Recombinant protein containing feline granulocyte colony-stimulating factor and serum albumin antigen-binding fragment, and its uses

Abstract

The present invention provides a composition for treating feline panpercytopenia, which relates to a recombinant protein comprising an antigen-binding fragment that binds to feline granulocyte colony-stimulating factor and serum albumin, a nucleic acid molecule, a vector, and a cell encoding the recombinant protein, and uses thereof.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims priority to KR No. 10 - 2020 - 0043606, filed on April 9, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [Reference to Electronically Submitted Sequence Listing] The content of the electronically submitted sequence listing of the ASCII text file (name: 2662 - 0002WO01_Sequence_Listing_ST25.txt, size: 48 KB, date of generation: April 9, 2021) submitted together with this application is incorporated herein by reference in its entirety.

[0003] [Technical Field] The present invention relates to a recombinant protein comprising an antigen - binding fragment that binds to feline granulocyte colony - stimulating factor and serum albumin, a nucleic acid molecule encoding the recombinant protein, a vector, a cell, a composition, and uses thereof.

Background Art

[0004] Feline panleukopenia is a viral enteritis induced by feline parvovirus (FPV), which is highly contagious, has a high mortality rate, and is one of the most fatal diseases for all cat breeds. Currently, the treatment of feline panleukopenia includes transfusion of whole blood to increase the white blood cell count, administration of granulocyte colony-stimulating factor (GCSF), or intravenous administration of infusions containing antibiotics and vitamins A, B, C, etc. to prevent dehydration due to sepsis. However, intravenous administration of infusions containing antibiotics, etc. is not a direct treatment for the disease, and transfusion also has the problem that it is difficult to secure a sufficient amount of whole blood. Administration of GCSF is widely used in the treatment of feline panleukopenia, but the recombinant GCSF used in the treatment is human GCSF (hGCSF) derived from humans, not cats. Therefore, if used for a long time, anti-drug antibodies (ADA) against hGCSF are generated, which not only reduces the drug efficacy and therapeutic efficacy, but also induces serious side effects such as acute immune reactions and autoimmune diseases.

[0005] In addition, feline GCSF, one of the hormones secreted in the cat's body, is known as a protein that regulates the production of blood cells circulating in the bone marrow. Specifically, the GCSF stimulates the proliferation and differentiation of neutrophils, increases the neutrophil value in the blood, shortens the neutropenia period, and contributes to the recovery of immunity. However, the half-life of the protein in vivo is only about 4 to 5 hours, and in order to maintain the therapeutic efficacy for a long time, it must be administered many times. For such reasons, PEGylation or hyperglycosylation, which conjugates a polymer such as a poly(alkylene glycol) derivative to GCSF, is widely used (Korean Patent Publication No. 10-2010-0052501). It also involves problems such as protein denaturation and the possibility of immunogenicity occurring during the chemical fusion process.

Summary of the Invention

Means for Solving the Problems

[0006] (a) An antigen-binding fragment comprising a heavy chain and a light chain, and (b) a recombinant protein comprising feline granulocyte colony-stimulating factor (fGCSF) are disclosed in the present application. Here, the heavy chain comprises a heavy chain variable domain and a feline heavy chain constant 1 domain, where the heavy chain variable domain (1) Heavy chain complementarity-determining region 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO: 51), Heavy chain complementarity-determining region 2 (CDR2) comprising the amino acid sequence of WINTYSGTKYAQKFQG (SEQ ID NO: 52), and Heavy chain complementarity-determining region 3 (CDR3) comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO: 53); (2) Heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO: 51), Heavy chain CDR2 comprising the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO: 54), and Heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO: 53); (3) Heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO: 55), Heavy chain CDR2 comprising the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO: 56), and Heavy chain CDR3 comprising the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO: 57); (4) Heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 58), Heavy chain CDR2 comprising the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO: 59), and Heavy chain CDR3 comprising the amino acid sequence of AGWLRQYGMDV (SEQ ID NO: 60); (5) Heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO: 61), Heavy chain CDR2 comprising the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO: 62), and Heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO: 63); or (6) Heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO: 64), A heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO: 65), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO: 66); wherein the light chain comprises a light chain variable domain and a feline light chain constant domain, and wherein the light chain variable domain (7) a light chain CDR1 comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO: 67), a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO: 68), and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO: 69); (8) a light chain CDR1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 70), a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO: 71), and a light chain CDR3 comprising the amino acid sequence of QQSYSTPPYT (SEQ ID NO: 72); (9) a light chain CDR1 comprising the amino acid sequence of RASQSIFNYVA (SEQ ID NO: 73), a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO: 74), and a light chain CDR3 comprising the amino acid sequence of QQRSKWPPTWT (SEQ ID NO: 75); (10) a light chain CDR1 comprising the amino acid sequence of RASETVSSRQLA (SEQ ID NO: 76), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 77), and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO: 78); (11) a light chain CDR1 comprising the amino acid sequence of RASQSVSSSSLA (SEQ ID NO: 79), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 77), and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO: 80); or (12) a light chain CDR1 comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO: 81), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO: 82), and It includes a light chain CDR3 containing the amino acid sequence of QQYYSFLAKT (SEQ ID NO: 83).

[0007] The recombinant protein may further include a linker that links the fGCSF to the antigen-binding fragment. In some embodiments, the cysteine(s) located in the interchain disulfide bond between the light chain and the heavy chain, (i) cysteine within the feline heavy chain constant 1 domain and / or (ii) cysteine within the feline light chain constant domain, are conserved, deleted, and / or substituted with amino acid residues other than cysteine.

[0008] In some embodiments of the recombinant protein disclosed in the present application, the heavy chain variable domain includes a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 64, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 65, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 66, and the light chain variable domain includes a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 81, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 82, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 83.

[0009] In some embodiments, the heavy chain variable domain includes an amino acid sequence having at least 80% identity to SEQ ID NOs: 1, 2, 3, 4, 5, or 6.

[0010] In some embodiments, the light chain variable domain includes an amino acid sequence having at least 80% identity to SEQ ID NOs: 7, 8, 9, 10, 11, 12, or 13.

[0011] In some embodiments, the heavy chain variable domain includes the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, and the light chain variable domain includes SEQ ID NOs: 7, 8, 9, 10, 11, 12, or 13.

[0012] In some embodiments, the heavy chain constant 1 domain of the cat comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 14. In some embodiments, the light chain constant domain of the cat comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 15.

[0013] In some embodiments, the fGCSF is modified from natural fGCSF by removing free cysteine groups and O-glycans. In some embodiments, the fGCSF comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 18. In some embodiments, the fGCSF comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 19. In some embodiments, the fGCSF comprises the amino acid sequence of SEQ ID NO: 19.

[0014] In some embodiments, the linker links fGCSF to the C-terminus of the cat heavy chain constant 1 domain, the N-terminus of the heavy chain variable domain, the C-terminus of the cat light chain constant domain, and / or the N-terminus of the light chain variable domain. In some embodiments, the linker comprises 1 to 50 amino acids, or 1 to 20 amino acids. In some embodiments, the linker comprises the formula (G p S s ) n or (S p G s ) n wherein G is glycine, S is serine, p is an integer from 1 to 10, s is 0, or an integer from 1 to 10, p + s is an integer of 20 or less, and n is an integer from 1 to 20.

[0015] Nucleic acid molecules encoding the recombinant proteins disclosed in the present application are disclosed in the present application. Expression vectors containing the nucleic acid molecules disclosed in the present application are disclosed in the present application. Cells transformed with the expression vectors disclosed in the present application are disclosed in the present application.

[0016] Compositions comprising the recombinant proteins disclosed in this application are disclosed in this application. Pharmaceutical compositions comprising the compositions disclosed in this application and pharmaceutically acceptable excipients are disclosed in this application. Kits comprising the compositions disclosed in this application and labels containing instructions for use thereof are disclosed in this application.

[0017] Disclosed herein is a method for treating feline panleukopenia, comprising administering to an individual in need thereof the compositions disclosed herein. In some embodiments, the composition increases the white blood cells in the blood of the individual. In some embodiments, the white blood cells are neutrophils, monocytes, basophils, or combinations thereof.

[0018] Also disclosed in this application is the use of the compositions disclosed in this application for treating feline panleukopenia in an individual in need thereof. Also disclosed in this application is the composition disclosed in this application for use in treating feline panleukopenia in an individual in need thereof. Also disclosed in this application is the use of the compositions disclosed in this application for the manufacture of a medicament for treating feline panleukopenia in an individual in need thereof.

[0019] The foregoing, as well as other aspects, features, and advantages of the specific embodiments of the present disclosure will become more apparent from the following description taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0041] As used herein, the terms “about” and “approximately,” when used to modify a numerical value or numerical range, indicate that a deviation of up to 10% more and up to 10% less than the recited numerical value or numerical range is intended to be maintained within the meaning.

[0042] As used herein, the term “prevent” means all acts of suppressing feline panleukopenia or delaying its onset by administration of the pharmaceutical composition.

[0043] As used herein, the term “treat” means all acts of improving, ameliorating, disappearing, or favorably changing the symptoms of feline panleukopenia by administration of the pharmaceutical composition.

[0044] As used herein, the term “individual” means an individual in need of treatment for feline panleukopenia, and more specifically, may refer to a feline, a cat, a domestic cat, for example, a pet cat.

[0045] “Feline panleukopenia,” which is the disease to be prevented or treated by the pharmaceutical composition, is a disease characterized by marked reduction of white blood cells, along with clinical symptoms such as bloody stools, diarrhea, severe dehydration, and malnutrition. It is a highly contagious and lethal disease, and is one of the diseases that are fatal to all cat breeds. The feline panleukopenia is also caused by infection with feline parvovirus (FPV). As a treatment method for the disease, administration of granulocyte colony-stimulating factor (G-CSF) is widely used, but there are treatment limitations such as the generation of anti-drug antibodies (ADA).

[0046] [Antibodies and Fragments Thereof] As used herein, it is a recombinant protein comprising (a) an antigen-binding fragment containing a heavy chain and a light chain, and (b) feline granulocyte colony-stimulating factor (fGCSF). Here, the heavy chain includes a heavy chain variable domain and a feline heavy chain constant 1 domain, where the heavy chain variable domain is (1) a heavy chain complementarity determining region 1 (CDR1) including the amino acid sequence of SYGIS (SEQ ID NO: 51), a heavy chain complementarity determining region 2 (CDR2) including the amino acid sequence of WINTYSGTKYAQKFQG (SEQ ID NO: 52), and a heavy chain complementarity determining region 3 (CDR3) including the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO: 53); (2) a heavy chain CDR1 including the amino acid sequence of SYGIS (SEQ ID NO: 51), a heavy chain CDR2 including the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO: 54), and a heavy chain CDR3 including the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO: 53); (3) a heavy chain CDR1 including the amino acid sequence of NYGIH (SEQ ID NO: 55), a heavy chain CDR2 including the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO: 56), and a heavy chain CDR3 including the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO: 57); (4) a heavy chain CDR1 including the amino acid sequence of SYAMS (SEQ ID NO: 58), a heavy chain CDR2 including the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO: 59), and a heavy chain CDR3 including the amino acid sequence of AGWLRQYGMDV (SEQ ID NO: 60); (5) a heavy chain CDR1 including the amino acid sequence of AYWIA (SEQ ID NO: 61), a heavy chain CDR2 including the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO: 62), and a heavy chain CDR3 including the amino acid sequence of LYSGSYSP (SEQ ID NO: 63); or (6) a heavy chain CDR1 including the amino acid sequence of AYSMN (SEQ ID NO: 64), a heavy chain CDR2 including the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO: 65), and comprising a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO: 66), wherein said light chain comprises a light chain variable domain and a feline light chain constant domain, wherein said light chain variable domain (7) a light chain CDR1 comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO: 67), a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO: 68), and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO: 69); (8) a light chain CDR1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 70), a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO: 71), and a light chain CDR3 comprising the amino acid sequence of QQSYSTPPYT (SEQ ID NO: 72); (9) a light chain CDR1 comprising the amino acid sequence of RASQSIFNYVA (SEQ ID NO: 73), a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO: 74), and a light chain CDR3 comprising the amino acid sequence of QQRSKWPPTWT (SEQ ID NO: 75); (10) a light chain CDR1 comprising the amino acid sequence of RASETVSSRQLA (SEQ ID NO: 76), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 77), and a light chain CDR3 comprising the amino acid sequence of QQYGSSEPRT (SEQ ID NO: 78); (11) a light chain CDR1 comprising the amino acid sequence of RASQSVSSSSLA (SEQ ID NO: 79), a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 77), and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO: 80); or (12) a light chain CDR1 comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO: 81), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO: 82), and comprising a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO: 83).

[0047] The recombinant protein may also be one that further includes a linker that links the fGCSF to an antigen-binding fragment. In some embodiments, the cysteine(s) located in the interchain disulfide bond between the light chain and the heavy chain, (i) within the feline heavy chain constant 1 domain, and / or (ii) within the feline light chain constant domain, is / are conserved, deleted, and / or substituted with an amino acid residue other than cysteine.

[0048] In some embodiments of the recombinant protein disclosed in the present application, the heavy chain variable domain includes a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 64, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 65, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 66, and the light chain variable domain includes a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 81, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 82, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 83.

[0049] In some embodiments, the heavy chain variable domain has at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, or 6.

[0050] In some embodiments, the light chain variable domain has at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13.

[0051] In some embodiments, the heavy chain variable domain includes the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6, and the light chain variable domain includes SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13.

[0052] In some embodiments, the heavy chain constant 1 domain of the cat has at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14.

[0053] In some embodiments, the light chain constant domain of the cat has at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15.

[0054] In some embodiments, the linker links fGCSF to the C-terminus of the heavy chain constant 1 domain of the cat, the N-terminus of the heavy chain variable domain, the C-terminus of the light chain constant domain of the cat, and / or the N-terminus of the light chain variable domain. In some embodiments, the linker comprises 1 to 50 amino acids, or 1 to 20 amino acids. In some embodiments, the linker comprises (G p S s ) n or (S p G s ) n wherein G is glycine, S is serine, p is an integer from 1 to 10, s is 0, or an integer from 1 to 10, p + s is an integer of 20 or less, and n is an integer from 1 to 20.

[0055] As used herein, the recombinant protein can be produced by fusing the FL355Fab antibody fragment, which is a feline chimeric antibody fragment, with feline GCSF. The recombinant protein has improved pharmacokinetic properties and has been demonstrated to increase white blood cells to therapeutically effective levels in the treatment of feline panleukopenia. Accordingly, disclosed herein is a recombinant protein comprising an antigen-binding fragment that binds to serum albumin and feline granulocyte colony-stimulating factor.

[0056] As used herein, the term "heavy chain (HC or CH: heavy chain)" means any full-length heavy chain and fragments thereof that include a variable region domain VH having an amino acid sequence with a sufficient variable region (VR: variable region) sequence to confer antigen specificity, and three constant region domains CH1, CH2, and CH3. As used herein, the term "light chain (LC or CL: light chain)" means any full-length light chain and fragments thereof that include a variable region domain VL having an amino acid sequence with a sufficient variable region sequence to confer antigen specificity, and a constant region domain CL.

[0057] In some embodiments, the antigen-binding fragment that binds to the albumin can be chimerized by including, for example, a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and a feline heavy chain constant 1 domain linked to said domain; and, for example, a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, and a feline light chain constant domain linked to said domain.

