Fc fragment peptides for the production of therapeutically active fusion peptides and conjugates

Abstract

This invention relates to the field of biotechnology, specifically to immunoglobulin Fc fragment polypeptides, which can be used for medical applications. The disclosed immunoglobulin Fc fragment polypeptides exhibit high binding capacity for FcRn and low binding capacity for FcγR, and can be used to produce therapeutically active fusion polypeptides and conjugates.

Description

[0001] Invention Field

[0002] Research has shown that by altering amino acid residues in Fc fragment peptides, the binding of Fc fragment peptide-based antibodies or fusion constructs to FcRn and FcγR can be modified. The Fc region peptide-based conjugates and fusion constructs disclosed herein can be used, for example, to treat diseases requiring targeted systemic administration for selected retention times. Background of the Invention

[0004] Immunoglobulin Fc domain receptors (FcRs) are divided into two main categories: those involved in effector functions, such as FcRs of IgG (FcγRI, II, and III), IgE (FcγRI), and IgA (FcαRI); and those responsible for transporting immunoglobulins across the epithelial surface, namely poly-IgA receptors (pIgRs) and neonatal IgG transporters FcRn.

[0005] The neonatal Fc receptor (FcRn) is crucial for the metabolic pathway of IgG antibodies in vivo. The function of FcRn is to protect wild-type IgG from lysosomal degradation, thereby reducing immunoglobulin clearance and prolonging its half-life. The neonatal Fc receptor is a heterodimeric protein composed of two polypeptides: a 50 kDa major histocompatibility complex class I-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 region of the Fc region of IgG antibodies. The interaction between IgG antibodies and FcRn is pH-dependent and occurs in a stoichiometric ratio of 1:2, meaning that one IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region peptides (see, for example, HUBER AH et al., Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc, Journal of Molecular Biology, 1993, Vol. 230, No. 3, pp. 1077-1083).

[0006] Therefore, the in vitro binding properties / characteristics of IgG molecules to FcRn can indicate their in vivo pharmacokinetics in blood.

[0007] Antibody Fc fragments can also interact with a variety of Fc receptors and ligands, thereby mediating the effector functions of immune cells. This receptor family includes FcγRI (CD64), including subtypes: FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including subtypes: FcγRIIa, FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including subtypes FcγRIIIa and FcγRIIIb (see NAGELKERKE SQ, KUIJPERS TW, Immunomodulation by IVIg and the role of Fc-gamma receptors: classic mechanisms of action after all?, Frontiers in immunology, 2015, Vol. 5, p. 674).

[0008] Fcγ receptors are known to possess an extracellular domain (mediating interaction with Fc), a membrane-binding domain, and an intracellular domain (mediating several intracellular signaling events). These receptors are expressed on a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. The formation of the Fc / FcγR complex mediates the interaction between these effector cells and antigens, typically leading to cell signaling and important downstream immune responses such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and antibody-dependent cytotoxicity. The ability to mediate phagocytic toxic effects is a potential mechanism by which antibodies exert their anti-infective defense function (NAGELKERKE SQ, KUIJPERS TW, Immunomodulation by IVIg and the role of Fc-gamma receptors: classic mechanisms of action after all?, Frontiers in immunology, 2015, Vol. 5, p. 674).

[0009] The interaction between FcRn and the Fc region of the IgG antibody involves multiple amino acid residues in the heavy chain CH2 and CH3 domains. The amino acid residues involved in the interaction with FcRn are located from approximately position 243 to position 261 according to EU numbers, from approximately position 275 to position 293 according to EU numbers, from approximately position 302 to position 319 according to EU numbers, from approximately position 336 to position 348 according to EU numbers, at position 408 according to EU numbers, and from approximately position 424 to position 440 according to EU numbers. More specifically, the following amino acid residues according to Kabat's EU numbers are involved in the interaction between the Fc region and FcRn: F243, P244, P245. P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E25 8. V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285 , N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I 336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C3 67. V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S38 3. N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440.

[0010] Studies using site-directed mutagenesis have shown that the key binding sites for FcRn within the IgG Fc region are histidine 310, histidine 435, and isoleucine 253; and to a lesser extent, histidine 433 and tyrosine 436 are also included (see, for example, Kim J. K. et al., Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn, European journal of immunology, 1999, Vol. 29, No. 9, pp. 2819-2825; Raghavan M. et al., Analysis of the pH dependence of the neonatal Fc receptor / immunoglobulin G interaction using antibody and receptor variants, Biochemistry, 1995, Vol. 34, No. 45, pp. 14649-14657; MEDESAN C. et al., Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgG1, The Journal of Immunology). 1997, Volume 158, Issue 5, pp. 2211-2217.

[0011] Methods to enhance the binding of IgG to FcRn by mutating multiple amino acid residues in IgG are known: threonine 250, methionine 252, serine 254, threonine 256, threonine 307, glutamic acid 380, methionine 428, histidine 433, and asparagine 434 (see, for example, KUO TT et al., Neonatal Fc receptor: from immunity to therapeutics, Journal of Clinical Immunology, 2010, Vol. 30, No. 6, pp. 777-789).

[0012] Studies on the interaction between immunoglobulins and FcγR have shown that the binding sites of C1q and FcγR are located within the IgGCH2 domain. Mutagenic analysis of IgG2b mAb identified glutamic acid, lysine, and lysine residues at positions 318, 320, and 322 as key motifs for its binding to C1q.

[0013] Furthermore, amino acid residues at positions 234-238 are involved in the high-affinity interaction between mouse IgG2a and FcγRI. In addition, studies have shown that replacing aspartic acid at position 265 with alanine (D265A) can eliminate the interaction between mouse IgG1 and low-affinity FcγRIIb and FcγRIII (BAUDINO L. et al., Crucial role of aspartic acid at position 265 in the CH2 domain for murine IgG2a and IgG2b Fc-associated effector functions, The Journal of Immunology, 2008, Vol. 181, No. 9, pp. 6664-6669).

[0014] Therefore, for certain therapeutic applications, it is best to avoid activating complement and FcγR. For example, when administering a therapeutically active peptide fused with an antibody Fc fragment, complement stimulation is undesirable (see, for example, CAPON DJ et al., Designing CD4 immunoadhesins for AIDS therapy, Nature, 1989, Vol. 337, No. 6207, pp. 525-531). Furthermore, because human IgG2 and IgG4 have relatively low affinity for FcγR and complement receptors (BRUHNS P. et al., Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses, Blood, The Journal of the American Society of Hematology, 2009, Vol. 113, No. 16, pp. 3716-3725), these two subclasses are used to develop therapeutic monoclonal antibodies (mAbs) (e.g., denosumab, natalizumab, panitumumab, and eculizumab) when their minimum effect potential is required.

[0015] Therefore, for example, fusion of glucagon-like peptide-1 (GLP-1) with human IgG2 avoids unwanted immunogenicity and has shown superior therapeutic and pharmacological properties compared to native GLP-1 in a type 1 diabetic mouse model (WANG Q. et al., Novel GLP-1 fusion chimera as potent long acting GLP-1 receptor agonist, PloS one, 2010, Vol. 5, No. 9, p.e12734). However, by simultaneously activating another effector system, it is also possible to reduce the effector function of one effector system. In particular, it has been observed that FcγR activation of mast cells can be inhibited by the synchronous activation of inhibitory FcγRIIb (DAËRON M. et al., The same tyrosine-based inhibitionmotif, in the intra-cytoplasmic domain of FcγRIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation, Immunity, 1995, Vol. 3, No. 5, pp. 635-646). This has stimulated the development of various anti-inflammatory Fc fusion proteins that could be used to treat allergic asthma.

[0016] In summary, it can be inferred that introducing specific modifications to the Fc fragment fused or conjugated with a bioactive spouse can enhance the binding ability of the fusion protein to FcRn and reduce its binding ability to the effector FcγR, thereby significantly improving the pharmacokinetic properties of the fusion protein.

[0017] This invention relates to generating immunoglobulin Fc fragment polypeptides with high binding capacity to FcRn and low binding capacity to FcγR, for use in creating therapeutically active fusion polypeptides and conjugates. Summary of the Invention

[0018] All terms and abbreviations used in this application have their common meanings in the art and are obvious to those skilled in the art.

[0019] The term "alteration" refers to a mutation (substitution), insertion (addition), or deletion of one or more amino acid residues in a parent antibody or fusion polypeptide (e.g., a fusion polypeptide containing an FcRn binding moiety of at least one Fc region), resulting in a modified antibody or fusion polypeptide. The term "mutation" refers to the substitution of one amino acid residue for another. For example, the L234A mutation refers to the substitution of a lysine residue at position 234 of the antibody's Fc region (peptide) for an alanine residue (lysine replaced by alanine) (numbered according to the EU index).

