Fusion proteins containing erythropoietin polypeptides

Abstract

The present invention relates to a fusion protein including an erythropoietin polypeptide fused to the Fc region of IgG derived from a mammal (elsewhere also referred to as an EPO-Fc fusion protein). The erythropoietin polypeptide has an amino acid sequence represented by SEQ ID NO: 9 or 10. The Fc region has an amino acid sequence represented by SEQ ID NO: 3 or 4. The fusion protein has superior physical properties and activity and can be acquired at a high yield rate / high yield.

Description

[Technical Field]

[0001] The present invention relates to fusion proteins comprising an erythropoietin polypeptide fused to the Fc region of IgG of canine or feline origin. [Background technology]

[0002] Erythropoietin (EPO), a glycoprotein with a molecular weight of 30,000–34,000, is a factor that promotes the production and differentiation of red blood cells. It is produced in the kidney and is essential for regulating the level of red blood cells in the circulation. This protein acts by binding to a receptor on red blood cell precursor cells, increasing intracellular calcium ion concentrations, increasing DNA biosynthesis, and stimulating hemoglobin production. Erythropoietin can be used to treat hematopoietic disorders or hematopoietic failure, but it has the drawback of having a short half-life in the blood.

[0003] On the other hand, because it is known that fusing an immunoglobulin constant region to a non-immunoglobulin protein significantly extends the half-life of the non-immunoglobulin protein, an approach has been attempted in which an immunoglobulin fragment is linked to an erythropoietin polypeptide. For example, Patent Document 1 describes the production and use of a fusion protein comprising an immunoglobulin Fc region and an erythropoietin polypeptide. In fact, it has been reported that a fusion protein comprising an immunoglobulin Fc region and an erythropoietin polypeptide extended the half-life of the erythropoietin polypeptide in vivo.

[0004] The neonatal Fc receptor (hereinafter referred to as FcRn) binds to the Fc region of IgG, recycling it into plasma and preventing IgG from being degraded in lysosomes. IgG binds to FcRn, resulting in a long plasma retention time. Binding of IgG to FcRn is only observed under acidic conditions (e.g., pH 6.0), and binding is barely observed under neutral conditions (e.g., pH 7.4). Normally, IgG is nonspecifically taken up by cells via endocytosis, but returns to the cell surface by binding to FcRn in endosomes under acidic conditions. Under neutral plasma conditions, IgG dissociates from FcRn and is recycled. As a result, IgG has a longer plasma retention time than other plasma proteins. IgG that does not bind to FcRn in endosomes proceeds to lysosomes, where it is degraded. A method for improving the plasma retention of IgG in humans has been reported in which the binding ability to FcRn under acidic conditions is increased. By introducing amino acid substitutions into the Fc region of IgG to increase the binding ability to FcRn under acidic conditions, the recycling efficiency from endosomes to plasma is increased, resulting in improved plasma retention (Patent Documents 2 and 3, Non-Patent Documents 1 to 3). In addition, cytokines and soluble membrane receptors fused with the Fc region of IgG (Fc fusion proteins) have been developed as human therapeutic drugs, and like IgG, these achieve long plasma retention through binding to FcRn. As mentioned above, biopharmaceuticals that modify and apply the binding between the Fc region of IgG and FcRn have been developed in humans, and there is a need to develop biopharmaceuticals with similarly improved plasma retention in non-human animals such as dogs and cats. However, no amino acid modifications in the Fc region that improve or enhance the plasma retention of antibodies in dogs and cats are known. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Publication WO99 / 02709 [Patent Document 2] WO2002 / 060919 publication [Patent Document 3] WO2012 / 083370 publication [Non-patent literature]

[0006] [Non-Patent Document 1] Yeung YA et al., J. Immunol. (2009) 182, 7663-71. [Non-patent document 2] Datta-Mannan A et al., J. Biol. Chem. (2007) 282, 1709-17. [Non-patent document 3] Dall'Acqua WF et al., J. Immunol. (2002) 169, 5171-80. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to obtain, in high yield / high quantity, a fusion protein containing an erythropoietin polypeptide fused to the Fc region of IgG (hereinafter also referred to as EPO-Fc fusion protein), which has superior physical properties, activity, and plasma retention. [Means for solving the problem]

[0008]

[0003] To achieve the above object, the present inventors investigated the effects of various amino acid modifications on the EPO portion, Fc region, and, if a hinge region is present between the EPO portion and Fc region, the hinge region of an EPO-Fc fusion protein. As a result, they found that specific mutations in the EPO region and hinge region can produce EPO-Fc fusion proteins with improved physical properties, activity, and yield. Furthermore, they discovered that the use of an Fc region with amino acid modifications at specific positions (so-called Fc region variants) that enhances FcRn binding ability under acidic conditions and improves plasma retention can produce EPO-Fc fusion proteins with improved physical properties, activity, yield, and plasma retention, leading to the completion of the present invention.

[0009] That is, the present invention is as follows. [1] A fusion protein comprising an erythropoietin polypeptide fused to the Fc region of IgG derived from a dog or a cat. [2] The fusion protein according to [1] above, wherein the fusion of the erythropoietin polypeptide to the Fc region is via the hinge region of IgG. [3] The fusion protein according to [1] or [2] above, wherein the erythropoietin polypeptide has at least one mutation, which is a substitution of at least one cysteine ​​in its wild-type sequence with arginine or proline. [4] The fusion protein according to [3] above, wherein the erythropoietin polypeptide having at least one mutation is derived from a feline and has the amino acid sequence represented by SEQ ID NO: 9 or 10. [5] The fusion protein according to any one of [2] to [4] above, wherein the hinge region has at least one mutation, and the mutation is a substitution of at least one cysteine ​​in the wild-type sequence with glycine. [6] The fusion protein according to [5] above, wherein the hinge region having at least one mutation is derived from a feline and has an amino acid sequence represented by any one of SEQ ID NOs: 5 to 8. [7] The fusion protein according to any one of [1] to [6] above, which has an amino acid modification in the Fc region. [8] The fusion protein according to [7] above, wherein the Fc region is derived from canine IgG. [9] The fusion protein according to [7] above, wherein the Fc region is derived from feline IgG.

[10] The amino acid modification of the Fc region is (i) substitution of leucine at position 252 with tyrosine or threonine; (ii) substitution of alanine at position 254 with threonine; (iii) substitution of threonine at position 256 with glutamic acid; (iv) substitution of isoleucine at position 308 with proline; (v) substitution of methionine at position 428 with leucine; (vi) substitution of histidine at position 433 with leucine; (vii) substitution of asparagine at position 434 with alanine, serine, tyrosine, or phenylalanine; (viii) substitution of tyrosine at position 436 with threonine; (ix) substitution of glutamine at position 438 with arginine, and (x) Substitution of serine at position 440 with glutamic acid The fusion protein according to [8] above, comprising at least one selected from the group consisting of: (wherein the amino acid numbering in the Fc region is according to the EU index of Kabat based on the Fc region of a human antibody).

[0010]

[11] The amino acid modification of the Fc region is (i) substitution of asparagine at position 434 with alanine; (ii) substitution of tyrosine at position 436 with threonine; (iii) substitution of glutamine at position 438 with arginine, and (iv) Substitution of serine at position 440 with glutamic acid The fusion protein according to

[10] above,

[12] The amino acid modification of the Fc region is (i) substitution of methionine at position 428 with leucine; (ii) substitution of asparagine at position 434 with alanine; (iii) substitution of tyrosine at position 436 with threonine; (iv) substitution of glutamine at position 438 with arginine, and (v) Substitution of serine at position 440 with glutamic acid The fusion protein according to

[10] above,

[13] The amino acid modification of the Fc region is (i) substitution of methionine at position 428 with leucine; (ii) substitution of asparagine at position 434 with alanine; (iii) substitution of glutamine at position 438 with arginine, and (iv) Substitution of serine at position 440 with glutamic acid The fusion protein according to

[10] above,

[14] The amino acid modification of the Fc region is (i) substitution of leucine at position 252 with tyrosine; (ii) substitution of alanine at position 254 with threonine, and (iii) Substitution of threonine at position 256 with glutamic acid The fusion protein according to

[10] above,

[15] The amino acid modification of the Fc region is (i) substitution of methionine at position 428 with leucine, and (ii) substitution of asparagine at position 434 with serine The fusion protein according to

[10] above,

[16] The amino acid modification of the Fc region is (i) substitution of isoleucine at position 308 with proline, and (ii) substitution of asparagine at position 434 with tyrosine The fusion protein according to

[10] above,

[17] The amino acid modification of the Fc region is (i) substitution of leucine at position 252 with threonine; (ii) substitution of alanine at position 254 with threonine; (iii) substitution of threonine at position 256 with glutamic acid; (iv) substitution of histidine at position 433 with leucine, and (v) Substitution of asparagine at position 434 with phenylalanine The fusion protein according to

[10] above,

[18] The amino acid modification of the Fc region is (i) substitution of serine at position 252 with tyrosine or threonine, (ii) substitution of serine at position 254 with threonine; (iii) substitution of threonine at position 256 with glutamic acid; (iv) substitution of valine at position 259 with isoleucine; (v) substitution of isoleucine at position 308 with proline or phenylalanine; (vi) substitution of serine at position 428 with leucine; (vii) substitution of histidine at position 433 with leucine; (viii) substitution of serine at position 434 with alanine, tyrosine, or phenylalanine, (ix) substitution of histidine at position 436 with threonine; (x) substitution of glutamine at position 438 with arginine; (xi) substitution of serine at position 440 with glutamic acid; (xii) substitution of leucine at position 235 with arginine; (xiii) substitution of glycine at position 236 with arginine, and (xiv) Substitution of serine at position 239 with lysine The fusion protein according to [9] above, comprising at least one selected from the group consisting of: (wherein the amino acid numbering in the Fc region is according to the EU index of Kabat based on the Fc region of a human antibody).

[19] The amino acid modification of the Fc region is (i) substitution of serine at position 434 with alanine; (ii) substitution of histidine at position 436 with threonine; (iii) substitution of glutamine at position 438 with arginine, and (iv) Substitution of serine at position 440 with glutamic acid The fusion protein according to

[18] above,

[20] The amino acid modification of the Fc region is (i) substitution of serine at position 428 with leucine; (ii) substitution of serine at position 434 with alanine; (iii) substitution of histidine at position 436 with threonine; (iv) substitution of glutamine at position 438 with arginine, and (v) Substitution of serine at position 440 with glutamic acid The fusion protein according to

[18] above,

[0011]

[21] The amino acid modification of the Fc region is (i) substitution of serine at position 428 with leucine; (ii) substitution of serine at position 434 with alanine; (iii) substitution of glutamine at position 438 with arginine, and (iv) Substitution of serine at position 440 with glutamic acid The fusion protein according to

[18] above,

[22] The amino acid modification of the Fc region is (i) substitution of serine at position 252 with tyrosine; (ii) substitution of serine at position 254 with threonine, and (iii) Substitution of threonine at position 256 with glutamic acid The fusion protein according to

[18] above,

[23] The amino acid modification of the Fc region is (i) substitution of isoleucine at position 308 with proline, and (ii) substitution of serine at position 434 with tyrosine The fusion protein according to

[18] above,

[24] The amino acid modification of the Fc region is (i) substitution of valine at position 259 with isoleucine; (ii) substitution of isoleucine at position 308 with phenylalanine, and (iii) substitution of serine at position 428 with leucine The fusion protein according to

[18] above,

[25] The amino acid modification of the Fc region is (i) substitution of serine at position 252 with threonine; (ii) substitution of serine at position 254 with threonine; (iii) substitution of threonine at position 256 with glutamic acid; (iv) substitution of histidine at position 433 with leucine, and (v) Substitution of serine at position 434 with phenylalanine The fusion protein according to

[18] above,

[26] The amino acid modification of the Fc region is (vi) substitution of serine at position 428 with leucine; (viii) substitution of serine at position 434 with alanine; (x) substitution of glutamine at position 438 with arginine; (xi) substitution of serine at position 440 with glutamic acid; (xii) substitution of leucine at position 235 with arginine; (xiii) substitution of glycine at position 236 with arginine, and (xiv) Substitution of serine at position 239 with lysine The fusion protein according to

[18] above,

[27] The fusion protein according to [7] above, wherein the Fc region of the IgG having the amino acid alteration is derived from a cat and has the amino acid sequence shown in SEQ ID NO: 3 or 4.

