RU2840969C1 - Versions of feline antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is modified IgG having high binding affinity properties with neonatal Fc receptor (FcRn), which contains a constant domain of feline IgG, comprising at least one amino acid substitution as compared to a constant domain of wild-type feline IgG, a method for its preparation and use. Said substitution is in amino acid residue 428 or 434. Also disclosed are: a pharmaceutical composition and a kit for increasing the half-life of IgG in blood serum of a cat, an expression vector, an isolated host cell, the use of immunoglobulin to increase its half-life in cat’s blood serum and a fused protein.

EFFECT: invention enables prolonging the half-life of feline antibody in cat’s blood serum.

17 cl, 6 tbl, 16 dwg, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/011,491, filed April 17, 2020, the contents of which are incorporated herein by reference in their entirety.

AREA OF TECHNOLOGY

[0002] The present invention generally relates to variants of feline antibodies and their uses. In particular, the present invention relates to a mutation in the Fc constant region of a feline antibody to improve its half-life and other characteristics.

LEVEL OF TECHNOLOGY

[0003] Feline IgG monoclonal antibodies (mAbs) are being developed as effective therapeutic agents in veterinary medicine. Several years ago, subclasses of feline IgG were identified and characterized (Strietzel et al., 2014, Vet Immunol Immunopathol., vol. 158(3-4), pages 214-223). However, little work has been done to extend the half-life of feline IgG.

[0004] Through a recycling mechanism, the neonatal Fc receptor (FcRn) extends the half-life of IgG in a pH-dependent interaction with its crystallizable fragment (Fc) region. Specifically, the Fc region spanning the interface of the CH2 and CH3 domains interacts with FcRn on the cell surface to regulate IgG homeostasis. This interaction is facilitated by an acidic interaction following pinocytosis of IgG, and thus IgG is protected from degradation. Endocytosed IgG is then recycled back to the cell surface and released into the bloodstream at alkaline pH, thereby maintaining sufficient IgG in the serum for proper function. Accordingly, the pharmacokinetic profile of IgGs depends on the structural and functional properties of their Fc regions.

[0005] Feline FcRn binds three subclasses of feline IgG, which have been compared with human IgG analogues. The half-life of feline IgG remains to be fully studied, since without any experimental support it cannot be expected or predicted whether they will accurately match human IgG.

[0006] The increased half-life of IgG allows for less frequent dosing and/or administration of a lower dose of the antibody drug product, which in turn reduces the number of visits to the veterinarian, improves patient compliance, and reduces concentration-dependent cytotoxicity/adverse events.

[0007] Accordingly, there is a need to identify mutations in Fc constant regions to improve half-life.

BRIEF DESCRIPTION OF THE ESSENCE OF THE INVENTION

[0008] The present invention relates to mutant feline IgGs that provide higher affinity for FcRn and a longer half-life compared to wild-type feline IgGs. In particular, the inventors of the present invention found that substitution of the amino acid residue serine (Ser or S) at position 428 or 434 with another amino acid surprisingly and unexpectedly increased the affinity for FcRn and, thus, increased the half-life of the IgG.

[0009] In one aspect of the invention, there is provided a modified IgG comprising: a constant domain of a feline IgG comprising at least one amino acid substitution compared to a constant domain of a wild-type feline IgG, wherein said substitution is at amino acid residue 428 or 434, numbered according to the EU index, as per Kabat. In an exemplary embodiment of the invention, said substitution is a substitution of serine at position 428 with leucine (S428L). In another exemplary embodiment of the invention, said substitution is a substitution of serine at position 434 with histidine (S434H). In some embodiments of the invention, said constant domain of a feline IgG comprises substitutions of serine at both position 428 and position 434 with leucine and histidine, respectively.

[00010] In another aspect of the invention, there is provided a polypeptide comprising: a constant domain of a feline IgG comprising one or more amino acid substitutions compared to a constant domain of a wild-type feline IgG, wherein said one or more substitutions are at amino acid residues 428, 434, or a combination thereof.

[00011] In another aspect of the invention, there is provided an antibody or molecule comprising: a constant domain of a feline IgG comprising one or more amino acid substitutions compared to a constant domain of a wild-type feline IgG, wherein said one or more substitutions are at amino acid residues 428, 434, or a combination thereof.