[0058] The heavy chain constant 1 domain of the cat and the light chain constant domain of the cat may be derived from the IgG1 antibody constant domain, and one or more of them may have a cysteine, which is an amino acid used for a disulfide bond between the light chain domain and the heavy chain domain, conserved, deleted, or substituted with another amino acid residue other than cysteine. For example, the heavy chain constant 1 domain of the cat may include the amino acid sequence of SEQ ID NO: 14, and the light chain constant domain of the cat may include the amino acid sequence of SEQ ID NO: 15. Deletion or substitution of cysteine within the domain may contribute to improving the expression level of the recombinant protein in the transformed cells during the process of producing the aforementioned recombinant protein. In some embodiments, the cysteine in (i) the heavy chain constant 1 domain of the cat and / or (ii) the light chain constant domain of the cat, which is located in the inter-chain disulfide bond between the light chain and the heavy chain, is conserved, deleted, or substituted with another amino acid residue other than cysteine.

[0059] In some embodiments, the cat chimeric antigen-binding fragment binding may also include a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identity to SEQ ID NO: 16, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identity to SEQ ID NO: 17.

[0060] The feline granulocyte colony-stimulating factor (fGCSF) may also be a non-mutated natural protein, which can be obtained from publicly available databases such as https: / / www.ncbi.nlm.nih.gov / , and may include, for example, but is not limited to, the amino acid sequence of SEQ ID NO: 18. In some embodiments, the feline granulocyte colony-stimulating factor may also be modified from the natural granulocyte colony-stimulating factor by removing free cysteine groups and O-glycans, and may include, for example, but is not limited to, the amino acid sequence of SEQ ID NO: 19. The removal of the free cysteine groups and O-glycans can provide convenience in the production, separation, and purification processes of recombinant proteins.

[0061] In some embodiments, the fGCSF includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18. In some embodiments, the fGCSF includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19. In some embodiments, the fGCSF includes the amino acid sequence of SEQ ID NO: 19.

[0062] In some embodiments, the antigen-binding fragment that binds to the serum albumin and the feline granulocyte colony-stimulating factor can be linked to each other via a linker. For example, the linker can link any one region selected from the C-terminus of the heavy-chain constant 1 domain of the antigen-binding fragment, the N-terminus of the heavy-chain variable domain, the C-terminus of the light-chain constant domain, and the N-terminus of the light-chain variable domain to the granulocyte colony-stimulating factor. Further, the linker can be appropriately modified and used as needed. For example, the linker can be a polypeptide consisting of 1 to 50, or 1 to 20 random or non-random amino acids. The peptide linker can contain Gly residues, Asn residues, and Ser residues, and can also contain neutral amino acids such as Thr and Ala. Amino acid sequences suitable for peptide linkers are known in the art. The copy number "n" can be adjusted in consideration of linker optimization to achieve appropriate separation between functional moieties or to maintain inter-moiety interactions as necessary. Other linkers are also known in the art. For example, in order to improve water solubility, not only polar amino acid residues are added, but also G linkers and S linkers with amino acid residues such as T and A added to maintain flexibility. Therefore, the linker can also be a flexible linker containing G, S residues and / or T, A residues. The linker can have a general formula selected from among (G p S s ) n and (S p G s ) n , in which case, independently, p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p + s is an integer of 20 or less, and n is an integer from 1 to 20. More specifically, examples of the linker are (GGGGS) n (SEQ ID NO: 40), (SGGG) n (SEQ ID NO: 41), (SRSSG) n (SEQ ID NO: 42), (SGSSC) n (SEQ ID NO: 43), (GKSSGSGSESKS) n (SEQ ID NO: 44), (RPPPPC)n (SEQ ID NO: 45), (SSPPPPC) n (SEQ ID NO: 46), (GSTSGSGKSSEGKG) n (SEQ ID NO: 47), (GSTSGSGKSEGSGSTKG) n (SEQ ID NO: 48), (GSTSGSGKPGSGEGSTKG) n (SEQ ID NO: 49) or (EGKSSGSGSESKEF) n It may also include (SEQ ID NO: 50), where n can be an integer from 1 to 20, or from 1 to 10.

[0063] In some embodiments, the linker links fGCSF to the C-terminus of the feline heavy chain constant 1 domain, the N-terminus of the heavy chain variable domain, the C-terminus of the feline light chain constant domain, and / or the N-terminus of the light chain variable domain. In some examples, the linker is (G p S s ) n or (S p G s ) n and includes the formula, where G is glycine, S is serine, p is an integer from 1 to 10, s is 0 or an integer from 1 to 10, p + s is an integer of 20 or less, and n is an integer from 1 to 20. Also, the linker may include the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, but is not limited thereto.

[0064] In some embodiments, the recombinant protein can also be composed of a combination of heavy chain recombinant proteins comprising antigen-binding fragments that bind to feline serum albumin, wherein the antigen-binding fragment comprises a heavy chain variable domain and a feline heavy chain constant 1 domain, and feline granulocyte colony-stimulating factor linked to the N-terminus of the feline heavy chain constant domain; and is bound to an antigen-binding fragment that binds to feline serum albumin, wherein the antigen-binding fragment is linked to a light chain variable domain and a feline light chain constant domain. Here, the heavy chain recombinant protein may also contain an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, 100% identity to SEQ ID NO: 22 or 23, and the antigen-binding fragment containing the light chain variable domain may also contain an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. The recombinant protein can have significantly improved pharmacokinetic properties while maintaining the intrinsic biological activity of feline granulocyte colony-stimulating factor.

[0065] As used herein, the terms "antibody" (antibody and antibodies) are terms in the art and can be used interchangeably to refer to a molecule having an antigen-binding site that specifically binds to an antigen. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, feline antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain - antibody heavy chain pairs, intrabodies, hetero - conjugate antibodies, single - domain antibodies, monovalent antibodies, single - chain antibodies or single - chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab’)2 fragments, disulfide - linked Fv (sdFv), anti - idiotype (anti - Id) antibodies (including, for example, anti - anti - Id antibodies), bispecific antibodies, and multispecific antibodies.

[0066] The antibody can be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b of an immunoglobulin molecule). In some embodiments, the antibody is a feline chimeric antibody.

[0067] As used herein, the terms "bioeffector moiety", "antigen - binding domain", "antigen - binding region", "antigen - binding site", and similar terms mean the portion of a recombinant protein that contains the amino acid residues that confer specificity for an antigen to the recombinant protein (e.g., complementarity - determining regions (CDRs)). The antigen - binding region can be derived from any animal species such as feline, rodents (e.g., mouse, rat or hamster), and humans.

[0068] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region typically refers to a part of an antibody, generally a part of the light or heavy chain, typically about 110 to 120 amino acids in the mature heavy chain, and about 90 to 115 amino acids in the mature light chain, which has particularly different sequences between antibodies and is used for the binding and specificity of a specific antibody to a specific antigen. The variability of the sequences is concentrated in regions called complementarity-determining regions (CDRs), while in the variable domain, more highly conserved regions are called framework regions (FRs). Without being bound by any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be the main cause of the interaction and specificity between the antigen and the antibody. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent CDRs or mouse CDRs and a human framework region (FR). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent CDRs or mouse CDRs and a primate (e.g., non-human primate) framework region (FR).

[0069] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody. The terms "VH" and "VH domain" are used interchangeably to refer to the variable region of the heavy chain of an antibody.

[0070] The terms "Kabat numbering" and similar terms are recognized in the art and are systems for numbering amino acid residues within the heavy chain variable region and the light chain variable region of an antibody, or within its antigen-binding portion. In certain embodiments, the CDRs of an antibody can be determined by the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically at amino acid positions 31 to 35 (optionally including one or two additional amino acids following position 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some embodiments, the CDRs of the antibodies described herein are determined by the Kabat numbering scheme.

[0071] As used herein, the terms "constant region" or "constant domain" are interchangeable and have a common meaning in the art. The constant region is the portion of the antibody, such as the carboxyl-terminal portion of the light chain and / or heavy chain, that does not directly participate in binding of the antibody to an antigen but can exhibit various effector functions such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have more conserved amino acid sequences compared to the immunoglobulin variable domains.

[0072] As used herein, the term "heavy chain," when used in connection with an antibody, can refer to any distinct type based on the amino acid sequence of the constant region, for example, alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which generate antibodies of the IgA, IgD, IgE, IgG, and IgM types, respectively, and include subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0073] As used herein, the term "light chain," when used in connection with an antibody, can refer to any distinct type, for example, kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0074] "Binding affinity" generally refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be indicated by the dissociation constant (K D ). The affinity can be measured and / or expressed in a variety of ways known in the art, including, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). The K D is calculated from the quotient of k off / k on , while the K A is calculated from the quotient of k on / k off . The k on refers to, for example, the association rate constant of an antibody for an antigen, and the k off refers to, for example, the dissociation rate constant of an antibody for an antigen. The k on and k off can be determined by techniques known to those of skill in the art, such as BIAcore® or KinExA.

[0075] As used herein, "conservative amino acid substitution" refers to an amino acid residue being substituted with an amino acid residue having a similar side chain. Amino acid residue families having side chains are defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within the CDR or framework region of an antibody can be replaced with amino acid residues having similar side chains.

[0076] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which an antibody can specifically bind. The epitope can be, for example, a continuous amino acid of a polypeptide (linear epitope or continuous epitope), or the epitope can, for example, come together from two or more non-contiguous regions of one polypeptide or a plurality of polypeptides (conformational, non-linear, discontinuous or discontinuous epitope). In certain embodiments, the epitope to which the antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning analysis, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).Antibody:antigen crystals are also studied using well-known X-ray diffraction techniques and refined to high precision using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be achieved using any method known to those skilled in the art. For an explanation of mutagenesis techniques including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394, and Cunningham BC & Wells JA (1989) Science 244: 1081-1085. In some embodiments, the epitope of the antibody is determined using alanine scanning mutagenesis studies.

[0077] As used herein, the terms “immunologically specifically bind,” “immunologically specifically recognize,” “specifically bind,” and “specifically recognize” are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope, an immune complex, or a binding partner of an antigen-binding site) as such binding is understood by one of ordinary skill in the art. For example, a molecule that specifically binds to an antigen generally binds to other peptides or polypeptides with a lower affinity, as determined, for example, by an immunoassay, BIAcore®, a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, a molecule that immunologically specifically binds to an antigen binds to the antigen with a K A that is at least 2 log, 2.5 log, 3 log, 4 log, or even greater than the K A for binding to that antigen compared to when the molecule binds to other antigens.

[0078] In other embodiments, a molecule that immunologically specifically binds to an antigen does not cross-react with other proteins under similar binding conditions. In some embodiments, a molecule that immunologically specifically binds to an antigen does not cross-react with other proteins. In some embodiments, provided herein are recombinant proteins that bind to a specific antigen with a higher affinity than to other, non-related antigens. In certain embodiments, provided herein are recombinant proteins that bind to a specific antigen (e.g., human serum albumin). The recombinant protein binds to the specific antigen with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more higher affinity than to other antigens or non-related antigens, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In some embodiments, the degree of binding of the recombinant proteins described herein to non-related proteins is less than 10%, 15%, or 20% of the binding of an antibody to a specific antigen as measured, for example, by radioimmunoassay.

[0079] In some embodiments, the present application provides recombinant proteins that bind to antigens of various species such as cats, rodents (e.g., mice, rats or hamsters) and humans. In some embodiments, the present application provides recombinant proteins that bind to feline antigens with a higher affinity than other species of antigens. In certain embodiments, for example, when measured by radioimmunoassay, surface plasmon resonance or kinetic exclusion analysis, the present application provides recombinant proteins that bind to feline antigens with an affinity 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more higher than other species. In some embodiments, the recombinant proteins described herein bind to feline antigens and bind to less than 10%, 15%, or 20% of the binding of antibodies to feline antigen proteins and bind to antigens of other species when measured, for example, by radioimmunoassay, surface plasmon resonance or kinetic exclusion analysis.

[0080] As used herein, the term "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, a cell derived from a cell line. In embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule, and the progeny, or potential progeny, of such a cell. The progeny of such a cell are not identical to the parental cell transfected with the nucleic acid molecule, for example, due to mutations or environmental effects that may occur in subsequent generations, or integration of the nucleic acid molecule into the host cell genome.

[0081] As used herein, the term "effective amount" refers to the amount of a treatment that achieves the desired prophylactic or therapeutic effect in the context of therapeutic administration to a subject.

[0082] In some embodiments, the Fab comprises a heavy chain variable region domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, or 6.

[0083] In some embodiments, the Fab comprises a light chain variable region domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13.

[0084] In some embodiments, the Fab comprises a heavy chain variable region domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, or 6, and a light chain variable region domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13, respectively, or any combination of the foregoing heavy chain variable region domain and light chain variable region domain. For example, the Fab may comprise a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96% or more, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6, and a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96% or more, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13.

[0085] In some embodiments, the Fab comprises a heavy chain domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96% or more, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16 (VHCH1 domain), and a light chain domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96% or more, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17 (VL-CL domain).

[0086] In certain embodiments, the recombinant proteins described herein may also be described by their VL domain alone, or their VH domain alone, or their three VL CDRs alone, or their three VH CDRs alone. For example, see Rader C et al., (1998) PNAS 95: 8910-8915, which is hereby incorporated by reference in its entirety, for identifying complementary light or heavy chains from libraries of human light or heavy chains, performing humanization of a mouse anti-αvβ3 antibody, which provides humanized antibody variants having an affinity that is similarly high or higher than that of the original antibody. Also, see Clackson T et al., (1991) Nature 352: 624-628, which is hereby incorporated by reference in its entirety, for library screening involving complementary variable domains and methods of producing antibodies that bind to a specific antigen using a specific VL domain (or VH domain). For a particular VH domain that is a strong binder as determined by ELISA, 13 new partners were generated, and for a particular VL domain, 14 new partners were generated. Also, see Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is hereby incorporated by reference in its entirety, for library screening (e.g., a human VL library) for complementary VL domains and methods of producing antibodies that bind to a specific antigen using a specific VH domain, and the selected VL domain can also be used to guide the selection of additional complementary (e.g., human) VH domains.

[0087] In certain embodiments, the CDRs of the antibody can be determined by the Chothia numbering system, which indicates the positions of the immunoglobulin structural loops (e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33 or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. When numbering using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system assigns insertions to H35A and H35B, and when neither 35A nor 35B is present, the loop ends at 32, when only 35A is present, the loop ends at 33, and when both 35A and 35B are present, the loop ends at 34).

[0088] In certain embodiments, provided herein are recombinant proteins that specifically bind to serum albumin (e.g., feline serum albumin) and comprise the Chothia VL CDR of VL. In certain embodiments, provided herein are antibodies that specifically bind to serum albumin (e.g., human serum albumin) and comprise the Chothia VH CDR of VH. In certain embodiments, provided herein are antibodies that specifically bind to serum albumin (e.g., human serum albumin), comprise the Chothia VL CDR of VL, and comprise the Chothia VH CDR of VH. In certain embodiments, an antibody that specifically binds to serum albumin (e.g., human serum albumin) comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are antibodies that specifically bind to serum albumin and comprise a combination of Kabat CDRs and Chothia CDRs.

[0089] In certain embodiments, the CDRs of the antibody can be determined by the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7: 132-136, and Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering system, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0090] In certain embodiments, the CDRs of the antibody can be determined by MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, for example, Martin A., "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering, Kontermann, and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0091] In certain embodiments, the CDRs of the antibody are determined by the AbM numbering system, which refers to the AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops and are used by the Oxford Molecular Group antibody modeling software (Oxford Molecular Group, Inc.).

