Igg fc mutant and fusion protein, and use thereof

Abstract

The present invention relates to the field of biomedicine. Specifically provided are an IgG Fc mutant and a fusion protein, and the use thereof. The IgG Fc mutant of the present invention comprises amino acid mutations at three or four sites in an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 16 or SEQ ID NO: 25, which sites are selected from: 1) M234A or M234F; 2) L235A or L235E; 3) G237A; and 4) P331S or P331G. The IgG Fc mutant of the present invention can completely eliminate or weaken the binding affinity for FcγRI, FcγRIII and a complement protein C1q. Further provided in the present invention is a fusion protein. The fusion protein comprises the IgG Fc mutant and an insulin polypeptide. The insulin polypeptide has an amino acid sequence as shown in SEQ ID NO: 39.

Description

IgG Fc mutants and fusion proteins and their applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an IgG Fc mutant and fusion protein and their applications. Background Technology

[0002] IgG binds to the neonatal Fc receptor (FcRn) in an acid-base dependent manner. In a weakly acidic environment (pH 5.0–6.5), amino acid residues His310 and His435 in the CH2-CH3 hinge region of the IgG-Fc fragment are protonated, readily binding to acidic amino acid residues of the FcRn, demonstrating a high affinity of IgG for FcRn. Conversely, under neutral or alkaline conditions, histidine residues are deprotonated, leading to decreased affinity. Vascular endothelium is the primary site for FcRn-mediated IgG recirculation. Cells take up extracellular IgG via endocytosis, fuse with early endosomes containing Fc receptors to avoid lysosomal degradation, and then bud back to the cell membrane. Upon contact with weakly alkaline plasma, IgG is released from the FcRn into the bloodstream, thus giving IgG a relatively long half-life. Based on this binding characteristic, the IgG Fc fragment is often used in drug design to prolong drug half-life and provide good biochemical and biophysical stability.

[0003] Immunoglobulin G (IgG) consists of two antigen-binding domains, Fab, linked by a hinge region to an Fc domain. The Fab domain possesses highly specific antigen recognition capabilities, while the Fc domain mediates a wide range of effector functions. Humoral immune responses are primarily controlled by proteolytic reactions involving complement protein C1q and related complement cascades. Cellular immune responses are mainly mediated by the interaction of antibodies with Fcγ receptors (FcγRs). FcγRI and FcγRIII can induce antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). FcγRI is a high-affinity receptor that plays a crucial role in initiating the endocytosis of soluble IgG, the phagocytosis of immune complexes, antibody-dependent cell killing, and the delivery of immune complexes to antigen-presenting pathways. FcγRIII interacts with antibodies to trigger NK cell-mediated ADCC. FcγRIIIA typically binds to the FcεRI-γ chain, CD3-ζ chain, or heterodimers of these two chains within the cell membrane. Both the cytoplasmic tails of the FcεRI-γ and CD3-ζ chains possess immunotyrosine-based activation motifs (ITAMs). ITAMs are phosphorylated and, through signal transduction mechanisms (binding to tyrosine kinases ZAP-70 and Syk, and activation of the PI3K, NF-κB, and ERK pathways), induce NK cell degranulation, cytokine secretion, and ultimately cell lysis.

[0004] Canine Immunoglobulin G (CIgG) has four subtypes: IgGA, IgGB, IgGC, and IgGD. IgGA is similar to human IgG2 and binds weakly to FcγR. IgGD is similar to human IgG4 and has virtually no affinity for complement protein C1q. IgGB, however, is similar to human IgG1 and has a strong affinity for both FcγR and complement protein C1q. The amino acid sequence of the WT canine IgGB Fc is as follows:

[0005] DCPKCPAPEM 234 L 235 GG 237 PSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFN 297 GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSP 331IERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 1).

[0006] Currently, feline immunoglobulin G (FIgG) has two subtypes: IgG1 and IgG2. IgG1 has two alleles, IgG1a and IgG1b. The amino acid sequence of WT feline IgG1b Fc is as follows:

[0007] DCPKCPPPEM 234 L 235 GG 237 PSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFN 297 STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSP 331 IERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 16)

[0008] The amino acid sequence of WT cat IgG 1aFc is as follows:

[0009] DCPKCPPPEM 234 L 235 GG 237 PSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFN 297 STYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSP 331 IERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 25)

[0010] Similar to human IgG1, it can bind to FcγR and complement protein C1q to induce strong effector functions, while IgG2 does not bind to FcγR or complement protein C1q.

[0011] Because some IgG subtypes of Fc can mediate immune effects, affecting drug safety and half-life, IgG Fc that do not have or weakly mediate immune effects are selected, such as human IgG2 Fc and human IgG4 Fc. Mutations in IgG Fc segments are also designed to eliminate or weaken their mediated immune effects.

[0012] Patent application CN115135669A discloses a canine Fc fragment, specifically disclosing the most preferred sequence 19 (HC-B_L235A_P331G) in the embodiments. Experimental results show that its binding with FcγI and C1q is essentially nonexistent. However, the affinity of the Fc fragment in this patent application was only verified with FcγI and C1q, and its affinity with FcγIII was not verified. FcγRIIIa (CD16a) is mainly responsible for triggering NK cell-mediated ADCC. When CD16a recognizes the target of IgG opsonization, NK cells release different cytotoxic molecules, thereby inducing the death of target cells. Therefore, the Fc mutant in this patent application has a greater impact on the C1q-mediated CDC effect, but a limited impact on the FcγR-mediated ADCC and ADCP effects.

