Fc mutant and use thereof

Abstract

The present application belongs to the field of biotechnology, and discloses an Fc mutant. At least one of position 23, position 21, and position 24 of the Fc mutant has a mutation. Position 23 is mutated to K or M. Position 21 is mutated to M, H, or W. Position 24 is mutated to R.

Description

Fc mutants and uses thereof

[0001] Priority information

[0002] The present application claims priority to the Chinese patent application No. 2024111110028, filed on August 13, 2024, entitled “Fc mutants and uses thereof”, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of biotechnology, and specifically relates to a method for increasing the purification efficiency of a target protein, more specifically to an Fc mutant, a protein, a nucleic acid molecule, a vector, a cell or host, and a method for improving the affinity of a target protein to protein A or the binding capacity of protein A filler. BACKGROUND

[0004] Affinity chromatography is a protein separation and purification technology. Some specific structural parts of biomolecules can recognize and bind to other molecules, such as the recognition and binding of enzymes and substrates, the recognition and binding of receptors and ligands, and the recognition and binding of antibodies and antigens. Such binding is specific and reversible, and can be removed by changing conditions. Affinity chromatography is designed based on this principle. Currently, the commonly used affinity chromatography protein fillers for purifying antibodies include protein A and protein G.

[0005] Protein G (Protein G) is a cell surface protein derived from group G streptococcus, also known as SPG, which includes a region that specifically binds to the Fc of IgG antibodies, and is composed of three homologous structures of 55 amino acid residues, labeled as C1, C2 and C3 three repeat sequences. Protein G can specifically bind to the Fc region of IgG, but there are several problems in purifying antibodies with protein G: 1) low elution pH, resulting in poor antibody stability; 2) not alkali-resistant, poor cleaning effect; 3) low binding capacity, difficult for industrial production.

[0006] Staphylococcus aureus protein A (SPA, protein A) is a cell wall associated protein of Staphylococcus aureus, which has the ability to specifically bind to the Fc segment of immunoglobulin molecules in human and other mammalian blood, and can adsorb IgG and IgG-containing immune complexes. Based on these characteristics, protein A affinity chromatography column has become a widely used affinity column for purifying antibodies in the field of biotechnology, but the purification efficiency of protein A needs to be further improved. SUMMARY

[0007] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides an Fc mutant.

[0008] In a first aspect, the present application provides an Fc mutant. According to embodiments of the present application, at least one of positions 23, 21, and 24 of the Fc mutant is mutated; the position 23 is mutated to K or M; the position 21 is mutated to M, H, or W; and the position 24 is mutated to R. The Fc mutant of the present application can improve at least one of expression, affinity to Protein A, and binding capacity to Protein A filler.

[0009] In a second aspect, the present application provides a protein. According to embodiments of the present application, the protein comprises the Fc mutant of the first aspect. As known from the foregoing, the Fc mutant can improve at least one of expression, affinity to Protein A, and binding capacity to Protein A filler. Thus, the protein comprising the Fc mutant can improve at least one of expression, affinity to Protein A, and binding capacity to Protein A filler, and can satisfy requirements for purity and thermal stability.

[0010] In a third aspect, the present application provides a nucleic acid molecule, a vector, a cell, or a host. According to embodiments of the present application, the nucleic acid molecule encodes the Fc mutant of the first aspect or the protein of the second aspect; the vector comprises the nucleic acid molecule; and the cell or host comprises the nucleic acid molecule or the vector, or expresses the Fc mutant of the first aspect or the protein of the second aspect. The nucleic acid molecule according to embodiments of the present application can encode the Fc mutant or the protein as described above, and the vector, the cell, or the host carrying the nucleic acid molecule can be used to express the Fc mutant or the protein.

[0011] In a fourth aspect, the present application provides a reagent or a kit. According to embodiments of the present application, the reagent or the kit comprises the protein as described above.

[0012] In a fifth aspect, the present application provides a method for improving expression of a target protein, improving affinity of the target protein to Protein A, improving binding capacity of the target protein to Protein A filler, preparing the target protein, or purifying the target protein, the target protein comprising an Fc fragment, the method comprising: mutating the Fc fragment, the mutation being consistent with the mutation of the Fc mutant of the first aspect.

[0013] In a sixth aspect, the present application provides use of the Fc mutant as described above in improving expression of a target protein comprising the Fc mutant, improving affinity of the target protein to Protein A, or improving binding capacity of the target protein to Protein A filler.

[0014] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0015] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0016] Figure 1 is a graph of the affinity test results of each mutant group in Example 1 of the present application, wherein Figure 1A is wild type, Figure 1B is mutant group 1, Figure 1C is mutant group 3, Figure 1D is mutant group 4, Figure 1E is mutant group 5, and Figure 1F is mutant group 7;

[0017] Figure 2 is a graph of the affinity test results of each mutant group in Example 2 of the present application, wherein Figure 2A is wild type, Figure 2B is mutant group 1, Figure 2C is mutant group 2, Figure 2D is mutant group 4, and Figure 2E is mutant group 5;

[0018] Figure 3 is a graph of the affinity test results of each mutant group in Example 3 of the present application, wherein Figure 3A is wild type, and Figure 3B is mutant group 1;

[0019] Figure 4 is a comparison of the partial amino acid sequences of the CH2 regions of IgG antibodies of different types of mice, rats, sheep, and cattle, and the annotation of the first binding region. DETAILED DESCRIPTION

[0020] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0021] It should be noted that the terms "first", "second" are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of the technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0022] For the purpose of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined otherwise in the present document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. The abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids.

[0023] In the present document, the terms "comprising" or "including" are open-ended terms, i.e. they include the indicated content but not exclude other aspects.

[0024] In this document, the terms“optionally,”“optional,”“may,”“might,”“may

[0025] In the present context, the terms "identity", "homology" or "similarity" are used when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, in terms of the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional means, see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that can be used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to perform the comparisons, including, but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program from Intelligenetics, Mountain View, California.

[0026] In the present text, the term "having at least 80% sequence identity" can be having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity. The sequence identity described in the present application can be measured using sequence analysis software, for example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present application are shown in the order N-terminal to C-terminal.

[0027] In the present text, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0028] In the present text, the term "vector" generally refers to a nucleic acid molecule capable of inserting itself into a suitable host and self-replicating, which transfers the inserted nucleic acid molecule to and / or between cells or hosts. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, the vector can produce a desired expression product by culturing a suitable cell or host containing the vector.

[0029] In the present text, the term "cell" generally refers to a cell having a unique trait with stable inheritance obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant vector can be introduced. The term "transformed" or "transfected" used herein means introducing a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present application, and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0030] The present application proposes an Fc mutant, a protein, a nucleic acid molecule, a vector, a cell or host, and a method for improving the affinity of a protein to Protein A or the binding capacity of Protein A filler, which will be described in detail below, respectively.