[0020] The term "amino acid mutation" refers to the substitution of at least one existing amino acid residue with another different amino acid residue (i.e., a substituted amino acid residue). The substituted amino acid residue can be a "natural amino acid residue" selected from the following: alanine (three-letter code: ala, single-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V). The substituted amino acid residue can also be a "non-natural amino acid residue," including but not limited to D-stereoisomers of "natural amino acid residues."

[0021] The term "amino acid insertion" refers to the insertion of at least one amino acid residue at a predetermined position in an amino acid sequence. In one embodiment of the invention, "insertion" means the insertion of one, two, three, or more amino acid residues. The inserted amino acid residue can be any natural or non-natural amino acid residue, or a combination thereof.

[0022] The term "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined position in an amino acid sequence.

[0023] The term "CH2 domain" refers to the portion of an antibody heavy chain polypeptide that extends from approximately position 231 according to the EU number (Kabat's EU numbering system) to position 340 according to the EU number.

[0024] The term "CH3 domain" refers to the portion of the antibody heavy chain polypeptide that extends from approximately position 341 according to the EU number to position 446 according to the EU number.

[0025] An antibody's "class" is related to the type of its heavy chain constant domain or constant region. Antibodies are mainly divided into five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further subdivided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

[0026] The term "derived from" refers to an amino acid sequence generated by introducing a change at at least one position in the parent amino acid sequence. This altered amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (the numbering scheme for the antibody Fc region uses the Kabat EU indexing system). In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs at one or more amino acid residues at corresponding positions. Similarly, the altered amino acid sequence has a high degree of amino acid sequence identity with the parent amino acid sequence. In one embodiment, the amino acid sequence identity derived from the parent amino acid sequence is 90% or higher. In one embodiment, the amino acid sequence identity derived from the parent amino acid sequence is 95% or higher.

[0027] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source but using a human antibody library or other human antibody encoding sequence. This definition of "human antibody" explicitly excludes humanized antibodies containing antigen-binding residues not found in human antibodies.

[0028] The term "human Fc fragment" refers to the C-terminal region of a human immunoglobulin heavy chain, which includes at least a portion of the hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the Fc region of the human IgG heavy chain extends from approximately Cys226 or Pro230 to the C-terminus of the heavy chain. The C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering scheme, also known as the EU index, as described in KABAT EA, Sequences of proteins of immunological interest, USDepartment of Health and Human Services, Public Health Service, National Institutes of Health, 1991, N. 91. The Fc region consists of two heavy chain Fc region polypeptides covalently linked to each other via hinge region cysteine ​​residues, which participate in the formation of disulfide bonds between the polypeptides.

[0029] The term "FcRn" refers to the human neonatal Fc receptor. The function of FcRn is to rescue IgG from lysosomal degradation, thereby reducing its clearance and prolonging its half-life. FcRn is a heterodimeric protein composed of two polypeptides: a 50 kDa major histocompatibility complex class I-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 region of the IgG Fc region. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a stoichiometric ratio of 1:2, with one IgG molecule binding two FcRn molecules via its two heavy chains (HUBER AH et al., Crystallization and stoichiometry of binding of a complex between a rat intestinal Fc receptor and Fc, Journal of Molecular Biology, 1993, Vol. 230, No. 3, pp. 1077-1083). FcRn binding occurs in endosomes at acidic pH (pH < 6.5), while IgG is released to the cell surface at neutral pH (pH ~ 7.4). This pH-dependent interaction promotes FcRn-mediated protection, allowing IgG molecules that enter the cell via endocytosis to bind to receptors under acidic endosome conditions, thus protecting them from intracellular degradation. Furthermore, FcRn can promote the return of IgG to the cell surface and its release into the bloodstream after the FcRn-IgG complex is exposed to an extracellular neutral pH environment.

[0030] The term "FcRn binding portion of the Fc region" refers to a portion of the antibody heavy chain polypeptide that extends from approximately position 243 of the EU designation to position 261 of the EU designation, from approximately position 275 of the EU designation to position 293 of the EU designation, from approximately position 302 of the EU designation to position 319 of the EU designation, from approximately position 336 of the EU designation to position 348 of the EU designation, from approximately position 367 of the EU designation to positions 393 and 408 of the EU designation, and from approximately position 424 of the EU designation to position 440 of the EU designation. In one embodiment, one or more of the following amino acid residues are varied according to the EU designation of Kabat: F243, P244, P245. P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258 , V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N 286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T3 07. V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336 , S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N3 84, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU numbers).

[0031] The term "human IgG Fc fragment polypeptide" refers to an amino acid sequence resulting from a change in at least one amino acid in the Fc region polypeptide of natural human IgG or the Fc region polypeptide of wild-type human IgG.

[0032] The term “heterodimer” or “heterodimer” refers to a molecule comprising two polypeptide chains (e.g., of comparable length) in which the amino acid sequences of the two polypeptide chains differ by at least one amino acid residue at a matching position, wherein the matching position is defined according to the Kabat EU index.

[0033] The terms “homodimer” and “homodimer” refer to a molecule containing two polypeptide chains of similar length, wherein the amino acid sequences of the two polypeptide chains are identical at matching positions, which are defined according to the Kabat EU index.

[0034] "Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0035] "Separated" nucleic acids are nucleic acid molecules that are separate from their components in their natural environment. "Separated nucleic acids" include nucleic acid molecules that are normally present in cells containing nucleic acid molecules, but which are located outside the chromosome or at a location on the chromosome that is different from their natural location.

[0036] According to the present invention, "nucleic acid" or "nucleic acid molecule" should be understood as a polynucleotide molecule, which may be of DNA or RNA type, preferably DNA type, and especially may be double-stranded. It may be natural or synthetic. Synthetic nucleic acids are generated in vitro. Examples of such synthetic nucleic acids are those in which the codons encoding polypeptides (especially the Fc fragment polypeptide of the present invention) have been optimized according to the host organism expressing them (e.g., by replacing codons with more or more preferred codons compared to the original host through a codon usage frequency table based on similar host organisms or the group to which such similar host organisms belong). Codon optimization methods are well known to those skilled in the art.

[0037] For the purposes of this invention, the terms "nucleic acid" and "polynucleotide" are used with the same meaning and are synonyms. For example, the phrase "encoding nucleic acid" is the same as the phrase "encoding polynucleotide".

[0038] As used herein, “containing” or “containing” a specific sequence “X” means a DNA or protein that contains or contains at least the sequence “X”. Therefore, other nucleotide or amino acid sequences may be included at the 5' (or N-terminus) and / or 3' (or C-terminus), such as optional markers and / or 5' leader sequences or 3' trailing sequences.

[0039] Similarly, the use of the term "comprising" (and its grammatical variations) in this specification or the appended claims is intended to include the specified number or step, or group of number or steps, but does not exclude any other number or step, or group of number or steps.

[0040] As used in this article, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures (including "cloning vectors"), as well as vectors that integrate into the host cell genome upon which they are introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are efficiently linked. Such vectors are referred to as "expression vectors" in this article.

[0041] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and all refer to cells in which exogenous nucleic acids have been introduced, including progeny cells of this type. Host cells include "transformers" and "transfected cells," which include primary transfected cells and their progeny cells, regardless of passage number. The nucleic acid composition of progeny cells may not be entirely identical to that of parent cells and may contain mutations. This invention covers mutant progeny cells that have the same function or biological activity as the original transfected cells screened or selected.

[0042] The "percentage of amino acid sequence identity (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to obtain the maximum percentage of sequence identity, excluding any conserved substitutions as part of the sequence identity. In the context of this document, the "percentage of identity" between two amino acid sequences refers to the percentage of identical amino acids in the two sequences to be compared, obtained through optimal alignment of the sequences; wherein the percentage is a purely statistical concept and the differences between the two sequences are randomly distributed across the amino acid sequences. In the context of this document, "optimal alignment" or "best alignment" refers to the alignment for which the specific percentage of identity (see below) reaches its highest value. Sequence comparisons between two amino acid sequences are typically performed by comparing sequences that have previously undergone optimal alignment; this comparison is performed at comparison sites to identify and compare local regions of sequence similarity. Besides manual alignment, local homology and general homology search algorithms can be used to achieve optimal alignment of the sequences to be aligned. These algorithms can be implemented using software (GAP, BESTFIT, BLASTP, BLAST N, FASTA, T FAST A) and the MUSCLE multiple sequence alignment algorithm (EDGAR RC, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleicacids research, 2004, Vol. 32, No. 5, pp. 1792-1797). For optimal alignment results, BLAST software and the BLOSUM 62 matrix are preferred. The percentage of identity between two given best-aligned sequences is determined by comparing them. The amino acid sequences may contain additions and deletions relative to the reference sequence. The percentage of identity is calculated by determining the number of identical positions in the two given sequences, dividing this number by the total number of positions compared, and then multiplying the result by 100.