[0013] This invention selects canine IgG B Fc fragments and feline IgG 1 Fc fragments (including feline IgG 1a Fc and feline IgG 1b Fc) that have similar functions to human IgG1 and modifies them to silence their effector functions.

[0014] Diabetes mellitus is a chronic disease caused by a deficiency of insulin secretion and / or impaired biological action, leading to disordered glucose metabolism. Diabetes is mainly divided into two types: type 1 diabetes and type 2 diabetes. Type 1 diabetes (formerly known as juvenile-onset or insulin-dependent diabetes) is caused by an autoimmune response triggered by environmental factors in genetically susceptible individuals, damaging pancreatic beta cells and resulting in insufficient insulin secretion. Type 2 diabetes is caused by insulin resistance, which affects beta cell function and leads to insufficient insulin secretion.

[0015] Diabetes is a relatively common internal medicine disease in companion dogs and cats, with a similar pathogenesis to human diabetes. In canine diabetes, β-cell loss is generally caused by immune-mediated destruction, vacuolar degeneration, or pancreatitis, similar to human type 1 diabetes. In felines, diabetes is caused by insulin resistance, islet amyloidosis, or chronic lymphocytic pancreatitis, similar to human type 2 diabetes. Clinical symptoms include polydipsia / polyuria (PU / PD), polyphagia (PP), and weight loss. Fat metastasis leads to hepatic lipid deposition, hepatomegaly, hypercholesterolemia, hypertriglyceridemia, and increased catabolism. If left uncontrolled or poorly controlled, it will eventually lead to ketoacidosis, ketonuria, and ketoacidosis. Currently, the FDA-approved insulin drugs for treating canine and feline diabetes include: Vestulin (Merck's Animal Health, a new suspension of porcine insulin), an intermediate-acting insulin with a duration of action close to 12 hours; and Prozinc (Boehringer Ingelheim's Animal Health, protamine zinc recombinant human insulin), a long-acting insulin with a duration of action of 8-24 hours. Frequent administration increases the burden on sick pets and their owners, thus requiring longer-acting insulin products to improve treatment adherence.

[0016] The main pathways of insulin clearance include insulin receptor-mediated clearance, lysosomal degradation, and renal clearance. When insulin fuses with the Fc fragment of immunoglobulin G (IgG), it can be rescued and its clearance slowed by the pH-dependent binding characteristic of its FcRn. However, some Fc fragments of immunoglobulin G (IgG) can interact with the immune system, mediating immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). For safety reasons, immune-silencing Fc fragments should be selected or developed and fused with insulin analogs for expression, enabling long-term and safe treatment of diabetes. Summary of the Invention

[0017] The present invention aims to provide a canine IgG B Fc mutant and a cat IgG 1a and IgG 1b Fc mutant that eliminate the affinity of canine IgG B Fc fragment or cat IgG 1 Fc fragment for FcγRI, FcγRIII, and complement protein C1q.

[0018] This invention provides an IgG Fc mutant containing a canine or feline Fc fragment. The IgG Fc mutant is a mutated amino acid polypeptide, which, relative to the wild-type Fc fragment, is a canine or feline IgG Fc mutant. The IgG Fc mutant includes amino acid sequences selected from: SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO: 25.

[0019] 1) M234A or M234F;

[0020] 2) L235A or L235E;

[0021] 3) G237A;

[0022] 4) Amino acid mutations at three or four sites in P331S or P331G.

[0023] In this invention, as one embodiment, the IgG Fc mutant includes amino acid mutations occurring at positions 235, 237, and 331. As another embodiment, the IgG Fc mutant includes amino acid mutations of L235A or L235E, G237A, and P331S. The aforementioned mutations refer to the occurrence of amino acid mutations of L235A, G237A, and P331S; or the occurrence of amino acid mutations of L235E, G237A, and P331S.

[0024] In this invention, as one embodiment, the IgG Fc mutant includes amino acid mutations at positions 234, 235, 237, and 331. As another embodiment, the IgG Fc mutant includes mutations in M234A or M234F, L235A or L235E, G237A, and P331S. The aforementioned mutations refer to mutations in M234A, L235A, G237A, and P331S; or mutations in M234A, L235E, G237A, and P331S; or mutations in M234F, L235A, G237A, and P331S; or mutations in M234F, L235E, G237A, and P331S.

[0025] In this invention, as one embodiment, the canine IgG Fc mutant comprises the amino acid sequence shown in SEQ ID NO: 1, wherein:

[0026] L235A / G237A / P331S;

[0027] L235E / G237A / P331S;

[0028] M234A / L235A / G237A / P331S;

[0029] M234A / L235E / G237A / P331S; or

[0030] Mutations in M234F / L235A / G237A / P331S.