[0031] Fc mutant

[0032] In a first aspect, the present application provides an Fc mutant. According to embodiments of the present application, at least one of position 23, position 21, and position 24 of the Fc mutant has a mutation; the position 23 is mutated to K or M; the position 21 is mutated to M, H, or W; and the position 24 is mutated to R. The Fc mutant of the present application can improve at least one of its expression, its affinity to Protein A, and its binding capacity to Protein A packing.

[0033] The numbering of the above mutation sites is obtained by sequentially numbering from the N-terminus to the C-terminus of the CH2 region of wild-type IgG1 of sheep.

[0034] The above "Y position" includes the Y site, and also includes equivalent sites of Y. For example, the above "position 23 is mutated to M" includes not only 23 being M, but also equivalent sites of 23 being M.

[0035] In the present application, the term "equivalent site" refers to a site that is different due to different antibody sources (e.g., different species), different subtypes (e.g., IgG can be divided into IgG1, IgG2, etc.), different numbering methods, or due to insertion or deletion, etc., but the overall sequence or structure is similar, which can be determined by sequence or structure homology alignment. For example, the 23rd position of Fc in wild-type IgG1 of sheep corresponds to the 21st position of Fc in wild-type IgG1 of mice, the 21st position of Fc in wild-type IgG1 of sheep corresponds to the 19th position of Fc in wild-type IgG1 of mice or wild-type IgG2a of rats, and the 24th position of Fc in wild-type IgG1 of sheep corresponds to the 22nd position of Fc in wild-type IgG1 of mice or wild-type IgG2a of rats.

[0036] In the present application, "native IgG" and "wild-type IgG" are synonymous.

[0037] In the present application, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0038] In the present application, the term "Fc mutant" or "Fc fragment" refers to an amino acid fragment containing CH2 region and CH3 region, and optionally hinge region, which can consist of CH2 region and CH3 region, and also can consist of hinge region, CH2 region and CH3 region.

[0039] In the present application, the mutation of position 23 to K can also be represented as "X23K".

[0040] In this context, without special indication, "X" represents an amino acid, which is determined according to the amino acid at the specific position in different types of wild-type Fc fragments. Exemplarily, for a wild-type IgG1 antibody of sheep, the amino acid at the 23rd position is S, i.e. the amino acid X is S (serine).

[0041] According to embodiments of the present application, the Fc mutant described above can further comprise at least one of the following technical features:

[0042] According to embodiments of the present application, the mutation of the Fc mutant is not X21M as the only mutation.

[0043] According to embodiments of the present application, the mutation of the Fc mutant is not X24R as the only mutation.

[0044] According to embodiments of the present application, one of the 23rd position, the 21st position, or the 24th position of the Fc mutant has a mutation.

[0045] According to embodiments of the present application, two of the 23rd position, the 21st position, and the 24th position of the Fc mutant have a mutation.

[0046] According to embodiments of the present application, the Fc mutant has a mutation selected from any one of the following a) to c):

[0047] a) the 21st position is mutated to M;

[0048] b) the 24th position is mutated to R;

[0049] c) the 21st position is mutated to M, and the 24th position is mutated to R.

[0050] Therefore, compared with the natural Fc fragment, the Fc mutant can further improve the affinity to protein A or the binding capacity of the protein A filler.

[0051] According to embodiments of the present application, the Fc mutant has a mutation selected from any one of the following d) to h):

[0052] d) the 21st position is mutated to W;

[0053] e) the 21st position is mutated to H;

[0054] f) the 23rd position is mutated to M;

[0055] g) the 23rd position is mutated to K;

[0056] h) the 21st position is mutated to W or H, and the 23rd position is mutated to M or K.

[0057] Thus, the Fc mutant can further improve the affinity to human serum albumin or the binding capacity of human serum albumin filler compared to the natural Fc fragment, and can also further improve the expression level.

[0058] According to an embodiment of the present application, the Fc mutant has mutations selected from any one of groups d) to h), and has mutations selected from any one of groups a) to c). Thus, the Fc mutant has an improved expression level compared to the natural Fc fragment, and further improved affinity to human serum albumin or binding capacity of human serum albumin filler.

[0059] According to an embodiment of the present application, the Fc mutant has mutations of any one of the following groups:

[0060] Thus, the Fc mutant has an improved expression level compared to the natural Fc fragment, and further improved affinity to human serum albumin or binding capacity of human serum albumin filler.

[0061] According to an embodiment of the present application, the mutation of the Fc mutant is X23K.

[0062] According to an embodiment of the present application, the mutation of the Fc mutant is X23M.

[0063] According to an embodiment of the present application, the mutation of the Fc mutant is X21H.

[0064] According to an embodiment of the present application, the mutation of the Fc mutant is X21W.

[0065] According to an embodiment of the present application, the mutation of the Fc mutant is X24R.

[0066] According to an embodiment of the present application, the mutation of the Fc mutant is X23K and one selected from X21M, X21H, X21W, X24R.

[0067] According to an embodiment of the present application, the mutation of the Fc mutant is X23K and X21M.

[0068] According to an embodiment of the present application, the mutation of the Fc mutant is X24R and X21M.

[0069] According to an embodiment of the present application, the Fc mutant is derived from IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4.

[0070] In the present context, "the Fc mutant is derived from IgGl" means that the Fc mutant is obtained by the above-mentioned mutations based on the Fc fragment in IgGl, wherein the species of origin of IgGl can be any animal or human, including but not limited to sheep, goat, mouse, rat, cow, rabbit, horse, pig, dog, cat, donkey, deer, mink, chicken, duck, goose or human. The same applies for Fc mutants derived from other subclasses.

[0071] According to embodiments of the present application, the species of origin of the Fc mutant is sheep, goat, mouse, rat, cow, rabbit, horse, pig, dog, cat, donkey, deer, mink, chicken, duck, goose or human.

[0072] According to embodiments of the present application, the Fc mutant is derived from sheep IgGl, mouse IgGl, rat IgG2a, rat IgGl, rat IgG2b, cow IgGl or cow IgG3.

[0073] According to embodiments of the present application, the Fc mutant is derived from sheep IgGl.

[0074] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups a1) to c1):

[0075] a1) T21M;

[0076] b1) G24R;

[0077] c1) T21M + G24R.

[0078] Thereby, the Fc mutant can further improve the affinity to Protein A or the binding capacity of Protein A packing compared to the native IgGl Fc fragment of sheep.

[0079] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups d1) to h1):

[0080] d1) T21W;

[0081] e1) T21H;

[0082] f1) S23M;

[0083] g1) S23K;

[0084] h1) combination of T21W / H and S23M / K.

[0085] Thereby, the Fc mutant can further improve the affinity to Protein A or the binding capacity of Protein A packing compared to the native IgGl Fc fragment of sheep, and can further improve the expression level.

[0086] According to embodiments of the present application, the Fc mutant has mutations selected from any one of d1) to h1), and has mutations selected from any one of a1) to c1). Thereby, the Fc mutant has improved expression, and further improved affinity to human serum albumin or binding capacity to human serum albumin packing compared to the native IgG1 Fc fragment of sheep.