[28] A pharmaceutical composition comprising the fusion protein according to any one of [1] to

[27] above.

[29] The pharmaceutical composition according to

[28] above, which is for treating a hematopoietic disease or hematopoietic failure. [Effects of the Invention]

[0012] The EPO-Fc fusion protein of the present invention has excellent physical properties and activity, and can be obtained in high yield / high amount according to the present invention. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a plasmid map showing the structure of pCAG. [Figure 2]FIG. 2 shows the results of Western blot analysis of feline EPO-Fc fusion proteins with modified EPO and hinge regions. [Figure 3] FIG. 3 shows the presence or absence of intermolecular disulfide bonds in feline EPO-Fc, as determined by SDS-PAGE under reducing and non-reducing conditions and CBB staining. [Figure 4A] 4A shows SEC analysis chromatograms of the EPO region- and hinge region-modified feline EPO-Fc fusion proteins (Table 8, items 1 to 3). The right panel is an enlarged version of the left panel. [Figure 4B] 4B shows SEC analysis chromatograms of the EPO region- and hinge region-modified feline EPO-Fc fusion proteins (Table 8, items 4 to 6). The right panel is an enlarged version of the left panel. [Figure 4C] 4C shows SEC analysis chromatograms of the EPO region- and hinge region-modified feline EPO-Fc fusion proteins (Nos. 7 to 9 in Table 8). The right panel is an enlarged version of the left panel. [Figure 4D] 4D shows SEC analysis chromatograms of the EPO region- and hinge region-modified feline EPO-Fc fusion proteins (10 to 12 in Table 8). The right panel is an enlarged version of the left panel. [Figure 4E] 4E shows SEC analysis chromatograms of the EPO region- and hinge region-modified feline EPO-Fc fusion proteins (13 to 15 in Table 8). The right panel is an enlarged version of the left panel. [Figure 4F] Figure 4F shows a merged chromatogram of the results of SEC analysis of the feline EPO-Fc fusion proteins wt catEPO-C2-Fc, C59P catEPO-C2-Fc, and C165R catEPO-C2-Fc (Table 8, items 1 to 3). [Figure 5] FIG. 5 is a graph showing the results of evaluating the biological activity of feline EPO-Fc fusion protein using the proliferation of TF-1 cells as an indicator. [Figure 6A] FIG. 6A shows the results of SDS-PAGE analysis of purified feline EPO-Fc fusion protein. [Figure 6B] Figure 6B shows the results of SEC analysis of the purified feline EPO-Fc fusion protein (lower panel). The SEC analysis chromatogram (upper panel) was quantified. [Figure 7] FIG. 7 is a graph showing the results of evaluating the in vitro activity of Fc-modified feline EPO-Fc fusion protein using TF-1 cell proliferation as an indicator. [Figure 8] FIG. 8 is a graph showing the results of evaluating the in vitro activity of feline EPO-Fc fusion protein using the proliferation of BaF3 / mouseEPOR cells as an indicator. [Figure 9] FIG. 9 is a graph showing the results of evaluating the in vitro activity of feline EPO-Fc fusion protein using the proliferation of BaF3 / catEPOR cells as an indicator. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides a fusion protein comprising an EPO polypeptide linked to the Fc region of mammalian IgG (hereinafter also referred to as an EPO-Fc fusion protein). The EPO-Fc fusion protein of the present invention is described in detail below.

[0015] 1. EPO-Fc fusion protein As used herein, the term "EPO-Fc fusion protein" refers to a protein comprising the Fc region of canine or feline IgG and an erythropoietin polypeptide. In a preferred embodiment of the present invention, the EPO-Fc fusion protein forms a homodimer and therefore typically has one or more disulfide bonds.

[0016] The mode of binding between the Fc region and the EPO polypeptide is not particularly limited, as long as they are functionally linked. In one embodiment, the EPO polypeptide is directly linked to the Fc region via a covalent bond. In another embodiment, the EPO polypeptide is indirectly linked to the Fc region. For example, an EPO-Fc fusion protein can include a linker between the Fc region and the erythropoietin polypeptide.

[0017] 2.Fc area In the present invention, the "Fc region" encompasses domains derived from the constant region of canine or feline IgG, preferably feline IgG, and includes fragments and variants of the constant region. IgG has isoforms, the number of which varies depending on the animal species. Four types, IgG1 to IgG4, are known in humans, mice, and rats. Four IgG immunoglobulins exist in dogs, defined as caIgG-A, caIgG-B, caIgG-C, and caIgG-D (Tang et al., Vet. Immunol. Immunopathol. 80(3-4), 259-270, 2001). Three types of IgG immunoglobulins exist in cats, and these have been reported to exist as IgG1a, IgG1b, and IgG2. 1)Kanai, TH, et al., 2000. Identification of two allelic IgG1 C(H)coding regions (Cgamma1) of cat. Vet. Immunol. Immunopathol. 73(1), 53-62. 2) Strietzel, CJ, et al., 2014. In Vitro functional characterization of feline IgGs, Vet. Immunol. Immunopathol. 158 (3-4), 214-233.

[0018] The constant region of an immunoglobulin is defined as a naturally or synthetically produced polypeptide homologous to the C-terminal region of an immunoglobulin, which may comprise the CH1, hinge, CH2, CH3, or CH4 domains, individually or in any combination. The Fc region of the heavy chain consists of the CH2 and CH3 domains. The hinge region is located between the CH1 and CH2 domains.

[0019] In the present invention, the Fc region of canine or feline IgG may be a variant Fc region in which amino acid alterations have been introduced into the Fc region of wild-type canine or feline IgG, and more preferably an Fc region variant that contains at least one amino acid alteration and whose activity of binding to canine or feline FcRn under acidic conditions (hereinafter also referred to as FcRn-binding activity) is higher than the FcRn-binding activity of the parent peptide before alteration.

[0020] "FcRn" is structurally similar to major histocompatibility complex (MHC) class I polypeptides, and in humans has 22 to 29% sequence identity with class I MHC molecules (human reference: Ghetie et al., Immunol. Today (1997) 18 (12), 592-598). FcRn is expressed as a heterodimer consisting of a soluble β-chain (or light chain), β2-microglobulin (sometimes referred to as β2m), and a transmembrane α-chain (or heavy chain, sometimes referred to as FCGRT). The α-chain of FcRn consists of three extracellular domains (α1, α2, and α3), and the α1 and α2 domains interact with the FcRn-binding domain in the Fc region of antibodies (Raghavan et al., Immunity (1994) 1, 303-315). FcRn forms a complex with β2-microglobulin in vivo. A complex of soluble FcRn and β2-microglobulin can be prepared using a conventional recombinant expression method (see the sections "Construction of FcRn expression vector" and "Expression and purification of FcRn protein" in the Examples), and this complex can be used to evaluate the FcRn-binding activity of the present invention. In the present invention, unless otherwise specified, FcRn is used as a complex with β2-microglobulin.

[0021] The term "parent polypeptide" refers to a polypeptide before an amino acid modification is introduced, as opposed to a polypeptide into which the modification has been introduced. Parent polypeptides containing an Fc region of canine or feline IgG include polypeptides containing the Fc region of natural canine or feline IgG, preferably antibodies, particularly polypeptides that constitute natural canine or feline IgG. Polypeptides having an Fc region in which an amino acid modification has been introduced into the Fc region of a parent polypeptide are also referred to as Fc region variants. Wild-type canine or feline IgG refers to a polypeptide that includes an amino acid sequence identical to a naturally occurring canine or feline IgG and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. Examples of amino acid modifications in the Fc region include amino acid substitution, insertion, deletion, etc., with amino acid substitution being preferred. The number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, or two or more amino acids may be modified. Preferably, two to several amino acids, more preferably about two to five amino acids, are modified.

[0022] For dogs or cats, the following modifications are preferred: In this specification, the alphabet displayed to the left of the number indicating the number of amino acid residues up to the substitution site indicates the single-letter notation of the amino acid before substitution, and the alphabet displayed to the right indicates the single-letter notation of the amino acid after substitution. The number of amino acid residues up to the substitution site is the Kabat EU numbering system of the Fc region of human IgG, adapted to the canine or feline Fc region. The "EU numbering system" or "EU index" is generally used when referring to residues in the heavy chain constant region of an antibody (e.g., Kabat et al., Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "Kabat EU numbering system" refers to the residue numbering of a human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers refer to the Kabat EU numbering system adapted to the canine or feline sequence.

[0023] (For dogs) (i) substitution of leucine at position 252 with tyrosine or threonine (L252Y or L252T), (ii) substitution of alanine at position 254 with threonine (A254T); (iii) substitution of threonine at position 256 with glutamic acid (T256E); (iv) substitution of isoleucine at position 308 with proline (I308P); (v) substitution of methionine at position 428 with leucine (M428L); (vi) substitution of histidine at position 433 with leucine (H433L); (vii) substitution of asparagine at position 434 with alanine, serine, tyrosine, or phenylalanine (N434A, N434S, N434Y, or N434F), (viii) substitution of tyrosine at position 436 with threonine (Y436T); (ix) substitution of glutamine at position 438 with arginine (Q438R), and (x) Substitution of serine at position 440 with glutamic acid (S440E) It has at least one such modification, and preferably two or more. Preferred examples of the modifications include the following DFV-1 to DFV-6 and DFV-8. DFV-1;N434A, Y436T, Q438R, S440E DFV-2;M428L, N434A, Y436T, Q438R, S440E DFV-3;M428L, N434A, Q438R, S440E DFV-4;L252Y, A254T, T256E DFV-5;M428L, N434S DFV-6;I308P, N434Y DFV-8;L252T, A254T, T256E, H433L, N434F

[0024] (In the case of cats) (i) substitution of serine at position 252 with tyrosine or threonine (S252Y or S252T), (ii) substitution of serine at position 254 with threonine (S254T); (iii) substitution of threonine at position 256 with glutamic acid (T256E); (iv) substitution of valine at position 259 with isoleucine (V259I); (v) substitution of isoleucine at position 308 with proline or phenylalanine (I308P or I308F), (vi) substitution of serine at position 428 with leucine (S428L); (vii) substitution of histidine at position 433 with leucine (H433L); (viii) substitution of serine at position 434 with alanine, tyrosine, or phenylalanine (S434A, S434Y, or S434F), (ix) substitution of histidine at position 436 with threonine (H436T), (x) substitution of glutamine at position 438 with arginine (Q438R), and (xi) Substitution of serine at position 440 with glutamic acid (S440E) (xii) substitution of leucine at position 235 with arginine (L235R), (xiii) substitution of glycine at position 236 with arginine (G236R), and (xiv) Substitution of serine at position 239 with lysine (S239K) It has at least one such modification, and preferably two or more. Preferred examples of the modifications include the following CFV-1 to CFV-4, CFV-6 to CFV-8, and siCFV3. CFV-1;S434A, H436T, Q438R, S440E CFV-2;S428L, S434A, H436T, Q438R, S440E CFV-3;S428L, S434A, Q438R, S440E CFV-4;S252Y, S254T, T256E CFV-6;I308P, S434Y CFV-7;V259I, I308F, S428L CFV-8;S252T, S254T, T256E, H433L, S434F siCFV-3;L235R, G236R, S239K, S428L, S434A, Q438R, S440E

[0025] When the human EU numbering is matched to the canine or feline sequences, and the amino acid at the start of the canine or feline Fc region is set to 1, the respective modifications are summarized in Table 1.

