Human IgG Fc variant, gene segment, expression vector, host cell, IgG antibody and preparation method and application of human IgG Fc variant

Abstract

The invention discloses a human IgG Fc variant, a gene segment, an expression vector, a host cell, an IgG antibody and a preparation method and application of the human IgG Fc variant, and belongs to the technical field of biological medicine. The affinity of the human IgG1 Fc variant T394C provided by the invention with FcRn is obviously superior to that of a wild type under the conditions of pH 6.0 and pH 6.5, and the human IgG1 Fc variant T394C keeps the characteristic of not being combined with FcRn under the condition of pH 7.0. As a fusion ligand unit of a protein drug, the fusion ligand unit can enhance the combination of the drug and FcRn, prolong the in-vivo circulation time of the drug, improve the in-vivo half-life period of the protein drug and reduce the administration frequency under an acidic pH condition, and is not combined with the FcRn under a neutral pH condition, so that the circulation of the protein drug in blood is not influenced. The human IgG1 Fc variant T394C disclosed by the invention has very good industrial popularization and application values.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a human IgG Fc variant, a gene fragment, an expression vector, a host cell, an IgG antibody, and a preparation method and application of the human IgG Fc variant. Background Art

[0002] Human immunoglobulins are tetrapeptide chains composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds. They can be divided into five categories, namely immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE), each of which plays a different role in the human immune system. Among them, IgG is the most abundant immunoglobulin in human blood, accounting for approximately 75% of the total serum immunoglobulin. It plays an important role in maintaining humoral immune function and mediating immune responses. Based on the differences in IgG heavy chains, human IgG is divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. These four subclasses share more than 90% identity at the amino acid level.

[0003] IgG recognizes target antigens through two antigen-binding fragments (Fab), which are linked by a hinge region to a constant crystallizable fragment (Fc). This fragment binds to the Fc receptor and induces a corresponding immune response. The neonatal Fc receptor (FcRn) differs from other Fc receptors in that its structure is more similar to MHC class I molecules. Mature FcRn is a heterodimer composed of N-glycosylated transmembrane MHC class I heavy chains non-covalently bound to soluble β2-microglobulin. FcRn is present in many tissues and organs, such as vascular endothelial cells and myeloid-derived antigen-presenting cells (APCs), such as monocytes, macrophages, and dendritic cells. Compared with other immunoglobulins, IgG has high circulating levels, a long half-life, and an enhanced ability to be transferred from mother to offspring, which is closely related to its interaction with FcRn.

[0004] Two FcRn molecules can bind to IgG by respectively binding to the symmetrical CH2-CH3 interface in Fc, which protects IgG from degradation in lysosomes: IgG can be absorbed by vascular endothelial cells and enter the cells through pinocytosis; then, IgG binds to FcRn in the endosomes (pH 6.0-6.5) in a pH-dependent manner, while antibodies not bound to FcRn fuse with lysosomes and are degraded by proteases in the lysosomes; and when the IgG-FcRn complex circulates to the cell surface, IgG quickly dissociates from FcRn in a slightly neutral environment (pH 7.0-7.4) and re-enters the circulation.

[0005] Based on the principle of FcRn-mediated recycling, Fc engineering can enhance the binding of drugs to FcRn, thereby prolonging the drug's circulation time in the body and reducing the frequency of dosing. For example, without affecting the binding to FcRn under neutral pH conditions, Fc variants that improve the binding to FcRn under acidic pH conditions can obtain a longer half-life than the parent. For example, Efgartigimod®, which was approved by the FDA for marketing in 2021, has five amino acid mutations that enhance its affinity to FcRn: M252Y / S254T / T256E / H433K / N434F. Among them, the affinity of Fc M252Y / S254T / T256E to FcRn at pH 6.0 is enhanced to 10 times that of the wild type. [1，2，5] The affinity of Fc H433K / N434F to FcRn at pH 6.0 was enhanced to 16 times that of the wild type. [3，5] Other mutations that enhance affinity for FcRn include Fc T307A / E380A / N434A, which increases affinity for cell-expressed FcRn to 3.3 times that of the wild type. [4，5] .

[0006] To date, most studies have focused on enhancing Fc affinity for FcRn by modifying the primary amino acid sequence of the Fc protein. It is rare to apply factors such as disulfide bonds, which inherently enhance protein stability, to Fc modification to enhance FcRn affinity. Furthermore, there is a continued need to discover new Fc variants with enhanced FcRn affinity to meet the development needs of various therapeutic antibodies and Fc fusion protein drugs.