[0043] As used herein, the term "peptide linker" refers to a peptide with a different amino acid sequence, in one embodiment of which the peptide is of synthetic origin. In one embodiment, the peptide linker is a peptide with an amino acid sequence length of at least 30 amino acids; in one embodiment, its amino acid sequence length is at least 32-50 amino acids. In one embodiment, the peptide linker is a peptide with an amino acid sequence length of 32-40 amino acids. In one embodiment, the peptide linker is (G... x S)n In this embodiment, G represents glycine and S represents serine (x is 3, n is 8, 9, or 10; or x is 4 and n is 6, 7, or 8). In one embodiment, x is 4 and n is 6 or 7. In one embodiment, x is 4 and n is 7. In one embodiment, the peptide linker is (G4S)6G2.

[0044] The term "physiologically active molecule" refers to a molecule that has any type of physiological activity in vivo, plays a role in regulating gene expression and physiological function, and can correct abnormal conditions caused by insufficient or excessive secretion of substances involved in the regulation of bodily functions.

[0045] The term "physiologically active peptide" as used in this article refers to peptides possessing any type of in vivo physiological activity, typically having a peptide structure and exhibiting different physiological activities. Physiologically active peptides include those that regulate gene expression and physiological function, and can correct abnormalities caused by insufficient or excessive secretion of substances involved in in vivo functional regulation. Physiologically active peptides may include conventional protein therapeutic agents.

[0046] Physiologically active peptides may be selected from (especially) the following: glucagon-like peptide-1 (GLP-1), granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, gastrin, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor, interleukin, interleukin receptor, enzyme, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell kinase, T cell kinase, protein A, allergy inhibitors, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor (tumor) Suppressor), transgender growth factor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein E, highly glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, coagulation regulatory protein, blood factors VII, VIIa, VIII, IX and XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, angiostatin, vasopressin The following are listed: glucagon, bone growth factor, bone growth stimulating protein, calcitonin, atrial peptide, chondroitin cartilage growth inducing factor, calcitonin-dependent calcitonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, growth mediator, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing hormone, thyroid-stimulating hormone, autolysin, lactoferrin, myostatin, cell surface antigen, viral vaccine antigen, monoclonal antibody, polyclonal antibody, and antibody fragments. Preferably, the physiologically active polypeptide may be selected from the following: glucagon-like peptide-1 (GLP-1), granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, gastrin modulatory factor, insulin, insulin analogs, and their derivatives.

[0047] For the purposes of this invention, the term "insulin" refers to the human insulin hormone. Human insulin consists of two polypeptide chains: chain A and chain B. Chain A is a peptide of 21 amino acids, and chain B is a peptide of 30 amino acids. The two chains are connected by disulfide bonds: the first pair of disulfide bonds is formed between cysteine ​​residues at position 7 of chain A and position 7 of chain B; the second pair of disulfide bonds is located between cysteine ​​residues at position 20 of chain A and position 19 of chain B; and the third pair of disulfide bonds is located between cysteine ​​residues at positions 6 and 11 of chain A.

[0048] For the purposes of this invention, the term "insulin analog" refers to modified insulin in which one or more amino acid residues of insulin are replaced by other amino acid residues, and / or one or more amino acid residues are removed from insulin, and / or one or more amino acid residues are added to and / or inserted into insulin. Preferred insulin analogs of this invention include, but are not limited to, insulin aspart (i.e., human insulin AspB28), insulin lispro (i.e., human insulin LysB28, ProB29), and insulin glutalis (LysB3, GluB29 human insulin), etc.

[0049] Furthermore, the term "physiologically active polypeptide" as used in this article includes not only naturally occurring physiologically active polypeptides, but also agonists, precursors, derivatives, fragments, or variants of each polypeptide.

[0050] As used in this article, the term "non-peptide linker" refers to a biocompatible polymer composed of two or more repeating units linked by arbitrary non-peptide covalent bonds. This non-peptide linker may have two or three ends.

[0051] For the purposes of this invention, the term "medicine" refers to a synthetic or naturally derived substance or mixture of substances presented in dosage forms including but not limited to tablets, capsules, solutions, ointments, aerosols, etc., for the prevention, diagnosis and treatment of diseases.

[0052] The term "pharmaceutical composition" refers to a formulation in which the active ingredient contained herein is in a form that facilitates the effective exertion of its biological activity and does not contain any additional components that would have unacceptable toxicity to a subject to whom the composition is to be administered.

[0053] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical composition that are different from the active ingredient and are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0054] As used herein, the term "treatment" (or "therapeutic approach") refers to a clinical intervention aimed at altering the natural course of a disease in an individual being treated. This intervention may be implemented for preventative purposes or as part of the treatment of a clinical condition. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, minimizing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing disease progression, improving symptoms or alleviating disease state, and mitigating or improving prognosis.

[0055] The term "instructions for use" refers to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings of such therapeutic products.

[0056] Embodiments of the present invention

[0057] This invention provides Fc fragment peptides that exhibit modified properties in binding to the human Fc receptor compared to the corresponding wild-type Fc fragments. The Fc fragment peptides contain specific amino acid mutations in their CH2 and / or CH3 domains. These mutations have been found to enable the generation of Fc fragment peptide variants with customized in vivo half-lives.

[0058] In one embodiment of the invention, the Fc fragment polypeptide is an Fc fragment of the human IgG class.

[0059] In one embodiment of the invention, the Fc fragment polypeptide is an Fc fragment of the human IgG1 class.

[0060] In one embodiment of the invention, the Fc fragment polypeptide is an Fc fragment of the human IgG2 class.

[0061] In one embodiment of the present invention, the Fc fragment polypeptide is an Fc fragment of the human IgG3 class.

[0062] In one embodiment of the invention, the Fc fragment polypeptide is an Fc fragment of the human IgG4 class.

[0063] In one embodiment of the present invention, a pair of Fc fragment polypeptides can form a functional dimer.

[0064] In one embodiment of the present invention, the human Fc receptor is selected from the human neonatal Fc receptor and the human Fcγ receptor.

[0065] In one embodiment of the present invention, the human Fcγ receptor is selected from the human FcγRI receptor, the human FcγRII receptor, and the human FcγRIII receptor.

[0066] In one embodiment of the present invention, the Fc fragment polypeptide differs from the wild-type Fc fragment polypeptide by one amino acid residue, or two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acid residues at the corresponding position in the Kabat EU indexing system.

[0067] In one embodiment of the invention, the Fc fragment polypeptide comprises one or more mutations located at positions 222, 223, 224, 225, 226-238, 226, 234, 235, 236, 237, 239, 250, 252, 252, 254, 256, 262, 264, 264, 264, 268, 272, 276 according to the Kabat-EU indexing system. 4, 283, 285, 286, 288, 294, 296, 297, 300, 302, 303, 308, 308, 309, 309, 312, 315, 327, 328, 330, 330, 331, 339, 355, 356, 358, 378, 397, 409, 419, 427, 428, 434, 434, 445, and 444-446.

[0068] In one embodiment of the present invention, the Fc fragment polypeptide comprises one or more of the following mutations or combinations thereof: According to the Kabat EU indexing system, K222D, C223K, C224T, V225H, E226-P238del, E226W, P234E, 234insF235, V235L, A236G, G237M, S239E, T250Q, M252Y, M252W, S254T, T256E, V262W, V264del, V264D, V264E, H268Q, E272Q, Q274K, E283Q, H285E, N286E, K288M, E294Q, F296Y, N297D, F300Y, V302W, V303del, V308W, V308F, V309del, V309L, D312N, N315D, G327A, L328G, A330S, A330F, P331S, T339A, R355Q, D356E, L358M, S378A, M397V, K409R, Q419E, V427L, M428L, N434Y, N434W, P445L, and S444-G446del.

[0069] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the mutations V262W, V264del, V302W, V303del, V308W, V309del, and S378A according to the Kabat EU numbering system.

[0070] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the mutations M252Y, S254T, T256E, and S378A according to the Kabat EU numbering system.

[0071] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the mutations T250Q, M428L, and S378A according to the Kabat EU numbering system.

[0072] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the mutations M252Y, T256E, V264D, N297D, V308F, S378A, and N434Y according to the Kabat EU numbering system.

[0073] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the following mutations according to the Kabat EU numbering system: P234E, 234insF235, V235L, A236G, T256E, V264D, H268Q, H285E, N286E, N297D, F300Y, V309L, A330S, P331S, R355Q, S378A, M397V, K409R, Q419E, M428L, and P445L.

[0074] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the mutations M252W, T256E, S378A, M428L, and N434Y according to the Kabat EU numbering system.

[0075] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the mutations S239E, T256E, V264E, A330F, S378A, and N434Y according to the Kabat EU numbering system.

[0076] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the Kabat EU numbering system mutations ins221D, V222K, ins223T, E224H, ins225W, G237M, T250Q, M252Y and V264D.