[0026] [Table 1]

[0027] When the EPO-Fc fusion protein of the present invention is derived from a feline, preferred Fc regions to be used include CFV-3 shown in SEQ ID NO:3 and siCFV-3 shown in SEQ ID NO:4.

[0028] In addition to binding to the EPO moiety, the polypeptides containing the IgG Fc region used in the present invention may be modified, for example, to enhance ADCC (antibody-dependent cellular cytotoxicity) activity or CDC (complement-dependent cytotoxicity), to increase protease resistance, to reduce effector function, to reduce complement binding activity, to improve antibody heterogeneity or stability, to promote antigen elimination, to allow repeated binding to multiple antigen molecules, to reduce the pI of the constant region to increase blood retention, or to confer binding ability to other antigens. For more details, please refer to the Fc engineering technology described in Current Pharmaceutical Biotechnology, 2016, 17, 1298-1314. The residue numbers in this document are based on the EU numbering system of Kabat. These modifications may be, for example, amino acid substitutions, deletions, additions, insertions, or modifications, or a combination thereof, with amino acid substitutions being preferred.

[0029] Such amino acid modifications (deletions, substitutions, insertions, and additions) into an amino acid sequence can be introduced by partially modifying the nucleotide sequence encoding the amino acid sequence. This partial modification of the nucleotide sequence can be achieved by known methods such as site-specific mutagenesis (Proc Natl Acad Sci USA., 1984 Vol. 81 5662-5666; Sambrook et al., Molecular Cloning A Laboratory Manual (1989) Second Edition, Cold Spring Harbor Laboratory Press) or overlap extension PCR. Furthermore, several known methods can be used to modify amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA to which an unnatural amino acid is bound is preferably used, such as a complementary amber suppressor tRNA for the UAG codon (amber codon), which is one of the termination codons. Furthermore, reference can be made to the techniques for modifying the Fc region of human IgG1 carried out in the following documents. Drug Metab Dispos. 2007 Jan;35(1):86-94, Int Immunol. 2006 Dec;18(12):1759-69, J Biol Chem. 2001 Mar 2;276(9):6591-604, J Biol Chem. 2007;282(3):1709-17, J Immunol. 2002;169(9):5171-80, J Immunol. 2009;182(12):7663-71, Molecular Cell, Vol. 7, 867-877, April, 2001, Nat Biotechnol. 1997 Jul;15(7):637-40, Nat Biotechnol. 2005 Oct;23(10):1283-8, Proc Natl Acad Sci US A. 2006 Dec 5;103(49):18709-14, EP2154157, US20070141052, WO2000 / 042072, WO2002 / 060919, WO2006 / 020114, WO2006 / 031370, WO2010 / 033279, WO2006 / 053301, WO2009 / 086320.

[0030] The modified Fc regions used in the present invention have FcRn-binding activity, and in particular exhibit higher FcRn-binding activity than the activity of the Fc region before modification, particularly higher FcRn-binding activity under acidic conditions. In the present invention, "having activity" means that in a system in which the activity can be measured, the measured value is higher than the background value in that system (or the value when a negative control is measured). For example, having binding activity means that the measured value is higher than the background value in a system in which the binding activity can be measured, such as ELISA, FACS, or Biacore. In the present invention, the measured value is preferably at least 2-fold higher than the background value, more preferably at least 3-fold higher, even more preferably at least 5-fold higher, and particularly preferably at least 10-fold higher. For example, in the present invention, the reciprocal of the KD (dissociation constant) can be used as the value of FcRn-binding activity. The KD value of the Fc region variants used in the present invention can be measured using, for example, a known method using Biacore (GE Healthcare). Specifically, in the case of Biacore, the Fc region variants provided by the present invention or antibody molecules containing such variants are immobilized on a sensor chip, and FcRn is applied as an analyte to measure the KD value. Measurements are performed using the Fc region of wild-type IgG (wild-type Fc) and the Fc region of a mutant IgG (Fc region variant) under acidic and neutral pH conditions, allowing the values ​​of KD(Fc region variant) / KD(wild-type Fc) and KD(acidic pH) / KD(neutral pH) to be calculated. It is also possible to use kd (Dissociation rate constant) instead of KD.

[0031] As used herein, the expression "canine or feline FcRn-binding activity is higher than that of the parent polypeptide before modification" means, for example, that the canine or feline FcRn-binding activity is 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, particularly preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more of that of the parent polypeptide. or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more.

[0032] If the Fc region variants of the present invention can be endowed with properties that make their binding activity to canine or feline FcRn stronger than that of native canine or feline IgG under acidic pH conditions, and if IgG can be constructed using these Fc region variants, then the FcRn binding of IgG under acidic conditions will be increased, thereby increasing the recycling efficiency from endosomes into plasma, and as a result, plasma retention will be improved or enhanced.

[0033] In the present invention, "binding activity to canine or feline FcRn under an acidic pH range condition" refers to FcRn-binding activity at pH 4.0 to pH 6.5, preferably at pH 5.0 to pH 6.5, more preferably at any of pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5, and particularly preferably at pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo. Furthermore, in the present invention, "binding activity to canine or feline FcRn under a neutral pH range condition" refers to FcRn-binding activity at pH 6.7 to pH 10.0. Preferably, it means FcRn-binding activity at pH 7.0 to pH 9.0, more preferably at any of pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, and particularly preferably at pH 7.4, which is close to the pH of plasma in vivo.

[0034] When the binding affinity to FcRn is too low at pH 7.4 to accurately measure the affinity, pH 7.0 can be used instead of pH 7.4. The binding affinity to FcRn may be measured at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity to FcRn. While not particularly limited, a temperature of 25°C is one preferred embodiment.

[0035] 3. Hinge area The hinge region is usually located at the C-terminus of the CH1 domain of the heavy chain constant region. In IgG isotypes, disulfide bonds typically occur in this hinge region. In the EPO-Fc fusion proteins of the present invention, the EPO polypeptide and the Fc region may be linked directly or via the hinge region. The hinge region can be derived from any immunoglobulin class. The hinge region of feline IgG has three cysteines, and the hinge region of canine IgG has three cysteines, at least one of which is involved in disulfide bonds between immunoglobulin heavy chains. Therefore, a preferred hinge region of the present invention is derived from IgG. In one embodiment, the first cysteine ​​in the hinge region of IgG is preferably substituted with another amino acid, preferably glycine. Another embodiment includes a substitution of the second or third cysteine ​​for glycine in addition to the substitution of the first cysteine ​​for glycine, and yet another embodiment includes a substitution of the second and third cysteines for glycine in addition to the substitution of the first cysteine ​​for glycine. When the Fc region is derived from canine IgG, a hinge region derived from canine IgG is preferably used, and when the Fc region is derived from feline IgG, a hinge region derived from feline IgG is preferably used. Examples of hinge regions derived from feline IgG include those having the amino acid sequence shown in any of SEQ ID NOs: 5 to 8. When the EPO-Fc fusion protein of the present invention is derived from a cat, the sequence of the hinge region to be used may be any of the sequences shown in SEQ ID NOs: 5 to 8.

[0036] 4. Erythropoietin Polypeptide (EPO Polypeptide) In the present invention, EPO polypeptides include wild-type or naturally occurring erythropoietin and recombinant erythropoietin derived from any animal species, preferably mammals, more preferably dogs or cats, and particularly preferably cats, as well as erythropoietin-like molecules including biologically active erythropoietin fragments and erythropoietin variants. When the Fc region is derived from canine IgG, a canine EPO polypeptide is preferably used, and when the Fc region is derived from feline IgG, a feline EPO polypeptide is preferably used. Examples of feline EPO polypeptides include those having the amino acid sequence set forth in SEQ ID NO: 9 or 10. Wild-type or native erythropoietin is a glycoprotein hormone that stimulates the growth and development of red blood cells from erythropoietin precursor cells. Wild-type or native erythropoietin can be routinely isolated and purified from blood or plasma or from urine.

[0037] Recombinant or chemically synthesized erythropoietin can be produced using techniques well known to those skilled in the art.

[0038] As used herein, the activity (particularly biological activity) of erythropoietin is defined as its ability to stimulate cell proliferation via interaction with the erythropoietin receptor. Functional assays of erythropoietin can be performed in vitro or in vivo. For example, the in vitro activity of erythropoietin can be tested in a cell-based assay. Specifically, erythropoietin activity can be evaluated by a cell proliferation assay using TF-1 cells expressing the EPO receptor. In vivo activity can be measured, for example, by hematocrit (HCT) assay or reticulocyte assay using an animal model.

[0039] The amino acid sequence of the erythropoietin-like molecule that can be used in the present invention is not particularly limited as long as it has biological activity or functional activity equivalent to that of wild-type or naturally occurring erythropoietin. However, the erythropoietin-like molecule typically has at least about 55%, about 65%, or about 75%, typically at least about 80%, about 85%, or about 90%, and most typically about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the corresponding sequence of wild-type or naturally occurring erythropoietin.

[0040] The erythropoietin of the present invention is understood to particularly include erythropoietin polypeptides having an amino acid sequence similar to that of wild-type erythropoietin. For example, the erythropoietin may contain one or more amino acid modifications in the amino acid sequence of wild-type erythropoietin, so long as its biological or functional activity is maintained. Examples of such amino acid modifications include addition, deletion, or substitution of amino acid residues.

[0041] When the EPO-Fc fusion protein of the present invention is derived from a feline, the sequence of the EPO polypeptide to be used may be the sequence shown in either SEQ ID NO:9 or SEQ ID NO:10.

[0042] Methods for introducing mutations into erythropoietin are well known in the art. For example, mutations can be introduced by site-directed mutagenesis techniques. A wide range of site-directed mutagenesis techniques are available.

