Linker" or "Linker" refers to a polypeptide sequence used to connect polypeptides (such as protein domains), usually with a certain flexibility, and the use of the linker will not cause the loss of the original structure and function of the polypeptide. Typical linkers contain about 1-30, 2-24 or 3-15 amino acids. In some embodiments, wherein the linker is (G ₄ S)n, wherein n is an integer from 1-20; preferably, n is 3.

The terms "Fc domain", "Fc region" or "fragment crystallizable region" are used to define the C-terminal region of an antibody heavy chain, including native sequence Fc regions and variant Fc regions. 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 carboxy terminus. The boundaries of the Fc region of an antibody heavy chain may also vary, for example deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region or deletion of the C-terminal glycine and lysine (according to the EU numbering system) of the Fc region. residues 446 and 447). Thus, in some embodiments, a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed. In some embodiments, a composition of intact antibodies may include a population of antibodies without removal of K447 residues and/or G446+K447 residues. In some embodiments, the composition of intact antibodies has a population of antibodies with and without a mixture of antibodies of K447 residues and/or G446+K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service , National Institutes of Health, Bethesda, MD, 1991.

The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a substantially similar structure to that of a native antibody or having a heavy chain containing an Fc region as defined herein. Antibody.

"Specifically binds", "specifically binds" or "binds" means that an antibody (or antibody fragment) binds to an antigen or epitope thereof with higher affinity than to other antigens or epitopes. Typically, antibodies are prepared at about 1 x ^10-7 M or less (eg, about 1 x ^10-8 M or less, about 1 x ^10-9 M or less, about 1 x 10-10 M or less, about 1 x ^10-10 M or less, about An equilibrium dissociation constant (KD) of 1 x ^10-11 M or less, or about 1 x ^10-12 M or less) binds an antigen or an epitope thereof, typically KD is the antibody binding to a non-specific antigen (such as BSA) , casein) at least one percent of the KD. KD can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope thereof may be cross-reactive to other related antigens; , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (common marmoset, marmoset)) homologous antigens are cross-reactive.

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

The term " _kassoc " or "ka" is intended to refer to the association rate of a particular antibody-antigen interaction. The term " _kdis " or "kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and expressed as molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Methods for determining antibody KD include measuring surface plasmon resonance using a biosensing system such as a system, or measuring affinity in solution by solution equilibrium titration (SET).

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA) ).

Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which one or more selected (or all) codons are replaced at the third position by a mixed-base ) and/or deoxyinosine residue substitutions (Batzer et al., Nucleic Acid Res [Nucleic Acids Research]. 19:5081, 1991; Ohtsuka et al., J.Biol.Chem [Journal of Biochemistry]. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

Sequence "identity" means that when two sequences are optimally aligned, gaps are introduced as necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered to be part of the sequence identity, the two sequences The degree (percent) to which amino acids/nucleic acids are identical at equivalent positions. To determine percent sequence identity, alignment can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). )software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

When applied to amino acid sequences, "conservatively modified variants" or "conservative substitutions" refer to the use of other amino acids with similar characteristics (eg, charge, side chain size, hydrophilicity/hydrophobicity, backbone structure and rigidity, etc.) Substituting amino acids in a protein such that such changes can often be made without statistically significantly altering the biological activity of the protein. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are shown in the table below.

Table 4. Exemplary conservative amino acid substitutions

original residue conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp;Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

The term "conservatively modified variant" when applied to nucleic acid sequences refers to those nucleic acids encoding the same or substantially the same amino acid sequence, or in the case of the nucleic acid not encoding an amino acid sequence, substantially the same the sequence of. Due to the degeneracy of the genetic code, any given protein can be encoded by multiple functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where a codon specifies an alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. The skilled artisan will recognize that every codon in a nucleic acid (except AUG, which is usually the only codon for methionine; and TGG, which is usually the only codon for tryptophan), can be modified to produce a functionally equivalent molecule. Thus, each silent variation of the nucleic acid encoding the polypeptide is implied in each such sequence.