[0092] In some embodiments, the positions of one or more CDRs in the VH (e.g., CDR1, CDR2, or CDR3) region and / or the VL (e.g., CDR1, CDR2, or CDR3) region of the antibodies described in the present application may differ by 1, 2, 3, 4, 5, or 6 amino acid positions within the range in which immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, the positions defining the CDRs of the antibodies described in the present application may differ from the CDR positions of a certain antibody described in the present application by moving the boundaries of the N-terminus and / or C-terminus of the CDR by 1, 2, 3, 4, 5, or 6 amino acids within the range in which immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other examples, the lengths of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) region and / or the VL (e.g., CDR1, CDR2, or CDR3) region of the antibodies described in the present application may differ by 1, 2, 3, 4, 5, or more amino acids (e.g., shorter or longer) within the range in which immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0093] In some embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described in the present application may be 1, 2, 3, 4, 5, or more amino acids shorter than one or more of the CDRs described in the present application, within the range in which immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other examples, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described in the present application may be 1, 2, 3, 4, 5, or more amino acids longer than one or more of the CDRs described in the present application, within the range in which immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other examples, the amino terminus of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described in the present application, compared to one or more of the CDRs described in the present application, within the range in which immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%), It can be extended by 1, 2, 3, 4, 5, or more amino acids. In other embodiments, the carboxy terminus of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be extended by 1, 2, 3, 4, 5, or more amino acids in a range where immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) as compared to one or more of the CDRs described herein. In other embodiments, the amino terminus of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids in a range where immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) as compared to one or more of the CDRs described herein. In other embodiments, the carboxy terminus of the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids in a range where immunospecific binding to the antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) as compared to one or more of the CDRs described herein. Any method known in the art is used to confirm whether immunospecific binding to the antigen is maintained, and for example, the binding assays and their conditions described in the "Examples" section of this application can be used.

[0094] The determination of percent identity between two sequences (e.g., amino acid or nucleic acid sequences) can be accomplished using a mathematical algorithm. Specific, non-limiting examples of mathematical algorithms used for comparing two sequences include the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403. The BLAST nucleotide search is performed with the NBLAST nucleotide program parameters, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. The BLAST protein search is performed with the XBLAST program parameters, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389 3402. Alternatively, PSI BLAST can be used to perform iterative searches to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, e.g., NCBI (National Center for Biotechnology Information) at worldwide web (ncbi.nlm.nih.gov)). Another specific, non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4: 11 17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0095] The percent identity between two sequences can be determined with or without allowing gaps and using techniques similar to those described above. When calculating the percent identity, typically only exact matches are counted.

[0096] The recombinant proteins disclosed in the present application can be fused or conjugated to a detectable label or substance (e.g., covalent or non-covalent linkage). Examples of detectable labels or substances include enzyme labels such as glucose oxidase; radioisotopes such as iodine ( 125 I), 121 carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and also include biotin. Such labeled antibodies can be used to detect antigen proteins.

[0097] [Antibody production] According to one exemplary embodiment, a recombinant protein (APB-F1) containing an antigen-binding fragment that binds to feline serum albumin is produced, where the antigen-binding fragment is linked to a feline heavy chain constant 1 domain and a feline light chain constant domain, and a mutant feline granulocyte colony-stimulating factor fused to the feline heavy chain constant 1 domain. It was confirmed that the recombinant protein could be obtained in a high yield while maintaining the biological activity of each factor.

[0098] Another aspect provides a method for producing a recombinant protein, comprising (a) culturing the cells, and (b) recovering the recombinant protein from the cultured cells. The cells can be cultured in a variety of media. Commercially available media can be used as culture media without limitation. All other essential supplements known to those skilled in the art can also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression and will be apparent to those skilled in the art. Recovery of the recombinant protein can be accomplished, for example, by removing impurities by centrifugation or ultrafiltration and purifying the resultant by, for example, affinity chromatography. Other additional purification techniques, such as anion exchange chromatography or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0099] The recombinant proteins disclosed in the present application can be produced by any method known in the art for antibody synthesis, such as chemical synthesis techniques or recombinant expression techniques. The methods described in the present application use techniques common in the relevant fields of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, synthesis and modification of oligonucleotides, nucleic acid hybridization, and related technical fields, unless otherwise indicated. Such techniques are described, for example, in the references cited herein and are fully explained therein.For example, see Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0100] In some embodiments, the recombinant proteins (e.g., recombinant antibodies) described in the present application are antibodies produced, expressed, generated, or isolated by any means including, for example, synthesis or generation via genetic engineering of DNA sequences. In certain embodiments, such antibodies contain sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur in the in vivo antibody germline repertoire of an animal or mammal (e.g., a human).

[0101] In some aspects, provided herein is a method for producing a recombinant protein described in the present application, which includes culturing a cell or host cell described in the present application. In some aspects, provided herein is a method for producing a recombinant protein, which includes expressing (e.g., recombinantly expressing) an antibody using a cell or host cell described in the present application (e.g., a cell or host cell containing a polynucleotide encoding an antibody described in the present application). In some embodiments, the cell is an isolated cell. In some embodiments, an exogenous polynucleotide has been introduced into the cell. In some embodiments, the method further includes purifying the antibody obtained from the cell or host cell.

[0102] Antibodies can be produced using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. For example, monoclonal antibodies are known in the art and are taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced via hybridoma technology. For example, such monoclonal antibodies can also be recombinantly produced from host cells that express the antibodies described herein exogenously.

[0103] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), where the antibody immunospecifically binds to an antigen (e.g., human serum albumin) as determined by, for example, ELISA or other antigen-binding assays known in the art or provided in the examples of the present application, or by a competitive binding assay. In certain embodiments, the monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain embodiments, the monoclonal antibody can be a monovalent antibody or a multivalent (e.g., divalent) antibody. In certain embodiments, the monoclonal antibody can be a Fab fragment or an F(ab’)2 fragment. The monoclonal antibodies described in the present application can be produced, for example, by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256:495, or can be isolated from, for example, a phage library using techniques such as those described in the present application. Other methods for the production of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0104] Methods of producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, a mouse, or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunized to induce lymphocytes capable of producing antibodies that specifically bind to an antigen used for immunization (e.g., human serum albumin). Alternatively, the lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Further, animals can be immunized using the RIMMS (Repeated Immunization Multiple Sites) technology (Kilpatrick KE et al., (1997) Hybridoma 16: 381-9, which is incorporated herein by reference in its entirety).

[0105] The antibodies described in the present application can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab’)2 fragments described in the present application can also be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (for generating Fab fragments) or pepsin (for generating F(ab’)2 fragments). The Fab fragment corresponds to one of the two equal arms of the tetrameric antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab’)2 fragment contains the two antigen-binding arms of the tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0106] Furthermore, the antibodies described in the present application can also be generated using various phage display methods known in the art. In such phage display methods, proteins are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH domain and the VL domain are amplified from an animal cDNA library (e.g., a human cDNA library or a murine cDNA library of affected tissues). The DNA encoding the aforementioned VH domain and VL domain is recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. Phages used in such methods are generally filamentous phages including fd and M13, and the VH domain and the VL domain are generally recombinantly fused to phage gene III or gene VIII. Phages expressing antibodies that bind to a specific antigen can be selected or identified using, for example, the antigen, such as a labeled antigen, or an antigen bound or captured on a solid surface or beads.Examples of phage display methods that can be used to produce the antibodies described in this application include Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT / GB91 / 001134; WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401 and WO97 / 13844; and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0107] As described in the above references, after phage selection, the antibody coding region derived from the phage is isolated and used to generate antibodies including human antibodies, and can be expressed in any desired host including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described below. Techniques for recombinantly producing antibodies such as Fab, Fab’ and F(ab’)2 fragments can also be utilized using methods known in the art such as those disclosed in WO92 / 22324; Mullinax RL et al., (1992) BioTechniques 12 (6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science.

[0108] In some embodiments, to generate an antibody, a PCR primer comprising a VH nucleotide sequence or a VL nucleotide sequence, a restriction site, and a flank sequence for protecting said restriction site can be used to amplify a VH sequence or a VL sequence from a template, such as an scFv. Utilizing cloning techniques known to those skilled in the art, the VH domain amplified by PCR can be cloned into a vector expressing a VH constant region, and the VL domain amplified by PCR can be cloned into a vector expressing a VL constant region, such as a human kappa constant region and a human lambda constant region. The aforementioned VH domain and VL domain can also be cloned into one vector expressing the necessary constant regions. Next, a heavy chain conversion vector and a light chain conversion vector are co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line that expresses an antibody, such as IgG.

[0109] A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, the chimeric antibody can include a variable region of a human monoclonal antibody fused to a constant region of a feline antibody. Methods for producing such chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397 and 6,331,415.

[0110] A feline chimeric antibody includes a framework region substantially having the amino acid sequence of a feline immunoglobulin and CDRs substantially having the amino acid sequence of a human immunoglobulin, and can bind to a predetermined antigen.

[0111] [Polynucleotide, Vector, and Cell] Nucleic acid molecules encoding the recombinant proteins disclosed in this application are disclosed in this application.

[0112] Expression vectors containing the nucleic acid molecules disclosed in this application are disclosed in this application.

[0113] Cells transformed with the expression vectors disclosed in this application are disclosed in this application.

[0114] Since the aforementioned nucleic acids, expression vectors, and transformed cells directly contain the aforementioned recombinant proteins or nucleic acids encoding the recombinant proteins, or use them, common descriptions will be omitted.

[0115] For example, in some embodiments, the recombinant protein can be produced by isolating the nucleic acid encoding the recombinant protein. The nucleic acid is inserted into an isolated and replicable vector for additional cloning (DNA amplification) or additional expression. Based on this, other embodiments relate to the vector containing the nucleic acid.

[0116] As used herein, the term "nucleic acid" comprehensively includes DNA (gDNA and cDNA) molecules and RNA molecules. Nucleotides, which are the basic units of nucleic acids, include not only natural nucleotides but also analogs having modified sugar or base moieties.

[0117] The nucleic acid is construed to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. Substantial identity means that the nucleotide sequence of the present disclosure and another selected sequence are aligned so as to correspond to each other as much as possible, and when the aligned sequences are analyzed using algorithms commonly used in the art, they exhibit at least 80% homology, more specifically at least 90% homology, and most specifically at least 95% homology.

[0118] The DNA encoding the recombinant protein is easily isolated or synthesized by using common procedures (e.g., using an oligonucleotide probe that can specifically bind to the DNA encoding the recombinant protein). Many vectors can be used. The vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0119] The term "vector" as used in the present application includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors as a means for expressing a target gene in a host cell. In the said vector, the nucleic acid encoding the recombinant protein is operably linked to a promoter.

[0120] "Operably linked" means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription regulatory factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence will regulate the transcription and / or translation of the said other nucleic acid sequence.

[0121] When a prokaryotic cell is used as a host, a strong promoter capable of directing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence are generally included. For example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter), or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) is utilized, and as a transcription termination sequence, a polyadenylation sequence is generally possessed. In some cases, the vector can be fused with other sequences to facilitate the purification of the recombinant protein expressed therefrom. The aforementioned fused sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine; Quiagen, USA), etc. The vector includes, as a selectable marker, an antibiotic resistance gene commonly used in the art, for example, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0122] In yet other aspects, the present disclosure provides cells transformed with the aforementioned vectors. The cells used to produce the recombinant proteins of the present invention can be, but are not limited to, prokaryotic cells, yeast cells, or higher eukaryotic cells. Prokaryotic host cells such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis strains, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be utilized. However, animal cells are of the greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO (GS null CHO-K1), CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, HepG2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S or HT1080.

[0123] As used herein, the term "transformation" means a molecular biology technique in which a DNA fragment or plasmid having a foreign gene of a different type from that originally possessed by a cell penetrates into the cell and binds to the DNA originally present in the cell, thereby changing the genetic traits of the cell. The transformation means that an expression vector containing the recombinant protein gene is inserted into a host cell.

[0124] The present application provides nucleic acid molecules and vectors comprising nucleotide sequences encoding the recombinant proteins described herein that immunospecifically bind to an antigen (e.g., variable light chain region and / or variable heavy chain region), e.g., vectors comprising polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). The present application provides not only polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, but also vectors comprising such polynucleotide sequences, e.g., expression vectors for efficient expression thereof in host cells (e.g., mammalian cells).

[0125] As used herein, the term “isolated” polynucleotide or nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule (e.g., mouse or human). Further, an “isolated” nucleic acid molecule, such as a cDNA molecule, when produced by recombinant techniques, may be substantially free of other cellular material or culture medium, or when chemically synthesized, may be substantially free of chemical precursors or other chemicals. For example, the phrase “substantially free of” includes preparations of polynucleotides or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of other substances, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals (particularly less than about 10%). In some embodiments, the nucleic acid molecules encoding the antibodies described herein are isolated or purified.

[0126] The present application provides polynucleotides comprising nucleotide sequences encoding antibodies that immunospecifically bind to an antigen polypeptide (e.g., human serum albumin) and comprise an amino acid sequence as described herein, as well as antibodies that compete with (e.g., in a dose-dependent manner) or bind to the same epitope as the antibody for binding to the antigen polypeptide.

[0127] The present application provides a polynucleotide comprising a nucleotide sequence encoding a light chain or a heavy chain of the antibody described in the present application. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising the VL FR and CDR of the antibody described in the present application. The polynucleotide may comprise a nucleotide sequence encoding a heavy chain comprising the VH FR and CDR of the antibody described in the present application.

[0128] The present application provides a polynucleotide comprising a nucleotide sequence encoding a recombinant protein comprising a Fab comprising three VL chain CDRs of the antibody against human serum albumin described in the present application, such as VL CDR1, VL CDR2, and VL CDR3, and three VH chain CDRs of the antibody against human serum albumin described in the present application, such as VH CDR1, VH CDR2, and VH CDR3.

[0129] The present application provides a polynucleotide comprising a nucleotide sequence encoding a recombinant protein comprising a VL domain.

[0130] In certain embodiments, the polynucleotide described in the present application comprises a nucleotide sequence encoding the recombinant protein provided in the present application comprising a light chain variable region comprising the amino acid sequence described in the present application (e.g., SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13), wherein the antibody immunospecifically binds to serum albumin.

[0131] In certain embodiments, the polynucleotide described in the present application comprises a nucleotide sequence encoding the antibody provided in the present application comprising a heavy chain variable region comprising the amino acid sequence described in the present application (e.g., SEQ ID NO: 1, 2, 3, 4, 5, or 6), wherein the antibody immunospecifically binds to serum albumin.

[0132] In certain aspects, polynucleotides are provided herein that include nucleotide sequences encoding antibodies that include light and heavy chains, such as separate light and heavy chains. With respect to the light chain, in some embodiments, the polynucleotides provided herein include a nucleotide sequence encoding a kappa light chain. In other embodiments, the polynucleotides provided herein include a nucleotide sequence encoding a lambda light chain. In still other embodiments, the polynucleotides provided herein include a nucleotide sequence encoding an antibody described herein that includes a human kappa light chain or a human lambda light chain. In some embodiments, the polynucleotides provided herein include a nucleotide sequence encoding an antibody that immunospecifically binds to serum albumin, wherein the antibody includes a light chain, wherein the amino acid sequence of the VL domain also includes the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13 as presented below, and wherein the constant region of the light chain includes the amino acid sequence of a feline kappa light chain constant region.

[0133] Also provided herein are polynucleotides encoding antibodies or fragments thereof that have been optimized, for example, by codon / RNA optimization, substitution with heterologous signal sequences, and removal of mRNA destabilizing elements. Methods for generating optimized nucleic acids encoding antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain or VL domain) for recombinant expression by introducing codon changes and / or removing inhibitory regions in mRNA can be accomplished, for example, by applying the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; 6,794,498. For example, potential splice sites and destabilizing elements within the RNA (e.g., A / T-rich elements or A / U-rich elements) can be mutated without altering the amino acids encoded by the nucleic acid sequence, increasing the stability of the RNA for recombinant expression. The changes can, for example, utilize alternative codons for the same amino acid and take advantage of the degeneracy of the genetic code. In some embodiments, it may be desirable to change one or more codons so as to encode similar amino acids having a similar chemical structure, properties and / or function to the original amino acid, such as conservative mutations.