[0031] In this invention, as one embodiment, the feline IgG Fc mutant comprises the amino acid sequence shown in SEQ ID NO: 16, wherein:

[0032] L235E / P331S / G237A;

[0033] M234A / L235A / G237A / P331S; or

[0034] Mutations in M234A / L235E / G237A / P331S.

[0035] In this invention, as one embodiment, the feline IgG Fc mutant comprises the amino acid sequence shown in SEQ ID NO: 25, wherein:

[0036] M234A / L235A / G237A / P331S;

[0037] M234A / L235E / G237A / P331S; or

[0038] The mutation of L235E / P331S / G237A.

[0039] In this invention, as one embodiment, the IgG Fc mutant is a canine IgG B Fc mutant, which contains the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0040] In this invention, as one embodiment, the IgG Fc mutant is a cat IgG 1b Fc mutant, which contains the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21.

[0041] In this invention, as one embodiment, the IgG Fc mutant is a cat IgG 1a Fc mutant, which contains the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28.

[0042] In this invention, as one embodiment, the canine Fc fragment contains the amino acid sequence shown in SEQ ID NO:1, and the feline Fc fragment contains the amino acid sequence shown in SEQ ID NO:16 or SEQ ID NO:25.

[0043] The IgG Fc mutant of the present invention, comprising the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27 or SEQ ID NO:28, can completely eliminate the binding affinity with Fc receptor and complement protein C1q, wherein the Fc receptor is FcγRI or FcγRIII.

[0044] The IgG Fc mutant of the present invention, comprising the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10, can completely eliminate the binding affinity with the Fc receptor and weaken the binding affinity with complement protein.

[0045] In one embodiment of the IgG Fc mutant described in this invention, the Fc receptor is FcγRI or FcγRIII, and the complement protein is C1q.

[0046] The present invention also provides a polynucleotide encoding the above-mentioned IgG Fc mutant, wherein the polynucleotide is DNA or RNA.

[0047] The present invention provides a fusion protein comprising the above-mentioned IgG Fc mutant or a fragment thereof.

[0048] This invention provides a pharmaceutical composition comprising the above-described IgG Fc mutant or fusion protein and a pharmaceutically acceptable carrier, excipient, diluent, or excipient. The pharmaceutical composition comprises the above-described IgG Fc mutant polypeptide, antibody, or antibody fragment.

[0049] The present invention provides the use of the above-mentioned IgG Fc mutant or fusion protein or pharmaceutical composition thereof in the preparation of medicaments for treating inflammatory diseases, autoimmune diseases, infectious diseases, cardiovascular and cerebrovascular dysfunction, pain, and eye diseases. Beneficial effects

[0050] Canine IgG B Fc mutant: It contains the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9; the three polypeptide mutants completely eliminate their affinity for FcγRI, FcγRIII, and complement protein C1q.

[0051] Canine IgG B Fc mutant: comprising the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10; the two polypeptide mutants completely eliminate affinity for FcγRI and FcγRIII and weaken affinity for complement protein C1q to 10.-4 .

[0052] The feline IgG 1Fc mutant contains the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27 or SEQ ID NO:28; the four polypeptide mutants completely eliminate affinity for FcγRI, FcγRIII and complement protein C1q.

[0053] The present invention also provides a fusion protein comprising any of the above-described IgG Fc mutants and an insulin polypeptide, wherein the insulin polypeptide has the amino acid sequence shown in SEQ ID NO:39.

[0054] In the fusion protein of the present invention, as one embodiment, the IgG Fc mutant is preferably the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:20 or SEQ ID NO:21.

[0055] In this invention, the IgG Fc mutant and insulin polypeptide of the above-mentioned fusion protein are linked by a peptide linker having the amino acid sequence shown in SEQ ID NO:40.

[0056] In this invention, as one embodiment, the fusion protein has the amino acid sequence shown in any one of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:38.

[0057] The present invention also provides a polynucleotide encoding the fusion protein described in any of the preceding claims, wherein the polynucleotide is DNA or RNA.

[0058] The present invention also provides a pharmaceutical composition comprising any of the fusion proteins described above and a pharmaceutically acceptable carrier, excipient, diluent, or excipient.

[0059] The present invention also provides the use of any of the above-described fusion proteins or the above-described pharmaceutical compositions in the preparation of medicaments for treating diabetes.

[0060] This invention expresses a fusion protein with a purity and dimer content both exceeding 90%.

[0061] The fusion protein in this invention was validated using a beagle dog model of type 1 diabetes. A single dose controlled blood glucose for at least 336 hours, allowing for once-weekly administration. It successfully and stably maintained fasting blood glucose below 8.3 mmol / L and median blood glucose within 6 hours below 13 mmol / L. Data disclosed by the FDA-approved insulin drug PZI indicates that a 10-hour MBG level <250 mg / dL is considered excellent for blood glucose control, and a fasting blood glucose level between 80-150 mg / dL is also considered excellent. Based on this data, the drug molecule of this invention can be considered to have excellent blood glucose control in diabetic model dogs.