The terms "about", "approximately" mean that the index value is within an acceptable error range for the particular value determined by one of ordinary skill in the art, which range of acceptable error depends on how the measurement or determination is made (ie, the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of ±30% of the specific numerical value indicated thereafter. Furthermore, particularly with respect to biological systems or processes, the term can mean up to one order of magnitude, or up to 5 times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for the specific value.

"GLP1-GCGR antibody fusion protein population" refers to a mixed population comprising different modified variants of GLP1-GCGR antibody fusion proteins, and the population includes not only hydroxylysine-based O-glycosylation modified variants, but no glycosylation The modified GLP1-GCGR antibody fusion protein also includes other modified variants, such as N-glycosylation modified variants.

"Pharmaceutical composition" means a mixture comprising one or more GLP1-GCGR antibody fusion protein glycosylation-modified variants described herein or a mixture of GLP1-glycosylation-modified variants and other chemical components, such as Physiological/Pharmaceutically Acceptable Carriers and Excipients.

The term "pharmaceutically acceptable carrier" means any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer with sodium chloride, dextrose, glycerol, polyethylene glycol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include isotonic agents such as sugars, polyols (eg, mannitol, sorbitol) or sodium chloride in the composition. Other examples of pharmaceutically acceptable substances are surfactants, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf-life or effectiveness of the antibody.

The pharmaceutical compositions of the present disclosure can be administered by various methods known in the art. The route and/or mode of administration will vary depending on the desired result. Preferably, administration may be intravitreal, intravenous, intramuscular, intraperitoneal or subcutaneous or near the target site. The pharmaceutically acceptable carrier should be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound (ie, antibodies, bispecific and multispecific molecules) can be coated in materials to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless indicated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

"Administering," "administering," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.

"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 serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , fluids in the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditions culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically excised tissue, organ cultures or cell cultures.

"Treatment/treatment" (and grammatical variants thereof) refers to clinical interventions that attempt to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or amelioration of the disease. 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.

An "effective amount" is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or underlying causes, prevent the appearance of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with a disease state amount.

In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

A "therapeutically effective amount" is sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or to otherwise prevent, retard, delay or reverse the disease state or any other irreversible disorder in any way associated with the disease state Amount of progression of desired symptoms.

A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined preventive effect, 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 therapeutic or prophylactic effect does not necessarily occur with the administration of a single dose, and may occur only after a series of doses have been administered. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations. A "therapeutically effective amount" and a "prophylactically effective amount" may vary depending on factors such as the individual's disease state, age, sex and weight, 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 in a patient.

Specific examples of the term "metabolic disorder" are metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemia hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia Symptoms, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerotic or prediabetic states.

The present disclosure is further described below in conjunction with the examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. The experimental methods for which specific conditions are not indicated in the disclosed examples or test examples are usually based on conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions suggested by raw material or commodity manufacturers; unspecified The reagent materials of specific sources are obtained from the market.

Example 1. Preparation and identification of O-glycosylated antibody fusion proteins

The anti-GLP1-GCGR antibody population was produced by Chinese hamster ovary (CHO) cells, and the GLP1-GCGR antibody fusion protein population was obtained by protein A affinity purification.

The molecular weight LC-MS analysis of GLP1-GCGR antibody fusion protein hu1803-9D was carried out by deglycosylated reduction. The specific experimental process is as follows:

Dilute the GLP1-GCGR antibody fusion protein sample to 0.5 mg/mL with pH 7.9 ammonium bicarbonate, take 100 μL of the sample, and add 1 μL of peptide N glycosidase F (PNGase F, new England lab, QPF-001-B), 45 ℃ water bath for 1 hour, add 2 μL of 1M DTT solution, and 37 ℃ water bath for 30 minutes; LC-MS analysis was performed after the reaction was completed.

LC conditions are as follows: Waters UPLC H-class for liquid chromatography; chromatographic column for Waters BEH C4 2.1×50mm, 1.7μm column; injection volume: 0.5μL; column temperature: 80°C; flow rate: 0.3mL/min; Degraded mobile phase B (0.1% formic acid in acetonitrile) was ramped from 5% to 30% over 15 minutes, then 30% to 90% over the next minute.

The mass spectrometer was Waters Xevo G2-XS QTOF mass spectrometer, and the positive ion mode was used for data acquisition, and the collected data was analyzed by Waters unifi software.