[00012] In a further aspect of the present invention, there is provided a method of making or creating an antibody or molecule, the method comprising: providing a vector or host cell comprising a constant domain of a feline IgG, wherein said constant domain of a feline IgG comprises one or more amino acid substitutions compared to a constant domain of a wild-type feline IgG, wherein said one or more substitutions are at amino acid residues 428, 434, or a combination thereof.

[00013] In another aspect of the present invention, there is provided a method for increasing the half-life of antibodies in the blood serum of a cat, the method comprising: administering to said cat a therapeutically effective amount of an antibody comprising a constant domain of a feline IgG, wherein said constant domain of a feline IgG comprises one or more amino acid substitutions compared to a constant domain of a wild-type feline IgG, wherein said one or more substitutions are at amino acid residues 428, 434, or a combination thereof.

[00014] Other features and advantages of the present invention will be apparent from the following detailed description and from the following drawings. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[00015] This patent or patent application contains at least one color image. Copies of this patent or this patent publication with the color image(s) will be provided by the authorized patent organization upon request and payment of the necessary fees.

[00016] Fig. 1 shows the domain structure of IgG. In the CH3 domain, Fc mutations S428L and/or N434H were introduced to increase the half-life of IgG by increasing the affinity for FcRn at pH6.

[00017] Figure 2A shows an alignment of the amino acid sequences of wild-type (WT) human IgG1, feline IgG1a WT, feline IgG1b WT, feline IgG2 WT, and a mutant feline IgG2 having a hinge mutation. Amino acid residues are numbered according to the EU index as per Kabat. The amino acid residues of CH1, hinge, CH2, and CH3 are highlighted in red, purple, blue, and green, respectively. Figure 2B shows the nucleotide sequence of Fc of feline IgG1a WT. Figure 2C shows the nucleotide sequence of Fc of feline IgG1a S434H. Figure 2D shows the nucleotide sequence of Fc of feline IgG1a S428L.

[00018] Fig. 3 shows individual serum concentrations of IgG DT mAb1 in 8 cats - 4 males (G00397, G00398, G00399, G00400) and 4 females (G00425, G00426, G00427, G00428) - after three injections (s.c., s.c., i.v.) at a dose of 2 mg/kg, measured over a period of 98 days.

[00019] Figure 4 shows individual serum concentrations of IgG DT mAb1 in 8 cats - 4 males (G00417, G00418, G00419, G00420) and 4 females (G00445, G00446, G00447, G0448) - after three injections (s.c., s.c., i.v.) at a dose of 2 mg/kg, measured over a period of 98 days.

[00020] Figure 5 shows individual serum mAb2 DT concentrations in 3 cats following a single subcutaneous injection of a 2 mg/kg dose, measured over a 30-day period.

[00021] Figure 6 shows individual serum concentrations of S428L mAb2 in 3 cats following a single subcutaneous injection of a 2 mg/kg dose, measured over a 30-day period.

[00022] Figure 7 shows individual serum concentrations of IgG DT mAb3 in 8 cats - 4 males (G00453, G00454, G00455, G00456) and 4 females (G00457, G00458, G00459, G00460) - after three injections (SC, SC, IV) at a dose of 2 mg/kg, measured over a period of 98 days.

[00023] Figure 8 shows individual serum concentrations of IgG S434H mAb3 in 8 cats - 4 males (G00469, G00470, G00471, G00472) and 4 females (G00473, G00474, G00475, G0476) - after three injections (SC, SC, IV) at a dose of 2 mg/kg, measured over a period of 98 days.

SEQUENCE LISTING SUMMARY

[00024] SEQ ID NO.: 1 is the amino acid sequence of the constant domain of a mutant IgG1a having the S428L mutation.

[00025] SEQ ID NO.: 2 is the amino acid sequence of the constant domain of a mutant IgG1a having the S434H mutation.

[00026] SEQ ID NO.: 3 is the amino acid sequence of the constant domain of wild-type IgG1a.

[00027] SEQ ID NO.: 4 is the nucleotide sequence of the constant domain of wild-type IgG1a.