[0043] 5. Linker The EPO-Fc fusion proteins of the present invention can include a linker molecule, preferably a peptide linker, between the Fc portion and the erythropoietin portion. Fusion proteins with linkers can have improved properties, such as increased biological activity. The linker generally contains 1 to 25 amino acids (e.g., 5 to 25 or 10 to 20 amino acids).

[0044] 6. In vitro activity and in vivo efficacy of EPO-Fc fusion proteins The in vitro activity of EPO-Fc fusion proteins can be tested by cell-based assays. In particular, the interaction between EPO-Fc fusion proteins and EPO receptor (EPOR) can be evaluated by a TF-1 cell proliferation assay.

[0045] The in vivo biological activity of EPO-Fc fusion proteins can be measured by assays performed in animal models, such as mice and rats. Examples of in vivo assays include, but are not limited to, the hematocrit (HCT) assay and the reticulocyte assay.

[0046] 7. Synthesis of EPO-Fc Fusion Protein The present invention provides a polynucleotide encoding the EPO-Fc fusion protein of the present invention. Polynucleotides are primarily composed of DNA, RNA, and other nucleic acid analogs. A polynucleotide encoding the EPO-Fc fusion protein of the present invention is constructed and inserted into an appropriate expression vector (two types of expression vectors may be used, if necessary). The polynucleotide is then incorporated into the expression vector so that it is expressed under the control of an expression control region, such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. Any appropriate combination of host and expression vector can be used.

[0047] The type of vector that can be used is not particularly limited as long as it stably retains the inserted gene, and various commercially available vectors can be used. Examples of vectors for gene cloning include M13-based vectors and pUC-based vectors. When using vectors for the purpose of producing the EPO-Fc fusion protein provided by the present invention, expression vectors are particularly useful. Expression vectors are not particularly limited as long as they express polypeptides in test tubes, E. coli, cultured cells, or whole organisms. For example, vectors for in vitro expression include the pBEST vector (Promega), vectors for E. coli expression include the pGEX, pET, and pBluescript vectors (Stratagene), vectors for cultured cell expression include the pME18S-FL3 vector (GenBank Accession No. AB009864), vectors for animal cell expression include pcDNA, and vectors for whole organism expression include the pME18S vector (Mol Cell Biol. 8:466-472 (1988)). The polynucleotide of the present invention can be inserted into a vector using, for example, the In-Fusion Advantage PCR Cloning Kit (Clontech).

[0048] There are no particular limitations on the host cells that can be used, and for example, Escherichia coli and various animal cells can be suitably used. Host cells can be used, for example, as a production system for producing and expressing the EPO-Fc fusion protein of the present invention. Production systems include in vitro and in vivo production systems. In vitro production systems include those using eukaryotic cells and those using prokaryotic cells.

[0049] Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108:94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero; amphibian cells such as Xenopus oocytes (Valle et al., Nature (1981) 291:338-340); and insect cells such as Sf9, Sf21, and Tn5. CHO-DG44, CHO-DX11B, COS7, HEK293, and BHK are preferred. CHO is particularly preferred for large-scale expression. Vectors can be introduced into host cells by techniques known to those skilled in the art, such as the calcium phosphate method, the DEAE-dextran method, a method using the cationic liposome DOTAP (Boehringer Mannheim), electroporation, lipofection, microinjection, etc. Alternatively, the FreeStyle 293 Expression System (Invitrogen) can be used to carry out processes from gene introduction to polypeptide expression.

[0050] The resulting EPO-Fc fusion protein can be isolated from inside or outside the host cells (culture medium, milk, etc.) and purified as a substantially pure, homogeneous molecule. Separation and purification of the EPO-Fc fusion protein can be performed using any separation and purification method commonly used for purifying polypeptides, and is not limited in any way. For example, separation and purification can be performed by appropriately selecting and combining methods such as column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0051] 8. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions containing the EPO-Fc fusion proteins produced according to the present invention. Pharmaceutical compositions can be used to treat diseases; for example, the pharmaceutical compositions provided by the present invention can be used to stimulate erythropoiesis and prevent and treat anemia. As used herein, "treatment" refers to achieving a pharmacological and / or physiological effect. The effect can be preventive, in that it completely or partially prevents the symptoms of a disease, or therapeutic, in that it completely or partially treats the symptoms of a disease. As used herein, "treatment" includes all treatments of diseases in mammals, particularly dogs or cats, or their closely related animal species.

[0052] The pharmaceutical compositions provided by the present invention can be formulated by methods known to those skilled in the art (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). They typically contain pharmaceutically acceptable additives that are conventional in the art and suitable for administration to a subject for therapeutic, diagnostic, or prophylactic purposes. For example, when formulated as a solid, fillers such as lactose, binders such as carboxymethylcellulose and gelatin, colorants, coating agents, etc. can be used, and such agents are suitable for oral administration. Furthermore, the compositions can be formulated as a topical agent by adding carriers or excipients such as white petrolatum, cellulose derivatives, surfactants, polyethylene glycol, silicone, olive oil, etc., and applying them to the affected area in the form of creams, emulsions, lotions, etc. When formulated as a liquid, they can contain commonly used physiologically acceptable solvents, emulsifiers, and stabilizers. Examples of solvents include water, PBS, isotonic saline, etc. Examples of emulsifiers include polyoxyethylene surfactants, fatty acid surfactants, silicone, etc. Examples of stabilizers include polyols such as canine serum albumin and gelatin, or sugars such as sorbitol and trehalose, etc. Compositions for oral administration can be in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, mouthwashes, or powders.

[0053] Although there is no particular limitation on the method of administration of the pharmaceutical composition of the present invention, the most effective treatment is expected by administration by injection, which may be any of intravenous, intramuscular, subcutaneous, intraperitoneal, and intrathoracic administration.

[0054] The dosage will be determined depending on the type of EPO-Fc fusion protein used, the size of the individual, the method of administration, the type of disease, symptoms, etc., but it is sufficient to administer an amount sufficient to demonstrate therapeutic and preventive effects.

[0055] All prior art documents cited herein are hereby incorporated by reference. [Example]

[0056] The present invention will now be further explained with reference to the following Reference Examples, Test Examples and Examples, but the present invention is not limited to these. Reference Example 1. Construction of an IgG expression vector having wild-type Fc Gene synthesis of the wild-type dog IgG H chain Fc region (SEQ ID NO: 1, hereinafter also referred to as "dog wild-type Fc" or "dog wtFc") registered in GenBank: AF354265.1 and the wild-type feline IgG H chain Fc region (SEQ ID NO: 2, hereinafter also referred to as "cat wild-type Fc" or "cat wtFc") registered in GenBank: AB016710.1 was requested to GenScript Japan Co., Ltd. based on the amino acid sequences. Gene synthesis was requested of GenScript Japan Inc. for the region from Fd to hinge of the IgG H chain (SEQ ID NO: 18; hereafter, Fd-Hinge) and the entire region of the IgG L chain (SEQ ID NO: 19; hereafter, L-Chain) based on the amino acid sequence of the humanized anti-human IgE antibody omalizumab registered at IMGT / mAb-DB ID: 77 in IMGT (reference URL: http: / / www.imgt.org / ). Gene synthesis was requested to be performed by GenScript Japan Inc. so that the amino acids MEFGLSWVFLVALFRGVQC (SEQ ID NO: 20) would be added as a secretory signal peptide to the N-terminus of Fd-Hinge and the amino acids MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 21) would be added to the N-terminus of the L chain. The synthesized omalizumab Fd-hinge gene containing the secretory signal peptide was amplified by PCR and ligated to the dog wtFc gene and cat wtFc gene, which had also been amplified by PCR, using the In-Fusion HD Cloning Kit (Clontech) (hereinafter referred to as the In-Fusion kit) so that the Fd-hinge was at the N-terminus and the wtFc was at the C-terminus. At the same time, this gene was inserted directly below the CMV promoter of pcDNA3.1(+) (Invitrogen), and Escherichia coli DH5α was transformed and the plasmid was extracted to obtain the H-chain expression vectors pcDNA3.1(+) / omalizumab Fd-dog wtFc and pcDNA3.1(+) / omalizumab Fd-cat wtFc. The omalizumab L-chain gene containing the synthesized secretory signal peptide was amplified by PCR and inserted directly below the CMV promoter of pcDNA3.1(+) (Invitrogen) using the In-Fusion kit. Escherichia coli DH5α was then transformed, and the plasmid was extracted to obtain the L-chain expression vector pcDNA3.1(+) / omalizumab Lch. In both cases, when using the In-Fusion kit, E. coli DH5α competent cells (TOYOBO) were transformed with the DNA solution after the In-Fusion reaction according to the method described in the attached instruction manual. The resulting transformants were cultured overnight at 37°C in LB liquid medium containing 100 μg / mL ampicillin, and plasmids were extracted from them using a NucleoBond Xtra Midi kit (Takara Bio Inc.). The nucleotide sequence of the resulting expression vector was determined by methods known to those skilled in the art, and it was confirmed that the protein with the desired amino acid sequence was encoded.

[0057] Amino acid sequence of the Fd-Hinge region of omalizumab EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP (SEQ ID NO: 18)

[0058] Amino acid sequence of wild-type canine IgG H chain Fc region APEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK (SEQ ID NO: 1)

[0059] Amino acid sequence of wild-type feline IgG H chain Fc region PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 2)

[0060] Omalizumab L-chain amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19)

[0061] Reference Example 2. Construction of an IgG H chain expression vector having a modified Fc Primers were designed to encode mutated amino acids so that the amino acid sequence of the Fc region would be substituted with the amino acids shown in Table 1 ("Positions of dog and cat Fc modifications"). Using the H chain expression vectors pcDNA3.1(+) / omalizumab Fd-dog wtFc and pcDNA3.1(+) / omalizumab Fd-cat wtFc prepared in Example 1 as templates, DNA fragments with mutations introduced into the Fc region were amplified by PCR using the designed primers, and the amplified DNA fragments were ligated using the In-Fusion kit to prepare H chain expression vectors with mutations introduced only at specific sites in the template Fc. In both cases, when using the In-Fusion kit, E. coli DH5α competent cells (TOYOBO) were transformed with the DNA solution after the In-Fusion reaction according to the method described in the attached instruction manual. The resulting transformants were cultured overnight at 37°C in LB liquid medium containing 100 μg / mL ampicillin, and plasmids were extracted from them using a NucleoBond Xtra Midi kit (Takara Bio Inc.). The nucleotide sequence of the resulting expression vector was determined by methods known to those skilled in the art, and it was confirmed that the protein with the desired amino acid sequence was encoded.

[0062] Reference Example 3. Antibody expression and purification The expression vectors obtained in Reference Examples 1 and 2 were transiently introduced into FreeStyle293 cells (Invitrogen) to express antibodies. The resulting culture supernatant was collected and then passed through a 0.22 μm Millex®-GP filter (Merck Millipore). The resulting culture supernatant was purified by affinity chromatography using MabSelect SuRe (GE Healthcare), eluting with 50 mM acetic acid and neutralizing with 1.5 M Tris-HCl, pH 7.5. The resulting antibody was subjected to buffer exchange using an ultrafiltration membrane (Merck Millipore) capable of 30 kDa cutoff in a buffer solution of 20 mM histidine-HCl, 150 mM NaCl, pH 6.5. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm using a spectrophotometer, and calculating the antibody concentration from the obtained value using the extinction coefficient calculated by the method of PACE et al. (Protein Science (1995); 4, 2411-2423).