[0062] This invention extends the half-life of insulin by fusing a target animal immunoglobulin G (IgG) Fc fragment, thereby reducing the frequency of administration and maintaining blood glucose levels. In dogs and cats, once-weekly administration was successfully implemented, and blood glucose was well controlled. Attached Figure Description

[0063] Figure 1: Sequence alignment of human IgG Fc and canine IgG Fc;

[0064] Figure 2: Sequence alignment of human FcγRI, canine FcγRI, and cat FcγRI;

[0065] Figure 3: Sequence alignment of human, canine, and cat FcγRIII;

[0066] Figure 4: Sequence alignment of human IgG Fc and cat IgG Fc;

[0067] Figure 5: FcRn receptor affinity results;

[0068] Figure 6: Results of IR receptor affinity;

[0069] Figure 7: Affinity results of FcγR and C1q for multiple fusion proteins;

[0070] Figure 8: Fasting blood glucose monitoring results in model animals;

[0071] Figure 9: Analysis of blood glucose fluctuation data after a single administration of the test substance SEQ ID NO:33;

[0072] Figure 10: Blood glucose monitoring results throughout the day after 6 weeks of drug administration;

[0073] Figure 11: Blood glucose control at different time points after 2 weeks of drug administration;

[0074] Figure 12: Fasting blood glucose monitoring results during drug administration;

[0075] Figure 13: Weight monitoring results after 6 weeks of drug administration. Detailed Implementation

[0076] The present invention will be further described in detail through the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0077] The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the invention in any way.

[0078] Example 1: Construction of IgG Fc mutant molecule

[0079] 1.1 Molecular construction of canine IgG B Fc mutant

[0080] Based on the results of multiple sequence alignment (human IgG Fc and canine IgG Fc sequence alignment, as shown in Figure 1; human FcγRI, canine FcγRI and feline FcγRI sequence alignment, as shown in Figure 2; human FcγRIII, canine FcγRIII and feline FcγRIII sequence alignment, as shown in Figure 3.) and protein structure analysis, molecular design was performed on wild-type canine IgG B, resulting in 13 mutants with the following amino acid sequences: Seq ID NO: 2, Seq ID NO: 3, Seq ID NO: 4, Seq ID NO: 5, Seq ID NO: 6, Seq ID NO: 7, Seq ID NO: 8, Seq ID NO: 9, Seq ID NO: 10, Seq ID NO: 11, Seq ID NO: 13, Seq ID NO: 14, and Seq ID NO: 15. The amino acid sequence of wild-type (WT) canine IgG B Fc is Seq ID NO: 1, and the amino acid sequence of WT canine IgG D Fc is Seq ID NO: 12. Referring to Molecular Cloning: A Laboratory Manual, 4th Edition, the aforementioned amino acid sequences were converted into nucleotide sequences, and the corresponding nucleotide fragments were synthesized by PCR amplification. The fragments were then digested and ligated with enzymes, cloned into a proprietary mammalian expression vector (such as pKS001), and positive clones were screened using a PCR reaction system for subsequent transfection and expression.

[0081] Seq ID NO: 1

[0082] Seq ID NO: 2

[0083] Seq ID NO: 3

[0084] Seq ID NO: 4

[0085] Seq ID NO: 5

[0086] Seq ID NO: 6

[0087] Seq ID NO: 7:

[0088] Seq ID NO: 8

[0089] Seq ID NO: 9

[0090] Seq ID NO: 10

[0091] Seq ID NO: 11

[0092] Seq ID NO: 12

[0093] Seq ID NO: 13

[0094] Seq ID NO: 14

[0095] Seq ID NO: 15

[0096] 1.2 Molecular construction of feline IgG1aFc and IgG1bFc mutants

[0097] Based on the results of multiple sequence alignment (sequence alignment of human IgG Fc and cat IgG Fc, as shown in Figure 4) and protein structure analysis, molecular design was performed on wild-type cat IgG1 Fc, resulting in 13 mutants with the following amino acid sequences: Seq ID NO: 17, Seq ID NO: 18, Seq ID NO: 19, Seq ID NO: 20, Seq ID NO: 21, Seq ID NO: 22, Seq ID NO: 23, Seq ID NO: 24, Seq ID NO: 27, Seq ID NO: 28, Seq ID NO: 29, Seq ID NO: 30, and Seq ID NO: 31. The amino acid sequence of WT cat IgG 1b Fc is Seq ID NO: 16, the amino acid sequence of WT IgG 1aFc is Seq ID NO: 25, and the amino acid sequence of WT IgG 2Fc is Seq ID NO: 26. Referring to the 4th edition of Molecular Cloning Laboratory Manual, the aforementioned amino acid sequence was converted into a nucleotide sequence, the corresponding nucleotide fragment was synthesized by PCR amplification, the fragment was digested and ligated with enzymes, and cloned into a proprietary mammalian expression vector (such as pKS001). Positive clones were screened through the PCR reaction system for subsequent transfection and expression.