[0077] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the Kabat EU numbering system mutations M252Y, T256E, V264E, N297D, V308F, A330F, S378A, and N434W.

[0078] In a preferred embodiment of the invention, the Fc fragment polypeptide comprises a combination of mutations corresponding to the Kabat EU numbering system mutations T250Q, M252Y, T256E, V264E, V308F, A330F, S378A, and N434W.

[0079] In one embodiment of the present invention, the Fc fragment polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of the human IgG2 Fc fragment.

[0080] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:1.

[0081] In a preferred embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where

[0082] X1 is either E or D. X2 is K or does not exist. X3 is T or does not exist. X4 is H or does not exist. X5 is W or does not exist. X 13 For P or E, X 14 F or not present. X 15 For V or L, X 16 It is either A or G. X 17 For G or M, X 19 For S or E, X 30 For T or Q, X 32 For M, Y, or W, X 34 For S or T, X 36 For T or E, X 44 For V, E, or D, X 48 For H or Q, X 65 For H or E, X 66 For N or E, X 77 For N or D, X 80 For F or Y, X 88 For V or F, X 89 For V or L, X 110 It can be A, F, or S. X 111 For P or S, X 119 For T or A, X 135 For R or Q, X 177 For M or V, X 189 For K or R, X 199 For Q or E, X 208 For M or L, X 214 It is M, Y or W, and X 225 It can be P or L.

[0083] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let Y and X be the numbers. 34 Let T, X 36 For E, X 44 Let V, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers.111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let P be the value.

[0084] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 For Q, X 32 Let M and X be the numbers. 34 Let S and X be the values ​​of S and X.36 Let T, X 44 Let V, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let L and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let P be the value.

[0085] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For D, X 48 For H, X 65 For H, X 66 Let N, X 77 For D, X 80 Let F, X 88 Let F, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let Y be X, and X be X 225 Let P be the value.

[0086] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 For E, X 14 Let F, X 15 Let L and X be the numbers. 16 For G, X 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let M and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For D, X 48 For Q, X 65 For E, X 66 For E, X 77 For D, X 80 Let Y and X be the numbers. 88 Let V, X 89 Let L and X be the numbers. 110 Let S and X be the values ​​of S and X. 111 Let S and X be the values ​​of S and X. 119 Let A and X be the two numbers. 135 For Q, X 177 Let V, X 189 Let R, X 199 For E, X 208 Let L and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let L be the value.

[0087] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 For W, X 34 Let S and X be the values ​​of S and X. 36 For E, X 44 Let V, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let L and X be the numbers. 214 Let Y be X, and X be X 225 Let P be the value.

[0088] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 For E, X 30 Let T, X 32 Let M and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For E, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let F, X 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers.214 Let Y be X, and X be X 225 Let P be the value.

[0089] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is D, X2 is K, X3 is T, X4 is H, X5 is W, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 Let M and X be the numbers. 19 Let S and X be the values ​​of S and X. 30 For Q, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 Let T, X 44 For D, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let P be the value.

[0090] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X.30 Let T, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For E, X 48 For H, X 65 For H, X 66 Let N, X 77 For D, X 80 Let F, X 88 Let F, X 89 Let V, X 110 Let F, X 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let W be the integer part of the integer part, and X be the integer part of the integer part. 225 Let P be the value.

[0091] In one embodiment of the present invention, the Fc fragment polypeptide comprises the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2. 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX 65 X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX214 HYTQKSLSLSX 225 G, where X1 is E, X2 does not exist, X3 does not exist, X4 does not exist, X5 does not exist, X 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 For Q, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For E, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let F, X 89 Let V, X 110 Let F, X 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let W be the integer part of the integer part, and X be the integer part of the integer part. 225 Let P be the value.

[0092] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3.

[0093] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4.

[0094] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5.

[0095] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6.

[0096] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:7.

[0097] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:8.

[0098] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:9.

[0099] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:10.

[0100] In one embodiment of the invention, the Fc fragment polypeptide has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:11.

[0101] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:3.

[0102] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:4.

[0103] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:5.

[0104] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:6.

[0105] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:7.

[0106] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:8.

[0107] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:9.

[0108] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:10.

[0109] In a preferred embodiment of the present invention, the Fc fragment polypeptide has the amino acid sequence of SEQ ID NO:11.

[0110] Binding to the Fc receptor can be measured using standard equipment such as the BIAcore instrument (GE Healthcare) via techniques such as ELISA or surface plasmon resonance (SPR), as well as using Fc receptors generated through recombinant expression technology.

[0111] Alternatively, cell lines known to express specific Fc receptors can be used to assess the binding affinity between the Fc domain or Fc-domain-containing antibodies and the Fc receptor, such cell lines being, for example, but not limited to, human NK cells expressing the FcγIIIa receptor.

[0112] Binding to the Fc receptor can also be determined, for example, using label-free biolayer interferometry (BLI) techniques on the ForteBio Octet system (see, for example, TOBIAS R., MA W., Analysis of FcRn-Antibody Interactions on the Octet Platform, ForteBio Appl Note 17, 2019, pp. 1-13; BAJARDI-TACCIOLI A. et al., Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function, Analyticalbiochemistry, 2020, Vol. 611, p. 113842).

[0113] The effector function of Fc domains or antibodies containing Fc domains can be assessed using methods well known in the art, such as by assessing the ADCC activity of the target molecule (CLYNES R. et al., Fc receptors are required inpassive and active immunity to melanoma, Proceedings of the National Academy of Sciences, 1998, Vol. 95, No. 2, pp. 652-656).

[0114] Alternatively, assessment can be performed using non-radioactive analytical methods. Effector cells suitable for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0115] Those skilled in the art are also aware of methods for analyzing FcRn binding and determining in vivo clearance or half-life (PETKOVA SB et al., Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease, International Immunology, 2006, Vol. 18, No. 12, pp. 1759-1769).

[0116] According to one aspect, this article provides a nucleic acid (polynucleotide) encoding the human IgG Fc fragment polypeptide described herein.

[0117] In some embodiments, the isolated nucleic acid (polynucleotide) encodes the full-length Fc region polypeptide of the present invention described herein. In other embodiments, the isolated nucleic acid (polynucleotide) encodes a polypeptide contained within the Fc region polypeptide of the present invention described herein.

[0118] In some embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0119] The Fc fragment polypeptide variants of the present invention can be generated by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or by recombinant technology. For recombinant production, one or more polynucleotides encoding the Fc fragment polypeptide (e.g., the polypeptide described above) are isolated and inserted into one or more vectors for subsequent cloning and / or expression in host cells. The polynucleotides can be easily isolated and sequenced using conventional methods. One subject of the invention is a vector (preferably an expression vector) comprising one or more polynucleotides according to the invention.

[0120] Methods well known to those skilled in the art can be used to construct expression vectors containing Fc fragment polypeptide coding sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, in vivo synthesis, and genetic recombination methods (see, for example, THOMASON LC et al., Recombineering: genetic engineering inbacteria using homologous recombination, Current protocols in molecular biology, 2014, Vol. 106, No. 1, Unit 1.16). The expression vector can be a plasmid or part of a virus, or it can be a nucleic acid fragment.

[0121] The expression vector contains an expression cassette in which a polynucleotide (i.e., the coding region) encoding an Fc fragment polypeptide is cloned to establish a functional link with a promoter and / or other transcriptional or translational control elements.

[0122] As used herein, a “coding region” refers to the portion of a nucleic acid consisting of codons that can be translated into amino acids. Although “stop codons” (TAG, TGA, or TAA) are not translated into any amino acids, their presence can still be considered part of a coding region. However, any flanking sequences (e.g., promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc.) are not considered coding regions. Two or more coding regions may exist in a single polynucleotide construct (e.g., a single vector) or in separate polynucleotide constructs (e.g., separate (different) vectors). Furthermore, any vector may contain one or more coding regions; for example, the vectors of this invention may encode one or more polypeptides that can be cleaved by proteolysis and separated into the final protein post-translation or during translation.