[0087] According to embodiments of the present application, the Fc mutant has mutations of any one of the following groups:

[0088] Thereby, the Fc mutant has improved expression, and further improved affinity to human serum albumin or binding capacity to human serum albumin packing compared to the native IgG1 Fc fragment of sheep.

[0089] According to embodiments of the present application, the Fc mutant has mutation S23K compared to the native IgG1 Fc fragment of sheep.

[0090] According to embodiments of the present application, the Fc mutant has mutation S23M compared to the native IgG1 Fc fragment of sheep.

[0091] According to embodiments of the present application, the Fc mutant has mutation T21H compared to the native IgG1 Fc fragment of sheep.

[0092] According to embodiments of the present application, the Fc mutant has mutation T21W compared to the native IgG1 Fc fragment of sheep.

[0093] According to embodiments of the present application, the Fc mutant has mutation G24R compared to the native IgG1 Fc fragment of sheep.

[0094] According to embodiments of the present application, the Fc mutant has mutation S23K and one selected from T21M, T21H, T21W, G24R compared to the native IgG1 Fc fragment of sheep.

[0095] According to embodiments of the present application, the Fc mutant has mutation S23K and T21M compared to the native IgG1 Fc fragment of sheep.

[0096] According to embodiments of the present application, the Fc mutant has mutation G24R and T21M compared to the native IgG1 Fc fragment of sheep.

[0097] According to embodiments of the present application, the Fc mutant is obtained based on the native IgG1 Fc fragment of sheep with the above mutations.

[0098] According to embodiments of the present application, the amino acid sequence of the sheep native IgG1 Fc fragment is shown as SEQ ID NO: 1.

[0099] According to embodiments of the present application, the Fc mutant has an amino acid sequence having at least 80% identity to SEQ ID NO: 1.

[0100] According to embodiments of the present application, the Fc mutant is derived from mouse IgG1.

[0101] It is noted that the amino acid positions in the mouse wild-type IgG1 Fc fragment are determined by homology alignment with the sheep wild-type IgG1 Fc fragment.

[0102] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups a2) to c2):

[0103] a2) T19M;

[0104] b2) L22R;

[0105] c2) T19M + L22R.

[0106] Thus, compared to the mouse native IgG1 Fc fragment, the Fc mutant can further improve the affinity to Protein A or the binding capacity of Protein A packing.

[0107] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups d2) to h2):

[0108] d2) T19W;

[0109] e2) T19H;

[0110] f2) T21M;

[0111] g2) T21K;

[0112] h2) combination of T19W / H and T21M / K.

[0113] Thus, compared to the mouse native IgG1 Fc fragment, the Fc mutant can further improve the affinity to Protein A or the binding capacity of Protein A packing, and can further improve the expression level.

[0114] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the groups d2) to h2), and has mutations selected from any one of the groups a2) to c2). Thus, compared to the mouse native IgG1 Fc fragment, the Fc mutant has improved expression level, and further improved affinity to Protein A or the binding capacity of Protein A packing.

[0115] According to embodiments of the present application, the Fc mutant has mutations of any one of the following groups:

[0116] Accordingly, the Fc mutant has improved expression, and further improved affinity to human serum albumin or binding capacity to human serum albumin packing compared to the native IgG1 Fc fragment of mouse.

[0117] According to embodiments of the present application, the Fc mutant has mutation T23K compared to the native IgG1 Fc fragment of mouse.

[0118] According to embodiments of the present application, the Fc mutant has mutation T23M compared to the native IgG1 Fc fragment of mouse.

[0119] According to embodiments of the present application, the Fc mutant has mutation T21H compared to the native IgG1 Fc fragment of mouse.

[0120] According to embodiments of the present application, the Fc mutant has mutation T21W compared to the native IgG1 Fc fragment of mouse.

[0121] According to embodiments of the present application, the Fc mutant has mutation L24R compared to the native IgG1 Fc fragment of mouse.

[0122] According to embodiments of the present application, the Fc mutant has mutations of T23K and one selected from T21M, T21H, T21W, L24R compared to the native IgG1 Fc fragment of mouse.

[0123] According to embodiments of the present application, the Fc mutant has mutations of T23K and T21M compared to the native IgG1 Fc fragment of mouse.

[0124] According to embodiments of the present application, the Fc mutant has mutations of L24R and T21M compared to the native IgG1 Fc fragment of mouse.

[0125] According to embodiments of the present application, the Fc mutant is obtained based on the native IgG1 Fc fragment of mouse by the above mutations.

[0126] According to embodiments of the present application, the amino acid sequence of the native IgG1 Fc fragment of mouse is shown in SEQ ID NO: 5.

[0127] According to embodiments of the present application, the amino acid sequence of the Fc mutant has at least 80% identity with SEQ ID NO: 5.

[0128] According to embodiments of the present application, the Fc mutant is derived from rat IgG2a.

[0129] It is noted that the amino acid positions in the rat wild-type IgG2a Fc fragment are determined by homology alignment with the sheep wild-type IgG1 Fc fragment.

[0130] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups a3) to c3):

[0131] a3) T19M;

[0132] b3) L22R;

[0133] c3) T19M + L22R.

[0134] Thus, the Fc mutant can further improve the affinity to Protein A or the binding capacity of Protein A packing compared to the rat native IgG2a Fc fragment.

[0135] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups d3) to h3):

[0136] d3) T19W;

[0137] e3) T19H;

[0138] f3) T21M;

[0139] g3) T21K;

[0140] h3) combination of T19W / H and T21M / K.

[0141] Thus, the Fc mutant can further improve the affinity to Protein A or the binding capacity of Protein A packing compared to the rat native IgG2a Fc fragment, and can further improve the expression level.

[0142] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the groups d3) to h3), and has mutations selected from any one of the groups a3) to c3). Thus, the Fc mutant has improved expression level, and further improved affinity to Protein A or the binding capacity of Protein A packing compared to the rat native IgG2a Fc fragment.

[0143] According to embodiments of the present application, the Fc mutant has mutations selected from any one of the following groups:

[0144] Thus, the Fc mutant has improved expression and further improved affinity to human serum albumin or binding capacity to human serum albumin packing compared to the rat native IgG2a Fc fragment.

[0145] According to an embodiment of the present application, the mutation of the Fc mutant is T23K compared to the rat native IgG2a Fc fragment.

[0146] According to an embodiment of the present application, the mutation of the Fc mutant is T23M compared to the rat native IgG2a Fc fragment.

[0147] According to an embodiment of the present application, the mutation of the Fc mutant is L24R compared to the rat native IgG2a Fc fragment.

[0148] According to an embodiment of the present application, the mutation of the Fc mutant is T21H compared to the rat native IgG2a Fc fragment.

[0149] According to an embodiment of the present application, the mutation of the Fc mutant is T21W compared to the rat native IgG2a Fc fragment.

