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

Abstract

The application discloses a human IgG Fc variant, a gene fragment, an expression vector, a host cell, an IgG antibody and a human IgG Fc variant preparation method and application, and belongs to the technical field of biological medicines. The human IgG1 Fc variant T394C provided by the application has a significantly better affinity to FcRn than a wild type under the conditions of pH 6.0 and pH 6.5, and 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 human IgG1 Fc variant T394C can enhance the combination of the drug with FcRn under the condition of an acidic pH, prolong the circulation time of the drug in the body, improve the half-life of the protein drug in the body, reduce the frequency of drug administration, and not be combined with FcRn under the condition of a neutral pH, thereby not affecting the circulation of the protein drug in the blood. The human IgG1 Fc variant T394C has very good industrial popularization and application values.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a human IgG Fc variant, gene fragment, expression vector, host cell, IgG antibody, and a method for preparing and applying the human IgG Fc variant. Background Technology

[0002] Human immunoglobulins are tetrapeptide chains composed of two identical light chains and two identical heavy chains linked by disulfide bonds. They can be divided into five classes: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE), each playing a different role in the human immune system. IgG is the most abundant immunoglobulin in human blood, accounting for approximately 75% of the total serum immunoglobulins, and plays a crucial role in maintaining humoral immunity and mediating immune responses. Based on differences in the IgG heavy chain, human IgG is divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. These four subclasses share over 90% amino acid identity.

[0003] IgG recognizes target antigens through two antigen-binding fragments (Fab), which are linked via a hinge region to a constant crystallizable fragment (Fc). This fragment binds to the Fc receptor and induces a corresponding immune response. Neonatal Fc receptors (FcRn) differ from other types of Fc receptors, with a structure more similar to MHC type 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 to other immunoglobulins, IgG exhibits enhanced circulation levels, a long half-life, and the ability to transfer from mother to offspring, which is closely related to its interaction with FcRn.

[0004] Two FcRn molecules can bind to IgG by binding to the symmetrical CH2-CH3 interface in Fc cells, which prevents IgG from being degraded in lysosomes: IgG can be absorbed by vascular endothelial cells and enter the cell via endocytosis; subsequently, IgG binds to FcRn in an incorpus in a pH-dependent manner (pH 6.0-6.5), while antibodies that do not bind to FcRn fuse with lysosomes and are degraded by proteases within the lysosomes; when the IgG-FcRn complex cycles to the cell surface, IgG rapidly dissociates from FcRn in a near-neutral environment (pH 7.0-7.4) and re-enters the cycle.

[0005] Based on the FcRn-mediated recycling principle, Fc engineering can enhance drug binding to FcRn, thereby prolonging drug circulation time in vivo and reducing dosing frequency. For example, without affecting FcRn binding under neutral pH conditions, improving Fc variants binding to FcRn under acidic pH conditions can achieve a longer half-life compared to the parent. For instance, Efgartigimod®, approved by the FDA in 2021, possesses five amino acid mutations that enhance FcRn affinity: M252Y / S254T / T256E / H433K / N434F. Specifically, Fc M252Y / S254T / T256E exhibits a 10-fold increased affinity for FcRn at pH 6.0 compared to the wild type. [1，2，5] The affinity of FcH433K / N434F for FcRn increased to 16 times that of the wild type at pH 6.0. [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 research has focused on modifying the Fc primary amino acid sequence to enhance affinity for FcRn. It is rare to find applications of factors that inherently improve protein stability, such as disulfide bonds, to enhance Fc affinity for FcRn. Furthermore, there is still a need to discover more novel Fc variants with enhanced affinity for FcRn to meet the needs of developing various therapeutic antibodies and Fc fusion protein drugs.

[0007] References:

[0008] 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.

[0009] 2. Dall’Acqua WF, Kiener PA, Wu H. Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn). J Biol Chem. 2006; 281:23514–23524.

[0010] 3. Vaccaro C, Bawdon R, Wanjie S, Ober RJ, Ward ES. Divergent activities of an engineered antibody in murine and human systems have implications for therapeutic antibodies. Proc Natl Acad Sci U S A. 2006; 103:18709–18714.

[0011] 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.