[0063] Reference Example 4. Construction of FcRn expression vector We commissioned GenScript Japan to synthesize the extracellular domains of canine FCGRT (SEQ ID NO: 22, hereafter referred to as "dog FCGRT") and feline FCGRT (SEQ ID NO: 23, hereafter referred to as "cat FCGRT"), registered at GenBank: XP_005616366.1, and XP_023100998.1, respectively, with a C-terminal His tag (HHHHHHHH) (SEQ ID NO: 24). The resulting expression vectors were designated pcDNA3.1(+) / dog FCGRT and pcDNA3.1(+) / cat FCGRT. Based on the amino acid sequences of dog β2m (SEQ ID NO: 25, hereafter referred to as dog β2m) registered in GenBank: NP_001271408 and feline β2m (SEQ ID NO: 26, hereafter referred to as cat β2m) registered in GenBank: NP_001009876, we commissioned GenScript Japan Co., Ltd. to synthesize the genes, clone the synthesized genes into pcDNA3.1(+) (Invitrogen), and extract the plasmids. The resulting expression vectors were named pcDNA3.1(+) / dog β2m and pcDNA3.1(+) / cat β2m.

[0064] Amino acid sequence of dog FCGRT extracellular domain MGVPRPRSWGLGFLLFLLPTLRAADSHLSLLYHLTAVSAPPPGTPAFWASGWLGPQQYLSYNNLRAQAEPYGAWVWENQVSWYWEKETTDLRTKEGLFLEALKALGDGGPYTLQGLLGCELGPDNTSVPVAKFALNGEDFMTFDPKLGTWNGDWPETETVSKRWMQQAGAVSKERTFLLYSCPQRLLGHLERGRGNLEWKEPPSMRLKARPGSPGFSVLTCSAFSFYPPELQLRFLRNGLAAGSGEGDFGPNGDGSFHAWSSLTVKSGDEHHYRCLVQHAGLPQPLTVELESPAKSS (SEQ ID NO: 22)

[0065] Amino acid sequence of cat FCGRT extracellular domain MGVPRPQPWGLGFLLFLLPTLRAAESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNNLRAQAEPCGAWVWENQVSWYWEKETTDLRNKQELFLEALKVLGEGGPYTLQGLLGCELGPDNASVPVAKFALNGEDFMDFDPKLGTWSGEWPETETISKRWMQEAGAVSKERTFLLNSCPQRLLGHLERGRGNLEWKEPPSMRLKARPGSPGFSVLTCSAFSFYPPELQLRFLRNGLAAGSGEGDFGPNGDGSFHAWSSLTVKSGDEHHYRCLVQHAGLPQPLTVELESPAKSS (SEQ ID NO: 23)

[0066] Amino acid sequence of dog β2m MAPRPALATAGFLALLLILLAACRLDAVQHPPKIQVYSRHPAENGKPNFLNCYVSGFHPPEIEIDLLKNGKEMKAEQTDLSFSKDWTFYLLVHTEFTPNEQDEFSCRVKHVTLSEPQIVKWDRDN (SEQ ID NO: 25)

[0067] Amino acid sequence of cat β2m MARFVVLVLLGLLYLSHLDAVQHSPKVQVYSRHPAENGKPNFLNCYVSGFHPPQIDITLMKNGKKMEAEQTDLSFNRDWTFYLLVHTEFTPTVEDEYSCQVNHTTLSEPKVVKWDRDM (SEQ ID NO: 26)

[0068] Reference Example 5. Expression and purification of FcRn protein The expression vectors obtained in Example 4, a combination of pcDNA3.1(+) / dog FCGRT and pcDNA3.1(+) / dogβ2m, and a combination of pcDNA3.1(+) / cat FCGRT and pcDNA3.1(+) / catβ2m, were cotransfected into FreeStyle293 cells (Invitrogen) to express canine and feline FcRn proteins. After culturing, the resulting culture supernatant was collected and then passed through a 0.22 μm Millex®-GP filter (Merck Millipore). The resulting culture supernatant was generally purified in the following two steps. The first step was His-tag affinity column chromatography (His Trap HP), followed by fractionation using a gradient of imidazole gradients (20 mM Tris, 0.5 M NaCl, 10 mM imidazole, pH 7.4 and 20 mM Tris, 0.5 M NaCl, 500 mM imidazole, pH 7.4). The second step was gel filtration column chromatography (Superdex 200), followed by buffer exchange with D-PBS(-), pH 7.0, and size fractionation. The purified protein was measured for absorbance at 280 nm using a spectrophotometer, and the concentration of the purified protein was calculated using the extinction coefficient calculated by the method of PACE et al. (Protein Science (1995); 4, 2411-2423).

[0069] Test Example 1: Interaction measurement using Biacore (binding analysis) Evaluation of the binding ability of the obtained antibodies to canine and feline FcRn The antibodies were evaluated using a BiacoreX100 (GE Healthcare) to determine their binding ability to canine and feline FcRn. Plasma conditions were set at pH 7.4. Endosomal conditions (acidic conditions) were set at pH 6.0. The target antibodies were captured on a Sensor chip Protein L (GE Healthcare), and canine and feline FcRn were used as antigens. Measurements were performed using three running buffers: 1; 50 mmol / L phosphate, 150 mmol / L NaCl, 0.05% (w / v) Tween-20, pH 7.4; 2; 50 mmol / L phosphate, 150 mmol / L NaCl, 0.05% (w / v) Tween-20, pH 7.0; 3; 50 mmol / L phosphate, 150 mmol / L NaCl, 0.05% (w / v) Tween-20, pH 6.0.

[0070] How to perform the measurement Antibodies diluted in running buffer were injected at a flow rate of 5 μL / min for 1 minute to capture them on the sensor chip. Subsequently, FcRn diluted to 1600, 800, 400, 200, and 100 nM in running buffer and running buffer (as a reference solution) were injected at a flow rate of 30 μL / min for 2 minutes to allow interaction with the captured antibodies. The running buffer was then further injected at a flow rate of 30 μL / min for 10 minutes to observe FcRn dissociation. Finally, the sensor chip was regenerated by two 1-minute injections of 10 mmol / L glycine-HCl, pH 1.7, at a flow rate of 30 μL / min. The regeneration step washed the captured antibodies from the sensor chip, and the sensor chip was reused. Because the binding affinity between wild-type IgG and FcRn is very low at pH 7.4, it is difficult to calculate the KD value. Therefore, when it was difficult to accurately measure the affinity, measurements were performed using pH 7.0 instead of pH 7.4.

[0071] Analysis method To calculate the dissociation constants (KD) (mol / L) of antibodies containing each Fc region variant with FcRn, kinetic analysis was performed as follows: First, the antibody of interest was captured on the sensor chip and allowed to interact with FcRn diluted in running buffer. The resulting sensorgram was then globally fitted using Biacore Evaluation Software with a 1:1 binding model to calculate the binding rate constant (ka) (L / mol / s) and dissociation rate constant (kd) (1 / s), and the dissociation constant (KD) (mol / L) was then calculated from these values. If the obtained sensorgram is box-shaped and quickly reaches equilibrium, the equilibrium value (=binding amount) during FcRn injection reflects the dissociation constant KD (M). The dissociation constant KD (mol / L) of each variant for FcRn was calculated by performing steady-state affinity analysis on the sensorgram obtained as the Biacore measurement result using Biacore Evaluation Software. The behavior of molecules interacting in a 1:1 binding model on Biacore can be expressed by the following equation 1. Req = C x Rmax / (KD + C) +RI (Equation 1) The meaning of each item in the above formula is as follows: Req(RU): steady state binding levels Rmax (RU): Affinity binding capacity of the surface RI(RU): Bulk refractive index contribution in the sample C(M): Analyte concentration KD(M): Equilibrium dissociation constant The results are shown in the table below.

[0072] [Table 2]

[0073] [Table 3]

[0074] [Table 4]

[0075] [Table 5]

[0076] Example 1. Construction of an expression plasmid for a fusion protein of wild-type feline erythropoietin and the IgG heavy chain Fc region An expression plasmid for a fusion protein of feline erythropoietin (hereinafter referred to as EPO) and the IgG heavy chain Fc region (hereinafter referred to as Fc) (hereinafter referred to as cat EPO-Fc or feline EPO-Fc) was constructed as follows. The amino acid sequence of feline EPO-Fc was determined (SEQ ID NO: 13) based on the amino acid sequence of wild-type feline EPO registered in GenBank: JQ413414.1 (SEQ ID NO: 11) and the amino acid sequence from the hinge region (SEQ ID NO: 12) to the Fc region (SEQ ID NO: 2) of the wild-type feline IgG H chain registered in GenBank: AB016710.1. The EPO and Fc regions retained their wild-type sequences, while one of the three cysteines in the hinge region on the N-terminal side was modified to glycine. A gene was synthesized based on the determined amino acid sequence. The in-house constructed plasmid pCAG (structure shown in Figure 1) was digested with the restriction enzymes SbfI and EcoRV (both from New England Biolabs) to prepare a circularized plasmid fragment directly under the CAG promoter. This was then ligated to a feline EPO-Fc gene fragment synthesized using the In-Fusion HD Cloning Kit (Clontech) (hereafter referred to as the In-Fusion kit) to construct the wild-type feline EPO-Fc expression plasmid pCAG / C2 cat wtEPO-Fc. Following the instructions in the In-Fusion kit's package insert, Escherichia coli DH5α competent cells (TOYOBO) were transformed with the DNA solution obtained after the In-Fusion reaction. The resulting transformants were cultured overnight at 37°C in LB liquid medium containing 25 μg / mL kanamycin, and the plasmid was extracted from the cultured E. coli using the NucleoBond Xtra Midi Kit (Takara Bio Inc.). The nucleotide sequence of the resulting plasmid was determined using methods known to those skilled in the art, and it was confirmed that the protein with the desired amino acid sequence was encoded.