[0150] According to an embodiment of the present application, the mutation of the Fc mutant is T23K and one selected from T21M, T21H, T21W, L24R compared to the rat native IgG2a Fc fragment.

[0151] According to an embodiment of the present application, the mutation of the Fc mutant is T23K and T21M compared to the rat native IgG2a Fc fragment.

[0152] According to an embodiment of the present application, the mutation of the Fc mutant is L24R and T21M compared to the rat native IgG2a Fc fragment.

[0153] According to an embodiment of the present application, the Fc mutant is obtained by the above mutation based on the rat native IgG2a Fc fragment.

[0154] According to an embodiment of the present application, the amino acid sequence of the rat native IgG2a Fc fragment is shown in SEQ ID NO: 9.

[0155] According to an embodiment of the present application, the amino acid sequence of the Fc mutant has at least 80% identity with SEQ ID NO: 9.

[0156] In an alternative embodiment of the present application, the Fc mutant is derived from rat IgG1 or rat IgG2b; and one skilled in the art can easily determine the above specific mutation sites or combinations thereof in combination with FIG. 4.

[0157] In an optional embodiment of the present application, the Fc mutant is derived from bovine IgG1, or bovine IgG3; in combination with FIG. 4 of the present application, one skilled in the art can easily determine each specific mutation site or combination thereof.

[0158] protein

[0159] In a second aspect of the present application, a protein is provided. According to embodiments of the present application, the protein comprises the Fc mutant of the first aspect. As known from the foregoing, the Fc mutant can improve at least one of its expression, its affinity to Protein A, and its binding capacity to Protein A filler. Thus, the protein containing the Fc mutant can improve at least one of its expression, its affinity to Protein A, and its binding capacity to Protein A filler, and meet the requirements for its purity and thermal stability.

[0160] According to embodiments of the present application, the protein can further comprise at least one of the following technical features:

[0161] According to embodiments of the present application, the protein is an antibody or a fusion protein.

[0162] According to embodiments of the present application, the protein further comprises a functional region, which is directly or indirectly connected to the Fc mutant.

[0163] According to embodiments of the present application, the protein is an antibody, and the functional region comprises a light chain variable region and / or a heavy chain variable region.

[0164] In this context, the term "antibody" is used in the broadest sense, which can be a naturally structured antibody or various engineered structured antibodies, and can be a monoclonal antibody, a multispecific antibody, a multivalent antibody, a nanobody, a heavy chain antibody, or a chimeric antibody, and the specific structure is not limited. A naturally structured antibody generally comprises a light chain with a relatively light molecular weight and a heavy chain with a relatively heavy molecular weight, and the antibody molecule is formed by the heavy chain (H chain) and the light chain (L chain) connected by a disulfide bond. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl-terminal (C-terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively. The H chain generally comprises a variable domain (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). The division of CH1, the hinge region, CH2, and CH3 is based on the IMGT website, see Lefranc, M.-P., et al., IMGT® the international ImMunoGeneTics database. Nucl. Acids Res., 29(1): 207-209 (2001). DOI: 10.1093 / nar / 29.1.207. PMID: 11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGHV, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Different methods of variable region division and IMGT division of variable region C terminal or constant region N terminal will have some differences in amino acids, other methods of variable region or constant region known in the art are also within the scope of the present application.

[0165] According to the embodiments of the present application, the protein is an antibody, and the functional region comprises a heavy chain variable region, and the heavy chain variable region is connected with the Fc mutant. When the antibody is a heavy chain antibody or a nanobody, the functional region only needs to contain a heavy chain variable region, and the heavy chain variable region can be combined with the target antigen to achieve the purpose of specific binding to the target antigen.

[0166] In the present disclosure, the term "nanobody" generally refers to a heavy chain variable region (VHH) portion of a heavy chain antibody naturally lacking a light chain, which is generally present in a Camelidae, and a conventional Fc mutant, which specifically binds to an antigen through the heavy chain variable region (VHH), and the heavy chain variable region (VHH) alone can achieve the effect of specific binding to the antigen.

[0167] According to an optional embodiment of the present application, the C-terminal of the heavy chain variable region is connected to the N-terminal of the Fc mutant, or the C-terminal of the Fc mutant is connected to the N-terminal of the heavy chain variable region.

[0168] According to an optional embodiment of the present application, the antibody further comprises a CH1 region, and the C-terminal of the CH1 region is connected to the N-terminal of the Fc mutant.

[0169] It should be noted that, according to the foregoing, the Fc mutant of the present application can comprise a hinge region or can not comprise a hinge region. In a preferred embodiment of the present application, the Fc mutant itself comprises a hinge region, and the hinge region is located at the N-terminal of the Fc mutant. The amino acid sequence of the hinge region is not particularly limited as long as the function of the hinge region can be achieved, and all are within the protection scope of the present application.

[0170] According to an optional embodiment of the present application, the heavy chain variable region is connected to the Fc mutant, or the heavy chain variable region is connected to the CH1 region.

[0171] According to an optional embodiment of the present application, the N-terminal of the heavy chain variable region is connected to the C-terminal of the Fc mutant.

[0172] According to an optional embodiment of the present application, the C-terminal of the heavy chain variable region is connected to the N-terminal of the CH1 region.

[0173] According to an optional embodiment of the present application, the antibody is not a nanobody, and the functional region comprises a light chain variable region and a heavy chain variable region, and the light chain variable region is connected to the heavy chain variable region.

[0174] It should be noted that the heavy chain variable region and the light chain variable region in the present application can be selected according to the antigen or the antigen epitope peptide to be combined, and the specific type is not limited, and all are within the protection scope of the present application. The connection mode of the heavy chain variable region and the light chain variable region to the Fc mutant is not limited, and can be adjusted according to the actual situation, and all are within the protection scope of the present application.

[0175] In an optional embodiment of the present application, the heavy chain variable region or the light chain variable region is connected to the N-terminal of the Fc mutant.

[0176] In an alternative embodiment of the present application, the heavy chain variable region or the light chain variable region is connected to the C-terminus of the Fc mutant.

[0177] In an alternative embodiment of the present application, the light chain variable region and the heavy chain variable region can be directly connected by an amide bond, can be connected by a connecting peptide, can be connected by a covalent bond, and can be connected by a disulfide bond, and the specific type is not limited, all of which are within the protection scope of the present application.

[0178] In an alternative embodiment of the present application, the heavy chain variable region and the light chain variable region exist in one of the following forms: F(ab')2 fragment, Fab fragment, Fv fragment, scFv fragment, dsFv fragment.

[0179] In the present text, the term "F(ab')2 fragment" refers to two antigen-binding F(ab') parts connected together by disulfide bonds.

[0180] In the present text, the term "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which is composed of VH and CH1 of the heavy chain and a complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0181] In the present text, the term "Fv fragment" generally refers to an antibody or fragment composed of only light chain variable region (VL) and heavy chain variable region (VH) connected by non-covalent bonds, which is the smallest functional fragment of the antibody molecule that retains the complete antigen binding site.