[0007] References: 1. Dall'Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, BrewahYA, Wu H, Kiener PA, Langermann S. Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. J Immunol. 2002; 169:5171–5180. 2. Dall'Acqua WF, Kiener PA, Wu H. Properties of human IgG1sengineered for enhanced binding to the neonatal Fc receptor (FcRn). J BiolChem. 2006; 281:23514–23524. 3. Vaccaro C, Bawdon R, Wanjie S, Ober RJ, Ward ES. Divergentactivities of an engineered antibody in murine and human systems haveimplications for therapeutic antibodies. Proc Natl Acad Sci US A. 2006; 103:18709–18714. 4. Petkova SB, Akilesh S, Sproule TJ, Christianson GJ, Al Khabbaz H, Brown AC, Presta LG, Meng YG, Roopenian DC. Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease. Int Immunol. 2006; 18:1759–1769. 5. Quinlin M. Hanson and Adam W. Barb*. A perspective on the structure and receptor-binding properties of immunoglobulin G Fc. Biochemistry. 2015; 54(19): 2931–2942.) Summary of the Invention In order to solve the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a human IgG Fc variant, gene fragment, expression vector, host cell, IgG antibody and human IgG Fc variant preparation method and application.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a human IgG Fc variant, wherein the human IgG Fc variant has an amino acid mutation in the wild-type human IgG Fc shown in the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2; the amino acid mutation is a mutation of threonine T at position 394 in the KabatEU numbering system to cysteine ​​C.

[0009] The present invention also provides a gene fragment encoding the human IgG Fc variant.

[0010] Furthermore, the nucleotide sequence of the gene fragment is shown in SEQ ID NO.4.

[0011] The present invention also provides an expression vector comprising the above gene fragment.

[0012] The present invention also provides a host cell, which contains the above gene fragment.

[0013] Furthermore, the host cell is an ovarian cell.

[0014] Furthermore, the host cell is a Chinese hamster ovary cell.

[0015] The present invention also provides an IgG antibody, comprising the above-mentioned human IgG Fc variant.

[0016] The present invention also provides a method for preparing the above-mentioned human IgG Fc variant, which comprises the following steps: (1) Connecting the above gene fragment to an expression vector; (2) Transform host cells with the expression vector obtained in step (1) and induce expression to obtain the product.

[0017] The present invention also provides the use of the human IgG Fc variant, gene fragment, expression vector, and host cell in the preparation of Fc fusion protein drugs.

[0018] The present invention has achieved the following beneficial effects: The human IgG1 Fc variant T394C provided by the present invention has significantly better affinity for FcRn than the wild-type at pH 6.0 and pH 6.5, while maintaining its FcRn-independent binding at pH 7.0. As a fusion ligand unit for protein drugs, it can enhance drug binding to FcRn at acidic pH conditions, prolong drug circulation in the body, increase the half-life of protein drugs, and reduce dosing frequency. Furthermore, it does not bind to FcRn at neutral pH, thus maintaining blood circulation. The human IgG1 Fc variant T394C provided by the present invention has excellent value for industrial application.

[0019] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0020] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. DETAILED DESCRIPTION

[0021] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0022] Two types of people IgG1 Fc The amino acid sequences encoded by the alleles are shown in SEQ ID NO. 1 (wild-type Fc 1) and SEQ ID NO. 2 (wild-type Fc 2). Wild-type Fc 1 and wild-type Fc 2 are two different wild-type Fcs, and numbers 1 and 2 are only for distinction; SEQ ID NO.1: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.2: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 1: Preparation of human IgG1 Fc variant T394C 1. Design of human IgG1 Fc disulfide bond mutants Human IgG1 Fc disulfide mutants were designed using the “Disulfide by Design” online tool.

[0023] The crystal structure of human IgG1 Sigma Fc fragment (RCSB, 5W5L) was used as a calculation template. The design results are shown in Table 1: Table 1 Design of human IgG1 Fc disulfide bond mutants The design results showed that the human IgG1 Fc variant T394C was able to form two new disulfide bonds between the two Fc peptide chains.

[0024] 2. Vector construction of human IgG1 Fc variant T394C Eukaryotic expression plasmids (shuttle plasmids) pRH It was established by Chengdu Rongsheng Pharmaceutical Co., Ltd., mainly by CMVp promoter, dhfr Genes, etc., can efficiently express foreign genes in mammalian cells.

[0025] The gene sequence of human IgG1 Fc expressing the amino acid sequence shown in SEQ ID NO.1 is as follows: (SEQ ID NO. 3) Among them, ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGG GTTCCAGGTTCCACTGGT (SEQ ID NO.5) is the mouse IgK signal sequence.

[0026] The above gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and then site-directed mutagenesis (three mutations, positions 580-582 from ACG to TGC) was performed on the above gene using overlapping PCR to obtain the Fc variant T394C expression gene as follows: (SEQ ID NO. 4) The above gene was cloned into plasmid after adding restriction endonuclease sites and Kozak sequence pRH Recombinant plasmids expressing human IgG1 wild-type Fc and Fc variant T394C were obtained. pRH- Fc and pRH-T394C .