[00028] SEQ ID NO.: 5 is the amino acid sequence of the CH1 domain of IgG1a.

[00029] SEQ ID NO.: 6 is the amino acid sequence of the hinge domain of IgG1a.

[00030] SEQ ID NO.: 7 is the amino acid sequence of the CH2 domain of IgG1a.

[00031] SEQ ID NO.: 8 is the amino acid sequence of the CH3 domain of IgG1a WT.

[00032] SEQ ID NO.: 9 is the nucleotide sequence of the CH1 domain of IgG1a.

[00033] SEQ ID NO.: 10 is the nucleotide sequence of the hinge domain of IgG1a.

[00034] SEQ ID NO.: 11 is the nucleotide sequence of the CH2 domain of IgG1a.

[00035] SEQ ID NO.: 12 is the nucleotide sequence of the CH3 domain of IgG1a WT.

[00036] SEQ ID NO.: 13 is the nucleotide sequence of the heavy chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00037] SEQ ID NO.: 14 is the amino acid sequence of the heavy chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00038] SEQ ID NO.: 15 is the amino acid sequence of the CDR1 of the heavy chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00039] SEQ ID NO.: 16 is the amino acid sequence of the CDR2 of the heavy chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00040] SEQ ID NO.: 17 is the amino acid sequence of the CDR3 variable region of the heavy chain of the anti-IL-31 antibody (ZTS-5864).

[00041] SEQ ID NO.: 18 is the nucleotide sequence of the light chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00042] SEQ ID NO.: 19 is the amino acid sequence of the light chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00043] SEQ ID NO.: 20 is the amino acid sequence of the CDR1 of the light chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00044] SEQ ID NO.: 21 is the amino acid sequence of the CDR2 of the light chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00045] SEQ ID NO.: 22 is the amino acid sequence of the CDR3 of the light chain variable region of the anti-IL-31 antibody (ZTS-5864).

[00046] SEQ ID NO.: 23 is the amino acid sequence of the heavy chain of an antibody against nerve growth factor (NGF) (ZTS768).

[00047] SEQ ID NO.: 24 is the amino acid sequence of the heavy chain CDR1 of the anti-NGF antibody (ZTS768).

[00048] SEQ ID NO.: 25 is the amino acid sequence of the heavy chain CDR2 of the anti-NGF antibody (ZTS768).

[00049] SEQ ID NO.: 26 is the amino acid sequence of the heavy chain CDR3 of the anti-NGF antibody (ZTS768).

[00050] SEQ ID NO.: 27 is the amino acid sequence of the light chain of the anti-NGF antibody (ZTS768).

[00051] SEQ ID NO.: 28 is the amino acid sequence of the light chain CDR1 of the anti-NGF antibody (ZTS768).

[00052] SEQ ID NO.: 29 is the amino acid sequence of the light chain CDR2 of the anti-NGF antibody (ZTS768).

[00053] SEQ ID NO.: 30 is the amino acid sequence of the light chain CDR3 of the anti-NGF antibody (ZTS768).

[00054] SEQ ID NO.: 31 is the amino acid sequence of the heavy chain of the anti-NGF antibody (NV02).

[00055] SEQ ID NO.: 32 is the amino acid sequence of the heavy chain CDR1 of the anti-NGF antibody (NV02).

[00056] SEQ ID NO.: 33 is the amino acid sequence of the heavy chain CDR2 of the anti-NGF antibody (NV02).

[00057] SEQ ID NO.: 34 is the amino acid sequence of the heavy chain CDR3 of the anti-NGF antibody (NV02).

[00058] SEQ ID NO.: 35 is the amino acid sequence of the kappa chain of the anti-NGF antibody (NV02).

[00059] SEQ ID NO.: 36 is the amino acid sequence of the CDR1 chain of the kappa chain of the anti-NGF antibody (NV02).

[00060] SEQ ID NO.: 37 is the amino acid sequence of the CDR2 chain of the kappa chain of the anti-NGF antibody (NV02).

[00061] SEQ ID NO.: 38 is the amino acid sequence of the CDR3 chain of the kappa chain of the anti-NGF antibody (NV02).