The results show (see Figure 2) that the heavy chain of the GLP1-GCGR antibody fusion protein hu1803-9D still has a variety of heterogenous bodies after de-N-glycosylation treatment; however, the main components of the first chain have been analyzed, including the first chain The hydroxylated or oxidized component (first chain + 16Da) and the first chain GCGR antibody heavy chain C-terminal lysine is not deleted (first chain + K). However, a high proportion (about 20%) of the 340Da increase over the normal first-strand component remained unresolved.

Example 2. Identification and Characterization of Glycosylation

1. Ides digestion and reduction molecular weight LC-MS analysis:

Ides enzyme can specifically cleave IgG, and after reduction treatment, two parts of Fc/2 and Fd will be obtained, which can be detected by LC-MS to preliminarily locate the position where the 340Da modification occurs.

The LC-MS analysis method of Ides digestion and reduction molecular weight is as follows:

Dilute the GLP1-GCGR antibody fusion protein hu1803-9D sample to 0.5 mg/mL with pH 7.9 ammonium bicarbonate, take 100 μL of the sample, add 50 units of Ides enzyme (Genovis, FabRICATOR) respectively, water bath at 37 °C for 4 hours, add 2 μL of 1M DTT solution, water bath at 37°C for 30 minutes; LC-MS analysis was performed after the reaction was completed.

LC conditions are as follows: Waters UPLC H-class for liquid chromatography; chromatographic column for Waters BEH C4 2.1×50mm, 1.7μm column; injection volume: 0.5μL; column temperature: 80°C; flow rate: 0.3mL/min; Degraded mobile phase B (0.1% formic acid in acetonitrile) was ramped from 5% to 30% over 15 minutes, then 30% to 90% over the next minute.

The mass spectrometer was Waters Xevo G2-XS QTOF mass spectrometer, and the positive ion mode was used for data acquisition, and the collected data was analyzed by Waters unifi software. The results are shown in Figures 3A and 3B.

The results showed that the modification to increase the molecular weight by 340 Da occurred in the Fd moiety.

2. Peptide map LC-MS analysis:

The sample was digested with Glu-c enzyme, and then analyzed by peptide map LC-MS. The specific experimental process is as follows:

The hu1803-9D sample was denatured by a 250 mM Tris-HCl (pH 7.4) solution of 6 M guanidine hydrochloride, and then reduced at 37°C by adding a final concentration of 20 mM DTT for 1 hour, followed by the addition of a final concentration of 50 mM IAM for alkylation. Then, Glu-c (Promega, V1651) was used for enzymatic digestion at 37°C for 7 hours, inactivated at 90°C for 5 minutes and returned to room temperature. After the reaction was completed, chymotrypsin (Promega, V1062) was added for analysis by LC-MS.

LC conditions are as follows: Liquid chromatography is Waters UPLC H-class, chromatographic column is Waters BEH C18 2.1×150mm, 1.7μm chromatographic column; injection volume: 0.5μL; column temperature: 65°C; flow rate: 0.3mL/min; wash Degraded mobile phase B (0.1% formic acid in acetonitrile) was ramped from 2% to 40% in 65 minutes.

The mass spectrometer was Waters XeVo G2-XS QTOF mass spectrometer, and the positive ion mode was used for data acquisition, and the collected data was analyzed by Waters unifi software. The mass spectrometry results are shown in Figure 4A and Figure 4B.

After mass spectrometry analysis, it was found that the GLP-1 polypeptide K28 of the first peptide chain of hu1803-9D was post-modified by 340 Da, as shown in FIG. 4A . The molecular weight of the fragment ion y20 (KGGGGGGGSGGGGSGGGGSE, SEQ ID NO: 27) is 1732.6911 Da, and the molecular weight of the fragment ion y19 (GGGGGGGSGGGGGSGGGGSE, SEQ ID NO: 28) is 1264.4795 Da, and the fragment ion y20 has one more K than the fragment ion y19. Theoretically Speaking, the molecular weight difference between the two should be 128Da, but in fact the difference between the two is 468Da, which is 340Da more than the theoretical molecular weight. And the secondary spectrum of LVKGGGGGGGSGGGGSGGGGSE peptide fragment (SEQ ID NO: 29) in CID mass spectrometry shows the common characteristic fragments of glycosylation, parent ions -162Da and -324Da (see Figure 4B), the above two glycosylation The characteristic fragment and molecular weight increase of 340Da proved that the post-translational modification of the peptide was Glucosyl-galactosyl hydroxylysine.