[0123] In one embodiment of the invention, the vector, polynucleotide, or nucleic acid of the invention may encode a heterologous coding region, which may or may not be fused with a polynucleotide encoding a polypeptide or a variant or derivative thereof of the Fc fragment of the invention. The heterologous coding region includes, but is not limited to, specific elements or motifs, such as secretory signal peptides or heterologous functional domains. A functional linkage exists when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences, thereby influencing or controlling the expression of the gene product by those regulatory sequences(s). If inducing promoter function leads to transcription of the mRNA encoding the desired gene product, and the linkage characteristics between the two DNA fragments do not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product, nor with the ability of the DNA template to be transcribed, then the two DNA fragments (e.g., the polypeptide coding region and the associated promoter) are "functionally linked" or "effectively linked." Therefore, if a promoter enables transcription of a nucleic acid encoding a polypeptide, then the promoter region is functionally linked to that nucleic acid. The promoter may be a cell-specific promoter that supports significant levels of DNA transcription only in pre-selected cells. Other transcriptional control elements besides promoters, such as enhancers, operons, repressors, and transcription termination signals, can also be effectively linked to polynucleotides to mediate cell-specific transcription. Suitable promoters and other transcriptional control regions are described herein. A wide variety of transcriptional control regions are well known to those skilled in the art. These control regions include, but are not limited to, those that function in vertebrate cells; for example (but not limited to), promoter and enhancer segments derived from cytomegaloviruses (e.g., combinations of early promoters and introns A), simian virus 40 (e.g., early promoters), and retroviruses (e.g., Raoult sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (e.g., actin, heat shock proteins, bovine growth hormone, and rabbit β-globin genes), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translational control elements are well known to those skilled in the art. These elements include, but are not limited to: ribosome binding sites, translation start and stop codons, and elements derived from the viral system (particularly internal ribosome entry sites, or IRES, also known as CITE sequences).

[0124] The expression cassette may also contain other specific structures, such as origin of replication and / or chromosomal integration elements, such as long terminal repeats (LTRs) of retroviruses or terminal inverted repeats (ITRs) of adeno-associated viruses (AAVs).

[0125] In one embodiment of the invention, the expression cassette may further comprise DNA or RNA encoding a short protein sequence that may facilitate further purification (e.g., a histidine tag) or be designed to label the fusion protein; the DNA or RNA encoding the short protein sequence may be contained within or at both ends of a polynucleotide encoding an Fc fragment polypeptide.

[0126] In one embodiment of the invention, the vector may further comprise (independent of each other) the following structural elements: a gene conferring tetracycline resistance to bacterial cells; a replication initiation site (ori); a ROP gene regulating plasmid copy number; a transcription promoter (e.g., Tac-T7); a transcription terminator (e.g., rrnB1 B2 T1 txn); and a LacI gene encoding a Tac promoter repressor protein.

[0127] This invention also provides a host cell comprising one or more polynucleotides of the present invention. Host cells comprising one or more vectors of the present invention are also within the scope of this invention. The polynucleotides and vectors may individually or in combination possess any of the features disclosed herein. In one embodiment of the invention, the host cell comprises a vector (e.g., transformed or transfected by the vector) containing a polynucleotide encoding the Fc fragment polypeptide of the present invention. In the context of this specification, the term "host cell" refers to any type of cell system that is engineered to produce the Fc fragment polypeptide or a fragment thereof of the present invention.

[0128] Suitable host cells for replicating and maintaining Fc fragment peptide expression are well known in the art. These cells can be transfected or transduced with specific expression vectors as needed, followed by the culture of larger quantities of cells containing that vector, which can then be seeded into large fermenters to produce sufficient quantities of Fc fragment peptides for clinical use. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO) and insect cells. For example, peptides can be produced within bacteria, especially without the need for glycosylation modification. After expression, the peptides can be isolated from bacterial cell sediment into soluble fractions for further purification.

[0129] In one embodiment of the invention, eukaryotes (e.g., filamentous fungi or yeast) can be used as hosts for cloning or expression vectors encoding the polypeptide, including fungal and yeast strains whose glycosylation pathways have been "humanized," thereby enabling the production of polypeptides with partially or fully humanized glycosylation patterns. (See GERNGROSS TU, Advances in the production of human therapeutic proteins in yeasts and filamentous fungi, Nature biotechnology, 2004, Vol. 22, No. 11, pp. 1409–1414; and LI H. et al., Optimization of humanized IgGs in glycoengineered Pichia pastoris, Nature biotechnology, 2006, Vol. 24, No. 2, pp. 210–215). Host cells suitable for expressing (glycosylated) polypeptides can also be obtained from multicellular organisms (invertebrates and vertebrates).

[0130] Examples of invertebrate cells include insect cells; plant cells can also serve as hosts. Numerous baculovirus strains and corresponding insect cells suitable as their hosts have been identified, particularly those suitable for the fall armyworm (P. spp.). Spodoptera frugiperda Cell-transfected strains. Plant cell cultures can also be used as hosts.

[0131] In one embodiment of the invention, vertebrate cells can also serve as a host. For example, mammalian cell lines adapted for suspension culture can be used. Other suitable mammalian host cell lines include: CV1 (COS-7) monkey kidney cell line transformed with SV40; human embryonic kidney cell lines, such as HEK293 (see GRAHAM FL et al., Characteristics of a human cell line transformed by DNA from human adenovirus type 5, Journal of general virology, 1977, Vol. 36, No. 1, pp. 59-72); young hamster kidney cells (BHK); mouse Sertoli cells; monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (see, for example, MATHER JP et al., Culture of testicular cells in hormone-supplemented serum-free). (medium, Annals of the New York Academy of Sciences, 1982, Vol. 383, pp. 44-68); MRC 5 cells and FS4 cells.

[0132] In one embodiment of the invention, the mammalian host cell line may be represented by Chinese hamster ovary (CHO) cells (including dhfr-CHO cells) (see URLAUB G. et al., Isolation of Chinese hamster cellmutants deficient in dihydrofolate reductase activity, Proceedings of the National Academy of Sciences, 1980, Vol. 77, No. 7, pp. 4216-4220) and myeloma cell lines (e.g., Sp2 / 0, NSO, P3X63, YO) (see, for example, STEINITZ M. et al. (eds.), Human monoclonal antibodies: Methods and Protocols, New York: Humana Press, 2014).

[0133] One subject of this invention is a method for producing the Fc fragment polypeptide of the present invention, wherein the method comprises culturing a host cell containing one or more polynucleotides encoding the Fc fragment polypeptide of the present invention, and optionally isolating the Fc fragment polypeptide from the host cell (or the culture medium of the host cell).

[0134] In one embodiment, the present invention also provides a method for producing the Fc fragment polypeptide of the present invention using recombinant DNA technology, the method comprising the following steps: 1) Provide the nucleotide sequence encoding the polypeptide of the Fc fragment; 2) Insert the nucleotide sequence of (1) into a suitable expression vector to generate a recombinant expression vector; 3) Introduce the recombinant expression vector from (2) into a suitable host cell; 4) Culture the transfected host cells under conditions suitable for expression; 5) Collect and purify the expression product.

[0135] This coding sequence can be introduced into host cells by a variety of methods known in the art, such as (but not limited to) calcium phosphate precipitation, liposome transfection, electroporation, microinjection, viral infection, and alkali metal ion methods.

[0136] Methods for culturing and expressing in host cells are well known in the art (see, for example, GRAHAM, 1977; LI, 2006). Cells and debris in the suspension can be removed by centrifugation, and the supernatant can be collected.

[0137] In one embodiment of the invention, the product may be expressed by cells in the form of inclusion bodies and may be further concentrated (e.g., by tangential flow filtration or percolation) and then purified by chromatography.

[0138] Methods for purifying recombinant proteins (including recombinant proteins containing antibody Fc fragments) are well known to those skilled in the art (see, for example, SHI Y. et al., Advantages of CE-SDS over SDS-PAGE in mAb purity analysis, Analytical Methods, 2012, Vol. 4, No. 6, pp. 1637-1642).

[0139] The Fc fragment peptides obtained by the above method can be purified to have essentially homogeneous properties, for example, appearing as a single band or specific bands in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel) electrophoresis. The supernatant is first concentrated. The concentrated supernatant can be further purified by gel chromatography or ion exchange chromatography, such as anion exchange chromatography or cation exchange chromatography. The gel matrix can be a commonly used protein purification matrix, such as agarose, dextran, polyamide, etc.

[0140] Finally, the purified product can be further purified by methods such as hydroxyapatite adsorption chromatography, metal chelation chromatography, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. All of the above purification steps can be combined in various ways to ultimately obtain a protein with essentially uniform purity.

[0141] The expressed Fc fragment peptide can be purified using an affinity chromatography column containing an antibody, receptor, or ligand specifically targeting the Fc fragment peptide. Depending on the characteristics of the affinity chromatography column used, the Fc fragment peptide bound to the column can be eluted using conventional methods (e.g., high-salt buffer, pH alteration, etc.).

[0142] In one embodiment of the invention, the chromatographic purification of Fc fragment peptides can be optimized by using auxiliary domains (e.g., His tags fused to the N-terminus or C-terminus of the peptide) (KOSOBOKOVA EN et al., Auxiliary domains in recombinant proteins (a review), Biochemistry, 2016, Vol. 81, No. 3, pp. 299-314).

[0143] In one embodiment of the invention, the auxiliary domain may be a SUMO peptide (see, for example, BUTTT.R. et al., SUMO fusion technology for difficult-to-express proteins, Protein expression and purification, 2005, Vol. 43, No. 1, pp. 1-9).