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[00062] The present invention can be better understood by reference to the following detailed description, which forms a part of this document. It is to be understood that the present invention is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is intended only to describe specific embodiments of the present invention by way of example and is not intended to limit the claimed invention.

[00063] Unless otherwise defined herein, scientific and technical terms used in connection with this application have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms include the plural, and plural terms include the singular.

[00064] As stated above and throughout this document, the following terms and abbreviations, unless otherwise specified, shall be understood to have the following meanings.

Definitions

[00065] As used herein, the singular forms "a" and "an" include a plural reference, and reference to a specific numerical value includes at least the specified specific value, unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" or "a compound" is a reference to one or more such molecules or compounds and equivalents thereof known to those skilled in the art, and so forth. As used herein, the term "a plurality" means more than one. When a range is recited, another embodiment of the invention includes a range from one specific value and/or to another specific value. Similarly, when values are recited as approximate values by the use of the preceding word "about," it is to be understood that the specific value forms another embodiment of the invention. All ranges are inclusive and may be combined.

[00066] In the specification and claims of the present invention, the numbering of amino acid residues in the immunoglobulin heavy chain corresponds to the EC index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EC index, as in Kabat" refers to the numbering of residues in an IgG antibody and is reflected herein in Fig. 2A.

[00067] The term "isolated" when used with respect to a nucleic acid is a nucleic acid that has been identified and separated from at least one contaminating nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid is in a form or environment that is different from that in which it is found in nature. Accordingly, isolated nucleic acid molecules are different from a nucleic acid molecule that exists in natural cells. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that normally express a polypeptide encoded therein, when, for example, said nucleic acid molecule is in a plasmid or in a chromosomal location different from that found in natural cells. An isolated nucleic acid may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is intended for use in protein expression, the corresponding oligonucleotide or polynucleotide will contain at least a sense or coding strand, but may contain both sense and antisense strands (i.e., may be double-stranded).

[00068] A nucleic acid molecule is "operably linked" or "functionally associated" when it is placed in a functional relationship with another nucleic acid molecule. For example, a promoter or enhancer is operably linked to a nucleic acid coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a nucleic acid coding sequence if it is positioned to facilitate translation thereof. A nucleic acid molecule encoding a variant Fc region is operably linked to a nucleic acid molecule encoding a heterologous protein (i.e., a protein or functional fragment thereof that, if naturally occurring, does not comprise an Fc region) if it is positioned such that the expressed fusion protein comprises said heterologous protein or functional fragment thereof adjacent at either the 5' or 3' position to said variant Fc region polypeptide; said heterologous protein may be directly adjacent to said variant Fc region polypeptide or may be separated therefrom by a linker sequence of any length and composition. Similarly, a polypeptide molecule (used herein as synonymous with the term "protein") is "functionally linked" or "functionally associated" when it is in a functional relationship with another polypeptide.

[00069] As used herein, the term "functional fragment" when used with respect to a polypeptide or protein (e.g., a variant Fc region or a monoclonal antibody) refers to fragments of such protein that retain at least one function of the full-length polypeptide. The fragments can range in size from six amino acids to the entire amino acid sequence of the full-length polypeptide minus one amino acid. A functional fragment of a variant Fc region polypeptide of the present invention retains at least one "amino acid substitution" as defined herein. A functional fragment of a variant Fc region polypeptide retains at least one function known in the art associated with an Fc region (e.g., ADCC, CDC, Fc receptor binding, C1q binding, downregulation of cell surface receptors, or can, for example, increase the in vivo or in vitro half-life of the polypeptide with which it is functionally associated).

[00070] The term "purified" or "purification" refers to the substantial removal of at least one contaminant from a sample. For example, an antigen-specific antibody may be purified by completely or substantially removing (at least 90%, 91%, 92%, 93%, 94%, 95%, or more, preferably at least 96%, 97%, 98%, or 99%) at least one contaminating non-immunoglobulin protein; it may also be purified by removing an immunoglobulin protein that does not bind the same antigen. Removing non-immunoglobulin proteins and/or removing immunoglobulins that do not bind a particular antigen results in an increased percentage of antigen-specific immunoglobulins in the sample. In another example, a polypeptide (e.g., an immunoglobulin) expressed in bacterial host cells is purified by completely or substantially removing host cell proteins; therefore, the percentage of said polypeptide in a given sample is increased.