[0077] Wild-type feline EPO MGSCECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAREAENVTMGCAEGCSFSENITVPDTKVNFYTWKRMDVGQQAVEVWQGLALLSEAILRGQALLANSSQPSETLQLHVDKAVSSLRSLTSLLRALGAQKEATSLPEATSAAPLRTFTVDTLCKLFRIYSNFLRGKLTLYTGEACRRGDR (SEQ ID NO: 11)

[0078] Hinge region of wild-type feline IgG H chain RKTDHPPGPKPCDCPKCP (SEQ ID NO: 12)

[0079] Wild-type feline IgG H chain Fc region PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 2)

[0080] Genetically synthesized feline EPO-Fc MGSCECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAREAENVTMGCAEGCSFSENITVPDTKVNFYTWKRMDVGQQAVEVWQGLALLSEAILRGQALLANSSQPSETLQLHVDKAVSSLRSLTSLLRALGAQKEATSLPEATSAAPLRTFTVDTLCKLFRIYSNFLRGKLTLYTGEACRRGDRRKTDHPPGPKPGDCPKCPPPEMLGGP SIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO: 13)

[0081] Example 2. Construction of expression plasmid for feline EPO-Fc with modified amino acids in the EPO region and hinge region To explore amino acid mutations that alter the physical properties and physiological activity of wild-type feline EPO-Fc, expression plasmids were constructed for amino acid mutants of feline EPO-Fc (hereinafter also referred to as feline EPO-Fc variants) in which the cysteines in the EPO domain, the hinge domain, and the presence or absence of the hinge domain were variously altered, as shown in Table 6. Details are described below. First, primers were designed that annealed to the mutation sites in the feline EPO-Fc DNA of pCAG / C2 cat wtEPO-Fc constructed in Example 1 and encoded amino acid sequences characteristic of each feline EPO-Fc variant shown in Table 6. Next, using pCAG / C2 cat wtEPO-Fc as a template, the mutated DNA fragments were amplified by PCR with the designed primers, and the amplified DNA fragments were ligated using the In-Fusion kit to produce the expression plasmids for the feline EPO-Fc variants: pCAG / C2 C59P cat EPO-Fc, pCAG / C2 C165R cat EPO-Fc, pCAG / C1a cat wtEPO-Fc, pCAG / C1a C59P cat EPO-Fc, pCAG / C1a C165R cat EPO-Fc, pCAG / C1b cat wtEPO-Fc, pCAG / C1b C59P cat EPO-Fc, pCAG / C1b C165R cat EPO-Fc, pCAG / C0 cat wtEPO-Fc, pCAG / C0 C59P cat EPO-Fc, and pCAG / C0 C165R cat EPO-Fc, pCAG / HL cat wtEPO-Fc, pCAG / HL C59P cat EPO-Fc, and pCAG / HL C165R cat EPO-Fc were constructed. The In-Fusion kit was used, and subsequent transformation and cultivation of Escherichia coli DH5α, plasmid extraction, and nucleotide sequencing were performed in the same manner as described in Example 1, confirming that the desired plasmids had been constructed.

[0082] [Table 6]

[0083] Example 3. Expression, purification, and analysis of wild-type feline EPO-Fc and EPO region and hinge region modified feline EPO-Fc proteins ExpiCHO containing cells, media, and transfection reagent TM Using the Expression System Kit (Gibco) and following the Max Titer protocol of the kit, each of the 15 feline EPO-Fc expression plasmids obtained in Examples 1 and 2 was transfected into Expi CHO-S TMThe cells were transfected with the vector and cultured for 14 days to allow protein expression. 30 mL of the culture supernatant after transfection was clarified through a 0.22 μm filter, Millex®-GP (Merck Millipore). Novex TM A portion of the clarified culture supernatant was electrophoresed by non-reducing SDS-PAGE using a 4-20% Tris-Glycine Mini gel (Invitrogen) and then transferred to a PVDF membrane (Invitrogen). The target proteins contained in the culture supernatant were analyzed by Western blotting using Rabbit Anti-Human Erythropietin Antibody (R&D Systems) as the primary antibody, Goat Anti-Rabbit IgG Antibody (AP) (Millipore) as the secondary antibody, and BCIP-NBT Solution Kit for Alkaline Phosphatase Stain, Nuclease Tested (Nacalai Tesque) as the colorimetric substrate (Figure 2). In non-reducing SDS-PAGE, feline EPO-Fc variants with C2, C1a, or C1b hinges showed a main band approximately twice as large (130-250 kDa) as the main band (55-70 kDa) of feline EPO-Fc variants with C0 or HL hinges. This indicates that disulfide bonds are formed between molecules in the feline EPO-Fc variants with C2, C1a, or C1b hinges. Furthermore, the feline EPO-Fc variant with the C165R mutation in the EPO domain showed a much fainter smear band than the other variants, indicating that it has fewer aggregates other than dimers and has superior physical properties.

[0084] The remaining clarified culture supernatant was loaded onto a column packed with MabSelect SuRe (GE Healthcare Lifesciences), and the expressed protein was purified by Protein A affinity chromatography using 50 mM acetic acid as the elution buffer and 1.5 M Tris-HCl, pH 7.5 as the neutralization buffer. The purified protein was then purified by gel filtration chromatography using a Superdex 200 gel filtration column (GE Healthcare Lifesciences) with 50 mM phosphate, 300 mM NaCl, pH 7.0 as the buffer. The main peak protein fraction was isolated and collected. The absorbance of the purified protein was measured at 280 nm using a spectrophotometer, and the yield of purified protein was calculated using the extinction coefficient calculated by the method of PACE et al. (Protein Science (1995); 4, 2411-2423) (Table 7).

[0085] [Table 7]

[0086] We found that the feline EPO-Fc variants with C2-, C1a-, or HL-type hinges had a higher protein yield after purification when the C165R mutation in the EPO domain was combined with the hinge mutation. When purifying the protein by gel filtration, we tried to isolate only the main peak as much as possible and avoided contaminating peaks that may represent protein aggregates or degradation products. This suggests that the excellent physical properties of the feline EPO-Fc with C165R in the EPO domain and C2-, C1a-, or HL-type hinges resulted in a high protein yield.

[0087] Purified feline EPO-Fc was purchased from Novex TM SDS-PAGE was performed using 4-20% Tris-Glycine Mini gels (Invitrogen) under non-reducing and reducing conditions, followed by SimplyBlue TM The presence or absence of intermolecular disulfide bonds in feline EPO-Fc was confirmed by CBB staining with SafeStain (Invitrogen) (Fig. 3). The main bands of feline EPO-Fc variants with C2-, C1a-, or C1b-type hinges were located between 55 and 70 kDa under reducing conditions, but were approximately twice as large (130 and 250 kDa) under non-reducing conditions, indicating that feline EPO-Fc variants with C2-, C1a-, or C1b-type hinges form dimers containing intermolecular disulfide bonds. Furthermore, the main bands of feline EPO-Fc variants with C0- and HL-type hinges were always located between 55 and 70 kDa, regardless of whether they were reduced or non-reduced, indicating that they do not form covalent dimers containing intermolecular disulfide bonds. On the other hand, since feline EPO-Fc variants with C0- and HL-type hinges still contain Fc, they are likely to form noncovalent dimers via Fc.

[0088] Furthermore, the purified feline EPO-Fc was analyzed by size exclusion chromatography (SEC) using a G3000SWXL column (Tosoh Corporation) as the carrier, 50 mM phosphate, 300 mM NaCl, pH 7.0 as the mobile phase, a flow rate of 0.7 mL / min, and a detection wavelength of 220 nm. Peaks eluting earlier than the main peak (dimer) were analyzed as aggregates and associations, while peaks eluting later were analyzed as monomers and degradation products. The content (%) of the main peak was calculated by the area percentage method and compared between samples along with the retention time (RT) (Table 8) (Figure 4).

[0089] [Table 8]

[0090] In Table 8, the retention times of the main peaks for all feline EPO-Fc variants were similar, strongly suggesting that feline EPO-Fc variants with C0-type and HL-type cysteine-free hinges also have molecular weights similar to those of variants with C2-type, C1a-type, and C1b-type cysteine-containing hinges, i.e., form dimers. Figure 4 shows that in the feline EPO-Fc variants with C2, C1a, or C1b hinges, the combination of the hinge mutation and the C165R or C59P mutation in the EPO domain reduces the subpeak, likely representing aggregates or aggregates, to the left of the main peak in SEC analysis compared to feline EPO-Fc with wild-type EPO. Figure 4F shows a merged chromatogram of the SEC analysis results for the feline EPO-Fc fusion proteins 1. wt catEPO-C2-Fc, 2. C59P catEPO-C2-Fc, and 3. C165R catEPO-C2-Fc (Table 8, items 1 to 3). Enlarging the merged chromatogram revealed that 3. C165R catEPO-C2-Fc had the fewest subpeaks. In particular, feline EPO-Fc, which has a C165R mutation in the EPO domain and a C2-type hinge, had fewer subpeaks, indicating that this variant had good physical properties.

[0091] As described above, it was found that all of the feline EPO-Fc variants evaluated in Example 3 mainly formed dimers, but the proportion of aggregates formed varied depending on the number of cysteines in the EPO domain and hinge region, which in turn led to differences in the yield of the native protein after purification. In particular, it was found that feline EPO-Fc variants having C165R in the EPO domain and a C2-type hinge region tended to suppress excessive multimerization such as aggregation, and the purified protein was found to be dimeric and to be excellent in both purity and yield.

[0092] Example 4. Construction of expression plasmids for fusion proteins of feline EPO and His tag, and mouse EPO and His tag Expression plasmids for a fusion protein of feline EPO with a His tag (hereafter referred to as feline EPO-His or cat EPO-His) and a fusion protein of mouse EPO with a His tag (hereafter referred to as mouse EPO-His or mouse EPO-His) were constructed for use in various experiments as a control for feline EPO-Fc protein, etc. Details are described below. First, a forward primer that anneals to the N-terminus of feline EPO and a reverse primer that anneals to the C-terminus and encodes a linker-containing His tag (GAAHHHHHHHHH (SEQ ID NO: 14)) were designed. Using pCAG / C2 cat wtEPO-Fc, pCAG / C2 C59Pcat EPO-Fc, and pCAG / C2 C165R cat EPO-Fc constructed in Examples 1 and 2 as templates, PCR was performed with the designed primers to prepare three types of DNA fragments covering the entire feline EPO-His region (wild-type, C59P, and C165R). Next, we determined the amino acid sequence of mouse EPO-His, which had a linker-containing His tag (GAAHHHHHHHHH) added to the C-terminus of the amino acid sequence of mouse EPO registered in GenBank: NM_007942.2 (SEQ ID NO: 15). Based on the amino acid sequence, we commissioned GenScript Japan Co., Ltd. to synthesize a DNA fragment of the entire mouse EPO-His region. Next, we cleaved the plasmid pCAG with restriction enzymes SbfI and EcoRV (both New England Biolabs) to prepare an open-circular plasmid fragment directly under the CAG promoter. Finally, using the In-Fusion kit, DNA fragments from three types of feline EPO-His (wild-type, C59P, and C165R) and mouse EPO-His were ligated to the pCAG plasmid fragment, resulting in a total of four EPO-His plasmids: pCAG / cat wtEPO-His, pCAG / C59P cat EPO-His, pCAG / C165R cat EPO-His, and pCAG / mouse wtEPO-His. The use of the In-Fusion kit, followed by transformation and cultivation of E. coli DH5α, plasmid extraction, and nucleotide sequencing were performed as described in Example 1 to confirm the construction of the desired plasmids.