[0134] In certain embodiments, the optimized polynucleotide sequences encoding the antibodies or fragments thereof (e.g., VL domain or VH domain) described herein can hybridize to the antisense (e.g., complementary) polynucleotides of the unoptimized polynucleotide sequences encoding the antibodies or fragments thereof (e.g., VL domain or VH domain) described herein. In certain examples, the optimized nucleotide sequences encoding the antibodies or fragments thereof described herein hybridize to the antisense polynucleotides of the unoptimized polynucleotide sequences encoding the antibodies or fragments thereof described herein under high stringency conditions. In some embodiments, the optimized nucleotide sequences encoding the antibodies or fragments thereof described herein hybridize to the antisense polynucleotides of the unoptimized nucleotide sequences encoding the antibodies or fragments thereof described herein under high stringency, intermediate or even lower stringency hybridization conditions. Information regarding hybridization conditions is described, for example, in US2005 / 0048549 (e.g., paragraphs 72-73), which is hereby incorporated by reference herein.

[0135] A polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. The nucleotide sequences encoding the antibodies described herein and modified versions of those antibodies can be determined using methods widely known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in a manner that generates a nucleic acid encoding the antibody. Such polynucleotides encoding the antibody can also be assembled from chemically synthesized oligonucleotides (e.g., similar to those described in Kutmeier G et al., (1994), BioTechniques 17: 242-246), which, briefly, includes the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of the oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0136] Alternatively, polynucleotides encoding the antibodies or fragments thereof described herein can also be generated from nucleic acids derived from a suitable source (e.g., hybridoma) using methods widely known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the antibody of interest. Such PCR amplification methods are also used to obtain nucleic acids containing sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods are also used to obtain nucleic acids containing sequences encoding the variable light chain region and / or variable heavy chain region of the antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, e.g., for chimeric antibody production and humanized antibody production.

[0137] Even if clones containing nucleic acids encoding specific antibodies or fragments thereof cannot be utilized, when the sequence of an antibody molecule or a fragment thereof is known, the nucleic acid encoding an immunoglobulin or fragment can be chemically synthesized or obtained by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence from a suitable source (any tissue or cells expressing the antibody, such as an antibody cDNA library or cDNA library generated from hybridoma cells selected to express the antibodies described in this application, nucleic acid molecules isolated from said tissue or cells, e.g., poly A+RNA), or by cloning using specific oligonucleotide probes related to a specific gene sequence, for example, to identify a cDNA clone derived from the cDNA encoding said antibody. The amplified nucleic acid generated by PCR can be cloned into a replicable cloning vector using any method well known in the art.

[0138] DNA encoding the recombinant proteins described in this application can be easily isolated and sequenced using general procedures (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the recombinant protein). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not normally produce immunoglobulin proteins, to obtain recombinant proteins synthesized in the recombinant host cells.

[0139] To generate an antibody, PCR primers containing a VH nucleotide sequence or a VL nucleotide sequence, a restriction site, and flanking sequences for protecting the restriction site can be used to amplify the VH sequence or the VL sequence in an scFv clone. The PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma 4 constant region, using cloning techniques known to those skilled in the art. The PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa constant region or the human lambda constant region. In certain embodiments, the vector for expressing the VH domain or the VL domain includes an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH domain and the VL domain can also be cloned into a single vector expressing the required constant regions. Next, the aforementioned heavy chain conversion vector and light chain conversion vector are co-transfected into a cell line to generate a stable cell line or a transient cell line expressing a full-length antibody, such as IgG, using techniques known to those skilled in the art.

[0140] Said DNA may also be modified, for example, by substituting the coding sequences related to the human heavy chain constant domain and the human light chain constant domain instead of the mouse sequences, or by covalently linking all or part of the coding sequences related to the non-immunoglobulin polypeptide to the immunoglobulin coding sequences.

[0141] Also provided herein are polynucleotides that hybridize to the polynucleotides encoding the antibodies described herein under high stringency, intermediate, or low stringency hybridization conditions. In certain embodiments, the polynucleotides described herein hybridize to the polynucleotides encoding the VH domain and / or VL domain provided herein under high stringency, intermediate, or even lower stringency hybridization conditions.

[0142] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50 - 65°C, and hybridization under very stringent conditions can involve hybridization to filter-bound nucleic acid in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art, see, for example, Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1 - 6.3.6 and 2.10.3.

[0143] An expression vector comprising the following is further disclosed herein: (a) a promoter, (b) a first nucleic acid molecule encoding an antigen-binding fragment (Fab) that binds to serum albumin, and (c) a second nucleic acid molecule encoding a bioactive effector moiety such as fGCSF and a linker Here, the promoter, the first nucleic acid sequence, and the second nucleic acid molecule are operably linked. The second nucleic acid molecule can encode two, three, four, five, six, or more bioactive effector moieties and linkers.

[0144] Also disclosed herein is an expression vector comprising: (a) a promoter, and (b) A nucleic acid molecule encoding a heavy chain variable domain as disclosed in the present application and a feline heavy chain constant 1 domain as disclosed in the present application.

[0145] Also disclosed in the present application is an expression vector comprising: (a) A promoter, and (b) A nucleic acid molecule encoding fGCSF as disclosed in the present application, a heavy chain variable domain as disclosed in the present application, and a feline heavy chain constant 1 domain as disclosed in the present application.

[0146] Also disclosed in the present application is an expression vector comprising: (a) A promoter, and (b) A nucleic acid molecule encoding a light chain variable domain as disclosed in the present application and a feline light chain constant domain as disclosed in the present application.

[0147] Also disclosed in the present application is an expression vector comprising: (a) A promoter, and (b) A nucleic acid molecule encoding fGCSF as disclosed in the present application, a light chain variable domain as disclosed in the present application, and a feline light chain constant domain as disclosed in the present application. One, two, three, or more expression vectors or nucleic acid molecules can be expressed to produce the desired recombinant protein.

[0148] In some embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a recombinant protein comprising an antigen-binding fragment comprising a heavy chain, wherein the heavy chain comprises a heavy chain variable domain and a feline constant 1 domain, wherein the heavy chain variable domain (1) A heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO: 51), A heavy chain complementarity determining region 2 (CDR2) comprising the amino acid sequence of WINTYSGTKYAQKFQG (SEQ ID NO: 52), and A heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO: 53); (2) a heavy-chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO: 51), a heavy-chain CDR2 comprising the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO: 54), and a heavy-chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO: 53); (3) a heavy-chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO: 55), a heavy-chain CDR2 comprising the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO: 56), and a heavy-chain CDR3 comprising the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO: 57); (4) a heavy-chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 58), a heavy-chain CDR2 comprising the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO: 59), and a heavy-chain CDR3 comprising the amino acid sequence of AGWLRQYGMDV (SEQ ID NO: 60); (5) a heavy-chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO: 61), a heavy-chain CDR2 comprising the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO: 62), and a heavy-chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO: 63); or (6) a heavy-chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO: 64), a heavy-chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO: 65), and a heavy-chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO: 66).

[0149] In some embodiments, the second nucleic acid molecule or vector comprises (a) a nucleic acid sequence encoding a recombinant protein comprising an antigen-binding fragment comprising a light chain, wherein the light chain comprises a light chain variable domain and a feline light chain constant domain, and wherein the light chain variable domain (7) a light-chain CDR1 comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO: 67), A light chain CDR2 containing the amino acid sequence of GASRLES (SEQ ID NO: 68), and a light chain CDR3 containing the amino acid sequence of QQSDSVPVT (SEQ ID NO: 69); (8) A light chain CDR1 containing the amino acid sequence of RASQSISSYLN (SEQ ID NO: 70), a light chain CDR2 containing the amino acid sequence of AASSLQS (SEQ ID NO: 71), and a light chain CDR3 containing the amino acid sequence of QQSYSTPPYT (SEQ ID NO: 72); (9) A light chain CDR1 containing the amino acid sequence of RASQSIFNYVA (SEQ ID NO: 73), a light chain CDR2 containing the amino acid sequence of DASNRAT (SEQ ID NO: 74), and a light chain CDR3 containing the amino acid sequence of QQRSKWPPTWT (SEQ ID NO: 75); (10) A light chain CDR1 containing the amino acid sequence of RASETVSSRQLA (SEQ ID NO: 76), a light chain CDR2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 77), and a light chain CDR3 containing the amino acid sequence of QQYGS SPRT (SEQ ID NO: 78); (11) A light chain CDR1 containing the amino acid sequence of RASQSVSSSSLA (SEQ ID NO: 79), a light chain CDR2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 77), and a light chain CDR3 containing the amino acid sequence of QKYSSYPLT (SEQ ID NO: 80); or (12) A light chain CDR1 containing the amino acid sequence of RASQSVGSNLA (SEQ ID NO: 81), a light chain CDR2 containing the amino acid sequence of GASTGAT (SEQ ID NO: 82), and a light chain CDR3 containing the amino acid sequence of QQYYSFLAKT (SEQ ID NO: 83).

[0150] For example, the nucleic acid molecule encoding the fGCSF can be linked to the aforementioned first or second nucleic acid molecule or vector.

[0151] In other embodiments, the first nucleic acid molecule comprises a heavy chain variable domain comprising the aforementioned (1) and a light chain variable domain comprising the aforementioned (7); a heavy chain variable domain comprising the aforementioned (2) and a light chain variable domain comprising the aforementioned (8); a heavy chain variable domain comprising the aforementioned (3) and a light chain variable domain comprising the aforementioned (9); a heavy chain variable domain comprising the aforementioned (4) and a light chain variable domain comprising the aforementioned (10); a heavy chain variable domain comprising the aforementioned (5) and a light chain variable domain comprising the aforementioned (11); a heavy chain variable domain comprising the aforementioned (6) and a light chain variable domain comprising the aforementioned (12); or any, or any or all combinations of the aforementioned heavy chain variable domain and light chain variable domain. In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab (FL335) comprising a heavy chain variable domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 66, and a light chain variable domain comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 83. The second nucleic acid molecule may encode fGCSF.

[0152] In other embodiments, the aforementioned first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the aforementioned second nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13. The nucleic acid molecule encoding the fGCSF can be linked to the first or second nucleic acid molecule or vector.

[0153] In some embodiments, the aforementioned first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, or 6, and a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13, respectively.

[0154] In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab (SL335) comprising a heavy chain domain comprising the amino acid sequence of SEQ ID NO: 16 (VH-CH1 domain) and a light chain domain comprising the amino acid sequence of SEQ ID NO: 17 (VL-CL domain).

[0155] In some embodiments, the bioactive effector moiety is fGCSF. For example, the second nucleic acid molecule can include a nucleotide sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one or more of SEQ ID NOs: 18 and 19.

[0156] Recombinant expression of an antibody or fragment thereof (e.g., a heavy or light chain of an antibody described herein) that specifically binds involves constructing an expression vector comprising a polynucleotide encoding the antibody or fragment. Once a polynucleotide encoding an antibody or fragment thereof (e.g., a heavy chain variable domain or a light chain variable domain) described herein is obtained, vectors for the production of antibody molecules can be produced by recombinant DNA techniques using techniques well known in the art. Accordingly, a method for producing a nucleotide sequence encoding a protein by expressing a polynucleotide comprising an antibody or antibody fragment (e.g., a light or heavy chain) is described herein. Methods well known to those skilled in the art can also be used to construct expression vectors comprising a coding sequence for an antibody or antibody fragment (e.g., a light or heavy chain), as well as appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Provided herein are replicable vectors comprising an antibody molecule, a heavy or light chain of an antibody, a heavy chain variable domain or a light chain variable domain of an antibody or fragment thereof, or a light chain CDR or heavy chain CDR described herein, operably linked to a promoter. Such vectors can, for example, include a nucleotide sequence encoding a constant region of an antibody molecule (see, e.g., WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0157] The expression vector can be introduced into cells (e.g., host cells) by common techniques, and the resulting cells can then be cultured by common techniques to produce the antibodies described in the present application.

[0158] To express the described antibody molecules, a variety of host expression vector systems can be utilized. Such host expression systems represent vehicles by which the coding sequence of interest can be generated and subsequently purified, but can also represent cells that are capable of expressing the antibody molecules described herein in situ when transformed or transfected with an appropriate nucleotide coding sequence. Here, bacteria (e.g., E. coli and B. subtilis) transformed with an expression vector of recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA containing an antibody coding sequence; yeast (e.g., Saccharomyces pichia) transformed with a recombinant yeast expression vector containing an antibody coding sequence; insect cell systems (e.g., baculovirus) infected with a recombinant virus expression vector containing an antibody coding sequence; plant cell systems (e.g., Chlamydomonas reinhardtii+ such as green algae) infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)), or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody coding sequence; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa cells, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM,, BSC1, BSC40, YB / 20, and BMT10 cells) possessing a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter), or from the genome of a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter), including but not limited to these. In some embodiments, the cells for expressing the antibodies described herein (e.g., an antibody containing any one of the CDRs of antibody pab1949 or antibody pab2044) are CHO cells, e.g., CHO cells of the CHO GS System™ (Lonza).In some embodiments, the cells for expressing the antibodies described herein are human cells, such as human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, particularly, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) for the expression of the whole recombinant antibody molecule are used for the expression of the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells combined with a vector such as the major intermediate early gene promoter element of human cytomegalovirus are an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105, and Cockett MI et al., (1990) Biotechnology 8: 662-667). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In some embodiments, the expression of the nucleotide sequence encoding the antibodies described herein is regulated by a constitutive promoter, an inducible promoter or a tissue-specific promoter.

[0159] In a bacterial system, a number of expression vectors can be advantageously selected depending on the intended use of the antibody molecule to be expressed. For example, if such an antibody has to be mass-produced, a vector that directs high-level expression of a fusion protein product that can be easily purified for generating a pharmaceutical composition of the antibody molecule may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 in which the antibody coding sequence is individually ligated into the vector in-frame with the lacZ coding region so that a fusion protein is produced (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794); pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G Schuster SM (1989) J Biol Chem 24: 5503-5509), etc. For example, pGEX vectors can also be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorbing and binding to matrix glutathione agarose beads and then eluting in the presence of free glutathione. The pGEX vectors are designed to contain a cleavage site for thrombin or factor Xa protease so that the cloned target gene product can be released from the GST moiety.

[0160] In mammalian host cells, a number of virus-based expression systems can be utilized. When adenovirus is used as an expression vector, the antibody-encoding sequence of interest can be linked to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. Such chimeric genes can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or region E3) can give rise to a recombinant virus that is viable and capable of expressing the antibody molecule in an infected host (see, for example, Logan J & Shenk T (1984) PNAS 81: 3655-3659). For efficient translation of the inserted antibody-encoding sequence, a specific initiation signal is also required. Such signals include an ATG start codon and adjacent sequences. Also, for ensuring translation of the entire insert, the start codon must be in phase with the reading frame of the desired coding sequence. Such exogenous translation control signals and start codons can have diverse origins, both natural and synthetic. The expression efficiency can be improved by including appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol 153: 516-544).

[0161] Alternatively, a host cell strain can be selected that modulates the expression of the inserted array or modifies and processes the gene product in a desired, specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Other host cells have characteristics and specific mechanisms for the post-translational processing and modification of proteins and gene products. To ensure the correct modification and processing of the expressed foreign protein, an appropriate cell line or host system can be selected. For this purpose, eukaryotic host cells that possess cellular mechanisms for appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O cells, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the recombinant proteins described herein (e.g., antibodies comprising CDRs) are produced in mammalian cells such as CHO cells.