[00071] The term "native," when used with respect to a polypeptide (e.g., an Fc region), is used herein to indicate that the polypeptide has an amino acid sequence consisting of the amino acid sequence of a polypeptide commonly found in nature, or a naturally occurring polymorphism thereof. A native polypeptide (e.g., a native Fc region) may be recombinantly produced or may be isolated from a natural source.

[00072] The term "expression vector" as used herein refers to a recombinant DNA molecule comprising a desired coding sequence and the corresponding nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.

[00073] As used in the context of this document, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, CHO cells, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether in vitro, in situ, or in vivo.

[00074] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the generally accepted boundaries of an immunoglobulin heavy chain Fc region may vary, the Fc region of a feline IgG heavy chain is generally defined as extending, for example, from amino acid residue position 231 to its carboxyl terminus. In some embodiments, variants comprise only portions of the Fc region and may or may not include the carboxyl terminus. An immunoglobulin Fc region typically comprises two constant domains, CH2 and CH3. In some embodiments, variants are provided that have one or more constant domains. In other embodiments, variants are provided that lack such constant domains (or have only portions of such constant domains).

[00075] The "CH2 domain" of the Fc region of feline IgG typically extends, for example, from about amino acid 231 to about amino acid 340 (see Fig. 2A). The CH2 domain is unique in that it is not closely associated with another domain. Two N-linked branched carbohydrate chains are located between the two CH2 domains of the intact native IgG molecule.

[00076] The "CH3 domain" of the Fc region of feline IgG is typically a stretch of residues located in a C-terminal position relative to the CH2 domain in the Fc region, extending, for example, from about amino acid residue 341 to about amino acid residue 447 (see Fig. 2A).

[00077] A "functional Fc region" has the "effector function" of a native sequence Fc region. At least one effector function of a polypeptide comprising a variant Fc region of the present invention may be enhanced or reduced compared to a polypeptide comprising a native Fc region or the parental region of the variant Fc region. Examples of effector functions include, but are not limited to: C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., BCR), etc. Such effector functions may require the Fc region to be operably linked to a binding domain (e.g., the variable domain of an antibody) and can be assessed using a variety of assays (e.g., Fc binding assay, ADCC assays, CDC assays, depletion of target cells from whole or fractionated blood samples, etc.).

[00078] A "native sequence Fc region" or "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of an Fc region typically found in nature. Exemplary native sequences of feline Fc regions are shown in Fig. 2A and include, for example, the native sequence of the Fc region of feline IgG1a.

[00079] A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region (or fragment thereof) by at least one "amino acid substitution" as defined herein. In preferred embodiments of the invention, said variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide, preferably 1, 2, 3, 4 or 5 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide. In an alternative embodiment of the invention, a variant Fc region can be generated according to the methods described herein, and said variant Fc region can be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a non-antibody polypeptide, such as a receptor or ligand binding domain.

[00080] As used herein, the term "derivative" in the context of polypeptides refers to a polypeptide that comprises an amino acid sequence that has been altered by the introduction of an amino acid residue substitution. The term "derivative" as used herein also refers to a polypeptide that has been modified by covalently attaching any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or another protein, etc. The derivative polypeptide may be prepared by chemical modifications using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative polypeptide has a function that is similar or identical to the function of the polypeptide from which it was derived. It should be understood that the polypeptide comprising a variant Fc region according to the invention may be a derivative as defined herein, preferably the derivatization occurs within the Fc region.

[00081] "Substantially of feline origin" when used herein with respect to a polypeptide (e.g., an Fc region or a monoclonal antibody), means that the polypeptide has an amino acid sequence that is at least 80%, at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, or even more preferably at least 95%, 95%, 97%, 98%, or 99% homologous to a native feline aminopolypeptide.