[0144] The present invention also relates to constructs comprising the disclosed Fc fragment polypeptide, wherein the Fc fragment polypeptide is linked to a therapeutically active molecule (including a polypeptide).

[0145] According to the present invention, in an Fc fragment polypeptide conjugated to a therapeutically active molecule, the Fc fragment polypeptide can be genetically fused to a physiologically active polypeptide or chemically conjugated to a physiologically active polypeptide. Genetic fusion of the Fc region polypeptide with the physiologically active polypeptide can be designed as a direct fusion of the Fc region polypeptide sequence to the polypeptide or an indirect fusion via a linker sequence. The composition and length of the linker can be determined using methods known in the art, and its effectiveness can be evaluated. Specific linkers are described herein. If desired, other sequences, such as endopeptidase recognition sequences, can also be included at the cleavage site to separate the individual fusion components. Furthermore, the fusion polypeptides of the present invention can be chemically synthesized using polypeptide synthesis methods known in the art (e.g., Merrifield solid-phase synthesis).

[0146] The Fc fragment peptides of this invention can be directly fused to physiologically active peptides, or fused via a linker comprising one or more amino acids (typically about 2-20 amino acids) to form fusion constructs. Linker peptides are known in the art and are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S). n (SG4) n (G4S) n Or G4 (SG4) n Where "n" generally represents an integer from 1 to 10, typically 2 to 4. In one embodiment of the invention, the adaptor peptide consists of at least 5 amino acids; in another embodiment, it consists of 5-100 amino acids; in yet another embodiment, it consists of 10-50 amino acids. In a specific embodiment of the invention, the adaptor peptide consists of 15 amino acids. In one embodiment of the invention, the adaptor peptide is (GxS). n Or (GxS) n G m Wherein G represents glycine, S represents serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3), in one embodiment of the invention, x=4 and n=2 or 3, and in another embodiment of the invention, x=4 and n=3. In a specific embodiment of the invention, the adaptor peptide is (G4S)3 (SEQ ID NO:12).

[0147] In one embodiment of the invention, the linker peptide has an amino acid sequence SEQ ID NO:12 (or is composed of therewith).

[0148] In one embodiment of the invention, the linker peptide has an amino acid sequence SEQ ID NO:13 (or is composed of therewith).

[0149] In one embodiment of the invention, the linker peptide has an amino acid sequence SEQ ID NO:14 (or is composed of therewith).

[0150] In one embodiment, the fusion polypeptide containing the Fc fragment polypeptide may also include a leader peptide at the N-terminus.

[0151] In one embodiment, the leader peptide is the SUMO peptide of SEQ ID NO:26.

[0152] Physiologically active peptides can be selected from: glucagon-like peptide-1 (GLP-1), granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, gastrin, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor, interleukin, interleukin receptor, enzyme, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell kinase, T cell kinase, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, α-1 antitrypsin, albumin, α-lactalbumin, apolipoprotein E, highly glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, coagulation regulatory protein, blood factors VII, VIIa, VIII, IX, and XIII Plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone growth stimulating protein, calcitonin, atrial peptide, chondroitin, calcitonin-dependent calcitonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, growth mediator, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing hormone, thyroid-stimulating hormone, autolysin, lactoferrin, myostatin, cell surface antigens, viral vaccine antigens, monoclonal antibodies, polyclonal antibodies and antibody fragments. Preferably, the physiologically active polypeptide may be selected from: glucagon-like peptide-1 (GLP-1), granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, gastrin-regulatory factor, insulin, insulin analogs and their derivatives, but is not limited to the above-mentioned polypeptides.

[0153] For the purposes of this invention, the term "insulin" refers to the human insulin hormone. Human insulin consists of two polypeptide chains: chain A and chain B. Chain A is a peptide of 21 amino acids, and chain B is a peptide of 30 amino acids. The two chains are connected by disulfide bonds: the first pair of disulfide bonds is formed between cysteine ​​residues at position 7 of chain A and position 7 of chain B; the second pair of disulfide bonds is located between cysteine ​​residues at position 20 of chain A and position 19 of chain B; and the third pair of disulfide bonds is located between cysteine ​​residues at positions 6 and 11 of chain A.

[0154] In the human body, this hormone is synthesized as a single-chain precursor to proinsulin, which consists of a 24-amino acid prepeptide followed by an 86-amino acid proinsulin, with the following conformation: prepeptide-B-Arg-Arg-C-Lys-Arg-A, where C is a 31-amino acid linker peptide. Arg-Arg and Lys-Arg are the sites used to cleave the linker peptide from the A and B chains, respectively.

[0155] For the purposes of this invention, the term "insulin analog" refers to modified insulin in which one or more amino acid residues of insulin are replaced by other amino acid residues, and / or one or more amino acid residues are removed from insulin, and / or one or more amino acid residues are added to and / or inserted into insulin. Preferred insulin analogs of this invention include, but are not limited to, insulin aspart (i.e., human insulin AspB28), insulin lispro (i.e., human insulin LysB28, ProB29), and insulin glutalis (LysB3, GluB29 human insulin), etc.

[0156] Furthermore, the term "physiologically active polypeptide" as used in this article includes not only naturally occurring physiologically active polypeptides, but also agonists, precursors, derivatives, fragments, or variants of each polypeptide.

[0157] The Fc fragment peptide of the present invention can be chemically conjugated to other physiologically active molecules (e.g., physiologically active peptides) using known chemical conjugation techniques. For this purpose, bifunctional cross-linking agents can be used, such as homofunctional or heterofunctional cross-linking agents known in the art. The type of cross-linking agent used depends on the characteristics of the molecule to be conjugated to the Fc fragment peptide and can be readily determined by those skilled in the art. In alternative or additional embodiments, the Fc region peptide and / or the molecule intended to be conjugated thereto can be chemically derivatized, thereby enabling conjugation by a separate reaction, as is also known in the art.

[0158] The Fc fragment polypeptide of the present invention can be chemically conjugated with other physiologically active molecules (e.g., physiologically active polypeptides) through non-peptide linkers.

[0159] The non-peptide linker used in this invention can be selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharides, dextran, polyethylene ether, biodegradable polymers (e.g., polylactic acid (PLA) and polylactic acid-glycolic acid copolymer (PLGA)), lipid polymers, chitin, hyaluronic acid and its derivatives and / or combinations thereof. Preferably, the linker is polyethylene glycol.

[0160] Furthermore, according to the present invention, the non-peptide linker connected to the Fc fragment polypeptide can be composed not only of a single polymer, but also of a combination of different types of polymers.

[0161] The non-peptide linker used in this invention has a reactive group capable of binding to Fc fragment peptides and protein drugs.

[0162] The reactive groups at both ends of the non-peptide polymer are preferably selected from reactive aldehyde groups, propionaldehyde groups, butyraldehyde groups, maleimide groups, and succinimidide derivatives. The succinimidide derivatives can be succinimidide propionate, hydroxysuccinimidide, succinimidide carboxymethyl ester, or succinimidide carbonate. In particular, if the non-peptide polymer has reactive aldehyde groups at both ends, physiologically active peptides and Fc fragment peptides can be effectively bound to both ends of the non-peptide linker, while minimizing nonspecific reactions. The final product formed by aldehyde reductive alkylation is significantly more stable than the product linked by amide bonds. The aldehyde reactive group can selectively bind to the N-terminus at low pH values ​​and can form covalent bonds with lysine residues at high pH values ​​(e.g., pH 9.0).

[0163] The reactive groups at both ends of the non-peptide linker can be the same or different. For example, one end of the non-peptide linker can have a maleimide group, and the other end can have an aldehyde, propionaldehyde, or butyraldehyde group. If polyethylene glycol with hydroxyl reactive groups at both ends is used as the non-peptide linker, the hydroxyl groups can be activated into a variety of reactive groups through known chemical reactions. Alternatively, commercially available polyethylene glycol with modified reactive groups can also be used to generate the conjugates of the present invention.

[0164] The present invention also relates to a method for producing a fusion construct based on the Fc fragment polypeptide described herein, wherein the method comprises culturing the host cells described herein to produce a fusion construct based on the Fc fragment polypeptide, the Fc fragment polypeptide being fused with a physiologically active polypeptide as described above.

[0165] The present invention also relates to a method for producing conjugates based on the Fc fragment polypeptide described herein, wherein the method comprises conjugating the Fc fragment polypeptide with a physiologically active polypeptide as described above.

[0166] The present invention also relates to the use of the Fc fragment of the present invention in the production of pharmaceuticals.

[0167] The present invention also relates to the use of the Fc fragment of the present invention for altering the half-life of physiologically active peptides.

[0168] The present invention also relates to the use of fusion constructs of Fc fragment peptides fused with physiologically active peptides as described herein for the production of medicaments comprising the construct as an active ingredient.

[0169] The present invention also relates to the use of conjugates of Fc fragment peptides conjugated with physiologically active peptides, as described herein, for the production of medicaments comprising the conjugates as active ingredients.