[0182] In the present text, the term "single-chain antibody" or "scFv fragment" refers to an antibody or fragment composed of antibody heavy chain variable region and light chain variable region connected by a short peptide.

[0183] In the present text, the term "dsFv fragment" refers to a disulfide-stabilized Fv fragment, which is formed by connecting the antibody heavy chain variable region (VH) and the light chain variable region (VL) by an interchain disulfide bond.

[0184] In an alternative embodiment of the present application, the antibody further comprises a CH1 region, and the C-terminus of the CH1 region is connected to the N-terminus of the Fc mutant.

[0185] In an alternative embodiment of the present application, the heavy chain variable region or the light chain variable region is connected to the N-terminus of the CH1 region, or the heavy chain variable region or the light chain variable region is connected to the C-terminus of the Fc mutant.

[0186] In an alternative embodiment of the present application, the heavy chain variable region and the light chain variable region are connected, and the C-terminus of the heavy chain variable region is connected to the N-terminus of the CH1 region.

[0187] In an alternative embodiment of the present application, the heavy chain variable region and the light chain variable region are connected, and the N-terminus of the heavy chain variable region is connected to the C-terminus of the Fc mutant.

[0188] According to an embodiment of the present application, the protein is a fusion protein.

[0189] In the present application, the term "fusion protein" refers to a protein formed by connecting two or more proteins or polypeptides with different functions together through genetic engineering or protein engineering. This connection can be direct or indirect (for example, through a short peptide sequence). In the present application, "fusion protein" should be interpreted in the broadest sense, i.e. a protein obtained by directly or indirectly connecting any polypeptide or protein to the Fc mutant described above.

[0190] According to an embodiment of the present application, the functional region comprises a functionally active fragment.

[0191] According to an embodiment of the present application, the N-terminus of the functionally active fragment is connected to the C-terminus of the Fc mutant, or the C-terminus of the functionally active fragment is connected to the N-terminus of the Fc mutant.

[0192] It should be noted that the functionally active fragment can be used to exert an effect in an animal body, or can be used to exert an effect in vitro. Exemplarily, when the functionally active fragment is used to exert an effect in an animal body, it can be used for preventing and / or treating diseases; when the functionally active fragment is used to exert an effect in vitro, it can be used for specifically binding to a certain substance, can be used for detecting the substance, or can be used for in vitro diagnosis of diseases.

[0193] According to an embodiment of the present application, the functionally active fragment comprises at least one of a cytokine, a toxin peptide, a growth factor, a tag protein, and a binding protein.

[0194] In the present application, the term "cytokine" should be interpreted in the broadest sense, which can refer to a class of proteins or small polypeptides capable of transmitting information between cells, having immune regulation and effector functions, which includes but is not limited to interleukins (ILs), interferons (IFNs), tumor necrosis factors (TNFs), colony-stimulating factors (CSFs), etc. For example, IL-12, IL-15, etc.

[0195] In the present application, the term "toxin peptide" refers to a polypeptide found in animal venom, which is chemically modified for the treatment of various diseases (such as cancer, cardiovascular disease, etc.). In addition, for example, in antibody-drug conjugates (ADCs), toxins are key payloads for precisely and efficiently killing cancer cells.

[0196] In some optional embodiments of the present application, the tag comprises at least one of His tag, Flag tag, GST tag, MBP tag, SUMO tag and C-Myc tag.

[0197] According to embodiments of the present application, the binding protein has target molecule binding activity.

[0198] According to embodiments of the present application, the target molecule comprises at least one of tumor antigen, virus, bacteria, endotoxin, cell receptor and ligand of cell receptor.

[0199] In this context, the term "tumor antigen" generally refers to an antigenic substance newly appearing or overexpressed in the process of tumorigenesis and development. According to the classification of tumor antigen specificity, it is classified into tumor specific antigen and tumor associated antigen, wherein the tumor specific antigen (TSA) is a new antigen specific to tumor cells or only present in certain tumor cells but not in normal cells, and the tumor associated antigen (TAA) refers to an antigen specific to non-tumor cells, which also exists on normal cells and other tissues, but the content is significantly increased when the cells become cancerous. Among them, it includes but is not limited to PD-L1, PD-1, TGF-β, CEA, GD2 and GD3, etc.

[0200] In this context, the term "cell receptor" or "receptor" should be understood broadly, which can refer to a molecule located on the cell membrane, which can recognize and bind to various signal molecules (ligands) outside the cell, including but not limited to growth factor receptor (such as VEGF receptor), (NKG2D polypeptide (receptor of MICA, MICB and ULB6), cytokine receptor (such as IL-13 receptor, IL-2 receptor, etc.), epidermal growth factor (EGF) receptor, Her2, CD27, natural cytotoxicity receptor (NCR) (such as NKP30 (NCR3 / CD337) polypeptide (receptor of HLA-B associated transcript 3 (BAT3) and B7-H6), etc.), T cell antigen receptor, dihydrofolate receptor, chimeric cytokine receptor, Fc receptor, extracellular matrix receptor (such as integrin), cell adhesion receptor (such as cadherin), immunomodulatory receptor (including positive co-receptor (such as CD28) and negative (immunosuppressive) co-receptor (such as PD1)), and receptor of immunomodulatory molecule (such as TGFβ), etc.

[0201] In the present context, the term "ligand for a cellular receptor" is to be interpreted broadly and can refer to a chemical substance, such as a polypeptide, a nucleic acid, a glycoprotein, a small molecule, a carbohydrate, a lipid, a glycolipid, a lipoprotein, a lipopolysaccharide, etc., capable of binding to, interacting with, and producing a specific biological effect on a cellular membrane receptor, including but not limited to a cytokine (e.g., IL-13, etc.), a growth factor (e.g., heregulin, vascular endothelial growth factor (VEGF), etc.), a peptide hormone, an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp), an N-glycan, etc. Illustratively, the ligand is VEGF and the receptor is a VEGF receptor; or the ligand is heregulin and the receptor is Her2.

[0202] According to embodiments of the present application, the target molecule comprises at least one of GFP, eGFP, CD19, ALPPL2, BCMA, SIRPa, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD273, CD274, CD276, CD279, CD295, CD339, CD340, EGFR, HER2, FGFR2, AFP, CA125, MSLN, GPC3, CEA, CLDN18.2, EpCAM, PSCA, GD2, IL-13, IL-13RA2, ROR1, MUC 1, PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1, and VEGFR-2.

[0203] According to embodiments of the present application, the binding active fragment is selected from at least one of a F(ab')2 fragment, a Fab fragment, a Fv fragment, a scFv fragment, a dsFv fragment, a receptor or a functional fragment thereof, a ligand of a receptor or a functional fragment thereof, a cell adhesion molecule or a functional fragment thereof.