[00082] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to an Fc region (e.g., an Fc region of an antibody). A preferred FcR is a native sequence FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, Fc gamma RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Another preferred FcR includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. hnmunol. 117: 587 (1976); and Kim et al., J. Immunol. 24: 249 (1994)). In this document, the term “FcR” includes other FcRs, including those that will be identified in the future.

[00083] The phrase "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated response in which non-specific cytotoxic cells (e.g., non-specific) that express FcR receptors (e.g., natural killer cells ("NK cells"), neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of those target cells. The primary cells that mediate ADCC, NK cells, express only Fc gamma RIII, whereas monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

[00084] As used herein, the phrase "effector cells" refers to leukocytes (preferably feline) that express one or more FcR receptors and perform effector functions. Preferably, the cells express at least Fc gamma RIII and perform an ADCC effector function. Examples of leukocytes that mediate ADCC include peripheral blood monoclonal cells (PBMCs), NK cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells may be isolated from a native source (e.g., from blood or from PBMCs).

[00085] A polypeptide variant with an "altered" binding affinity for FcRn is a polypeptide that has either an increased (i.e., increased, greater, or higher) or a decreased (i.e., decreased, less, or lower) binding affinity for FcRn compared to the parent polypeptide of the variant or to a polypeptide comprising a native Fc region, when measured at pH 6.0. A polypeptide variant that exhibits increased binding or increased binding affinity for FcRn binds FcRn with a higher affinity than the parent polypeptide. A polypeptide variant that exhibits decreased binding or decreased binding affinity for FcRn binds FcRn with a lower affinity than its parent polypeptide. Such variants that exhibit reduced binding to FcRn may have little or no detectable binding to FcRn, such as 0-20% binding to FcRn, compared to the parent polypeptide. A polypeptide variant that binds FcRn with "increased affinity" compared to its parent polypeptide is one that binds FcRn with a higher binding affinity than the parent polypeptide when the amounts of said polypeptide variant and said parent polypeptide in a binding assay are substantially the same and all other conditions are identical. For example, a variant polypeptide with enhanced binding affinity to FcRn may exhibit from about 1.10-fold to about 100-fold (more typically from about 1.2-fold to about 50-fold) increased binding affinity to FcRn compared to the parent polypeptide, wherein the binding affinity to FcRn is determined, for example, in an enzyme-linked immunosorbent assay (ELISA) or other method available to one of ordinary skill in the art.

[00086] As used herein, the term "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence with a different, "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring replacement amino acid residues" (i.e., encoded by the genetic code) and selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Substitution with one or more unnatural amino acid residues is also encompassed by the definition of amino acid substitution set forth herein. "Unnatural amino acid residue" refers to a residue, other than the naturally occurring amino acid residues listed above, that is capable of covalently linking adjacent amino acid residue(s) in a polypeptide chain. Examples of unnatural amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman et al. Meth. Enzym. 202: 301-336 (1991).

[00087] The term "assay signal" refers to the output signal of any method for detecting protein-protein interactions, including but not limited to absorbance measurements in colorimetric assays, fluorescence intensity, or cpm. Assay formats may include ELISA, fluorescence-activated cell sorting (FACS), or other methods. A change in "assay signal" may reflect a change in cell viability and/or a change in a kinetic measure of the reverse reaction rate, a kinetic measure of the forward reaction rate, or both. A "higher assay signal" refers to a measured output number that is greater than another number (e.g., a variant may have a higher (greater) measured number in an ELISA assay compared to the parent polypeptide). A "lower" assay signal refers to a measured output number that is less than another number (e.g., a variant may have a lower (lower) measured number in an ELISA assay compared to the parent polypeptide).

[00088] The term "binding affinity" refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each Fc receptor-Fc binding interaction. Binding affinity is directly related to the kinetic rate constant of the reverse reaction (typically reported in units of reciprocal time, e.g., seconds ^-1 ) divided by the kinetic rate constant of the forward reaction (typically reported in units of concentration per unit time, e.g., mol/second). In general, it is not possible to definitively state whether changes in equilibrium dissociation constants are due to differences in the rates of the forward reaction, the reverse reaction, or both, unless each of these parameters is determined experimentally (e.g., using BIACORE or SAPIDYNE measurements).