[0098] Seq ID NO: 16

[0099] Seq ID NO: 17

[0100] Seq ID NO: 18

[0101] Seq ID NO: 19

[0102] Seq ID NO: 20

[0103] Seq ID NO: 21

[0104] Seq ID NO: 22

[0105] Seq ID NO: 23

[0106] Seq ID NO: 24

[0107] Seq ID NO: 25

[0108] Seq ID NO: 26

[0109] Seq ID NO: 27

[0110] Seq ID NO: 28

[0111] Seq ID NO: 29

[0112] Seq ID NO: 30

[0113] Seq ID NO: 31

[0114] Example 2: Expression and purification of recombinant proteins

[0115] 2.1. Expression and Purification

[0116] Plasmids were extracted from the sequenced bacterial culture and electroporated into CHO cells. Cells from the electroporation tubes were aliquoted into pre-prepared shake flasks containing culture medium and incubated statically for 40 min. After incubation, the shake flasks were placed in a 37°C, 110 rpm, 8% CO2 environment for 24 h. Feed / sodium butyrate / antibiotics were added, and culture continued for 3-7 days. The cell culture supernatant was harvested, and proteins were purified using Protein A affinity chromatography (GE Healthcare). The method is as follows:

[0117] 1) Equilibration column: 1×PBS, flow rate 1ml / min

[0118] 2) Sample loading: Flow rate 1 ml / min

[0119] 3) Washing: 1×PBS, flow rate 1ml / min

[0120] 4) Elution: Sodium acetate buffer (pH 3.4), 1 ml / min, collected in separate tubes, and the absorbance value at 280 nm was read using a NanoDrop instrument.

[0121] 5) Dialysis: Aspirate high-concentration protein into a dialysis bag and place it in a beaker containing 1×PBS for dialysis.

[0122] 2.2. Purity Testing

[0123] SEC experiments were performed using a high-performance liquid chromatograph (LC-20AT) and a gel chromatography column. The experimental conditions were as follows:

[0124] 1) Flow rate 1 ml / min

[0125] 2) Injection volume: 20 μL for concentrations ≤ 4 mg / ml; 50 μg for concentrations > 4 mg / ml.

[0126] 3) Column temperature 35℃

[0127] 4) Detection wavelengths: 214nm, 280nm

[0128] 5) Data collection time: 15 min

[0129] Replace the water with the mobile phase, and slowly increase the flow rate to 1.0 ml / min until the baseline stabilizes. Transfer 25 μL of antibody to the corresponding numbered vial, place the vial in the appropriate position on the instrument, and inject for 15 min. Analyze, process, and save the data. Replace the mobile phase with deionized water and rinse for 1.5 h.

[0130] Example 3 Receptor Affinity Assessment

[0131] 3.1 Canine FcγRI receptor affinity assessment

[0132] Canine FcγRI was immobilized in HBS-EP+ buffer at room temperature. The sensor chips in flow cells 1 and 2 were activated with fresh 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Canine FcγRI was diluted with 10 mmol / L NaAC and injected into flow cell 2 to couple appropriate resonant units, while flow cell 1 was set as a blank. Remaining active coupling sites on the chip surface were blocked with 1 mol / L ethanolamine hydrochloride. The test sample was diluted and injected onto the surfaces of flow cells 1 and 2 as the associative phase, followed by the injection of buffer as the dissociation phase. The association time was set to 120 s, the dissociation time to 360 s, and the flow rate to 30 μL / min. All data were processed using Biacore 8K evaluation software version 4.0.

[0133] 3.2 Human FcγRIIIA receptor affinity assessment

[0134] Human FcγRIIIA was immobilized at room temperature using HBS-EP+ as buffer. Sensor chips in flow cells 1 and 2 were activated with fresh 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Human FcγRIIIA was diluted with 10 mmol / L NaAC and injected into flow cell 2 to couple appropriate resonant units, while flow cell 1 was set as a blank. Remaining active coupling sites on the chip surface were blocked with 1 mol / L ethanolamine hydrochloride. The test sample was diluted and injected onto the surfaces of flow cells 1 and 2 as the associative phase, followed by the injection of buffer as the dissociation phase. The association time was set to 120 s, the dissociation time to 360 s, and the flow rate to 30 μL / min. All data were processed using Biacore 8K evaluation software version 4.0.

[0135] 3.3 Complement protein Human C1q affinity assessment

[0136] The test sample was immobilized on a CM3 chip as a ligand, and human C1q protein was used as the analyte (mobile phase) for detection. The test sample was coupled at 25°C using HBS-EP+ as the run buffer. Flow cells 1 and 2 on the chip surface were activated using freshly mixed N-hydroxysulfosuccinimide (NHS) to a final concentration of 50 mmol / L and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to a final concentration of 200 mmol / L. Then, the analyte was diluted with 10 mM sodium acetate and injected into flow cell 2 to achieve the appropriate resonance unit (coupling level). Flow cell 1 served as a blank control. After the amino coupling reaction was complete, the remaining activation sites on the chip surface were blocked using 1 mol / L ethanolamine hydrochloride. For the affinity assay of the test sample with human C1q protein, the human C1q protein was diluted with run buffer and injected into flow cells 1 and 2 on the chip surface; this process served as the binding phase. Run buffer was then injected immediately afterward; this process served as the dissociation phase. All data were processed using Biacore 8K evaluation software version 4.0.

[0137] The affinity results for the canine IgG B Fc mutant are as follows: (NA indicates not detected)

[0138] Table 1. Affinity results of wild-type and mutant canine molecules with Canine CD64 receptor.