[0204] In the present context, the term "functional fragment" is a fragment comprising part or all of a full-length chain of a biologically active protein (e.g. an antibody, a receptor, a ligand of a receptor, a cell adhesion molecule, etc.) which lacks at least some of the amino acids present in the full-length chain but which is still capable of the performance activity of specifically binding to an antigen. Exemplarily, for a functional fragment of an antibody, the fragment can comprise part or all of the CDRs of the antibody; such a fragment is biologically active in that it binds to an antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope; such a fragment is selected from a Fab, a Fv, a scFv or a single domain antibody.

[0205] In the present context, a "cell adhesion molecule" or "CAM" can mean a polypeptide that binds to a component of the extracellular matrix (ECM) or to a cell surface molecule. For example, the cell adhesion molecule can be an extracellular region of a CAM, wherein the CAM can be a calcium-independent adhesion molecule, e.g. the CAM is an immunoglobulin superfamily CAM, the CAM can also be a calcium-dependent adhesion molecule, e.g. the CAM is an integrin, a cadherin or a selectin; the cell adhesion molecule can be an integrin, e.g. a cadherin, e.g. an E-cadherin, a P-cadherin, an N-cadherin, an R-cadherin, an M-cadherin, etc.; the cell adhesion molecule can also be a selectin, e.g. an E-selectin, an L-selectin or a P-selectin.

[0206] Nucleic acid molecule, vector, cell or host

[0207] In a third aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the Fc mutant of the first aspect or the protein of the second aspect. The nucleic acid molecule according to embodiments of the present application can encode the Fc mutant or the protein as described above.

[0208] According to embodiments of the present application, the nucleic acid molecule comprises DNA or RNA.

[0209] It is to be understood that, for the nucleic acid molecules mentioned herein, the skilled person understands that either of the complementary double strands is actually included, or both. For convenience, in the present context, although in most cases only one strand is given, the other strand complementary thereto is actually disclosed. In addition, the sequence of the molecule in the present application includes DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0210] In the fourth aspect of the present application, a vector is provided. According to embodiments of the present application, the vector comprises the nucleic acid molecule. In linking the nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the vector, as long as the control elements can control the translation and expression of the nucleic acid molecule. Of course, the control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid molecule can be operably linked to the control elements.

[0211] "Operably linked" herein refers to linking of an exogenous gene to a vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In some embodiments, the vectors are suitable for transforming cells, eukaryotic cells such as fungal cells, microbial cells such as yeast or prokaryotic cells. In a preferred embodiment, the vectors are suitable for stable transformation of bacterial cells, for example, to transcribe the nucleic acid molecule of the present application.

[0212] According to some specific embodiments of the present application, after the vector is introduced into a suitable recipient cell, the expression of the protein of the first aspect can be effectively realized under the mediation of the regulation system, and thus the in vitro mass acquisition of the protein can be realized.

[0213] In an embodiment of the present application, the vector can contain a marker gene and a replication origin to ensure replication in a selected host, a promoter and a transcription termination signal. Preferably, between the promoter and the termination signal, there is at least one restriction site capable of inserting a nucleic acid sequence / molecule to be expressed.

[0214] According to embodiments of the present application, the vector comprises a eukaryotic expression vector or a prokaryotic expression vector.

[0215] In an alternative embodiment of the present application, the vector is a plasmid expression vector, a viral expression vector, for example, a lentivirus expression vector. Preferably, the vector of the present application is selected from a pET-based expression vector, a pGEX-based expression vector, a pcDNA-based expression vector, a pCMV-based expression vector, for example, a pMF09 expression vector.

[0216] In the fifth aspect of the present application, a cell or host is provided. According to embodiments of the present application, the cell or host comprises the nucleic acid molecule or the vector. Using the cell, the Fc mutant or the protein described above can be effectively expressed in the cell under suitable conditions.

[0217] According to embodiments of the present application, the cell or host comprises the Fc mutant of the first aspect or the protein of the second aspect.

[0218] According to embodiments of the present application, the cell or host is obtained by introducing the vector of the fourth aspect into the cell or host.

[0219] It should be noted that the cell or host of the present application is not particularly limited, and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cell includes Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, etc. fungal cells, grasshopper cells, tobacco cells, BHK cells, CHO cells, COS cells, myeloma cells, etc. mammalian cells.

[0220] In an optional embodiment of the present application, the cell is a mammalian cell, including BHK cells, CHO cells, NSO cells or COS cells, and does not include animal reproductive cells, fertilized eggs or embryonic stem cells.

[0221] It should be noted that the "suitable conditions" in the present application refer to conditions suitable for the expression of the Fc mutant or protein of the present application. It is easily understood by those skilled in the art that the conditions suitable for the expression of the Fc mutant or protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the Fc mutant or protein according to the specific environment of the laboratory.

[0222] Reagent or kit

[0223] In the fourth aspect of the present application, a reagent or kit is provided. According to embodiments of the present application, the reagent or kit comprises the aforementioned protein. The protein comprises a functional region. Those skilled in the art can select fragments of the functional region according to the needs of the reagent and kit. For example, the functional region can be a fragment (such as a heavy chain variable region and a light chain variable region) that specifically binds to the A antigen, and the protein containing the above functional region can be used for detecting the A antigen.

[0224] It should be noted that in this document, the functional region in the present application can be selected according to the actual situation, and the specific type is not limited, all of which are within the protection scope of the present application.

[0225] Method

[0226] In a fifth aspect, the present application provides a method for improving the expression of a target protein, improving the affinity of the target protein to Protein A, improving the binding capacity of the target protein to Protein A filler, preparing the target protein, or purifying the target protein, wherein the target protein comprises an Fc fragment, the method comprising: mutating the Fc fragment, wherein the mutation is consistent with the mutation of the Fc mutant of the first aspect. Thus, at least one of the expression of the target protein, the affinity of the target protein to Protein A, and the binding capacity of the target protein to Protein A filler can be improved, and the purity and thermal stability of the target protein meet the requirements, which is more suitable for industrial production.

[0227] Use

[0228] In a sixth aspect, the present application provides the use of the aforementioned Fc mutant in improving the expression of a target protein comprising the Fc mutant, improving the affinity of the target protein to Protein A, or improving the binding capacity of the target protein to Protein A filler.

[0229] The solutions of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0230] Example 1: Design of mutation site of sheep IgG1

[0231] 1. Mutation site design

[0232] The specific sites on the antibody Fc were selected for mutation, and the mutation sites and mutated amino acids are shown in Table 1. The amino acid sequence of the Fc fragment (CH2 fragment and CH3 fragment) of sheep wild-type IgG1 is shown in SEQ ID NO: 1.

[0233] Table 1: Mutation sites of sheep IgG1

[0234] 2. Plasmid construction for expression

[0235] In this embodiment, the restriction enzymes and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. The BD SMART TMRACE cDNA Amplification Kit was purchased from Takara Company. pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were completed by Gene Sequencing Company. The hybridoma cell strain secreting Anti-CA153 monoclonal antibody was an existing hybridoma cell strain, which was recovered for use.