[0162] In some embodiments, the antibodies described herein have a reduced fucose content or no fucose content. Such antibodies can be generated using techniques known to those of skill in the art. For example, the antibodies can be expressed in cells that lack or have reduced fucosylation ability. In certain embodiments, cell lines in which both alleles of α1,6-fucosyltransferase are knocked out can be used to produce antibodies with a reduced fucose content. The Potelligent® system (Lonza) is an example of a system that can be used to produce antibodies with a reduced fucose content.

[0163] For the long-term and high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines that stably express the recombinant proteins disclosed in the present application can be manipulated. In certain embodiments, the cells provided herein stably express the light chain / light chain variable domain and the heavy chain / heavy chain variable domain associated with forming the antibodies (e.g., antibodies containing CDRs) described herein.

[0164] In certain embodiments, instead of using an expression vector containing a viral replication origin, the host cells can also be transformed with DNA regulated by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introducing the foreign DNA / polynucleotide, the manipulated cells can be allowed to grow in enriched medium for 1-2 days and then transferred to selective medium. The selectable marker of the recombinant plasmid confers resistance to selection, allowing the cells to grow such that the plasmid stably integrates into the chromosome to form foci, which can then be cloned and expanded into cell lines. The method can advantageously be used to manipulate cell lines that express the antibodies or fragments thereof described herein. Such manipulated cell lines are particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0165] A variety of selection systems are used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11 (1): 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska EH Szybalski W(1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22 (3): 817-823) genes, which can be used in tk-, hgprt- or aprt- cells respectively. Also, antimetabolite resistance can be used as the basis for selection against the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77 (6): 3567-3570; O’Hare K et al., (1981) PNAS 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-215)); hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-156).

[0166] The expression level of antibody molecules can be increased by vector amplification (for reference, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker of the vector system expressing the antibody is amplifiable, an increase in the level of the inhibitor present in the culture of the host cells will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66).

[0167] The host cell can be co-transfected with two or more expression vectors described in the present application, a first vector encoding a heavy chain-derived polypeptide, and a second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable marker that enables the co-expression of the heavy chain polypeptide and the light chain polypeptide. The host cell can be co-transfected with two or more expression vectors having different contents. For example, the host cell can be transfected at any one of the following ratios for the first expression vector and the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

[0168] Alternatively, a single vector capable of encoding and expressing a heavy chain polypeptide and a light chain polypeptide can be used. In such situations, the light chain should be arranged before the heavy chain to prevent an excess of toxic free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Koehler G (1980) PNAS 77: 2197-2199). The coding sequences for the heavy and light chains can include cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. A polycistronic nucleic acid construct can encode a range of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or 2-5, 5-10 or 10-20 gene / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in order, a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein). In such an expression vector, transcription of the two genes is driven by a promoter, while translation of the mRNA from the first gene is by a cap-dependent scanning mechanism and translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.

[0169] The vector can include a first nucleic acid molecule encoding an antigen-binding fragment (Fab) that binds to serum albumin, and a second nucleic acid molecule encoding a bioactive effector moiety and a linker.

[0170] If the antibody molecules described herein are produced by recombinant expression, they can also be purified by any method known in the art for the purification of immunoglobulin molecules, e.g., chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after affinity protein A, and size column chromatography), centrifugation, salting out solubility, or other standard techniques for protein purification. Furthermore, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0171] In certain embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is one in which substantially no other antibodies having different antigen specificities from the aforementioned isolated antibodies are present. For example, in some embodiments, a preparation of an antibody described herein is substantially free of cellular material and / or chemical precursors. The phrase "substantially free of cellular material" includes antibody preparations in which the antibody has been separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material has less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the antibody, e.g., antibodies having different post-translationally modified forms. When the aforementioned antibody or fragment is recombinantly produced, the protein preparation is generally substantially free of culture medium, e.g., the culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody or fragment is produced by chemical synthesis, the aforementioned antibody or fragment is generally substantially free of chemical precursors or other chemicals, i.e., separated from the chemical precursors or other chemicals involved in protein synthesis. Thus, such a formulation of the antibody or fragment has less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody or fragment of interest. In some embodiments, the antibodies described herein are isolated or purified.

[0172] [Composition] Still other aspects provide a composition, e.g., a pharmaceutical composition for the prevention or treatment of feline panleukopenia, a pharmaceutical composition comprising the recombinant protein as an active ingredient; a method of treating feline panleukopenia, a method of administering the composition to an individual; and the pharmaceutical use of the recombinant protein for the prevention or treatment of feline panleukopenia.

[0173] For example, the pharmaceutical composition may comprise (a) a pharmaceutically effective amount of the recombinant protein, and (b) a pharmaceutically acceptable carrier.

[0174] In some embodiments, the in vivo half-life of the pharmaceutical composition can show an increase of 3 to 20 times compared to that of feline GCSF. The in vivo half-life can increase, for example, by about 3.5 to about 6 times, about 4 to about 6 times, about 4.5 to about 6 times, about 5 to about 6 times, about 5.5 to about 6 times, about 3 to about 5.5 times, about 3.5 to about 5.5 times, about 4 to about 5.5 times, about 4.5 to about 5.5 times, about 5 to about 5.5 times, about 3 to about 5 times, about 3.5 to about 5 times, about 4 to about 5 times, about 4.5 to about 5 times, about 3 to about 4.5 times, about 3.5 to about 4.5 times, or about 4 to about 4.5 times compared to feline GCSF. In some examples, further, the in vivo half-life of feline GCSF can be evaluated after subcutaneous injection of the mutant fGCSF.

[0175] In some examples, the pharmaceutical composition can increase the blood leukocyte level. The leukocytes can be, for example, neutrophils, monocytes, basophils, or combinations thereof. The aforementioned elevated leukocyte level can be sustained and maintained up to 20 days, 15 days, 12 days, 10 days, 8 days, or 7 days after administration.

[0176] The pharmaceutically acceptable carrier included in the pharmaceutical composition can include those commonly used in formulations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition of the present invention can further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc. in addition to the aforementioned components.

[0177] The pharmaceutical composition can be administered orally or parenterally. Specifically, the pharmaceutical composition is administered parenterally, and in that case, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration, rectal administration, etc. In some embodiments, they can be administered in the form of subcutaneous injection. Since proteins or peptides are digested during oral administration, oral compositions need to be formulated by coating the active ingredient or protecting it from degradation in the gastrointestinal tract. Also, the pharmaceutical composition can be administered by any device capable of delivering the active substance to the target cells.

[0178] The pharmaceutical composition is administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" used in this application is a reasonable benefit / risk ratio applicable to any medical treatment and refers to an amount sufficient to treat a disease. The effective dosage level can be determined by the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and the excretion ratio, the treatment period, the drugs administered in combination, and other factors well-known in the medical field. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered simultaneously with, individually, or sequentially to existing therapeutic agents, once or in several divided doses. Considering all the above factors, it is important to administer at the minimum dose that can achieve the maximum effect without side effects, and such an amount can be easily determined by those skilled in the art. The term "pharmaceutically effective amount" used in this application refers to an amount sufficient to prevent or treat feline panleukopenia.

[0179] The pharmaceutical composition is manufactured in unit dosage form or can also be manufactured by incorporating it into a multi-dose container by formulating it using pharmaceutically acceptable carriers and / or excipients by a method that can be easily implemented by those skilled in the technical field to which the invention belongs. In that case, the dosage form may be in the form of a solution, suspension or emulsion in an oily solvent or an aqueous medium, or may also be in the form of an extract, suppository, powder, granule, tablet or capsule, and the dosage form may further contain a dispersant or a stabilizer.

[0180] In the present application, a composition containing the recombinant protein described in the present application having a desired degree of purity is provided in a physiologically acceptable carrier, excipient or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Further, a pharmaceutical composition containing the recombinant protein described in the present application and a pharmaceutically acceptable excipient is disclosed in the present application. The acceptable carrier, excipient or stabilizer is non-toxic to the recipient at the dosages and concentrations used.

[0181] The pharmaceutical composition of the present disclosure can provide rapid, sustained or delayed release of the active ingredient after administration to an individual and can be formulated using methods well known to those skilled in the art. The dosage form may also be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, sterile powders, etc. Examples of suitable carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. Further, the dosage form may further contain a filler, an anti-aggregation agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, etc.

[0182] The pharmaceutical composition described in the present application can be useful for improving, inducing or activating the activity of the recombinant protein described in the present application and treating diseases or conditions.

[0183] Compositions for in vivo administration can be sterilized. This can be readily achieved, for example, by filtration through a sterile filtration membrane.

[0184] [Use and Method] Also disclosed in the present application is a method for treating feline panleukopenia in an individual in need thereof, which comprises administering the pharmaceutical composition disclosed herein. The individual is a feline (e.g., a domestic cat or a wild cat, lion, tiger, jaguar, cheetah, leopard cat, etc.).

[0185] Also disclosed in the present application is the use of the composition disclosed in the present application for treating feline panleukopenia in an individual in need thereof. Also disclosed in the present application is the composition disclosed in the present application for use in treating feline panleukopenia in an individual in need thereof. Also disclosed in the present application is the use of the composition disclosed in the present application for the manufacture of a medicament for treating feline panleukopenia in an individual in need thereof.

[0186] Feline panleukopenia virus (FPLV) is a species of parvovirus that can infect wild and captive individuals of the feline (cat) family worldwide. It is a highly contagious and severe infectious disease that induces diseases in the gastrointestinal tract, immune system and nervous system. Its main effect is to reduce the number of white blood cells.

[0187] In some embodiments, the composition increases the white blood cells in the blood of the individual. In some embodiments, the white blood cells are neutrophils, monocytes, basophils, or combinations thereof.

[0188] In one example, the recombinant protein (APB-F1) obtained by fusing the FL355Fab antibody fragment, which is a feline chimeric antibody fragment, with feline granulocyte colony-stimulating factor has a half-life in the feline body of about 13.3 hours, which is approximately 4.9 times longer than that of fGCSF, which showed 2.7 hours. The increase in the blood lymphocyte and neutrophil counts following administration of the recombinant protein persisted until the 20th day and the 11th day after administration, respectively. Therefore, it was confirmed that the recombinant protein disclosed in the present application can also be used as an active ingredient of a pharmaceutical composition for the prevention or treatment of feline panleukopenia.

[0189] In some embodiments, the elimination half-life (T1 / 2) of the recombinant protein is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7-fold, at least about 10-fold, or any multiple or range of multiples derived therefrom, compared to that of feline granulocyte-stimulating factor (fGCSF). In some embodiments, the recombinant protein has an elimination half-life (T1 / 2) of about 8 hours to about 20 hours, about 10 hours to about 18 hours, about 12 hours to about 15 hours, or any half-life or range derived therefrom. In some embodiments, the Tmax of the recombinant protein is at least about 10% to about 200% higher, about 50% to 100% higher, about 50% to 75% higher, or any % or range of % derived therefrom, compared to the Tmax of fGCSF. In some embodiments, a dose of about 360 μg / kg of the recombinant protein to the subject provides a Tmax of about 8 hours to about 20 hours, about 10 hours to about 15 hours, about 12 hours to about 14 hours, or any Tmax or range derived therefrom. In some embodiments, the Cmax of the recombinant protein is about 10% or more higher, about 20% or more higher, about 30% or more higher, or any % or range of % derived therefrom, compared to the Cmax of fGCSF. In some embodiments, a dose of about 360 μg / kg of the recombinant protein to the subject provides a Cmax of about 700 ng / ml to about 1,000 ng / ml, about 750 ng / ml to about 900 ng / ml, about 800 ng / ml to about 850 ng / ml, or any dose or range of doses derived therefrom. In some embodiments, the AUClast of the recombinant protein is at least about 2-fold greater, at least about 3-fold greater, at least about 4-fold greater, at least about 5-fold greater, or any multiple or range of multiples derived therefrom, compared to the AUClast of fGCSF.In some embodiments, a dose of about 360 μg / kg of the recombinant protein to the subject provides an AUClast of about 8,000 hr*ng / ml to about 25,000 hr*ng / ml, about 16,000 hr*ng / ml to about 22,000 hr*ng / ml, about 18,000 hr*ng / ml to about 20,000 hr*mg / ml, or any concentration or range of concentrations derived therefrom.

[0190] In some aspects, the present application relates to a method of modulating one or more immune functions or immune responses in an individual, comprising administering to the individual in need thereof an antibody or composition thereof as described herein. Disclosed herein is a method of activating, enhancing or inducing one or more immune functions or immune responses in an individual, comprising administering to the individual in need thereof an antibody or composition thereof. In some embodiments, provided herein is a method of preventing and / or treating a disease in which it is desirable to activate or enhance one or more immune functions or immune responses, comprising administering to the individual in need thereof an antibody or composition thereof as described herein. In certain embodiments, provided herein is a method of treating an autoimmune disease or condition thereof, comprising administering to the individual in need thereof an antibody or composition thereof.

[0191] In some embodiments, the antibody described herein, using assays well known in the art, such as ELISPOT, ELISA, and cell proliferation assays, activates, enhances or induces one or more immune functions or immune responses in an individual by at least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%, or in the range of 10% to 25%, 25% to 50%, 50% to 75%, or 75% to 95% compared to the immune function of an individual not administered the recombinant protein described herein.

[0192] [Route of Administration and Dosage] The pharmaceutical compositions of the present disclosure can be administered to an individual via a variety of routes of administration, including oral, transdermal, subcutaneous, intravenous, and intramuscular routes of administration.

[0193] The amount of the recombinant protein or composition disclosed in the present application that is effective for the treatment and / or prevention of a condition depends on the characteristics of the disease and can be determined by standard clinical techniques.

[0194] The amount of the recombinant protein disclosed in the present application that is actually administered is determined in light of a variety of relevant factors, including the disease to be treated, the selected route of administration, the age, sex, and weight of the patient, the severity of the disease, and the type of bioactive polypeptide as the active ingredient. Since the recombinant protein of the present invention has excellent persistence in the blood, the number of administrations and the frequency of the peptide preparation containing the recombinant protein of the present invention can be significantly reduced.

[0195] The exact dosage used in the composition also depends on the route of administration and the severity of the disease and is determined by the judgment of the practitioner and the circumstances of each individual. For example, the effective dosage can also vary depending on the means of administration, the target site, the physiological state of the patient (including age, weight, and health), other drugs being administered, or whether the treatment is prophylactic or therapeutic. Generally, the patient is a cat, a pet cat, or a domestic cat. The therapeutic dosage is optimally adjusted to optimize safety and efficacy.

[0196] In certain embodiments, in vitro assays are used to help identify the optimal dosage range. The effective dosage can be inferred from a dose-response curve derived from in vitro or animal model test systems.

[0197] [Kit] A kit is provided herein that includes one or more recombinant proteins described in the present application, or a conjugate thereof. Also provided herein are kits that include the compositions disclosed in the present application and labels that include instructions for use thereof. In some embodiments, provided herein is a pharmaceutical pack or kit that includes one or more containers filled with one or more of the components of the pharmaceutical compositions described in the present application, such as one or more of the recombinant proteins provided herein. In some embodiments, the kit includes the pharmaceutical composition described in the present application and any prophylactic or therapeutic agent as described in the present application. Optionally, a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products may accompany such containers, and the notice reflects approval by the manufacturing, using, or selling agency for administration to cats. Also provided herein are kits that can be used in the methods described above. In some embodiments, the kit includes one or more recombinant proteins described in the present application, such as purified recombinant proteins, in one or more containers. In some embodiments, the kits described in the present application include a substantially isolated antigen (e.g., feline serum albumin) that can also be used as a control. In other embodiments, the kits described in the present application further include a control antibody that does not react with the serum albumin antigen. In other embodiments, the kits described in the present application include one or more elements for detecting the binding of a recombinant protein to the serum albumin antigen (e.g., the recombinant protein can be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In certain embodiments, the kits provided herein can also include a recombinantly produced or chemically synthesized serum albumin antigen. The serum albumin antigen provided in the kit may be attached to a solid support. In some embodiments, the detection means of the aforementioned kit includes a solid support to which the serum albumin antigen is attached. Such a kit can also include an unbound reporter-labeled anti-feline antibody or anti-mouse / rat antibody.In the binding of an antibody to serum albumin, the antigen can be detected by binding to the reporter-labeled antibody.