[0139] Table 2. Affinity results of canine wild-type molecules and mutants with the Human CD16a(V176) receptor.

[0140] Table 3. Affinity results of wild-type and mutant canine molecules and complement protein Human C1q.

[0141] Comparative analysis revealed that sites 235, 237, 238, 239, 329, 330, and 331 in the Fc fragments of canine IgGA, IgG B, and IgG D are highly conserved. Structural comparison showed that site L235 is highly similar to site L235 in human IgG1, and the amino acids in the hydrophobic pocket region of human and canine FcγRI are highly conserved. It is speculated that the binding mechanism of canine IgG B to FcγRI also involves the formation of a hydrophobic interaction between the L235 side chain and the receptor's hydrophobic pocket. Sites P329, S330, and P331 are also highly conserved, suggesting that they bind to FcγRIII by forming a proline sandwich, disrupting the hydrophobic binding at this site, and thus reducing the affinity for FcγRIII. However, experiments showed that the single mutant P331S (Seq ID NO: 14) could not reduce the affinity for FcγRIII, while the combination of P331S and L235 to form the mutant Seq ID NO: 3 eliminated the affinity for C1q and reduced the affinity for FcγRI by approximately 10%. 2 Even after increasing the molecular weight by 10 times, the affinity for FcγRIII could not be eliminated. This result also differs from the reported results of human IgG1Fc modification, suggesting that the main sites affecting the binding of canine IgG B Fc to C1q are 331 and 235. Furthermore, it was found that the simple mutation L235A (Seq ID NO: 2) only reduced the affinity for FcγRI by approximately 10 times. 2 This further demonstrates that canine IgG B Fc and human IgG 1 Fc differ structurally in their binding regions to FcγRI, FcγRIII, and complement protein C1q. Further experiments with M234A, M234F, L235E, and G237A mutants unexpectedly revealed that the G237A molecule eliminates affinity for FcγRIII and reduces affinity for FcγRI by approximately 10-fold. The Seq ID NO: 5 and Seq ID NO: 11 mutants completely eliminate affinity for receptors FcγRI and FcγRIII; while the Seq ID NO: 4 and Seq ID NO: 10 mutants, in addition to eliminating affinity for receptors FcγRI and FcγRIII, also reduce affinity for C1q by approximately 10-fold. 4The Seq ID NO: 8 mutant completely eliminates affinity for FcγRI, FcγRIII, and complement protein C1q, maximally silencing the immune response. Furthermore, it was unexpectedly found that when the leucine at L235 is mutated to glutamate, the mutants Seq ID NO: 6 and Seq ID NO: 9, formed by mutating only G237A and P331S, also completely eliminate affinity for FcγRI, FcγRIII, and complement protein C1q. Deglycosylated canine IgG BFc molecules, Seq ID NO: 13, only eliminate affinity for FcγRIII. The L235 and P331 sites have a significant impact on the affinity for complement protein C1q, while the L235 and G237 sites have a significant impact on the affinity for FcγRI and FcγRIII.

[0142] The affinity results for feline IgG1aFc and IgG1bFc mutants are as follows: (NA indicates not detected)

[0143] Table 4. Affinity results of wild-type feline molecules and mutants with Canine CD64 receptor.

[0144] Table 5. Affinity results of wild-type feline molecules and mutants with the Human CD16a(V176) receptor.

[0145] Table 6. Affinity results of wild-type and mutant cat molecules with complement protein Human C1q.

[0146] An unexpected discovery was made: unlike canine IgGB Fc, the M234 and L235 sites in feline IgG 1 Fc are equally important for binding to FcγRI and complement protein C1q. Mutating only L235A, G237A, and P331S to form mutants Seq ID NO: 18 and Seq ID NO: 31 only weakens the binding to complement protein C1q, eliminates the affinity for FcγRIII, and retains a high affinity for FcγRI. Mutating L235 to glutamate to form mutants Seq ID NO: 19 and Seq ID NO: 29 retains a high affinity for complement protein C1q but eliminates the affinity for FcγRI and FcγRIIIA. Based on the above molecules, mutating M234 to alanine, forming mutants Seq ID NO: 20, Seq ID NO: 21, Seq ID NO: 27, and Seq ID NO: 28, completely eliminates the affinity for FcγRI, FcγRIIIA, and complement protein C1q. However, when M234 is mutated to phenylalanine, a hydrophobic amino acid with a larger side chain, it retains a high affinity for complement protein C1q. Whether the affinity for FcγRI is reduced or eliminated depends on the amino acid mutated at L235. This indicates that the mutation of M234 in feline IgG 1Fc to a non-hydrophobic amino acid, as well as the combined mutation with L235, has a significant impact on the binding to FcγRI and complement protein C1q.