[0236] 2.1, Anti-VD antibody gene preparation

[0237] The mRNA was extracted from the hybridoma cell strain secreting Anti-VD monoclonal antibody, the DNA product was obtained by RT-PCR method, and it was inserted into pMD-18T vector, transformed into DH5α competent cells, and after the colonies were grown, 4 colonies of positive clones of Heavy Chain and Light Chain genes were sent to the gene sequencing company for sequencing.

[0238] 2.2, Sequence analysis of Anti-VD antibody variable region gene

[0239] The gene sequence obtained by the above sequencing was placed in the IMGT antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the amplified genes by the heavy chain and light chain primer pairs were correct. Among them, the VL gene sequence in the Light Chain amplified gene fragment is 330bp, and there is a 57bp leader peptide sequence in front of it; the VH gene sequence in the Heavy Chain primer pair amplified gene fragment is 369bp, which belongs to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.

[0240] 2.3, Construction of recombinant antibody expression plasmid

[0241] pcDNA TM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector, which is modified to introduce multiple cloning enzyme digestion sites, and is hereinafter referred to as 3.4A expression vector; according to the antibody variable region gene sequencing results in the above pMD-18T, Anti-VD antibody VL and VH gene specific primers were designed, which were respectively labeled with restriction endonuclease digestion sites and protection bases at both ends. The 0.72KB Light Chain gene fragment and the 1.40kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0242] The heavy chain and light chain gene fragments were double digested by restriction enzymes, the 3.4A vector was double digested by restriction enzymes, and the fragments and the vector were purified and recovered, and then the heavy chain gene and the light chain gene were connected into the 3.4A expression vector, respectively, to obtain the recombinant expression plasmids of the heavy chain and the light chain.

[0243] The amino acid sequence of the light chain in the Anti-VD antibody is shown in SEQ ID NO: 2.

[0244] The heavy chain in the Anti-VD antibody is sequentially composed of a heavy chain variable region, a CH1 and hinge region, and an Fc fragment (or Fc mutant) from N-terminal to C-terminal, and the Fc mutants in each mutation group are shown in Table 1; the amino acid sequence of the heavy chain variable region in the Anti-VD antibody is shown in SEQ ID NO: 3.

[0245] The amino acid sequence of the CH1 and hinge region in the Anti-VD antibody is shown in SEQ ID NO: 4.

[0246] ASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSS VVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPDPCKHCRCPP (SEQ ID NO: 4). (The underlined part is the hinge region, and the same applies to other sequences)

[0247] 2.4, Expression of the sample of the recombinant antibody

[0248] The HEK293 cells were recovered in advance, subcultured in a 200ml system, and the cell density was adjusted to (3-5)×10 6 cells / ml, and the cell viability was >95%; the cells were centrifuged and washed, resuspended with the culture medium, and the cell density was adjusted to 2.9×10 6 cells / ml as a cell diluent. The plasmid DNA and the transfection reagent diluent were prepared with the culture medium, respectively. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and placed at room temperature for 15min; the mixture was slowly added to the cell diluent within 1min, mixed, sampled, counted, and recorded and observed the cell viability after transfection, and placed in a 35℃ constant temperature incubator for culture at a speed of 120rmp and a CO2 content of 8%, and sampled by centrifugation after 13 days.

[0249] 3. HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography)

[0250] Take 20 μg of purified antibody, determine the SEC purity of the sample by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC), and the chromatographic conditions of HPLC-SEC are shown in Table 2. The results show that the expression of mutant group 8, mutant group 2 and mutant group 6 is decreased, and the yield of other mutant groups in Table 1 is increased, and part of the test results are shown in Table 3.

[0251] Table 2: Chromatographic conditions of HPLC-SEC

[0252] Table 3: Yield and HPLC-SEC results after expression

[0253] 4. Thermal stability Tm determination

[0254] Dilute the antibody to 1 mg / ml, and use Panta's DSF to measure the Tm (unfolding temperature) of the sample, Turbidity aggregation temperature, DLS to determine the hydrodynamic radius Cumulant Radius with PDI, and the specific parameter settings are as follows:

[0255] Sample volume: 400 μL;

[0256] Measurement range: 25-95°C;

[0257] Temperature rising rate: 0.3°C / min.

[0258] The results show that each mutant group in Table 1 has no significant effect on thermal stability, and part of the test results are shown in Table 4.

[0259] Table 4: Thermal stability Tm results

[0260] 5. Affinity analysis

[0261] Use protein A sensor chip (cytiva, 10315483) sensor, and gradient dilute the purified antibody with PBST: 200 nmol / ml, 150 nmol / ml, 50 nmol / ml, 25 nmol / ml, 12.5 nmol / ml, 6.25 nmol / ml, 3.125 nmol / ml, 1.5625 nmol / ml, 0.78125 nmol / ml.

[0262] The detection process is carried out at 25°C, and the buffer is 1x HBS-EP+, and the analysis process and each step Flow cell 1 and Flow cell 2 mobile phase are as follows:

[0263] Binding: Flow cell 2: Antibody after dilution;

[0264] Dissociation: Flow cell 1 & Flow cell 2: 1x HBS-EP+;

[0265] Regeneration: Flow cell 1 & Flow cell 2: 10 mM Glycine-HCl, pH=1.5 (KD represents the equilibrium dissociation constant, i.e. affinity; kon represents the association rate; kdis represents the dissociation rate).

[0266] The results show that each mutation group in Table 1 improves the affinity, and part of the test results are shown in Table 5 and Figure 1, and the affinity of mutation group 7 is the highest.

[0267] Table 5: Affinity data

[0268] 6. Capacity test

[0269] Take 40 mg of the protein eluted from the protein A filler described above and replace it in PBS buffer, with the same buffer and pH, and adjust it to a similar concentration (about 1 mg / ml). Re-calculate the protein A column, with a retention time of 1 min, to obtain test data. It is found that compared with the wild type, the mutation groups in Table 1 above can improve the capacity of the same protein A binding, and part of the test data is shown in Table 6, and the capacity of mutation group 7 is the highest.

[0270] Table 6: Mouse IgG1 protein A binding capacity

[0271] Example 2: Design of mouse IgG1 mutation site

[0272] 1. Mutation site design

[0273] Some superior mutation points and combined mutations of sheep IgG1 tested are put on mouse (house mouse) IgG1 for testing, see Table 7, wherein the amino acid sequence of the Fc fragment (CH2 fragment and CH3 fragment) of the wild type mouse IgG1 is shown in SEQ ID NO: 5.