[0198] Hereinafter, the present invention will be described in more detail through exemplary embodiments. However, those exemplary embodiments are only for illustrative purposes of the present invention, and the scope of the present disclosure is not limited thereto.

Example

[0199] [Production Example 1. Production of Expression Vector] All DNA cloning experiments were carried out according to standard procedures. Feline IgG CH1 (delta C, IMGT AB016710 IGHG1*01, Felis catus, CH1), Feline CL kappa (delta C, IMGT000050 IGKC*01, Felis catus, C-region), feline serum albumin (FSA, UniProtKB P49064), feline granulocyte colony-stimulating factor (fGCSF, UniProtKB Q9GJU0)) and APB-F1 (v2) (FL335 Fd + mutant fGCSF) genes were obtained from Cosmogenetech Inc. (Korea). Also, in the polymerase chain reaction (PCR), pyrobest DNA polymerase (Takara, Japan) was used as the polymerase, and PCR was performed using a T100 Thermal Cycler (Bio-Rad, Hercules, California) machine. In addition, the T4 DNA ligase (NEB Bio Lab, Ipswich, Massachusetts) was used to insert the gene into the expression vector. On the other hand, the sequence information regarding the primers is shown in Table 1 below.

Table 1

[0200] (1) Production of FL335 Expression Vector

[0201] To produce FL335, a chimeric antibody fragment of the human anti-serum albumin Fab antibody fragment SL335, V, the variable region of the SL335 Fab antibody fragment H The amplification of the gene was carried out via PCR under the conditions of 30 cycles of 5°C for 30 seconds, 60°C for 20 seconds, and 72°C for 50 seconds, using primers 1 and 2. Also, for the amplification of the V L gene, primers 5 and 6 were used, and PCR was carried out under the same conditions as described above. Also, for the SL335 V H gene and feline IgG CH1, using primers 1 and 4, and by performing linking PCR under the conditions of 30 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, a chimeric Fd (V H +CH1) gene was produced. Additionally, for the SL335 V L gene and feline light chain (kappa) C L using primers 5 and 8, and performing linking PCR under the same conditions as described above, a chimeric L (V L +C L k) gene was obtained.

[0202] Thereafter, as shown in Figure 1, after treating the chimeric Fd gene with the BbsI (Takara, Japan) restriction enzyme, it was inserted into the expression vector pd2535nt (ATUM, Newark, California) to produce the FL335 Fd pd2535NT vector. After treating the chimeric L gene with the BbsI restriction enzyme and the BsrGI (Takara, Japan) restriction enzyme, it was inserted into the expression vector pd2539 (ATUM, Newark, California) to produce the APB-F1L pd2539 vector.

[0203] (2) Production of an expression vector for feline serum albumin or natural feline granulocyte colony-stimulating factor

[0204] As shown in Figure 2, the feline serum albumin gene was treated with the restriction enzymes EcoRI (Takara, Japan) and ApaI (Takara, Japan), and then inserted into the pJK expression vector to produce the Feline serum albumin pJK-dhfr vector. Also, as shown in Figure 3, for the native fGCSF gene, primers 1, 9, and 10 were used, and PCR was carried out under the same conditions as described above to ligate a myc tag to the N-terminus of fGCSF and a His tag to the C-terminus. After treating it with the BbsI restriction enzyme, it was inserted into the expression vector pd2535nt to produce the Feline GCSF pd2535NT vector.

[0205] (3) Production of the APB-F1 expression vector

[0206] APB-F1 was prepared in two versions, APB-F(v1) and APB-F(v2). Specifically, APB-F(v1) is in the form where the naturally occurring ("native") fGCSF with O-glycans is fused to FL335, and APB-F(v2) is in the form where a mutant fGCSF from which the free cysteine group (C17S) and O-glycans have been removed is fused to FL335, derived from APB-F(v1).

[0207] To produce APB-F(v1)Fd, using natural fGCSF as a template, primers 14 and 15 were used, and PCR was carried out under the same conditions as described above to obtain a natural fGCSF gene partially containing a linker sequence (GSAGSAPAPAGSGEF (SEQ ID NO: 84)). Using the FL335 Fd gene as a template, primers 1 and 2 were utilized to carry out PCR to produce an FL335 Fd gene partially containing the linker sequence. Subsequently, using the produced natural f-GCSF gene and the FL335 Fd gene as templates, primers 1 and 15 were used, and linking PCR was carried out under the same conditions as described above to produce an APB-F(v1)Fd (FL335 Fd-GSAGSAPAPAGSGEF(SEQ ID NO: 84)-natural fGCSF) gene. Then, as shown in Figure 4A, after treating the above-produced gene with BbsI restriction enzyme, it was inserted into the pd2535nt expression vector to produce an APB-F1(v1) Fd pd2535NT vector. On the other hand, as shown in Figure 4B, for the light chain of APB-F1, the light chain of FL335 inserted into the pd2539 expression vector was used.

[0208] Also, to produce APB-F(v2)Fd, in the same manner as described above, primers 1, 11, and 12 were used to ligate a His tag to the C-terminus of fGCSF, and an APB-F(v2)Fd (FL335 Fd-GGGGSGGGGS(SEQ ID NO: 85)-mutant fGCSF) gene was synthesized and produced. Similar to the above, as shown in Figure 4A, after treating the above-produced gene with BbsI restriction enzyme, it was inserted into the pd2535nt expression vector to produce an APB-F1(v2) Fd pd2535NT vector.

[0209] Subsequently, to obtain a large amount of the gene for CHO cell transformation, DH5 cells (RBC royal bank, Canada) and the gene mixture were heat-shocked at 42°C for 45 seconds to insert plasmid DNA into the cells. Then, a plasmid DNA prep kit (GeneAll, South Korea) was used to obtain plasmid DNA.

[0210] [Production Example 2. Construction of a transient protein expression system] For the expression of proteins such as FL335 Fab, feline serum albumin, native fG-CSF, APB-F1 (v1), and APB-F1 (v2), the ExpiCHO Expression System was utilized in this experiment. Specifically, ExpiCHO-S (trademark) cells (ThermoFisher Scientific) were cultured in an Erlenmeyer flask at 37°C, 5% CO2, and 140 rpm using ExpiCHO expression media (Gibco, ThermoFisher Scientific). Then, 10 μl of the cell culture solution was mixed with 10 μl of trypan blue, and 10 μl of the mixture was added to Cell Counting Chamber Slides. Subsequently, changes in cell number and cell viability were measured using a Countess II Automated Cell Counter (Invitrogen) machine. Then, the transformation of cells using the expression vector prepared in Production Example 1 was carried out using OptiPRO SFM medium (ThermoFisher Scientific) and ExpiFectamine (trademark) CHO reagent (ExpiFectamine (trademark) CHO Transfection Kit). 18 - 22 hours after the time of transformation (day 0), ExpiFectamine (trademark) CHO Enhancer and ExpiCHO (trademark) Feed were added to the culture medium, and when the cell concentration reached 2x10 6 cells / mL, the culture conditions were changed to 32°C, 5% CO2, and 140 rpm, and the transformed cells were cultured. On the 5th day after the time of transformation, ExpiCHO (trademark) Feed was further added to the culture medium, and the cells were cultured until the cell viability reached 80%, and then the culture solution containing the transformed cells was collected.

[0211] The protein expression pattern and level in the recovered culture solution were confirmed via Western blotting. First, the protein sample was loaded at 1 μg / well onto a 4 - 15% gradient gel and then electrophoresed at 150 V for 50 minutes. Then, using a transfer buffer (Tris base, glycine, SDS, 20% methanol), the protein was transferred to a nitrocellulose transfer membrane at a constant current of 400 mA for 60 minutes. After that, a 3% skim milk solution at pH 7 was added to the membrane on which the protein transfer was completed, and then it was shaken at room temperature for 1 hour for blocking and washed while shaking with a washing buffer (0.1% tween in 1xPBS). Then, the detection antibody was diluted in a 3% skim milk solution at pH 7 and reacted with the blocked membrane at room temperature for 1 hour. The detection antibodies used for each protein are as follows. For APB - F1 (v1, v2) and fG - CSF (wild - type, mutant type), rabbit anti - Fe GCSF pAb (1 st Ab) (Aprilbio, Korea) and donkey anti - rabbit IgG (H + L) HRP (2 nd Ab) (Jackson Immuno Research, West Grove, Pennsylvania) were used. For FL335 Fab, 1 st Ab goat anti - cat light chain Ab (Bethyl Laboratories, Montgomery, TX), 2 nd Ab goat IgG - Fc fragment cross antibody (Bethyl), or 1 st Ab goat anti - cat F(ab)2 - biotin (Jackson Immuno Research, Bar Harbor, Maine), 2 ndStreptavidin-HRP (GE Healthcare, Chicago, Illinois) was used, and finally, for FSA, anti-His Tag HRP (BioLegend, San Diego, California) was used. Thereafter, after the membrane was washed with a washing buffer, 1 mL of TMB (Surmodics, Eden Prairie, Minnesota) was added thereto, and the substrate reaction was allowed to proceed for 1 to 5 minutes. Thereafter, triple distilled water was added thereto to terminate the substrate reaction, and after discarding the mixed solution in which TMB and triple distilled water were mixed, the protein expression pattern and expression level were confirmed.

[0212] [Production Example 3. Preparation of a stabilized cell line for the production of recombinant proteins] To prepare a stabilized cell line that produces FL335 Fab, native fGCSF, APB-F1 (v1), APB-F1 (v2), etc., GS null CHO-K1 cells (HD-BIOP3 cells, Horizon Discovery, UK) were used, and the genes prepared by DNA cloning were used to perform stable transformation as follows. As the basal medium and production medium, CD FortiCHO (trademark) Media (Life technologies, Carlsbad, California) was used, and subculture was performed using an Erlenmeyer flask (Corning) under the conditions of 37 °C, 5% CO2, and 125 rpm. Then, after mixing 10 μl of the cell culture solution and 10 μl of trypan blue, 10 μl of the mixture was added to Cell Counting Chamber Slides. Then, changes in the cell number and cell viability were measured using a Countess II Automated Cell Counter (Invitrogen) machine. For transformation of GS null CHO-K1 cells, two tubes containing 600 μl of OptiPRO SFM medium were prepared. Then, 37.5 μg of plasmid DNA was added to one of them, and 37.5 μl of Freestyle (trademark) Max reagent (Invitrogen, ThermoFisher Scientific) was added to the other. Then, the two tubes were carefully mixed while shaking, and then incubated at room temperature for 5 minutes. Then, the SFM medium containing the Freestyle (trademark) Max reagent was transferred into the tube containing the plasmid DNA, carefully mixed while shaking, and then reacted at room temperature for 20 - 25 minutes. Then, the mixture was carefully dispensed into GS null CHO-K1 cells, and it was cultured under the conditions of 37 °C, 5% CO2, and 125 rpm. Two days later, the culture medium was replaced with CD FortiCHO (trademark) Media without added glutamine, and then pool selection was advanced using 50 M of L-methionine sulfoximine and 10 μg / mL of puromycin.

[0213] After completing the pool selection of the stabilized cell lines, ELISA was performed to confirm the productivity of the recombinant protein in each cell line. Human serum albumin (Sigma Aldrich, Saint Louis, Missouri) was used as the capture Ag for APB-F1 (v1, v2) and FL335, and rat anti-human GCSF Ab (SouthenBiotech, Canada) was used as the capture Ag for fGCSF (wild type, mutant type). 100 μl of the capture Ag related to each sample was added to a MaxiSorp NUNC Immuno ELISA plate (Thermo Fisher, Waltham, Massachusetts) at a concentration of 1 μg / mL, and then the capture Ag was coated on the plate by culturing it overnight at 4°C. Then, PBST [PBS (phosphate buffered saline) containing 0.1% (v / v) Tween (Thermo Fisher)] with 3% BSA (bovine serumal bumin) was added to each well at 300 μl each and blocked at room temperature for 3 hours. 300 μl of washing buffer (PBST 0.1%) was dispensed into each well and washed, and such a process was repeated 3 times. 0.3% PBA (in 0.1% PBST) was used as the dilution solution for the samples and antibodies. The samples diluted with the dilution solution were dispensed into each well at 100 μl each and reacted at room temperature for 1 hour. After washing 3 times by the method described above, the detection antibodies were dispensed into each well at 100 μl / well and reacted at room temperature for 1 hour. Here, rabbit anti-Fe GCSF pAb (1 st Ab) (Aprilbio) and donkey anti-rabbit IgG (H+L) HRP (Jackson Immuno Research, West Grove, Pennsylvania) were used as the detection antibodies for APB-F1 (v1, v2) and fGCSF, and fGCSF, goat anti-cat IgG (H+L)-HRP was used as the detection antibody for FL335.

[0214] Furthermore, the production amounts of the respective purified proteins were measured using them as standards. Specifically, the binding signal was measured as the absorbance at 450 nm using an ELISA reader with a 3,3,5,5 - tetramethylbenzidine (TMB) substrate.

[0215] [Production Example 4. Separation and Purification of Recombinant Protein] The recombinant protein was separated and purified using equipment of AKTA pure 150L (GE Healthcare, Chicago, Illinois) by the following method.

[0216] (1) Separation and Purification of Native fG - CSF or Mutant fG - CSF

[0217] Native fGCSF or mutant fGCSF was purified using immobilized metal affinity chromatography (IMAC) purification technique and two-step ion exchange chromatography purification technique. First, 300 ml of the culture solution produced using CD FortiCHO (trademark) Media was centrifuged at 4,000 rpm for 20 minutes under refrigerated conditions to recover the supernatant, which was then filtered through a 0.2 μm membrane filter to prepare the supernatant. The prepared 300 ml of culture supernatant was flowed through a 5 ml prepacked Ni-NTA His·Bind (registered trademark) Resin column equilibrated with 10 CVs of 20 mM sodium phosphate and 500 mM NaCl buffer at pH 7.4 at a flow rate of 45 cm / h to bind the sample. Then, the column was washed at a flow rate of 60 cm / h using 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole buffer at pH 7.4 until the UV at 280 nm was below 10 mAU. After the column washing was completed, 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole buffer at pH 8.0, which is the protein elution buffer, was used to perform stepwise elution according to the elution buffer concentration at a flow rate of 60 cm / h, and each elution fraction by concentration was collected. After confirming the results of separation and purification using SDS-PAGE analysis, dialysis was carried out overnight under refrigerated conditions using 20 mM sodium citrate buffer at pH 5.5 to proceed to the subsequent purification step. The fGCSF sample alternated with 20 mM sodium citrate buffer at pH 5.5 was flowed through a prepacked Hitrap SP HP 5 ml column equilibrated with 10 CVs of 20 mM sodium citrate buffer at pH 5.5 at a flow rate of 60 cm / h to bind the sample. Then, the column was washed at a flow rate of 60 cm / h using 20 mM sodium citrate buffer at pH 5.5 until the UV at 280 nm was below 10 mAU. After the column washing was completed, 20 mM sodium citrate, 1 M NaCl buffer at pH 6.5, which is the protein elution buffer, was used to perform stepwise elution according to the elution buffer concentration at a flow rate of 60 cm / h, and the elution fractions with UV at 280 nm above 10 mAU were collected.Subsequently, after confirming the results of separation and purification using SDS-PAGE analysis, in order to proceed to the subsequent purification step, dialysis was carried out overnight under refrigeration conditions using 20 mM sodium citrate buffer at pH 5.5. The fGCSF sample alternated with 20 mM sodium citrate buffer at pH 5.5 was flowed through a prepacked POROS XQ 5 ml column equilibrated with 10 CVs of 20 mM sodium citrate buffer at pH 5.5 at a flow rate of 60 cm / h, and the sample passing through without binding to the column was collected. Subsequently, after confirming the results of separation and purification using SDS-PAGE analysis, impurities were removed with a 0.2 μm filter, protein quantification was performed using UV protein quantification method, and after protein quantification, it was stored at -20 °C until use.