[0093] Wild-type mouse EPO MGVPERPTLLLLLSLLLIPLGLPVLCAPPRLICDSRVLERYILEAKEAENVTMGCAEGPRLSENITVPDTKVNFYAWKRMEVEEQAIEVWQGLSLLSEAILQAQALLANSSQPPETLQLHIDKAISGLRSLTSLLRVLGAQKELMSPPDTTPPAPLRTLTVDTFCKLFRVYANFLRGKLKLYTGEVCRRGDR (SEQ ID NO: 15)

[0094] Example 5. Protein expression and purification by transfection of mammalian cells with feline EPO-His and mouse EPO-His expression plasmids ExpiCHO containing cells, media, and transfection reagent TM Using the Expression System Kit (Gibco) and following the Max Titer protocol of the kit, pCAG / cat wtEPO-His, pCAG / C59P cat EPO-His, pCAG / C165R cat EPO-His, and pCAG / mouse wtEPO-His obtained in Example 4 were each transformed into Expi CHO-S TM The transfection was carried out in cells and cultured for 7 days to allow protein expression. 30 mL of the culture supernatant was clarified through a 0.22 μm Millex®-GP filter (Merck Millipore). The culture supernatant was loaded onto a column packed with HisTrap HP (GE Healthcare Lifesciences), and the His-tagged protein was affinity purified by imidazole gradient elution. The purified protein was then purified by gel filtration chromatography using a Superdex 200 gel filtration column (GE Healthcare Lifesciences) with D-PBS(-) as the buffer. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer, and the concentration of the purified protein was calculated using the extinction coefficient calculated by the method of PACE et al. (Protein Science (1995); 4, 2411-2423).

[0095] Example 6. Evaluation of in vitro activity of feline EPO-Fc with modified EPO and hinge regions using TF-1 cells Using TF-1 cells, a human erythroleukemia cell line that exhibits human EPO-dependent cell proliferation, the biological activities of the 15 types of feline EPO-Fc prepared in Example 3, the 3 types of feline EPO-His prepared in Example 5, and purchased human EPO (PeproTech) were evaluated in vitro. Details are described below. After washing BaF3 / cat EPOR and TF-1 cells three times with RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific) and 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific), 4 × 10 5 The cell number was adjusted to 1000 cells / mL with the same medium, and 50 μL of each was seeded into each well of a 96-well plate (CORNING). Eight 4-fold dilutions of ligands (various EPO-Fc, EPO-His, and EPO) were prepared starting from 13.16 nM using the same medium as the diluent, and 50 μL of each was dispensed into each well of the 96-well plate containing the seeded cells. The cells were cultured at 37°C and 5% CO2 for 3 days. Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL / well, and after 3 hours of culture, the absorbance at 450 nm (reference wavelength 620 nm) was measured again to evaluate the biological activity of each ligand. The results are shown in Figure 5. The monomeric feline EPO-His clearly has lower activity in inducing TF-1 cell proliferation than human EPO. This is thought to be due to the difference in affinity between feline EPO and human EPO expressed by TF-1 cells. However, feline EPO-Fc clearly has higher activity than feline EPO-His. In particular, mutants with C59P and C165R mutations in the EPO domain and C2 and C1a mutations in the hinge domain tended to exhibit higher activity than other feline EPO-Fc variants. In particular, the activity of a modified feline EPO-Fc with a C165R mutation in the EPO domain and a C2-type hinge domain is high.

[0096] Example 7. Construction of an expression plasmid for Fc-modified feline EPO-Fc In Examples 3 and 6, feline EPO-Fc with the C165R mutation in the EPO domain and the C2 mutation in the hinge domain tended to have improved physical properties and physiological activity. To confirm whether the properties of the C165R mutation in the EPO domain and the C2 mutation in the hinge domain were impaired in feline EPO-Fc in which mutations had been introduced into wild-type Fc, an expression plasmid for feline EPO-Fc in which mutations had also been introduced into the Fc domain was constructed. The sequence characteristics of the EPO domain, hinge domain, and Fc domain of the newly constructed feline EPO-Fc variants are shown in Table 9. Details of the plasmid construction are described below. Primers were designed to anneal to the desired site of mutation in wild-type feline Fc DNA and encode the amino acid sequence characteristic of the Fc variant (referred to as CFV3) shown in SEQ ID NO: 3. Next, using pCAG / C2 C165R cat EPO-Fc constructed in Example 2 as a template, the mutated DNA fragments were amplified by PCR using the designed primers, and the amplified DNA fragments were ligated using the In-Fusion kit to construct the expression plasmid pCAG / C2 C165R cat EPO-CFV3. Primers were designed to anneal to the desired site of mutation in CFV3 DNA and encode the amino acid sequence characteristic of the Fc variant (referred to as siCFV3) shown in SEQ ID NO: 4. Next, using pCAG / C2 C165R cat EPO-CFV3 as a template, the mutated DNA fragment was amplified by PCR using the designed primers, and the amplified DNA fragments were ligated using the In-Fusion kit to construct the expression plasmid pCAG / C2 C165R cat EPO-siCFV3. Primers were designed to amplify only the Fc region, and a DNA fragment of siCFV3 was prepared by PCR using pCAG / C2 C165R cat EPO-siCFV3 as a template. Primers were also designed to amplify the EPO region and the region from EPO to the hinge region. DNA fragments of wild-type feline EPO containing the C2 hinge region (cat wtEPO-C2 hinge) and C165R feline EPO without the hinge region (cat C165R EPO) were prepared by PCR using pCAG / C2 cat wtEPO-Fc constructed in Example 1 and pCAG / HL C165R cat EPO-Fc constructed in Example 2 as templates. Finally, pCAG / C2 cat wtEPO-siCFV3 and pCAG / HL C165R cat EPO-siCFV3 were constructed by ligating PCR-prepared EPO (cat wtEPO-C2 hinge, cat C165R EPO) and Fc (siCFV3) DNA fragments with the pCAG plasmid fragment prepared by restriction enzyme digestion (SbfI and EcoRV) described in Example 1 using the In-Fusion kit. The use of the In-Fusion kit, followed by transformation and cultivation of Escherichia coli DH5α, plasmid extraction, and nucleotide sequencing were performed in the same manner as described in Example 1, to confirm the construction of the desired plasmids.

[0097] Cat Fc variant CFV3 PPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVLHEALHAHHTRKELTQSPGK (SEQ ID NO: 3)

[0098] Cat Fc variant siCFV3 PPEMRRGPKIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVLHEALHAHHTRKELTQSPGK (SEQ ID NO: 4)

[0099] [Table 9]

[0100] Example 8. Protein expression and purification of Fc-modified feline EPO-Fc ExpiCHO containing cells, media, and transfection reagent TM Using the Expression System Kit (Gibco) and following the Max Titer protocol of the kit, five types of feline EPO-Fc expression plasmids (pCAG / C2 C165R cat EPO-Fc constructed in Example 2 and four types of feline EPO-Fc expression plasmids constructed in Example 7, pCAG / C2 C165R cat EPO-siCFV3, pCAG / C2 cat wtEPO-siCFV3, pCAG / C2 C165R cat EPO-CFV3, and pCAG / HL C165R cat EPO-siCFV3) were each expressed in Expi CHO-S TM The cells were transfected with the vector and cultured for 7 days to allow protein expression. 30 mL of the culture supernatant after transfection was clarified through a 0.22 μm filter, Millex®-GP (Merck Millipore). The clarified culture supernatant was purified and analyzed in the same manner as in Example 3. FIG. 6 shows the results of SDS-PAGE analysis and SEC analysis of the purified protein, and Table 10 shows the results of the yield of the purified protein.

[0101] [Table 10]

[0102] We found that even when the siCFV3 mutation or CFV3 was introduced into the Fc region based on a feline EPO-Fc variant with a C165R mutation in the EPO region and a C2-type hinge region, the expressed protein formed a dimer, and the purified protein had good purity and yield.

[0103] Example 9. Evaluation of in vitro activity of Fc-modified feline EPO-Fc using TF-1 cells The biological activities of feline EPO-Fc prepared in Example 8 and purchased human EPO (PeproTech) were evaluated in vitro using TF-1 cells, a human erythroleukemia cell line that exhibits human EPO-dependent cell proliferation. Details are described below. TF-1 cells were washed three times with RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific) and 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific), and then cultured at 4 × 10 5 The cell number was adjusted to 1000 cells / mL with the same medium, and 50 μL of each was seeded into each well of a 96-well plate (Corning). Eight 4-fold dilutions of ligands (various EPO-Fc, EPO-His, and EPO) were prepared starting from 13.16 nM using the same medium as the diluent, and 50 μL of each was dispensed into each well of the 96-well plate containing the seeded cells. The cells were cultured at 37°C and 5% CO2 for 3 days. Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL / well, and after 3 hours of culture, the absorbance at 450 nm (reference wavelength 620 nm) was measured again to evaluate the biological activity of each ligand. The results are shown in Figure 7. It was confirmed by comparison with EPO-Fc variants with wild-type EPO domains or no hinge domains that feline EPO-Fc variants with a C165R mutation in the EPO domain and a C2-type hinge domain maintain a tendency for high activity even when the siCFV3 mutation or CFV3 is further introduced into the Fc domain.

[0104] Example 10. Construction of expression plasmids for feline and mouse EPO receptors Expression plasmids for the feline EPO receptor (hereafter referred to as cat EPOR) and mouse EPO receptor (hereafter referred to as mouse EPOR) were constructed, which are necessary for establishing cell lines to be used in assessing the biological activity of feline EPO. Details are described below. Genes were synthesized based on the amino acid sequence of feline EPOR (SEQ ID NO: 16) registered in GenBank: XM_023245578.1 and the amino acid sequence of mouse EPOR (SEQ ID NO: 17) registered in GenBank: NM_010149.3, to obtain DNA fragments for feline and mouse EPOR. Plasmid pBApo-EF1α Pur (TaKaRa Bio) was then digested with restriction enzymes XbaI and HindIII (both New England Biolabs) to prepare open-loop plasmid fragments directly under the EF1α promoter. Finally, the feline and mouse EPOR DNA fragments were ligated to the pBApo-EF1α Pur plasmid fragment using the In-Fusion kit to construct the expression plasmids for feline and mouse EPOR, i.e., pBApo-EF1α Pur / cat EPOR and pBApo-EF1α Pur / mouse EPOR. When using the In-Fusion kit, E. coli DH5α competent cells (TOYOBO) were transformed with the DNA solution after the In-Fusion reaction according to the method described in the attached instruction manual. The resulting transformants were cultured overnight at 37°C in LB liquid medium containing 100 μg / mL ampicillin, and a plasmid was extracted from the cultured E. coli using a NucleoBond Xtra Midi kit (Takara Bio Inc.). The nucleotide sequence of the resulting plasmid was determined by a method known to those skilled in the art, and it was confirmed that the protein with the desired amino acid sequence was encoded.