[0147] Example 4: Expression and purification of fusion protein

[0148] The amino acid sequence of the insulin polypeptide is as follows:

[0149] SEQ ID NO: 39:

[0150] The amino acid sequence of the peptide linker is as follows:

[0151] SEQ ID NO: 40:

[0152] The amino acid sequence of the insulin peptide-Fc fusion protein is as follows:

[0153] SEQ ID NO: 32 (fusion protein 1)

[0154] SEQ ID NO: 33 (fusion protein 2)

[0155] SEQ ID NO: 34 (fusion protein 3)

[0156] SEQ ID NO: 35 (fusion protein 4)

[0157] SEQ ID NO: 36 (fusion protein 5)

[0158] SEQ ID NO: 37 (fusion protein 6)

[0159] SEQ ID NO: 38 (fusion protein 7)

[0160] 4.1 Expression: Referring to Molecular Cloning Laboratory Manual, 4th Edition, the above amino acid sequence was converted into a nucleotide sequence. The corresponding nucleotide fragment was synthesized by PCR amplification, digested and ligated with enzymes, and cloned into a proprietary mammalian expression vector (e.g., pKS001). Positive clones were screened using the PCR reaction system for subsequent transfection and expression. Plasmids were extracted from the sequenced bacterial culture and electroporated into CHO cells. Cells from the electroporation tubes were aliquoted into shake flasks containing culture medium and incubated statically for 40 min. After incubation, the shake flasks were placed in a 37°C, 110 rpm, 8% CO2 environment for 24 h. Afterward, feed, sodium butyrate, and penicillin-streptomycin mixture were added, and the culture continued for 3-7 days.

[0161] 4.2 Purification: The protein was purified by Protein A affinity chromatography (GE Healthcare). Molecular purity was assessed by SDS-PAGE and SEC to determine protein concentration.

[0162] 1) Equilibration chromatography column: 1×PBS, flow rate 1ml / min, 20ml;

[0163] 2) Sample loading: Flow rate 1 ml / min;

[0164] 3) Washing: 1×PBS, flow rate 1ml / min, 20ml;

[0165] 4) Elution: Sodium acetate buffer (pH 3.4), 1 ml / min, collected in aliquots, approximately 500 μL per tube. A total of 10 tubes were collected, and the absorbance at 280 nm was read using a NanoDrop instrument.

[0166] 4.3 Dialysis: Aspirate high-concentration protein into a dialysis bag and place it in a beaker containing 1×PBS for dialysis.

[0167] Example 5 Detection

[0168] 5.1 Purity Testing:

[0169] SEC experiments were performed using a high-performance liquid chromatograph (LC-20AT) and a gel chromatography column. The experimental conditions were as follows:

[0170] Flow rate: 1 ml / min

[0171] Injection volume: 50ug

[0172] Column temperature: 35℃

[0173] Detection wavelength: 280nm

[0174] Data collection time: 15 min

[0175] Replace the water with the mobile phase, and slowly increase the flow rate to 1,000 ml / min until the baseline stabilizes. Take 25 μL of the sample to be tested into the corresponding numbered vial, place the vial in the corresponding position on the instrument, and inject the sample for 15 min. Analyze, process, and save the data. Replace the mobile phase with deionized water and rinse for 1.5 h.

[0176] The SEC results are shown in Table 7. The proportion of dimers in the purified samples was higher than 90%, and the proportion of effective components in the samples met expectations.

[0177] Table 7

[0178] 5.2 Receptor Affinity Detection

[0179] Human FcγRIIIA, Canine FcγRI, complement protein C1q, Canine FcRn, Feline FcRn, and human IR were immobilized at room temperature using HBS-EP+ as buffer. Sensor chips in flow cells 1 and 2 were activated with fresh 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Human FcγRIIIA was diluted with 10 mmol / L NaAC and injected into flow cell 2 to couple appropriate resonance units, while flow cell 1 was set as a blank. The remaining active coupling sites on the chip surface were blocked with 1 mol / L ethanolamine hydrochloride. The test sample fusion proteins SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 were diluted and injected onto the surfaces of flow cells 1 and 2 as the associative phase, followed by the injection of buffer as the dissociation phase. The association time was set to 120 s, the dissociation time to 360 s, and the flow rate to 30 μL / min. All data were processed using Biacore 8K evaluation software version 4.0.

[0180] The results of FcRn receptor affinity are shown in Figure 5, and the results of IR receptor affinity are shown in Figure 6.

[0181] Figure 7 shows the affinity results between FcγR and C1q, where FcγRIIIA corresponds to the left Y-axis, and FcγRI and complement protein C1q correspond to the right Y-axis.

[0182] Receptor affinity results showed that after the wild-type Fc fragment was fused with the insulin peptide (SEQ ID NO:32 and SEQ ID NO:36), it still retained its binding to the FcγR receptor and complement protein C1q, which may induce immune effects such as ADCC and CDC. However, after the mutant Fc fragment was fused with the insulin peptide (SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38), it did not bind to FcγR and complement protein C1q, minimizing the possible immune effects and having almost no impact on the binding to FcRn and insulin receptor IR.

[0183] Example 6 Pharmacodynamic Evaluation

[0184] Eight beagle dogs were used in the experiment. After passing quarantine, they were weighed and administered alloxan at a dose of 50 mg / kg via superficial venous injection in the leg to destroy the pancreas and establish a diabetic model. Day 0 (D0) was the day of model establishment. Fasting and random blood glucose levels were monitored daily after model establishment. Successful modeling was defined as a blood glucose level ≥11.1 mmol / L that remained at this level for two weeks. The successfully modeled animals were randomly assigned to two groups and administered the test substance SEQ ID NO:33 for blood glucose control once a week. Blood glucose levels were monitored daily, and weight and complete blood counts were performed every two weeks. After six weeks of administration, blood glucose was monitored throughout the day under normal dietary conditions to observe the drug's effect on blood glucose control.