[0027] 3. Recombinant CHO cell expression of human IgG1 wild-type Fc and Fc variant T394C The dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line (CHO-DG44) was purchased from Invitrogen. The cationic liposome transfection reagent Lipofectamine 2000 was also purchased from Invitrogen; CDM4 PERMAb serum-free medium was purchased from HyClone; and human IgG1 Fc content was determined using a human Fcγ (IgG Fc fragment) enzyme-linked immunosorbent assay kit (Wuhan Elarite Biotechnology Co., Ltd.).

[0028] CHO-DG44 cells were revived from liquid nitrogen in 30 ml CDM4PERMAb serum-free medium and suspended at 37°C with 5% CO2 on a shaker at 100 rpm. One day before transfection, CHO-DG44 cells in the logarithmic growth phase were cultured in 6-well cell plates with antibiotic-free medium CDM4PERMAb, 2 ml / well, supplemented with 10% FBS and HT (final working concentrations of 100 μM hypoxanthine and 16 μM thymidine). When the cells grew to 90% confluency, the recombinant plasmids were taken. pRH- Fc and pRH-T394C Gently mix 4 μg of the protein and 10 μl of the eukaryotic cell lipofectamine 2000 reagent (Invitrogen) in 200 μl of the above-mentioned antibiotic-free medium. Let it stand for 5 minutes. Then, add the expression plasmid and lipofectamine mixture to the cell wells, gently shake, and incubate at 37°C, 5% CO2 for 6 hours. After 6 hours, replace the medium with fresh CDM4PERMAb supplemented with 10% FBS and HT, and continue incubation at 37°C for 24 hours. Disintegrate the cells with 0.25% trypsin, seed them into 96-well plates, and incubate them at 37°C with HT-free selection medium. Fresh medium is replaced every 3 days until colonies emerge. Fc production is measured.

[0029] The clones with excellent yield in 96-well plates were gradually scaled up and cultured in shake flasks. After batch culture in CDM4PERMAb serum-free medium for 5 days, the supernatants expressing wild-type Fc and Fc variant T394C were collected for determination of affinity with FcRn.

[0030] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0031] Experimental Example 1: Affinity determination of human IgG1 wild-type Fc and Fc variant T394C to FcRn The cell culture supernatant containing wild-type Fc and Fc variant T394C harvested in Example 1 was treated using a Merk 10KD ultrafiltration centrifuge tube and buffer exchanged with 1×PBS-EP.

[0032] Affinity detection was performed using a Biacore T200 (Cytiva) capture method. Anti-histidine antibody (Cytiva) was immobilized on a CM5 chip, and histidine-tagged human FcRn protein (Sino biologicals) was captured on a CM5 chip coupled with anti-histidine tag antibody as the stationary phase.

[0033] Table 2 Affinity of wild-type Fc and Fc variant T394C to FcRn The results are shown in Table 2. At pH 6.0 and pH 6.5, the affinity of human IgG1 Fc variant T394C to FcRn was 3.02 times and 1.95 times that of the wild type, respectively; while at pH 7.0, neither human IgG1 Fc variant T394C nor wild type Fc bound to FcRn.

[0034] In summary, the human IgG1 Fc variant T394C provided by the present invention has significantly better affinity for FcRn than the wild-type at pH 6.0 and pH 6.5, while maintaining its FcRn-independent properties at pH 7.0. As a fusion ligand unit for protein drugs, it can enhance drug binding to FcRn at acidic pH conditions, prolong drug circulation in the body, increase the half-life of protein drugs in vivo, and reduce dosing frequency. Furthermore, it does not bind to FcRn at neutral pH conditions, thus maintaining its blood circulation. The human IgG1 Fc variant T394C of the present invention has excellent value for industrial application.

Claims

1. A human IgG Fc variant, characterized in that The human IgG Fc variant has an amino acid mutation in the wild-type human IgG Fc as shown in the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2; the amino acid mutation is a mutation of threonine T at position 394 in the Kabat EU numbering system to cysteine ​​C.

2. A gene fragment encoding the human IgG Fc variant according to claim 1.

3. The gene fragment according to claim 2, characterized in that The nucleotide sequence of the gene fragment is shown in SEQ ID NO.

4.

4. An expression vector, characterized in that The vector comprises the gene fragment according to claim 2 or 3.

5. A host cell, characterized in that The host cell comprises the gene fragment according to claim 2 or 3.

6. The host cell according to claim 5, characterized in that The host cell is an ovarian cell.

7. An IgG antibody, characterized in that The IgG antibody comprises the human IgG Fc variant of claim 1.

8. A method for preparing the human IgG Fc variant according to claim 1, characterized in that: The method comprises the following steps: (1) Connecting the gene fragment according to claim 2 or 3 to an expression vector; (2) Transform host cells with the expression vector obtained in step (1) and induce expression to obtain the product.

9. Use of the human IgG Fc variant according to claim 1, the gene fragment according to claim 2 or 3, the expression vector according to claim 4, and the host cell according to claim 5 or 6 in the preparation of an Fc fusion protein drug.

Images