[0105] NekoEPOR MDHLWAPLWPGVGSLCLLLAGAAWAPPPNPLDPKFESKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVGPDNYSFFYQLEGEPWKPCSLHQAPTARGAVRFWCSLPTADASSFVPLELRVTAVSS GAPRYHRIIHINEVVLLDPPAGLLARRADEGGHVVLRWLPPPGAPVASLIRYEVNISSGNVAGGAQKVEILDGRTECALSNLRGRTRYTFVMVRARMAEPSFGGFWSAWSEPASLLTASDLDPLILTLSL ILVLILLLLAVLALLSHRRTLKQKIWPGIPSPESEFEGLFTTHKGNFQLWLYQNEGCLWWSPCAPFAEDPPSPLEVLSERCWGATQAAEPGAEEGPLLEPLGSEHTQDTYLVLDKWLLPRNPPSEDLPRPDGSLDMVAMHKGSEASSCSSALSLKPGPEGALGASFEYTILDPSSQLLRPRALPPELPPTPPHIKYLYLMVSDSGISTDYSSGGSQEAQGDSSTGPYLNPYENSLIPATETSPPSYVACS (SEQ ID NO: 16)

[0106] Mouse EPOR MDKLRVPLWPRVGPLCLLLAGAAWAPSPSLPDPKFESKAALLASRGSEELLCFTQRLEDLVCFWEEAASSGMDFNYSFSYQLEGESRKSCSLHQAPTVRGSVRFWCSLPTADTSSFVPLELQVTEASG SPRYHRIIHINEVVLLDAPAGLLARRAEEGSHVVLRWLPPPGAPMTTHIRYEVDVSAGNRAGGTQRVEVLEGRTECVLSNLRGGTRYTFAVRARMAEPSFSGFWSAWSEPASLLTASDLDPLILTLSLLI LVLISLLLTVLALLSHRRTLQQKIWPGIPSPESEFEGLFTTHKGNFQLWLLQRDGCLWWSPGSSFPEDPPAHLEVLSEPRWAVTQAGDPGADDEGPLLEPVGSEHAQDTYLVLDKWLLPRTPCSENLSGPGGSVDPVTMDEASETSSCPSDLASKPRPEGTSPSSFEYTILDPSSQLLCPRALPPELPPTPPHLKYLYLVVSDSGISTDYSSGGSQGVHGDSSDGPYSHPYENSLVPDSEPLHPGYVACS (SEQ ID NO: 17)

[0107] Example 11. Establishment of feline EPOR and mouse EPOR-expressing Ba / F3 cell lines To obtain cell lines showing feline EPO- and mouse EPO-dependent growth, we established Ba / F3 cell lines expressing feline EPOR and mouse EPOR, respectively. Details are given below. 10 μg of pBApo-EF1α Pur / cat EPOR and pBApo-EF1α Pur / mouse EPOR constructed in Example 10 were aliquoted and transfected into Ba / F3 cells (0.8×10 7The resulting mixture was mixed with feline EPO-His and 13.16 nM EPO-His. The resulting mixture was mixed with feline EPO-His and 13.16 nM EPO-His. The resulting mixture was pulsed at 0.33 kV and 950 μFD using a Gene Pulser (Bio-Rad). The Ba / F3 cells transfected by electroporation were cultured overnight in RPMI1640 medium (Thermo Fisher Scientific) containing 0.4 ng / mL mouse interleukin-3 (R&D Systems), 10% fetal bovine serum (FBS, Thermo Fisher Scientific), and 100 U / mL penicillin / streptomycin. Wild-type feline EPO-His or mouse EPO-His (prepared in Example 5) was added to the cells to select for EPO-His-dependent proliferation. Two final concentrations of EPO-His were tested: feline EPO-His for feline EPOR gene transfection and mouse EPO-His for mouse EPOR gene transfection. Using the above procedure, we established feline EPOR-expressing Ba / F3 cell lines (BaF3 / cat EPOR) and mouse EPOR-expressing Ba / F3 cell lines (BaF3 / mouse EPOR). We confirmed that the established BaF3 / cat EPOR proliferated in a feline EPO-His-dependent manner, and the BaF3 / mouse EPOR proliferated in a mouse EPO-His-dependent manner.

[0108] Example 12. Evaluation of in vitro activity of feline EPO-Fc using BaF3 / mouse EPO We confirmed whether feline EPO-Fc (C165R cat EPO-siCFV3) prepared in Example 8 can induce the proliferation of BaF3 / mouse EPO established in Example 11 in a concentration-dependent manner. As controls for feline EPO-Fc, the activities of wild-type feline EPO-His prepared in Example 5, wild-type mouse EPO-His, and purchased wild-type human EPO (PeproTech) were also evaluated. Details are described below. After washing the BaF3 / mouse EPOR three times with RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific) and 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific), 1.5 × 10 5 The cell number was adjusted to 1000 cells / mL with the same medium, and 50 μL of each was seeded into each well of a 96-well plate (CORNING). Eight 4-fold dilutions of ligands (feline EPO-Fc, feline EPO-His, mouse EPO-His, and human EPO) were prepared starting from 13.16 nM using the same medium as the diluent, and 50 μL of each was dispensed into each well of the 96-well plate containing the seeded cells. The cells were cultured at 37°C and 5% CO for 2 days. Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL / well, and after 2 hours of culture, the absorbance at 450 nm (reference wavelength 620 nm) was measured again to evaluate the biological activity of each ligand. The results are shown in Figure 8. We were able to confirm in vitro that feline EPO-His and feline EPO-Fc act on mouse EPO in a concentration-dependent manner. We also believe that feline EPO-Fc may exhibit physiological activity in in vivo experiments in which it is administered to individual mice.

[0109] Example 13. Evaluation of in vitro activity of feline EPO-Fc using BaF3 / cat EPOR We confirmed whether feline EPO-Fc (C165R cat EPO-siCFV3) prepared in Example 8 could induce the proliferation of BaF3 / cat EPOR established in Example 11 in a concentration-dependent manner. As controls for feline EPO-Fc, the activities of wild-type feline EPO-His prepared in Example 5 and purchased wild-type human EPO (PeproTech) were also evaluated at the same time. Details are described below. After washing the BaF3 / cat EPOR three times with RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific) and 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific), 1.5 × 10 5The cell number was adjusted to 1000 cells / mL with the same medium, and 50 μL of each was seeded into each well of a 96-well plate (CORNING). Eight 4-fold dilutions of the ligands (feline EPO-Fc, feline EPO-His, and human EPO) were prepared starting from 13.16 nM using the same medium as the diluent, and 50 μL of each was dispensed into each well of the 96-well plate containing the seeded cells. The cells were cultured at 37°C and 5% CO for 2 days. Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL / well, and after 2 hours of culture, the absorbance at 450 nm (reference wavelength 620 nm) was measured again to evaluate the biological activity of each ligand. The results are shown in Figure 9. We were able to confirm that feline EPO-Fc induces proliferation of BaF3 / cat EPOR cells in a concentration-dependent manner. Furthermore, feline EPO-Fc was shown to be more active than feline EPO-His. This suggests that feline EPO-Fc may also exhibit physiological activity in in vivo experiments in which it is administered to individual cats. [Industrial Applicability]

[0110] The EPO-Fc fusion protein of the present invention has excellent physical properties and activity, and can be obtained in high yield / high amount according to the present invention. This application is based on Japanese Patent Application No. 2020-101237, filed on June 10, 2020, the contents of which are incorporated herein by reference in their entirety.

Claims

1. A fusion protein comprising an erythropoietin polypeptide fused to an Fc region of IgG from a feline, a fusion protein, wherein the erythropoietin polypeptide is of feline origin and has at least 90% sequence identity with the corresponding sequence of wild-type erythropoietin and has at least one mutation, C165R; Here, the fusion to the Fc region of the erythropoietin polypeptide is via a hinge region of a feline IgG, and the hinge region has at least one mutation, which is a substitution of at least one cysteine ​​in its wild-type sequence with a glycine.

2. A fusion protein described in claim 1, wherein the erythropoietin polypeptide has an amino acid sequence represented by sequence number 10.

3. The fusion protein according to claim 1 or 2, wherein the hinge region having at least one mutation has an amino acid sequence represented by any one of SEQ ID NOs: 5 to 8.

4. The fusion protein according to any one of claims 1 to 3, which has an amino acid modification in the Fc region.

5. The amino acid modification of the Fc region is (i) substitution of serine at position 252 with tyrosine or threonine; (ii) substitution of serine at position 254 with threonine; (iii) substitution of threonine at position 256 with glutamic acid; (iv) substitution of valine at position 259 with isoleucine; (v) substitution of isoleucine at position 308 with proline or phenylalanine; (vi) substitution of serine at position 428 with leucine; (vii) substitution of histidine at position 433 with leucine; (viii) substitution of serine at position 434 with alanine, tyrosine, or phenylalanine; (ix) substitution of histidine at position 436 with threonine; (x) substitution of glutamine at position 438 with arginine, and (xi) substitution of serine at position 440 with glutamic acid; (xii) substitution of leucine at position 235 with arginine, (xiii) substitution of glycine at position 236 with arginine, and (xiv) substitution of serine at position 239 with lysine 5. The fusion protein of claim 4, comprising at least one selected from the group consisting of: wherein the numbering of amino acids in the Fc region is according to the EU index of Kabat based on the Fc region of a human antibody.

6. The amino acid modification of the Fc region is (i) substitution of serine at position 434 with alanine; (ii) substitution of histidine at position 436 with threonine; (iii) substitution of glutamine at position 438 with arginine, and (iv) substitution of serine at position 440 with glutamic acid The fusion protein according to claim 5,

7. The amino acid modification of the Fc region is (i) substitution of serine at position 428 with leucine; (ii) substitution of serine at position 434 with alanine; (iii) substitution of histidine at position 436 with threonine; (iv) substitution of glutamine at position 438 with arginine, and (v) substitution of serine at position 440 with glutamic acid The fusion protein according to claim 5,

8. The amino acid modification of the Fc region is (i) substitution of serine at position 428 with leucine; (ii) substitution of serine at position 434 with alanine; (iii) substitution of glutamine at position 438 with arginine, and (iv) substitution of serine at position 440 with glutamic acid The fusion protein according to claim 5,

9. The amino acid modification of the Fc region is (i) substitution of serine at position 252 with tyrosine; (ii) substitution of serine at position 254 with threonine, and (iii) substitution of threonine at position 256 with glutamic acid The fusion protein according to claim 5,

10. The amino acid modification of the Fc region is (i) substitution of isoleucine at position 308 with proline, and (ii) substitution of serine at position 434 with tyrosine The fusion protein according to claim 5,

11. The amino acid modification of the Fc region is (i) substitution of valine at position 259 with isoleucine; (ii) substitution of isoleucine at position 308 with phenylalanine, and (iii) substitution of serine at position 428 with leucine The fusion protein according to claim 5,

12. The amino acid modification of the Fc region is (i) substitution of serine at position 252 with threonine; (ii) substitution of serine at position 254 with threonine; (iii) substitution of threonine at position 256 with glutamic acid; (iv) substitution of histidine at position 433 with leucine, and (v) substitution of serine at position 434 with phenylalanine The fusion protein according to claim 5,

13. The amino acid modification of the Fc region is (vi) substitution of serine at position 428 with leucine; (viii) substitution of serine at position 434 with alanine; (x) substitution of glutamine at position 438 with arginine, (xi) substitution of serine at position 440 with glutamic acid; (xii) substitution of leucine at position 235 with arginine, (xiii) substitution of glycine at position 236 with arginine, and (xiv) substitution of serine at position 239 with lysine The fusion protein according to claim 5,

14. The fusion protein according to claim 4, wherein the Fc region of the IgG having the amino acid modification has the amino acid sequence represented by SEQ ID NO: 3 or 4.

15. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 14.

16. 16. The pharmaceutical composition according to claim 15, which is for treating a hematopoietic disease or hematopoietic deficiency.

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