[0012] 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

[0013] In order to solve the above-mentioned problems in the prior art, the purpose of this invention is to provide a human IgG Fc variant, gene fragment, expression vector, host cell, IgG antibody, and a method for preparing and applying the human IgG Fc variant.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] 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 with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; wherein the amino acid mutation is a mutation of threonine T at position 394 of the KabatEU numbering system to cysteine ​​C.

[0016] The present invention also provides a gene fragment encoding the above-mentioned human IgG Fc variant.

[0017] Furthermore, the nucleotide sequence of the gene fragment is shown in SEQ ID NO.4.

[0018] The present invention also provides an expression vector comprising the above-mentioned gene fragment.

[0019] The present invention also provides a host cell containing the above-described gene fragments.

[0020] Furthermore, the host cell is an ovarian cell.

[0021] Furthermore, the host cell is a Chinese hamster ovary cell.

[0022] The present invention also provides an IgG antibody comprising the above-described human IgG Fc variant.

[0023] The present invention also provides a method for preparing the above-mentioned human IgG Fc variant, the method comprising the following steps:

[0024] (1) Ligate the above gene fragments to the expression vector;

[0025] (2) Transform host cells with the expression vector obtained in step (1) and induce expression to obtain the expression vector.

[0026] This invention also provides the application of the above-mentioned human IgG Fc variant, gene fragment, expression vector, and host cell in the preparation of Fc fusion protein drugs.

[0027] The present invention has achieved the following beneficial effects:

[0028] The human IgG1 Fc variant T394C provided by this invention exhibits significantly superior affinity for FcRn compared to the wild type at pH 6.0 and pH 6.5, while remaining non-binding to FcRn at pH 7.0. As a fusion ligand unit for protein drugs, it enhances drug binding to FcRn under acidic pH conditions, prolonging drug circulation time in vivo, increasing the in vivo half-life of protein drugs, and reducing dosing frequency. Furthermore, it does not bind to FcRn under neutral pH conditions, thus not affecting the circulation of protein drugs in the blood. This human IgG1 Fc variant T394C has excellent potential for industrial application.

[0029] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0030] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0031] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0032] 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. The numbers 1 and 2 are only for differentiation.

[0033] SEQ ID NO.1:

[0034] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0035] SEQ ID NO.2:

[0036] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0037] Example 1: Preparation method of human IgG1 Fc variant T394C

[0038] 1. Design of human IgG1 Fc disulfide mutant

[0039] Human IgG1 Fc disulfide mutants were designed using the "Disulfide by Design" online tool.

[0040] The crystal structure of the human IgG1 Sigma Fc fragment (RCSB, 5W5L) was used as a computational template, and the design results are shown in Table 1:

[0041] Table 1. Design of human IgG1 Fc disulfide mutants

[0042]

[0043] The design results show that the human IgG1 Fc variant T394C is capable of forming two new disulfide bonds between the two Fc peptide chains.

[0044] 2. Vector construction of human IgG1 Fc variant T394C

[0045] Eukaryotic expression plasmids (shuttle plasmids) pRH Constructed by Chengdu Rongsheng Pharmaceutical Co., Ltd., mainly composed of CMVp promoter, dhfr Composed of genes and other components, it can efficiently express exogenous genes in mammalian cells.

[0046] The gene sequence of human IgG1 Fc expressing the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0047] (SEQ ID NO.3)

[0048] Among them, ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGG GTTCCAGGTTCCACTGGT (SEQ ID NO.5) is a mouse... IgK signal sequence.

[0049] The above-mentioned gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd. Subsequently, site-directed mutagenesis (three mutations, with ACG to TGC at positions 580-582) was performed using overlap PCR to obtain the Fc variant T394C expression gene, as follows:

[0050] (SEQ ID NO.4)

[0051] The above gene was cloned into a plasmid after adding restriction endonuclease sites and a kozak sequence. pRH Recombinant plasmids expressing human IgG1 wild-type Fc and Fc variant T394C were obtained. pRH- Fc and pRH-T394C .