[0185] The modeling results are shown in Figure 8. Approximately 10 days after alloxan injection, the blood glucose levels of all 8 animals were above 11.1 mmol / L, indicating successful model establishment. Figure 9 shows the blood glucose control after a single dose. Blood glucose reached its lowest point at 24 hours, followed by a gradual plateau. After 312 hours, blood glucose rose again, indicating that the test substance could lower blood glucose and maintain this level for approximately 312 hours. Figure 11 shows the blood glucose control at different time points after 2 weeks of administration. Data from multiple administrations (see Figures 10 and 12) show that, compared to the model group, the test substance effectively reduced both fasting and 24-hour blood glucose. After 4 weeks of administration, blood glucose control was relatively stable, with fasting blood glucose maintained below 7.5 mmol / L and postprandial blood glucose also stably controlled. Figure 13 shows the body weight results. Compared to the model group, the test substance controlled the body weight of the affected animals, preventing weight loss due to diabetes. These results are consistent with the clinical expectations for canine and feline diabetes management.

Claims

1. A canine or feline IgG Fc mutant, characterized in that, The IgG Fc mutant includes amino acid sequences selected from those shown in SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO:

25. 1) M234A or M234F; 2) L235A or L235E; 3) G237A; 4) Amino acid mutations at three or four sites in P331S or P331G.

2. The canine or feline IgG Fc mutant according to claim 1, characterized in that, The IgG Fc mutant includes amino acid mutations at positions 235, 237, and 331, preferably L235A or L235E, G237A, and P331S.

3. The IgG Fc mutant according to claim 1, characterized in that, The IgG Fc mutant includes amino acid mutations at positions 234, 235, 237 and 331, preferably amino acid mutations of M234A or M234F, L235A or L235E, G237A and P331S.

4. The IgG Fc mutant according to claim 1, characterized in that, The canine IgG Fc mutant includes the amino acid sequence shown in SEQ ID NO: 1, which occurs in: L235A / G237A / P331S; L235E / G237A / P331S; M234A / L235A / G237A / P331S; M234A / L235E / G237A / P331S; or Mutations in M234F / L235A / G237A / P331S.

5. The IgG Fc mutant according to claim 1, characterized in that, The feline IgG Fc mutant comprises the amino acid sequence shown in SEQ ID NO: 16, which occurs in: L235E / P331S / G237A; M234A / L235A / G237A / P331S; or Mutations in M234A / L235E / G237A / P331S.

6. The IgG Fc mutant according to claim 1, characterized in that, The feline IgG Fc mutant comprises the amino acid sequence shown in SEQ ID NO: 25, which occurs in: M234A / L235A / G237A / P331S; M234A / L235E / G237A / P331S; or The mutation of L235E / P331S / G237A.

7. The IgG Fc mutant according to claim 4, characterized in that, The canine IgG BFc mutant contains the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:

10.

8. The IgG Fc mutant according to claim 5, characterized in that, The cat IgG 1bFc mutant contains the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:

21.

9. The IgG Fc mutant according to claim 6, characterized in that, The cat IgG 1aFc mutant contains the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:

28.

10. A polynucleotide encoding the IgG Fc mutant of any one of claims 1-9, wherein the polynucleotide is DNA or RNA.

11. A fusion protein comprising the IgG Fc mutant or a fragment thereof as described in any one of claims 1-9.

12. A pharmaceutical composition comprising the IgG Fc mutant or a fragment thereof as described in any one of claims 1-9 or the fusion protein as described in claim 11, and a pharmaceutically acceptable carrier, excipient, diluent, or excipient.

13. The use of the IgG Fc mutant of any one of claims 1-9, the fusion protein of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating inflammatory diseases, autoimmune diseases, infectious diseases, cardiovascular and cerebrovascular dysfunction, pain, and eye diseases.

14. A fusion protein comprising an IgG Fc mutant according to any one of claims 1-9 and an insulin polypeptide having the amino acid sequence shown in SEQ ID NO:

39.

15. The fusion protein according to claim 14, characterized in that, The IgG Fc mutant has an amino acid sequence as shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:20 or SEQ ID NO:

21.

16. The fusion protein according to any one of claims 14 or 15, characterized in that, The fusion protein further includes a peptide linker having the amino acid sequence shown in SEQ ID NO:

40.

17. The fusion protein according to any one of claims 14, 15, or 16, characterized in that, The fusion protein has the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:

38.

18. A polynucleotide encoding the fusion protein of any one of claims 14-17, wherein the polynucleotide is DNA or RNA.

19. A pharmaceutical composition comprising the fusion protein of any one of claims 14-17 and a pharmaceutically acceptable carrier, excipient, diluent, or excipient.

20. Use of the IgG Fc mutant of any one of claims 1-9, the fusion protein of any one of claims 14-17, or the pharmaceutical composition of claim 19 in the preparation of a medicament for treating diabetes.

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