3. Amino acid mutation experiment:

Through amino acid mutation, it was further verified that Glucosylgalactosyl hydroxylysine modification occurred at K28 of the heavy chain of hu1803-9D;

1) adding a serine (S) to the V27-K28-G29 sequence in the GLP-1 polypeptide of the first peptide chain of hu1803-9D, and mutating the final amino acid sequence into V-K-S-G to obtain hu1803-9D mutant 1;

2) K at position 28 of the first peptide chain of hu1803-9D was mutated to R (arginine) to obtain hu1803-9D mutant 2.

The mutated GLP1-GCGR antibody fusion protein gene sequence was transfected into CHO cells, expressed and purified to obtain GLP1-GCGR antibody fusion protein mutants 1 and 2. Then use LC-MS analysis of sugar reduction molecular weight, and the specific experimental process is as follows:

Dilute the GLP1-GCGR antibody fusion protein and its mutant samples to 0.5 mg/mL with pH 7.9 ammonium bicarbonate, take 100 μL of the sample, add 1 μL PNGase F (new England lab, QPF-001-B) to each, and place in a 45°C water bath. After 1 hour, 2 μL of 1M DTT solution was added, and the water was bathed at 37°C for 30 minutes; LC-MS analysis was performed after the reaction was completed.

LC conditions are as follows: Waters UPLC H-class for liquid chromatography; chromatographic column for Waters BEH C4 2.1×50mm, 1.7μm column; injection volume: 0.5μL; column temperature: 80°C; flow rate: 0.3mL/min; Degraded mobile phase B (0.1% formic acid in acetonitrile) was ramped from 5% to 30% over 15 minutes, then 30% to 90% over the next minute.

The mass spectrometer was Waters XeVo G2-XS QTOF mass spectrometer, and the positive ion mode was used for data acquisition, and the collected data was analyzed by Waters unifi software.

The results are shown in Figure 5A to Figure 5C, relative to the unmutated GLP1-GCGR antibody fusion protein (Figure 5A, peaks 53512, 53852), in hu1803-9D mutant 1 (Figure 5B, peak 53600) and mutant 2 ( Figure 5C, peak 53540) did not detect the component with an increase of 340Da, which further verified that the modification of the fusion protein with an increased molecular weight of 340Da occurred at the K28 position.

Example 3. Effect of glycosylation modification on the stability of GLP1-GCGR fusion protein

In order to study the effect of glucose-galactose-hydroxylysine modification on the fusion protein, a high temperature experiment at 25°C was carried out. The GLP1-GCGR fusion protein hu1803-9D was placed at 25°C for 4 days or 7 days, and then the molecular weight LC-MS analysis experiment of deglycosylation reduction was performed. Stability data for the first-strand components that undergo glucose-galactose-hydroxylysine (+340 Da) modification and those that do not undergo such modification are classified and counted. The results are shown in Table 5.

The results showed that the GLP1-GCGR fusion protein hu1803-9D had glucose-galactose-hydroxylysine (+340Da) modified large fragments of the first chain (including K28 The proportion of fragments generated by the fragmentation at position, W25 and F22 positions) was significantly lower than that of the large fragments of the first strand without this modification.

This result further confirms that the glucose-galactose-hydroxylysine modification at K28 can inhibit the generation of large fragments of the first chain of the GLP1-GCGR antibody fusion protein, which helps to improve the stability of the GLP1-GCGR antibody fusion protein.

Table 5. Effects of glucose-galactose-hydroxylysine modification on fragmentation of GLP1-GCGR fusion protein

NOTE: Large fragments contain fragments resulting from breaks at positions K28, W25 and F22 of the first strand.