[0274] Table 7: Mutation site of mouse IgG1

[0275] 2. Plasmid construction, expression of recombinant antibody sample, HPLC-SEC purification, thermal stability Tm value, affinity analysis

[0276] Plasmid construction, expression of samples of recombinant antibodies, HPLC-SEC purification, thermal stability Tm value, affinity analysis are all referred to Example 1, wherein the antibody is Anti-CA153 antibody, and the relevant amino acid sequences are as follows:

[0277] The amino acid sequence of the light chain in the Anti-CA153 antibody is as shown in SEQ ID NO: 6:

[0278] The heavy chain in the Anti-CA153 antibody is in turn a heavy chain variable region, a CH1 and hinge region, and a Fc fragment (or Fc mutant) from N-terminal to C-terminal, and the Fc mutant in each mutation group is shown in Table 7; the amino acid sequence of the heavy chain variable region in the Anti-CA153 antibody is as shown in SEQ ID NO: 7:

[0279] The amino acid sequence of the CH1 and hinge region in the Anti-CA153 antibody is as shown in SEQ ID NO: 8:

[0280] The detection results of HPLC-SEC show that the mutation group 3 causes the expression amount to decrease, and other mutation groups have no obvious effect on the expression amount, and the results are shown in Table 8. The affinity of each mutation group to protein A is improved, and part of the detection results are shown in Table 10 and FIG. 2.

[0281] Table 8: HPLC-SEC results after expression

[0282] Table 9: Tm results

[0283] Table 10: Affinity data

[0284] 3. Load test

[0285] Take 50 mg of the protein eluted from the above protein A filler and replace it in PBS buffer, with the same buffer and PH, and adjust to a similar concentration (about 1 mg / ml), re-apply protein A chromatography, and the retention time is 1 min. The results show that compared with the wild type, the binding load of each mutation group to the protein A filler is improved, and part of the detection results are shown in Table 11.

[0286] Table 11: Protein A binding load of mouse IgG1

[0287] Example 3: Design scheme of rat IgG2a mutation site

[0288] 1. Mutation site design

[0289] The best case of the sheep antibody was applied to the rat (Norway rat) IgG2a, and the specific mutation sites are shown in Table 12. The amino acid sequence of the Fc fragment (CH2 fragment and CH3 fragment) of the wild-type rat IgG2a is shown in SEQ ID NO: 9.

[0290] Table 12: Mutation sites of rat IgG2a

[0291] 2. Plasmid construction, expression of recombinant antibody samples, HPLC-SEC purification, thermal stability Tm value, affinity analysis

[0292] The plasmid construction, expression of recombinant antibody samples, HPLC-SEC purification, thermal stability Tm value, and affinity analysis are all referred to Example 1, wherein the antibody is Anti-PCT antibody, and the relevant amino acid sequences are as follows:

[0293] The amino acid sequence of the light chain in the Anti-PCT antibody is shown in SEQ ID NO: 10:

[0294] The heavy chain of the Anti-PCT antibody from N-terminal to C-terminal is in turn heavy chain variable region, CH1 and hinge region, Fc fragment (or Fc mutant); the amino acid sequence of the heavy chain variable region in the Anti-PCT antibody is shown in SEQ ID NO: 11:

[0295] The amino acid sequence of the CH1 and hinge region in the Anti-PCT antibody is shown in SEQ ID NO: 12:

[0296] The detection results of HPLC-SEC are shown in Table 13, the detection results of affinity analysis are shown in Table 14 and Figure 3, and the detection results of loading test are shown in Table 15.

[0297] Table 13: Yield and HPLC-SEC results after expression

[0298] Table 14: Affinity data

[0299] Note: N / A means not binding.

[0300] Table 15: Protein A binding load

[0301] Example 4:

[0302] The present example compares the CH2 region partial amino acid sequences of different types of IgG antibodies, such as rat (Norway rat) IgG1, rat IgG2b, bovine IgG1, bovine IgG3, etc. See FIG. 4. The first amino acid in the first binding region of the gray shaded portion in FIG. 4 is mutated to M, and the third amino acid is mutated to K, and the thermal stability, purity, and protein A filler binding capacity are detected. See Example 1 for specific steps. The results show that the above mutations have little effect on thermal stability and purity, but can significantly improve the protein A filler binding capacity and facilitate a significant increase in expression.

[0303] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0304] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. An Fc mutant, characterized in that, at least one of positions 23, 21, and 24 of the Fc mutant is mutated; the position 23 is mutated to K or M; the position 21 is mutated to M, H, or W; the position 24 is mutated to R.

2. The Fc mutant according to claim 1, characterized in that, the Fc mutant has mutations selected from any one of groups a) to c): a) the position 21 is mutated to M; b) the position 24 is mutated to R; c) the position 21 is mutated to M and the position 24 is mutated to R.

3. The Fc mutant according to claim 1, characterized in that, the Fc mutant has mutations selected from any one of groups d) to h): d) the position 21 is mutated to W; e) the position 21 is mutated to H; f) the position 23 is mutated to M; g) the position 23 is mutated to K; h) the position 21 is mutated to W or H and the position 23 is mutated to M or K.

4. The Fc mutant according to any one of claims 1 to 3, characterized in that, the Fc mutant has mutations selected from any one of groups d) to h) and has mutations selected from any one of groups a) to c).

5. The Fc mutant according to any one of claim 4, characterized in that, The Fc mutant has mutations of any one of the following sets:

6. The Fc mutant according to any one of claim 5, characterized in that, the Fc mutant is derived from IgGl, IgG2a, IgG2b, IgG2c, IgG3, or IgG4.

7. The Fc mutant of claim 1, wherein the Fc mutant is derived from a species selected from the group consisting of sheep, goat, mouse, rat, bovine, rabbit, horse, pig, dog, cat, donkey, deer, mink, chicken, duck, goose, and human.

8. The Fc mutant according to claim 7, characterized in that, the Fc mutant is derived from sheep IgGl, mouse IgGl, rat IgG2a, rat IgGl, rat IgG2b, bovine IgGl, or bovine IgG3.

9. A protein, characterized in that, an Fc mutant according to any one of claims 1 to 8.

10. The protein of claim 9, wherein, the protein is an antibody or a fusion protein.

11. The protein of claim 10, wherein the protein further comprises a functional region, which is directly or indirectly linked to the Fc mutant.

12. The protein of claim 11, wherein, the functional region comprises a light chain variable region and / or a heavy chain variable region.

13. A nucleic acid molecule, vector, cell or host, characterized in that, the nucleic acid molecule encodes an Fc mutant according to any one of claims 1 to 8, or a protein according to any one of claims 9 to 12; the vector comprises the nucleic acid molecule; the cell or host comprises the nucleic acid molecule or the vector.

14. A reagent or kit characterized in that, a protein according to any one of claims 9 to 12.

15. A method for increasing the expression amount of a target protein, increasing the affinity of a target protein to Protein A, increasing the binding capacity of a target protein to a Protein A filler, producing a target protein, or purifying a target protein, the target protein containing an Fc fragment, characterized in that, comprising: mutating the Fc fragment so that the mutations are identical to the mutations of the Fc mutant according to any one of claims 1 to 8.

16. Use of an Fc mutant according to any one of claims 1 to 8 for increasing the expression of a protein of interest comprising the Fc mutant, for increasing the affinity to protein A, or for increasing the binding capacity of a protein A filler.

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