[0052] 3. Expression of human IgG1 wild-type Fc and Fc variant T394C in recombinant CHO cells

[0053] Dihydrofolate reductase (DHFR) deficient Chinese hamster ovary cell line (CHO-DG44) was purchased from Invitrogen. Cationic liposome transfection reagent Lipofectamine 2000 was purchased from Invitrogen; CDM4PERMAb serum-free medium was purchased from HyClone; human IgG1 Fc content was determined using a human Fcγ (IgG Fc fragment) enzyme-linked immunosorbent assay kit (Wuhan Yilairuit Biotechnology Co., Ltd.).

[0054] CHO-DG44 cells were resuscitated from liquid nitrogen and cultured in 30 ml of serum-free CDM4PERMAb medium at 37°C and 100 rpm on a 5% CO2 shaker. One day before transfection, CHO-DG44 cells in logarithmic growth phase were cultured in 6-well plates (2 ml / well) with antibiotic-free CDM4PERMAb medium, supplemented with 10% FBS and HT (final working concentrations of 100 μM hypoxanthine and 16 μM thymidine). When cells reached 90% confluence, the recombinant plasmid was harvested. pRH- Fc and pRH-T394C 4 μg of the expression plasmid and 10 μl of eukaryotic cell liposome transfection reagent (Lipofectamine 2000, Invitrogen) were gently mixed in 200 μl of the above antibiotic-free medium, and allowed to stand for 5 minutes. The mixture of expression plasmid and liposome transfection reagent was then added to the cell wells, gently shaken, and incubated at 37°C with 5% CO2 for 6 h. After 6 h, the medium was replaced with fresh CDM4PERMAb, and 10% FBS and HT were added. The cells were then incubated at 37°C for another 24 h. Cells were digested with 0.25% trypsin, seeded into 96-well plates, and cultured at 37°C with HT-free selection medium, changing the medium every 3 days until clones appeared. Fc yield was then measured.

[0055] Clones with high yields in 96-well plates were scaled up and cultured into 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 the determination of affinity with FcRn.

[0056] The following experimental examples demonstrate the beneficial effects of the present invention.

[0057] Experimental Example 1: Affinity determination of human IgG1 wild-type Fc and Fc variant T394C with FcRn

[0058] The cell culture supernatant containing wild-type Fc and Fc variant T394C harvested in Example 1 was processed using Merk 10KD ultrafiltration centrifuge tubes and replaced with 1×PBS-EP buffer.

[0059] Affinity was determined using the Biacore T200 (Cytiva) capture method. Anti-histidine antibody (Cytiva) was immobilized on a CM5 chip, and human FcRn protein (Sino Biologicals) with a histidine tag was captured as a stationary phase onto a CM5 chip conjugated with an anti-histidine tag antibody.

[0060] Table 2. Affinity of wild-type Fc and Fc variant T394C to FcRn

[0061]

[0062] The results are shown in Table 2. Under pH 6.0 and pH 6.5 conditions, the affinity of human IgG1 Fc variant T394C for FcRn was 3.02 times and 1.95 times that of wild type, respectively; while under pH 7.0 conditions, neither human IgG1 Fc variant T394C nor wild type Fc bound to FcRn.

[0063] In summary, the human IgG1 Fc variant T394C provided by this invention exhibits significantly superior affinity for FcRn compared to the wild type under pH 6.0 and pH 6.5 conditions, while maintaining its non-binding property under pH 7.0 conditions. As a fusion ligand unit for protein drugs, it can enhance drug binding to FcRn under acidic pH conditions, prolonging drug circulation time in vivo, increasing the in vivo half-life of protein drugs, and reducing dosing frequency. Furthermore, it does not bind to FcRn under neutral pH conditions, thus not affecting the circulation of protein drugs in the blood. The human IgG1 Fc variant T394C of this invention has excellent potential for industrial application.

Claims

1. Use of a human IgG Fc variant in the manufacture of a protein drug fusion ligand unit that enhances the binding of the protein drug to FcRn at acidic pH, characterized in that, The human IgG Fc variant is subjected to an amino acid mutation on a wild-type human IgG Fc with an amino acid sequence shown in SEQ ID NO. 1; the amino acid mutation is that a threonine T at position 394 in the Kabat EU numbering system is mutated into a cysteine C.

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