Example 4. Effect of GLP1-GCGR glycosylation modification on the function of fusion protein

1. Isolation and purification of GLP1-GCGR glycosylation variants:

In order to further obtain glycosylation variants of the GLP1-GCGR antibody fusion protein rich in glucose-galactose-hydroxylysine modification, HIC (hydrophobic chromatography) was used to separate and purify the samples. The specific process is as follows:

Instrument: Agilent 1260; Column: MAbPacTM HIC-10 (Thermo Cat. No. 088481); Mobile Phase A: 50mM PB and 2M Ammonium Sulfate (pH=7.0); Mobile Phase B: 50mM PB (pH=7.0); Flow Rate: 0.8 mL/min; column temperature: 30℃; detection wavelength 280nm;

The chromatographic gradient was as follows: 67.0% mobile phase B equilibrated in 0-5 minutes; mobile phase B changed from 67% to 85.4% in 5-40 minutes; mobile phase B changed from 85.4 to 100 in 40-40.1 minutes %; mobile phase B was maintained at 100% in 40.1-45 minutes; mobile phase B changed from 100 to 67% in 45-45.1 minutes; mobile phase B was maintained at 67% in 45.1-60 minutes. Subsequent fraction collection was performed using an Agilent fraction collector.

The typical HIC spectrum of the GLP-1-GCGR antibody fusion protein hu1803-9D is shown in Figure 6. Subsequently, the two components of peak 3 and peak 4 marked in Figure 6 were collected. Analysis, the results are shown in Figure 7A and Figure 7B.

The results showed that peak 4 was mainly GLP1-GCGR fusion protein hu1803-9D without glucose-galactose-hydroxylysine modification, and peak 3 was mainly GLP1-GCGR fusion protein hu1803-9D with one Glucosylgalactosyl hydroxylysine modification.

2. The effect of glucose-galactose-hydroxylysine post-translational modification on the activity of GLP1-GCGR:

The effect of glycosylation modification on the activity of GLP1-GCGR antibody fusion protein was detected by detecting fusion protein blocking GCGR ligand binding to GCGR and cell-based GLP-1R binding and activation experiments. For the specific experimental process, refer to Test Example 2 and Test Example 6 of WO2020125744.

2.1 Antibody fusion protein blocking the binding of GCGR ligand to GCGR test:

(1) Test purpose:

The antagonistic activity of the antibody fusion protein was evaluated by the GLP1-GCGR antibody fusion protein hu1803-9D blocking the binding of the GCGR ligand glucagon to GCGR.

(2) Test principle:

The combination of cAMP and CRE can initiate the expression of the downstream luciferase gene (luciferase) of CRE, luciferase emits fluorescence after binding to its substrate, and the inhibition efficiency is reflected by the change of fluorescence signal. CRE was cloned upstream of the luciferase gene, and CHO-K1 cells were co-transfected with a plasmid containing the GCGR gene to select monoclonal cells that highly expressed both CRE and GCGR. GLP-1/GCGR antibody fusion protein and glucagon can competitively bind to GCGR, block the downstream signal transmission of GCGR, and affect the expression of downstream cAMP. The GLP-1/GCGR antibody fusion protein can be evaluated by measuring the change of fluorescence signal. Antagonistic activity against GCGR.

(3) Test sample:

①. The GLP1-GCGR antibody fusion protein hu1803-9D modified with glucose-galactose-hydroxylysine was obtained by purifying and collecting peak 3 in Section 1 of this example (referred to as the peak 3 sample);

②. The GLP1-GCGR antibody fusion protein hu1803-9D without glucose-galactose-hydroxylysine modification was obtained by purifying and collecting peak 4 in Section 1 of this example (referred to as peak 4 sample).

(4) Experimental steps:

a. Prepare a cell suspension with fresh cell culture medium, add 20,000 cells/well to a 96-well cell culture plate of 80 μL culture system, and culture at 37°C for 16 hours with 5% carbon dioxide.

b. Add 10 μL of the prepared fusion protein to be tested to each well, and then add 10 μL of the prepared glucagon, 5% carbon dioxide, and incubate at 37° C. for 5 hours.

c. Add 100 μL of detection solution ONE Glo (Promega) to each well, and place at room temperature for 7 minutes in the dark.

d. Detect fluorescence on a microplate reader Victor3, and calculate the IC50 value of blocking the binding of GCGR chimeric antibody fusion to human GCGR ligand glucagon.