[0218] (2) Separation and purification of APB-F1

[0219] APB-F1(v1, v2) was purified using an affinity chromatography purification technique with Capto L resin and a two-step ion exchange chromatography purification technique. First, 900 ml of the APB-F1(v1, v2) culture solution produced using CD FortiCHO (trademark) Media was centrifuged at 4,000 rpm for 20 minutes under refrigerated conditions to recover the supernatant, which was then filtered through a 0.2 μm membrane filter to prepare the supernatant. The prepared 900 ml of culture supernatant was passed through a prepacked Capto L 34 ml column equilibrated with 1xPBS 10 CVs at a flow rate of 120 cm / h to bind the sample. After that, a buffer of 20 mM sodium citrate at pH 6.0 and 1% D-mannitol was used to wash the column at a flow rate of 120 cm / h until the UV at 280 nm was below 10 mAU. After the column washing was completed, a protein elution buffer of 50 mM sodium citrate at pH 3.0 and 3% D-mannitol was used, and 100% of the elution buffer was flowed at a flow rate of 120 cm / h to collect the protein elution fractions with a UV at 280 nm above 10 mAU. To the collected elution fractions, a 1M Tris-Cl solution at pH 8.0 was added, and after weakly acidifying the pH to 6.0, impurities were removed with a 0.2 μm filter. The sample obtained by AC purification was passed through a CM sepharose F-F 10 ml column equilibrated with 20 mM sodium citrate buffer at pH 6.0 10 CVs at a flow rate of 153 cm / h to bind the sample. After that, a 20 mM sodium citrate buffer at pH 6.0 was used to wash the column at a flow rate of 153 cm / h until the UV at 280 nm was below 10 mAU. After the column washing was completed, a protein elution buffer of 20 mM sodium citrate at pH 6.0 and 1M NaCl was used, and elution was carried out stepwise with different concentrations of the elution buffer at a flow rate of 153 cm / h to collect the elution fractions with a UV at 280 nm above 10 mAU. After confirming the separation and purification results using SDS-PAGE analysis, in order to proceed to the subsequent purification step, dialysis was carried out overnight under refrigerated conditions using a 20 mM sodium citrate buffer at pH 6.0.The APB-F1 (v1, v2) sample alternated with 20 mM sodium citrate buffer at pH 6.0 was flowed through a prepacked POROS 50HQ 35 ml column equilibrated with 10 column volumes (CVs) of 20 mM sodium citrate buffer at pH 6.0 at a flow rate of 34 cm / h, and the sample passing through without binding to the column was collected. Then, after confirming the separation and purification results using SDS-PAGE analysis, impurities were removed with a 0.2 μm filter.

[0220] [Production Example 5. SDS-PAGE Analysis] The protein sample was treated under a total of three conditions: (1) reducing conditions (10% glycerol, 1% lithium dodecyl sulfate (LDL), 0.2 M triethanolamine-Cl at pH 7.6, 1% Ficoll (registered trademark)-400, 0.000625% phenol red, 0.000625% Coomassie G250, 0.5 mM disodium EDTA, 1.25% 2-mercaptoethanol: boiled for 10 minutes), (2) non-reducing conditions (10% glycerol, 1% lithium dodecyl sulfate (LDL), 0.2 M triethanolamine-Cl at pH 7.6, 1% Ficoll (registered trademark)-400, 0.000625% phenol red, 0.000625% Coomassie G250, 0.5 mM disodium EDTA, boiled for 10 minutes), and (3) non-reducing conditions [without boiling] (10% glycerol, 0.375 M triethanolamine-Cl at pH 6.8, 0.005% bromophenol blue: without boiling). Then, the protein sample was loaded onto a precast 4-15% gradient gel at a concentration of 1 μg / well, and electrophoresis was performed under the conditions of a constant voltage of 150 V for 50 minutes. After electrophoresis, the separated gel was stained with Ez Gel Staining Solution for 1 hour and then decolorized with water. Then, protein analysis was performed by comparing with Excelband (trademark) Enhanced 3-color high range protein marker.

[0221] [Production Example 6. Size Exclusion Chromatography] To confirm the purity of APB-F1(v1,v2), SE-HPLC was performed using an Agilent 1260 InfinityII system. As the SE-HPLC column, a TSK gel UltraSW aggregate 7.8 x 300 mm (Tosoh Biosciences, Japan) column was used. Specifically, (1) column temperature: 20 °C, (2) mobile phase A: 20 mM sodium citrate pH 5.5, 100 mM NaCl, (3) flow rate: 0.7 ml / min, (4) wavelength: 280 nm, (5) injection: 40 μg, (6) gradient: performed under isocratic conditions.

[0222] [Example 1. Expression and production of FL335 and feline serum albumin] In this example, the expression and production of the FL335 antibody fragment and feline serum albumin according to the above production example were confirmed. As shown in Figure 5, FL335 contains the human V H ,V L sequence, feline IgG1 CH1 (delta C) and CL-kappa. To prepare FL335, SL335V H and feline IgG1 CH1 were ligated via linking PCR to prepare the FL335 Fd gene. In the same manner as described above, SL335V L and feline CL-kappa were ligated to prepare the FL335 L gene. Subsequently, FL335 Fd, FL335 L, and feline serum albumin produced and purified according to the above production example were confirmed by SDS-PAGE.

[0223] After inserting FL335 Fd or FL335 L into the expression vector respectively, the protein expression by the expression vector was confirmed through transient expression using an ExpiCHO expression system. Subsequently, for the culture solution obtained by flask production of the stable cell line, a purification process including affinity chromatography and two-step ion exchange chromatography was carried out, and the protein purified from the GS null CHO-K1 cell culture supernatant was confirmed by SDS-PAGE on a 4 - 15% gradient gel.

[0224] In addition, feline serum albumin was also directly prepared via gene synthesis. For separation, purification, and analysis, an myc tag was linked to the N-terminus and a his tag was linked to the C-terminus, and it was inserted into the expression vector pJK plasmid. Subsequently, the protein expression by the said expression vector was confirmed via transient expression using the ExpiCHO expression system. Also, the culture solution obtained via the flask production of the stable cell line was separated and purified, and the protein purified therefrom was confirmed by SDS-PAGE.

[0225] Figures 6A and 6B show the confirmation of FL335 Fd, FL335 L, and feline serum albumin in the culture solution after the expression and purification process according to an embodiment. Figure 6A is the result of confirming FL335 via SDS-PAGE, and Figure 6B is the result of confirming feline serum albumin via SDS-PAGE.

[0226] As shown in Figure 6A, protein bands corresponding to FL335 Fd having a theoretical molecular weight of 23.492 kDa and FL335 L having a theoretical molecular weight of 23.877 kDa were confirmed. Similarly, a protein band corresponding to FL335 Fab having a theoretical molecular weight of 47.352 kDa was also confirmed. Also, as shown in Figure 6B, a protein band corresponding to feline serum albumin having a theoretical molecular weight of 67.8 kDa was confirmed.

[0227] [Example 2. Confirmation of the binding ability of FL335 to feline serum albumin] In this example, the binding ability of FL335 to feline serum albumin is confirmed. Specifically, feline serum albumin was diluted to 1 μg / mL with a carbonate coating buffer at pH 9.6, and 100 μL of the diluted solution was added to each well of a MaxiSorp NUNC Immuno ELISA plate. Then, it was left overnight at 4 °C to coat the plate with feline serum albumin. As the blocking buffer, PBST [PBS containing 0.1% (v / v) Tween] containing 3% bovine serum albumin was used, and 300 μL of the blocking buffer was added to each well and blocked at room temperature. Thereafter, 300 μL of a washing buffer (PBST) was dispensed into each well for washing, and this was repeated 3 times. As a solution for diluting the antibody fragment or sample, 0.3% PBA (in 0.1% PBST) was used, and 100 μL of the diluted solution was dispensed into the wells coated with feline serum albumin and reacted at room temperature for 1 hour. Washing was performed 3 times using the same method as described above. Thereafter, SL335 detection 1 st As the antibody, goat anti-human kappa-HRP was diluted 1:3,000 and added to the wells, FL335 detection 1 st As the antibody, goat anti-feline light chain Ab was diluted 1:3,000, FL335 detection 2 nd As the antibody, goat IgG-Fc fragment cross antibody was diluted 1:5,000 and added to the wells. The antigen-antibody reaction was allowed to proceed at room temperature for 1 hour, and the binding signal was measured using 3,3,5,5-tetramethylbenzidine (TMB) as a substrate and measuring the absorbance at 450 nm using an ELISA reader. On the other hand, in this example, in order to confirm the functionality of the FL335 antibody fragment that binds to albumin, the binding level to human serum albumin or feline serum albumin was compared with SL335, which is a Fab fragment that specifically binds to human albumin.

[0228] Figures 7A and 7B show the binding ability of FL335 to serum albumin. Figure 7A shows the result of confirming the binding ability of human and feline serum albumin to SL335 via ELISA, and Figure 7B shows the result of confirming the binding ability of human and feline serum albumin to FL335 via ELISA.

[0229] As shown in Figure 7, the FL335 antibody fragment shows a binding ability similar to that of SL335 for all serum albumins. Such experimental results indicate that when an existing antibody that specifically binds to anti-serum albumin, that is, the heavy chain variable region domain or the light chain variable region domain of SL335, is combined with the feline-derived heavy chain constant 1 domain or the light chain constant domain (CH1, Cκ), the binding ability to albumin is maintained as it is.

[0230] [Example 3. Expression and production of feline granulocyte colony-stimulating factor] In this example, it is to confirm the expression and production of the fGCSF protein according to the above production example. In order to prevent misfolding that may be caused during the process of protein production and for the convenience of the purification process and the treatment process, a total of two types of feline granulocyte colony-stimulating factors, native feline granulocyte colony-stimulating factor (native fGCSF) and mutant feline granulocyte colony-stimulating factor (mutant fGCSF), were produced together. Specifically, the mutant fGCSF was prepared in a form in which C17S was substituted for the removal of free cysteine and T133A was substituted for the removal of O-glycans from the native fGCSF. In order to prepare cell lines that stably express native fGCSF and mutant fGCSF, after transforming GS null CHO-K1 cells with them, L-methionine sulfoximine and puromycin were used and selection was carried out over about 4 weeks to prepare a stabilized cell line. Thereafter, with respect to the culture solution obtained by flask production of the stabilized cell line, a purification process including immobilized metal affinity chromatography and two-step ion exchange chromatography was advanced, and the protein purified from the GS null CHO-K1 cell culture supernatant was confirmed by SDS-PAGE on a 4-15% gradient gel.

[0231] A of FIG. 8 and B of FIG. 8 show the confirmation of native fG-CSF and mutant fGCSF in the culture solution after the expression and purification process according to an example. A of FIG. 8 is the result of confirming native fGCSF via SDS-PAGE, and B of FIG. 8 is the result of confirming mutant fGCSF via SDS-PAGE.

[0232] As shown in FIG. 8, protein bands corresponding to native fGCSF and mutant fGCSF were confirmed, and it was confirmed that under non-reducing (without boiling) conditions, due to the removal of free cysteine and O-glycans, the protein band size of mutant fGCSF became smaller than that of native fGCSF.

[0233] [Example 4. Expression and Production of APB-F1] In this example, the expression and production of APB-F1 according to the above production example were confirmed. As shown in Fig. 9, APB-F1 was prepared as two types of proteins by fGCSF. That is, APB-F1 (v1) is a protein in which natural fGCSF is linked to the C-terminus of FL335 Fd, and APB-F1 (v2) is a protein in which mutant fGCSF is linked to the C-terminus of FL335 Fd. Thereafter, in order to prepare cell lines that stably express APB-F1 (v1) and APB-F1 (v2), GS null CHO-K1 cells were transformed with them, and then selected using L-methionine sulfoximine and puromycin over about 4 weeks to prepare stabilized cell lines. Thereafter, with respect to the culture solution obtained by flask production of the stabilized cell line, a purification process including affinity chromatography and two-step ion exchange chromatography was carried out, and the protein purified from the supernatant of the GS null CHO-K1 cell culture solution was confirmed by SDS-PAGE on a 4-15% gradient gel.

[0234] A of Fig. 10 and B of Fig. 10 show the confirmation of APB-F1 in the culture solution after the expression and purification process according to an example. A of Fig. 10 is the result of confirming APB-F1 (v1) via SDS-PAGE, and B of Fig. 10 is the result of confirming APB-F1 (V2) via SDS-PAGE. Fig. 11 shows the confirmation of the purity of the APB-F1 sample after the expression and purification process according to an example. Fig. 11A is the SEC-HPLC analysis result for the APB-F1 (v1) sample, and Fig. 11B is the SEC-HPLC analysis result for the APB-F1 (v2) sample.

[0235] As shown in FIG. 10, protein bands corresponding to APB-F1(v1) and APB-F1(v2) were confirmed. Similar to the above results, under non-reducing (without boiling) conditions, due to the removal of free cysteine and O-glycans, the protein band size of APB-F1(v2) containing mutant fGCSF was confirmed to be smaller than that of APB-F1(v1) containing circular fGCSF. Also, as shown in FIG. 11, it was confirmed that the samples of APB-F1(v1) and APB-F1(v2) obtained through the above series of manufacturing and purification processes had a high purity of approximately 98%.

[0236] [Example 5. Evaluation of Biological Activity of APB-F1] In this example, the EC of APB-F1 50 was measured to evaluate their biological activities. Specifically, a cell proliferation assay was performed on M-NFS60 cells (ATCC No. 1838). FL335 was used as the negative control group, and human granulocyte colony-stimulating factor (hGCSF I.S, rDNA Derived, 2 nd International Standard, NIBSC code: 09 / 136), which is the biological activity standard substance designated by WHO, was used as the positive control group. Filgrastim, PEG-Filgrastim, and fGCSF were used as the comparison groups. First, M-NFS60 cells were cultured in RPMI-1640 medium supplemented with 1% FBS, and the culture conditions were maintained at 37°C and 5% CO2. Then, the cells were adjusted to 6×10 4The concentration per cell / ml was diluted with RPMI-1640 (Gibco) medium, and 100 μl of the diluted solution was added to a 96-well plate. Next, APB-F1, the control substance, and the control substance were diluted in RPMI-1640 medium to concentrations of 0.01 - 1,000 pM, and then they were added in triplicate at 100 μl per well each, and cultured at 37 °C under 5% CO2 conditions for 42 hours. Thereafter, to the previously cultured cells, Cell Counting Kit-8 solution was added at a concentration of 20 μl / well, and the reaction was further allowed to proceed for 6 hours. Thereafter, in order to confirm the cell viability, the absorbance at 450 nm was measured using a microplate reader.