[0170] In one aspect of the invention, the Fc fragment polypeptide has insulin or an agonist, precursor, derivative, fragment or variant thereof as a fusion or conjugate fusion construct or conjugate fusion ...

[0171] In one aspect of the invention, the Fc fragment polypeptide is intended for use in a method of producing a medicament, wherein the use is as a fusion or conjugated partner of insulin or an agonist, precursor, derivative, fragment or variant thereof, and wherein the medicament is intended for treating insulin-sensitive conditions such as type 1 diabetes, type 2 diabetes and hyperglycemia.

[0172] In one aspect of the invention, the Fc fragment polypeptide is intended for use in pharmaceutical formulations as a fusion or conjugated partner of insulin or an agonist, precursor, derivative, fragment, or variant thereof, and wherein the pharmaceutical preparation is intended to treat insulin-sensitive conditions such as type 1 diabetes, type 2 diabetes, and hyperglycemia. Brief description of the attached diagram

[0174] Figure 1 General structure of expression plasmids for proteins fused with Fc fragment peptides.

[0175] Implementation of the invention

[0176] The following embodiments illustrate the present invention, but are not limited thereto.

[0177] Example 1. Generation of the construct

[0178] To investigate the effect of specific modifications in the amino acid sequence of the Fc fragment polypeptide on its binding affinity to FcRn and FcγR, the following structures were generated: GP20091.01-L1-01 (SEQ ID NO:16), GP20091.01-L2-05 (SEQ ID NO:17), GP20091.01-L2-06 (SEQ ID NO:18), GP20091.01-L2-07 (SEQ ID NO:19), GP20091.01-L2-08 (SEQ ID NO:20), GP20091.01-L2-09 (SEQ ID NO:21), GP20091.01-L2-10 (SEQ ID NO:22), GP20091.01-L2-12 (SEQ ID NO:23), GP20091.01-L2-13 (SEQ ID NO:24). ID NO:24), GP20091.01-L2-14 (SEQ ID NO:25), wherein the Fc fragment polypeptide variant is fused to the C-terminus of the single-chain insulin of SEQ ID NO:17 via the polypeptide linker of SEQ ID NO:15 or SEQ ID NO:16.

[0179] To obtain the constructs, *E. coli* BL21 cells were transfected with suitable plasmids capable of expressing constructs GP20091.01-L1-01, GP20091.01-L2-05, GP20091.01-L2-06, GP20091.01-L2-07, GP20091.01-L2-08, GP20091.01-L2-09, GP20091.01-L2-10, GP20091.01-L2-12, GP20091.01-L2-13, and GP20091.01-L2-14 fused with the SUMO leader peptide, as well as expressing a ULP protease capable of selectively hydrolyzing the SUMO leader peptide. The coding sequences of the proteins studied are shown in SEQ ID NO.:27-35.

[0180] The general structure of a plasmid used to generate the target protein is as follows: Figure 1 As shown.

[0181] Each plasmid contains: 1. Genes that confer tetracycline resistance to bacterial cells, replication origin (ori) genes, and ROP genes that regulate plasmid copy number; 2. The tac / T7 transcription promoter for the expression of recombinant proteins at the initiation site; 3. The gene sequences encoding the Met-SUMO-GP20091.01-L2-XX molecule and the gene sequence encoding the ULP protease are both controlled by the Tac-T7 promoter and form an operon; 4.rrnB1 B2 T1 txn transcription terminator; 5. The LacI gene, which encodes the Tac promoter repressor protein; 6. EcoRI and SpeI are restriction sites for gene insertion; 7. Pr1082 and Pr166 sequencing primers.

[0182] Cell transfection was performed using electroporation.

[0183] Following the standard protocol (20 min, 8000 rpm), cell biomass was separated by centrifugation using an Avanti J-HC floor-standing centrifuge and a JLA-8.1000 rotor. The cell precipitate was placed in labeled polyethylene resealable bags and stored in a refrigerator at a temperature not exceeding -18°C.

[0184] To disrupt the bacterial cell walls, the biomass was subjected to two consecutive disruptions using a high-pressure homogenizer. Prior to cell disruption, the required amount of biomass needed to be resuspended. For this purpose, the cell pellet was thawed at room temperature and then resuspended in disruption buffer (20 mM Tris; 5 mM EDTA, pH 8.0) cooled to 4°C using a Polytron 3100D immersion homogenizer; homogenized for 10 minutes until the solution appeared visually homogeneous.

[0185] Inclusion bodies were separated by centrifugation using an Avanti J-HC floor-standing centrifuge and a JLA-8.1000 rotor, according to the standard procedure (20 min, 8000 rpm).

[0186] To dissolve the constructs, place the inclusion body subsamples into a container of appropriate volume, add the required amount of dissolution buffer (7 M guanidine hydrochloride, 100 mM Tris, 15 mM DTT, pH 9.5), and stir until the protein aggregates are completely dissolved.

[0187] To generate the fusion protein under study in dimer form, we used a buffer solution (2 M urea, 300 mM arginine, 50 mM Tris, pH 8.25) with 0.55 mM L-cysteine ​​as a reducing agent and 0.55 mM L-cystamine as an oxidizing agent. The refolding process was carried out at 2–8 °C for 16 hours with a stirring rate of 200 rpm.

[0188] Purification of the obtained protein construct

[0189] The resulting protein constructs were purified by chromatography, using Protein A affinity chromatography, hydrophobic interaction chromatography (using Butyl Sepharose™ 4 Fast Flow adsorbent), and anion exchange chromatography (using QSepharose 4FF adsorbent) in sequence.

[0190] Example 2. FcRn combination

[0191] The binding of the Fc fragment peptide to FcRn was determined using label-free biolayer interferometry. Following the manufacturer's recommendations, the binding kinetics of the prepared biotinylated neonatal Fc receptor (Immunitrack) were evaluated using an Octet Red96 microplate interferometer (ForteBio) and a sensor kit (streptavidin(sa) biosensor, Sartorius). Rituximab from Mabxience was used as a reference. All measurements were performed at pH 6.0. Binding curves after reference values ​​were analyzed using a 1:1 interaction model with ForteBio Acquisition Manager software according to standard procedures. The results are shown in Table 1, where R^2 is the coefficient of determination.

[0192] Table 1. FcRn binding results

[0193] The results showed that the obtained Fc fragment peptide variants had similar or improved binding affinity to human FcRn compared to the wild-type Fc fragment. Similar results were obtained at pH 7.4.

[0194] Example 3. FcγRI binding

[0195] The binding kinetics of biotinylated FcγRI (CD64 / FCGR1A) were evaluated using label-free biolayer interferometry on a 96-well Octet Red96 microplate interferometer (Pall ForteBio). Rituximab solution (MB01, Mabxience) was used as a positive control.

[0196] This assay used a streptavidin sensor (Sartorius, catalog number 18–5019) hydrated in 0.05% PBST solution for 10 min. The biotinylated FcγRI receptor concentration was 2.5 μg / mL. The studied samples were diluted with 0.05% PBST solution at pH 7.4. For the rituximab positive control and the GP20091.01-L2 test sample, results were measured at equimolar concentrations of 1.75 nmol / mL, 0.44 nmol / mL, 0.11 nmol / mL, 0.03 nmol / mL, and 0.007 nmol / mL. A 1:1 interaction model was used for data processing. The results are shown in Table 2, where R^2 is the coefficient of determination.

[0197] Table 2. FcγRI binding results

[0198] FcγRII binding

[0199] The binding kinetics of biotinylated FcγRII (CD32a / FCGR2A) were evaluated using label-free biolayer interferometry on a 96-well Octet Red96 microplate interferometer (Pall ForteBio). Rituximab solution (MB01, Mabxience) was used as a positive control.

[0200] This assay used a streptavidin sensor (Sartorius, catalog number 18–5019) hydrated in 0.05% PBST solution for 10 min. The biotinylated FcγRII receptor concentration was 2.5 µg / ml. The studied samples were diluted with 0.05% PBST solution at pH 7.4. For the rituximab positive control and the GP20091.01-L2 test sample, results were measured at equimolar concentrations of 1.75 nmol / ml, 0.44 nmol / ml, 0.11 nmol / ml, 0.03 nmol / ml, and 0.007 nmol / ml. Binding curves after reference subtraction were analyzed using a 1:1 interaction model with ForteBio Acquisition Manager software according to standard procedures. The results are shown in Table 3, where R^2 is the coefficient of determination.

[0201] Table 3. FcγRII binding results

[0202] FcγRIII binding

[0203] The binding kinetics of biotinylated FcγRIII (CD16a / FCGR3A) were evaluated using label-free biolayer interferometry on a 96-well plate-compatible Octet Red96 interferometer (Pall ForteBio). Rituximab solution (MB01, Mabxience) was used as a positive control.