Table 6. Antagonistic activity of GLP1-GCGR antibody fusion proteins

sample IC50(nM) Peak 3 sample 49.16 Peak 4 sample 56.36

The results showed that the glucose-galactose-hydroxylysine modification had little effect on the GCGR antagonistic activity of the antibody fusion protein hu1803-9D.

2.2 Cell-based GLP-1R binding and activation experiments

(1) Test purpose:

The activating activity of the GLP-1 portion of the GLP1-GCGR antibody fusion protein hu1803-9D on GLP-1R was evaluated.

(2) Test principle:

The combination of cAMP and CRE can initiate the expression of the luciferase gene downstream of CRE, and the luciferase emits fluorescence after binding to its substrate, and the inhibition efficiency is reflected by the change of the fluorescent signal. CRE was cloned upstream of the luciferase gene, and the CHO-K1 cells were co-transfected with a plasmid containing the GLP-1R gene to select monoclonal cells that highly expressed both CRE and GLP-1R. The GLP-1/GCGR antibody fusion protein can bind to GLP-1R, activate the downstream signaling of GLP-1R, and stimulate the expression of downstream cAMP. The effect of the GLP1-GCGR antibody fusion protein hu1803-9D on GLP-1R can be assessed by measuring the change of fluorescence signal. activation activity.

(3) Test sample:

①. The GLP1-GCGR antibody fusion protein hu1803-9D with glycosylation modification was obtained by purifying and collecting peak 3 in Section 1 of this example (referred to as the peak 3 sample);

②. The GLP1-GCGR antibody fusion protein hu1803-9D without glycosylation was obtained by collecting peak 4 through purification in Section 1 of this example (referred to as peak 4 sample).

(4) Experimental steps:

a. Prepare a cell suspension with fresh cell culture medium, add 25,000 cells/well to a 90 μL culture system in a 96-well cell culture plate, and culture at 37° C. for 16 hours with 5% carbon dioxide.

b. Add 10 μL of the prepared fusion protein to be tested to each well, 5% carbon dioxide, and incubate at 37 degrees for 5 hours.

c. Add 100 μL of detection solution ONE Glo (Promega) to each well, and place at room temperature for 7 minutes in the dark.

d. Detect fluorescence on a microplate reader Victor3, and calculate the EC50 value of the binding activation of GLP1-GCGR antibody fusion protein to GLP-1R.

Table 7. Activation activity of fusion protein on GLP-1R

sample EC50(nM) Peak 3 sample 0.31 Peak 4 sample 0.17

The results showed that the glucose-galactose-hydroxylysine modification had little effect on the GLP-1R activation activity of the antibody fusion protein hu1803-9D.

Claims (20)