[0237] Figure 12 shows the results of the cell proliferation assay analysis of APB-F1 performed on M-NFS60 cells. As shown in Figure 12, the EC 50 of hGCSF I.S, fGCSF, APB-F1 (v1) and APB-F1 (v2) were 2.45 pM, 3.59 pM, 8.32 pM and 5.67 pM respectively, and the EC 50 of filgrastim and PEG-filgrastim were confirmed to be 3.94 pM and 3.07 pM respectively. Specifically, the activity of filgrastim measured in this experiment was 0.6×10 8 U / mg, which is within the known activity level range of filgrastim (0.6 ± 1.0)×10 8 U / mg, and the effectiveness of this experiment could be confirmed. Also, the activities of fGCSF and PEG-filgrastim were measured to be 6.5×10 6 U / mg and 3.6×10 7 U / mg respectively, and the activities of APB-F1 (v1) and APB-F1 (v2) were 7.8×10 6 U / mg and (8.7 ± 0.3)×10 6It was confirmed that it showed a similar level of activity in U / mg. This indicated that the biological activity of APB-F1(v2) was maintained despite the removal of free cysteine and O-glycans in fGCSF. Considering the ease of the production, separation, and purification processes of the recombinant protein, in the following examples, APB-F1(v2) was selected as the APB-F1 protein.

[0238] [Example 6. Intact Mass Analysis] In this example, in order to confirm the exact molecular weight of APB-F1, intact mass analysis was performed under reducing conditions. In this experiment, a Dionex UHPLC and Q-TOF5600+ MS / MS system were used. The mass of APB-F1 was measured using an Acquity UPLC (registered trademark) BEH130 C4, 1.7 μm column, with acetonitrile as the mobile phase, at a flow rate of 0.3 ml / min.

[0239] Figures 13A and 13B show the molecular weight of APB-F1 confirmed through intact mass analysis. Figure 13A shows the result of confirming the mass of APB-F1 Fd, and Figure 13B shows the result of confirming the mass of APB-F1L. As shown in Figure 13, the mass of APB-F1 Fd was measured to be 47.743 kDa, and the mass of APB-F1 FdL was 23.872 kDa.

[0240] [Example 7. N-Terminal Sequencing] In this example, the N-terminal sequence of APB-F1 was specifically confirmed. After the expression of the recombinant protein, in order to confirm the accuracy of processes such as the removal of the signal sequence, N-terminal sequencing analysis was performed. This experiment was carried out by entrusting ProteomeTech Inc., and the Edman degradation method and the LC-MS / MS method were utilized. The Edman degradation method utilized a protein sequencer equipment, and based on the relative retention time (dptu) of a standard substance (PTH-AA, 8.0 pM), with an exposure time of 3.5 - 18.0 minutes and a detector scale condition of 0.005 AUFS, the N-terminal sequence of APB-F1 (10.0 pM) was analyzed. Table 2 shows the result of confirming the N-terminal sequence of APB-F1 through the Edman degradation method.

Table 2

[0241] As shown in Table 2, it was confirmed that the N-terminal sequence of APB-F1L is DIVLT. On the other hand, for APB-F1 Fd, the reaction was blocked and its sequence could not be confirmed, which was judged to be the result of the biological conversion of the first residue, glutamine (Gln), to polyglutamate (pGlu).

[0242] Subsequently, LC-MS / MS utilized NanoUPLC, LTQ-orbitrap-mass spectromete equipment, and under the conditions of peptide mass tolerance (±10 ppm) in peptide mass matching, fragment mass tolerance (±0.8 Da) in fragment mass matching, Max Missed Cleavages (2), and a range of 300 - 2,000 m / z, an analysis was performed on the QVQLVQSGGGPVKPGGSLRLSCAAS base sequence (the N-terminals of SEQ ID NO: 22 and 23). Subsequently, the analysis was carried out using Proteome Discoverer and MASCOT software. Table 3 shows the results of LC-MS / MS analysis of the N-terminal sequence of APB-F1 Fd using Proteome Discoverer software, and Figure 14 shows the results of LC-MS / MS analysis of the N-terminal sequence of APB-F1 Fd using MASCOT software.

Table 3

[0243] As shown in Table 3 and Figure 14, it was confirmed that the N-terminal sequence of APB-F1 Fd (SEQ ID NOs: 22 and 23) is Q(pyro-glu)VQLV.

[0244] [Example 8. Pharmacokinetic Evaluation of APB-F1] In this example, a pharmacokinetic evaluation was performed to confirm the absorption, distribution, metabolism, and excretion of APB-F1. Specifically, after subcutaneous injection of the test substance of APB-F1 into healthy cats, blood samples were collected and analyzed. Each group consisted of a total of 4 cats, 2 male cats and 2 non-pregnant female cats. A total of 15 times over time, 3 ml of whole blood was obtained via jugular vein blood sampling at 2 days or 4 days before the administration of the test substance, immediately after the administration of the test substance, and at the time points when 2, 6, or 12 hours had elapsed after the administration of the test substance. The whole blood was centrifuged at 3,000 rpm for 5 minutes immediately after being obtained, and after separating the plasma, it was stored in an ultra-low temperature freezer (about -70 °C) until use. In this experiment, as the experimental groups, a group administered with 100 μg / kg of mutant fGCSF protein and a group administered with 360 μg / kg of APB-F1 were set up.

[0245] Thereafter, the obtained sample was analyzed by the ELISA method. First, using a carbonate coating buffer at pH 9.6, a rat anti-human GCSF Ab solution diluted to a concentration of 1 μg / mL was loaded into each well of the ELISA plate at 100 ng / well, 100 μL each, and then incubated at 2-8°C overnight to coat the antibody. After removing the solution in the plate, 300 μL of washing buffer was added per well and washed once. Thereafter, blocking buffer was dispensed into each well at 300 μL each and blocked at room temperature for 3 hours. The solution in the plate was removed, the plate was inverted at room temperature for 1 hour, and the remaining solution was removed. Then, 100 μL of the standard substance and the diluted test solution were added to the wells in triplicate. Thereafter, the plate was covered with a sealer and reacted at room temperature and 450 rpm for 90 minutes, and washed 4 times by the method as described above using the washing buffer. Thereafter, rabbit anti-fGCSFpAb, the primary antibody, was diluted 1:1,000 and dispensed into all wells at 100 μL each, and then the plate was covered with a sealer and reacted at room temperature for 1 hour. Next, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), the secondary antibody, was added to induce an antigen-antibody reaction. Specifically, after washing 4 times by the method as described above, the secondary antibody diluted 1:10,000 was added to all wells at 100 μL each and reacted at room temperature and in the dark for 1 hour. Thereafter, after washing 5 times by the method as described above, BioFX (registered trademark) TMB Super Sensitive One Component HRP Microwell Substrate was added to each well at 100 μL / well and reacted at room temperature for 5-10 minutes. Thereafter, 1N HCL was added to terminate the substrate reaction. Next, the absorbance was measured at a measurement wavelength of 450 nm and a reference wavelength of 650 nm using a SPECTROstar Nano Microplate reader, and statistical analysis was performed using Phoenix WinNonlin software.

[0246] Table 4 and Figure 15 show the results of the pharmacokinetic evaluation of APB-F1 in cats. As shown in Table 4 and FIG. 15, the time to reach the maximum blood concentration (T max ) is 6 hours in the case of fGCSF, while APB-F1 shows 12 hours. The AUClast of APB-F1 is 19,025.4 h·ng / mL, which is approximately 3.1 times increased compared to fGCSF that showed a value of 6,050.03 h·ng / mL. Also, the maximum blood concentration (C max ) is 576.3 ng / mL in the case of fGCSF, while APB-F1 is 823.9 ng / mL, showing a value 1.4 times increased compared to fGCSF. The elimination half-life (T1 / 2) is 13.3 hours in the case of APB-F1, which is approximately 4.9 times increased compared to fGCSF that showed 2.7 hours. From such experimental results, it was found that APB-F1 according to one embodiment has improved pharmacokinetic properties compared to the mutant fGCSF protein. [Table 4]

[0247] [Example 9. Pharmacodynamic Evaluation of APB-F1] In this example, to confirm the physiological, physicochemical actions and effects of APB-F1, a pharmacodynamic evaluation was carried out. Specifically, after subcutaneous injection of a test substance such as APB-F1 into healthy cats, the white blood cell counts in the blood were measured and analyzed. Each group was composed of a total of 4 cats, 2 male cats and 2 non-pregnant female cats. Based on the administration day (day 0), 3 ml of whole blood was obtained each time via jugular venipuncture over time. Then, hematological examinations using an automatic blood analyzer and blood biochemical examinations using a blood biochemical analyzer and an electrolyte automatic analyzer were carried out on the obtained samples. In this experiment, as the experimental groups, a 36 μg / kg APB-F1 administration group, a 360 μg / kg APB-F1 administration group, a 10 μg / kg fGCSF administration group, a 10 μg / kg filgrastim (Grasin) administration group, a 100 μg / kg PEG-filgrastim (Neulasta) administration group, and a 26 μg / kg FL335 (Fab) administration group were set, and as the control group, a vehicle administration group was set.

[0248] Figure 16 shows the results of the pharmacodynamic evaluation of APB-F1 on cats, which is the result of checking the numerical values of blood leukocytes. As shown in Figure 16, the leukocyte numerical values in the APB-F1 administration group were significantly higher than those before administration from the 1st day to the 11th day after administration, and were also observed to be higher than the normal range level (4.9×10 3 cells / μl 0~2.0×10 4 cells / μl) on the 20th day after administration (not shown). In addition, the leukocyte numerical values in the fGCSF administration group showed a significant difference compared to those before administration on the 1st day after administration, but decreased to the normal range level from the 5th day after administration. In the filgrastim administration group, the leukocyte numerical values also increased to a significant level on the 1st day after administration, but decreased to the normal range level on the 2nd day after administration. Moreover, the leukocyte numerical values in the PEG-filgrastim administration group were significantly higher than those before administration until the 7th day after administration, and it was confirmed that they maintained a level higher than the normal range until the 10th day after administration.

[0249] Figure 17 is the result of checking the numerical values of blood neutrophils. As shown in Figure 17, the blood neutrophil numerical values in the fGCSF administration group and the APB-F1 administration group were both significantly higher than those before administration on the 1st day after administration, but the blood neutrophil numerical values in the APB-F1 administration group were observed to be even higher than those in the fGCSF administration group. In addition, the blood neutrophil numerical values in the APB-F1 administration group maintained a significantly high level from the 1st day to the 11th day. Similarly, compared with the filgrastim administration group, a pattern was observed in which the blood neutrophil numerical values in the PEG-filgrastim administration group were higher and maintained for a longer period. On the other hand, the other administration groups showed no significant difference from the control group.

[0250] Figure 18 is the result of checking the numerical values of blood monocytes. As shown in Figure 18, the blood monocyte numerical values in the APB-F1 administration group were significantly higher than those before administration from the 2nd day to the 9th day after administration, and the blood monocyte numerical values in the PEG-filgrastim administration group were higher than those before administration from the 2nd day to the 5th day after administration. On the other hand, the other administration groups showed no significant difference from the control group.

[0251] Figure 19 shows the results of checking the numerical values of blood basophils. As shown in Figure 19, the numerical values of blood basophils in the APB-F1 administration group were significantly higher than the normal range levels from the second day to the seventh day after administration.

[0252] Figure 20 shows the results of checking the numerical values of blood lymphocytes, and Figure 21 shows the results of checking the numerical values of blood eosinophils. As shown in Figures 20 and 21, no significant difference was observed between the experimental group and the control group for blood lymphocytes and blood eosinophils.

[0253] From such a series of experimental results, it was found that APB-F1 according to one embodiment contributes to increasing and maintaining for a long period the numerical values of blood leukocytes, specifically, blood neutrophils, blood monocytes, and blood basophils.

[0254] A recombinant protein containing an antigen-binding fragment that binds to serum albumin and feline granulocyte colony-stimulating factor can improve pharmacokinetic properties including an increase in the in-vivo half-life and can continuously show the effect of raising the blood leukocyte level to a therapeutically effective level.

[0255] Therefore, the recombinant protein disclosed herein is also used as an active ingredient of a composition for preventing or treating feline panleukopenia.

[0256] The foregoing description of specific embodiments can fully show the general characteristics of the present invention. Thus, others can, by applying the knowledge within the art of this technical field, easily make various adaptations and / or modifications for various applications without departing from the general concept of the invention. Therefore, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the embodiments disclosed based on the teachings and guidelines presented herein. It is understood that the terms or phrases in this specification are for purposes of explanation rather than limitation, and the terms or phrases in this specification should be interpreted by those skilled in the art in light of the teachings and guidelines.

[0257] The width and scope of the present invention are not limited by any of the foregoing exemplary embodiments, but are defined only by the claims and their equivalents. All of the various aspects, embodiments, and options described herein may be combined in any and all variations.

[0258] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein.

Claims

1. A recombinant protein comprising (a) an antigen-binding fragment comprising a heavy chain and a light chain, and (b) feline granulocyte colony-stimulating factor (fGCSF), wherein the heavy chain comprises a heavy chain variable domain and a feline heavy chain constant 1 domain, and the heavy chain variable domain comprises a heavy chain CDR1 consisting of the amino acid sequence of AYSMN (SEQ ID NO: 64), a heavy chain CDR2 consisting of the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO: 65), and a heavy chain CDR3 consisting of the amino acid sequence of ETVMAGKALDY (SEQ ID NO: 66), the light chain comprises a light chain variable domain and a feline light chain constant domain, and the light chain variable domain comprises a light chain CDR1 consisting of the amino acid sequence of RASQSVGSNLA (SEQ ID NO: 81), a light chain CDR2 consisting of the amino acid sequence of GASTGAT (SEQ ID NO: 82), and a light chain CDR3 consisting of the amino acid sequence of QQYYSFLAKT (SEQ ID NO: 83), the recombinant protein.

2. The recombinant protein according to claim 1, further comprising a linker that links fGCSF to the antigen-binding fragment.

3. The recombinant protein according to claim 1, wherein the cysteine in (i) the feline heavy chain constant 1 domain and / or (ii) the feline light chain constant domain located at the interchain disulfide bond between the light chain and the heavy chain is conserved, deleted, and / or substituted with an amino acid residue other than cysteine.

4. The recombinant protein according to claim 1, wherein the fGCSF is modified by removing free cysteine groups and O-glycans from natural fGCSF.

5. The recombinant protein according to claim 1, wherein the fGCSF consists of the amino acid sequence of SEQ ID NO:

18.

6. The recombinant protein according to claim 1, wherein the fGCSF consists of the amino acid sequence of SEQ ID NO:

19.

7. The recombinant protein according to claim 2, wherein the linker links the fGCSF to the C-terminus of the feline heavy chain constant 1 domain, the N-terminus of the heavy chain variable domain, the C-terminus of the feline light chain constant domain, and / or the N-terminus of the light chain variable domain.

8. The recombinant protein according to claim 7, wherein the linker comprises 1 to 50 amino acids.

9. A nucleic acid molecule encoding the recombinant protein according to claim 1.

10. An expression vector comprising the nucleic acid molecule according to claim 9.

11. A cell transformed with the expression vector according to claim 10.

12. A composition comprising the recombinant protein according to claim 1.

13. A pharmaceutical composition comprising the composition according to claim 10 and a pharmaceutically acceptable excipient.

14. A kit comprising the composition according to claim 12 and a label containing a usage manual.

15. A method for treating feline panleukopenia in a cat, comprising administering the pharmaceutical composition according to claim 13 to an individual in need thereof.

16. The method according to claim 15, wherein the composition increases white blood cells in the blood of an individual.

17. The method according to claim 16, wherein the white blood cells are neutrophils, monocytes, basophils, or a combination thereof.

18. The method according to claim 15, wherein the elimination half-life (T1 / 2) of the recombinant protein is at least twice greater than that of feline granulocyte colony-stimulating factor (fGCSF).

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