[0204] This assay utilized a streptavidin sensor (Sartorius, catalog number 18–5019) hydrated in 0.05% PBST solution for 10 min. The biotinylated FcγRIII receptor concentration was 2.5 μg / mL. Test samples were diluted with 0.05% PBST solution at pH 7.4. For the rituximab positive control, the following concentrations were evaluated: 1.2 nmol / mL, 0.3 nmol / mL, 0.08 nmol / mL, 0.02 nmol / mL, and 0.005 nmol / mL. GP20091.01-L2 test samples were measured at equimolar concentrations of 25 nmol / mL, 8.3 nmol / mL, 2.8 nmol / mL, 0.93 nmol / mL, and 0.309 nmol / mL. Binding curves, after reference subtraction, were analyzed using a 1:1 interaction model with ForteBio Acquisition Manager software according to standard procedures. The results are shown in Table 4, where R^2 is the coefficient of determination.

[0205] Table 4. FcγRIII binding results

[0206] Experimental results showed that, compared with the wild-type Fc fragment, the resulting variant Fc fragment peptides bound significantly less or not at all to various human Fcγ receptors.

[0207] Example 4.

[0208] To confirm whether the bioactivity of the fusion partner was preserved, glucose uptake induced by the resulting fusion protein was evaluated.

[0209] Glucose uptake was studied using the L6J1 rat myoblast cell line (Institute of Cell Biology, Russian Academy of Sciences). Working cell suspensions were seeded at a density of 8000 cells / well in 96-well plates in DMEM + 2% horse serum (HS). Cells dispersed in DMEM + 10% FBS served as controls. After 72 hours, the experimental wells were replaced with DMEM + 2% HS, and the control wells with DMEM + 10%. On day 7, DMEM (4.5 g / L glucose, phenol red-free, serum-free) was added to all wells, and the cells were starved for 24 hours.

[0210] Test samples were added to wells at concentrations of 2 nM, 20 nM, 100 nM, 200 nM, 500 nM, 800 nM, 1600 nM, 3200 nM, and 5000 nM. Rinsulin R was added as a control at concentrations of 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 400 nM, 800 nM, 1600 nM, and 2500 nM. Cells were then incubated with the test samples and controls for 72 hours.

[0211] Glucose concentration was measured using the GOD-PAP glucose kit by adding culture medium to the GOD-PAP working glucose solution. Detection was performed on a CLARIOstar multi-functional microplate reader. Results are shown in Table 5.

[0212] Table 5. Measurement of induced glucose uptake

[0213] All the fusion proteins studied resulted in decreased glucose levels and exhibited biological activity comparable to recombinant human insulin.

Claims

1. An immunoglobulin Fc fragment polypeptide having the amino acid sequence X1X2X3X4X5CPPCPAPX as shown in SEQ ID NO:2 13 X 14 X 15 X 16 X 17 PX 19 VFLFPPKPKDX 30 LX 32 IX 34 RX 36 PEVTCVVX 44 DVSX 48 EDPEVQFNWYVDGVEVX65X 66 AKTKPREEQFX 77 STX 80 RVVSVLTX 88 X 89 HQDWLNGKEYKCKVSNKGLPX 110 X 111 IEKTISKX 119 KGQPREPQVYTLPPSX 135 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPX 177 LDSDGSFFLYSX 189 LTVDKSRWQX 199 GNVFSCSVX 208 HEALHX 214 HYTQKSLSLSX 225 G, where X1 is either E or D. X2 is K or does not exist. X3 is T or does not exist. X4 is H or does not exist. X5 is W or does not exist. X 13 For P or E, X 14 F or not present. X 15 For V or L, X 16 It is either A or G. X 17 For G or M, X 19 For S or E, X 30 For T or Q, X 32 For M, Y, or W, X 34 For S or T, X 36 For T or E, X 44 For V, E, or D, X 48 For H or Q, X 65 For H or E, X 66 For N or E, X 77 For N or D, X 80 For F or Y, X 88 For V or F, X 89 For V or L, X 110 It can be A, F, or S. X 111 For P or S, X 119 For T or A, X 135 For R or Q, X 177 For M or V, X 189 For K or R, X 199 For Q or E, X 208 For M or L, X 214 For M, Y, or W, and X 225 It can be P or L.

2. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let Y and X be the numbers. 34 Let T, X 36 For E, X 44 Let V, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let P be the value.

3. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 For Q, X 32 Let M and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 Let T, X 44 Let V, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let L and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let P be the value.

4. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For D, X 48 For H, X 65 For H, X 66 Let N, X 77 For D, X 80 Let F, X 88 Let F, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let Y be X, and X be X 225 Let P be the value.

5. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 For E, X 14 Let F, X 15 Let L and X be the numbers. 16 For G, X 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let M and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For D, X 48 For Q, X 65 For E, X 66 For E, X 77 For D, X 80 Let Y and X be the numbers. 88 Let V, X 89 Let L and X be the numbers. 110 Let S and X be the values ​​of S and X. 111 Let S and X be the values ​​of S and X. 119 Let A and X be the two numbers. 135 For Q, X 177 Let V, X 189 Let R, X 199 For E, X 208 Let L and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let L be the value.

6. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 For W, X 34 Let S and X be the values ​​of S and X. 36 For E, X 44 Let V, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let L and X be the numbers. 214 Let Y be X, and X be X 225 Let P be the value.

7. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 For E, X 30 Let T, X 32 Let M and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For E, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let F, X 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let Y be X, and X be X 225 Let P be the value.

8. The polypeptide of claim 1, wherein X1 is D, X2 is K, X3 is T, X4 is H, X5 is W, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 Let M and X be the numbers. 19 Let S and X be the values ​​of S and X. 30 For Q, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 Let T, X 44 For D, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let V, X 89 Let V, X 110 Let A and X be the two numbers. 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let N be the number of elements, and X be the number of elements. 225 Let P be the value.

9. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 Let T, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For E, X 48 For H, X 65 For H, X 66 Let N, X 77 For D, X 80 Let F, X 88 Let F, X 89 Let V, X 110 Let F, X 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let W be the integer part of the integer part, and X be the integer part of the integer part. 225 Let P be the value.

10. The polypeptide of claim 1, wherein X1 is E, X2 is absent, X3 is absent, X4 is absent, X5 is absent, and X... 13 Let P, X 14 Does not exist, X 15 Let V, X 16 Let A and X be the two numbers. 17 For G, X 19 Let S and X be the values ​​of S and X. 30 For Q, X 32 Let Y and X be the numbers. 34 Let S and X be the values ​​of S and X. 36 For E, X 44 For E, X 48 For H, X 65 For H, X 66 Let N, X 77 Let N, X 80 Let F, X 88 Let F, X 89 Let V, X 110 Let F, X 111 Let P, X 119 Let T, X 135 Let R, X 177 Let M and X be the numbers. 189 Let K, X 199 For Q, X 208 Let M and X be the numbers. 214 Let W be the integer part of the integer part, and X be the integer part of the integer part. 225 Let P be the value.

11. An immunoglobulin Fc fragment polypeptide having an amino acid sequence selected from SEQ ID NO:3 to 11.

12. A nucleic acid encoding a polypeptide according to any one of claims 1-11.

13. An expression cassette comprising the nucleic acid according to claim 12.

14. A vector comprising the nucleic acid of claim 12 or the expression cassette of claim 13.

15. A host cell for expressing a polypeptide according to any one of claims 1 to 11, said cell comprising the nucleic acid of claim 12, the expression cassette of claim 13, or the vector of claim 14.

16. The host cell of claim 15, wherein the cell is Escherichia coli.

17. A method for producing a polypeptide according to any one of claims 1-11, wherein the method comprises culturing the cell of claim 15 under conditions capable of expressing the polypeptide of any one of claims 1-11.

18. Use of the polypeptide according to any one of claims 1-11 in the production of a pharmaceutical product.

19. The use as described in claim 18, wherein the drug is a polypeptide of any one of claims 1-11, conjugated to a physiologically active molecule.

20. The use as described in claim 18, wherein the drug is a polypeptide of any one of claims 1-11, fused with a physiologically active polypeptide.

21. The use as claimed in claim 20, wherein the drug comprises a peptide linker through which the polypeptide of any one of claims 1-11 is fused to a physiologically active polypeptide.

22. A method for producing a pharmaceutical product, wherein the method comprises producing the polypeptide according to any one of claims 1-11 by the method of claim 17.

23. The method of claim 22, wherein the drug is a polypeptide of any one of claims 1-11, which is conjugated to a physiologically active molecule.

24. The method of claim 22, wherein the drug is a polypeptide of any one of claims 1-13, fused with a physiologically active polypeptide.

25. The method of claim 24, wherein the drug comprises a peptide linker through which the polypeptide of any one of claims 1-13 is fused to a physiologically active polypeptide.

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