A glycosylation modification variant of a GLP1-GCGR antibody fusion protein, comprising: Glycosylation-modified variants of GLP1, and Anti-GCGR antibody; Wherein, the glycosylation modification variant of described GLP1 comprises O-glycosylation modification based on hydroxylysine; Preferably, wherein said anti-GCGR antibody comprises: a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; and A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively. The glycosylation modification variant of GLP1-GCGR antibody fusion protein according to claim 1, wherein the glycosylation modification variant of GLP1 comprises galactose-hydroxylysine modification or glucose-galactose-hydroxylysine modification amino acid modification. The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to claim 1 or 2, wherein the glycosylation modification variant of GLP1 comprises the group consisting of SEQ ID NOs: 4, 1, 2, 3 and The amino acid sequence shown in any one of 5; preferably, the hydroxylysine-based O-glycosylation modification site is at K28 of GLP1. The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to any one of claims 1 to 3, wherein the anti-GCGR antibody comprises: A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18, 16 or 17 . The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to claim 4, wherein the anti-GCGR antibody comprises: The heavy chain constant region as set forth in SEQ ID NO: 19, and A light chain constant region as shown in SEQ ID NO: 20; Preferably, wherein said anti-GCGR antibody comprises: The heavy chain as set forth in SEQ ID NO: 21, and A light chain as shown in SEQ ID NO:22. The glycosylation-modified variant of the GLP1-GCGR antibody fusion protein according to any one of claims 1 to 5, wherein the C-terminus of the glycosylation-modified variant of GLP1 is linked to the anti-GCGR antibody through a linker N-terminal of the heavy chain variable region. The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to claim 6, comprising two first peptide chains with the same sequence and two second peptide chains with the same sequence, wherein: a first peptide chain comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25 or 26; and A second peptide chain comprising the amino acid sequence shown in SEQ ID NO:22. The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to claim 7, which is: i) homodimeric variants, wherein both first peptide chains comprise hydroxylysine-based O-glycosylation modifications; or ii) Heterodimeric variants in which only one of the first peptide chains contains a hydroxylysine-based O-glycosylation modification. The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to claim 8, wherein the hydroxylysine-based O-glycosylation modification is glucose-galactose-hydroxylysine modification; preferably Ground, wherein said hydroxylysine-based O-glycosylation modification occurs at K28 of the first peptide chain. The glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to claim 9, which has 340 Da±5 Da relative to the GLP1-GCGR antibody fusion protein without hydroxylysine-based O-glycosylation modification or a molecular weight increase of 680 Da ± 10 Da, preferably with a molecular weight increase of 340 Da or 680 Da. The glycosylation-modified variant of the GLP1-GCGR antibody fusion protein of any one of claims 7 to 10, which is relative to a GLP1-GCGR antibody fusion that does not contain a hydroxylysine-based O-glycosylation modification The protein is not susceptible to peptide chain cleavage; preferably, the first chain of the glycosylation variant is not susceptible to cleavage at positions K28, W25 and/or F22. A GLP1-GCGR antibody fusion protein population comprising a glycosylation modification variant of the GLP1-GCGR antibody fusion protein of any one of claims 1 to 11. The GLP1-GCGR antibody fusion protein population according to claim 12, wherein the ratio of the glycosylation modification variant in the GLP1-GCGR antibody fusion protein population is at least 0.1%; preferably, the ratio is at least 0.1% more preferably, the ratio is at least 10%; optionally, the content of the glycosylation-modified variant is measured by liquid chromatography-mass spectrometry. A glycosylation modification variant of GLP1 comprising a hydroxylysine-based O-glycosylation modification. The glycosylation-modified variant of GLP1 according to claim 14, which comprises a galactose-hydroxylysine modification or a glucose-galactose-hydroxylysine modification. The glycosylation-modified variant of GLP1 according to claim 14 or 15, wherein said GLP1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 1, 2, 3 and 5. The glycosylation modification variant of GLP1 according to claim 14, wherein the hydroxylysine-based O-glycosylation modification site is at K28 of GLP1. A pharmaceutical composition comprising: A glycosylation modification variant of a GLP1-GCGR antibody fusion protein according to any one of claims 1 to 11, or a population of GLP1-GCGR antibody fusion proteins according to claim 12 or 13, or according to claim 14 To the glycosylation-modified variant of GLP1 of any one of 17; and One or more pharmaceutically acceptable carriers, diluents, buffers or excipients. A method of reducing blood glucose concentration in a subject, the method comprising administering to the subject a therapeutically effective amount of the glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to any one of claims 1 to 11 , or the GLP1-GCGR antibody fusion protein population according to claim 12 or 13, or the glycosylation modification variant of GLP1 according to any one of claims 14 to 17, or the GLP1 according to claim 18 pharmaceutical composition. A method of treating a metabolic disorder, the method comprising administering to a subject a therapeutically effective amount of the glycosylation modification variant of the GLP1-GCGR antibody fusion protein according to any one of claims 1 to 11, or according to The GLP1-GCGR antibody fusion protein population of claim 12 or 13, or the glycosylation modification variant of GLP1 according to any one of claims 14 to 17, or the pharmaceutical composition of claim 18 ; Preferably, the metabolic disorder is selected from the group consisting of: metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemia hyperosmolar syndrome, perioperative hyperglycemia, hyperglycemia Insulinemia, insulin resistance syndrome, impaired fasting glucose, dyslipidemia, atherosclerosis, and prediabetic states.

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