[00089] As used herein, the term "hinge region" refers to a region of amino acids, such as in feline IgG1a (e.g., the region extending from position 216 to position 230 of feline IgG1a). Hinge regions of other IgG isotypes can be aligned with the IgG sequence by placing the first and last cysteine residues that form interheavy chain (S-S) disulfide bonds at the same positions.

[00090] "Clq" is a polypeptide that includes a binding site for the Fc region of immunoglobulin. C1q, together with two serine proteases, C1r and C1s, forms the C1 complex, the first component of the CDC pathway.

[00091] As used herein, the term "antibody", when used interchangeably with the term "immunoglobulin" or "Ig", is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or functional activity. Single-chain antibodies and chimeric, feline, or feline-derived antibodies, as well as chimeric or CDR-grafted single-chain antibodies and the like, containing portions derived from different species, are also encompassed by the present invention and the term "antibody". The various portions of such antibodies may be joined together chemically by conventional techniques, synthetically, or may be produced as a continuous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or felinized chain can be expressed to produce a continuous protein. See, for example, U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; WO 86/01533; U.S. Patent No. 5,225,539; and U.S. Patent Nos. 5,585,089 and 5,698,762. See also Newman, R. et al. BioTechnology, 10: 1455-1460, 1993, regarding a primatized antibody and Ladner et al., U.S. Patent No. 4,946,778 and Bird, R. E. et al., Science, 242: 423-426, 1988, regarding single-chain antibodies. It should be understood that all forms of antibodies containing an Fc region (or a portion thereof) are encompassed by the term "antibody" herein. In addition, the antibody may be labeled with a detectable label, immobilized on a solid phase, and/or conjugated to a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[00092] As used herein, the term "antibody fragment" refers to a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments, and multispecific antibodies formed from antibody fragments. Antibody fragments preferably retain at least a portion of the hinge and optionally the CH1 region of an IgG heavy chain. In other preferred embodiments of the invention, antibody fragments comprise at least a portion of the CH2 region or the entire CH2 region.

[00093] As used herein, the term "functional fragment" when used in relation to a monoclonal antibody is intended to refer to a portion of the monoclonal antibody that still retains functional activity. The functional activity can be, for example, antigen-binding activity or specificity, receptor-binding activity or specificity, effector functional activity, and the like. Functional fragments of a monoclonal antibody include, for example, individual heavy or light chains and fragments thereof, such as VL, VH, and Fd; monovalent fragments such as Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single-chain Fv (scFv); and Fc fragments. These terms are described, for example, in Harlowe and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178: 497-515 (1989); and Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The term "functional fragment" is intended to include, for example, fragments obtained by protease cleavage or reconstitution of a monoclonal antibody and by recombinant DNA techniques known to those skilled in the art.

[00094] As used herein, the term "fragment" refers to a polypeptide comprising an amino acid sequence of at least 5, 15, 20, 25, 40, 50, 70, 90, 100 or more contiguous amino acid residues from the amino acid sequence of another polypeptide. In a preferred embodiment of the invention, a fragment of a polypeptide retains at least one function of the full-length polypeptide.

[00095] As used herein, the term "chimeric antibody" includes monovalent, bivalent, or multivalent immunoglobulins. A monovalent chimeric antibody is a dimer formed by a chimeric heavy chain linked via disulfide bridges to a chimeric light chain. A bivalent chimeric antibody is a tetramer formed by two heavy chain-light chain dimers linked via at least one disulfide bridge. A chimeric heavy chain antibody for use in cats comprises an antigen-binding region derived from a heavy chain of a non-feline antibody that is linked to at least a portion of a constant region of a feline heavy chain, such as CH1 or CH2. A chimeric light chain antibody for use in cats comprises an antigen-binding region derived from a light chain of a non-feline antibody linked to at least a portion of a constant region of a feline light chain (CL). Antibodies, fragments or derivatives having chimeric heavy chains and light chains with the same or different variable region binding specificities can also be prepared by appropriately combining the individual polypeptide chains according to known method steps. In this approach, host organisms expressing the chimeric heavy chains are cultured separately from host organisms expressing the chimeric light chains, and the immunoglobulin chains are separately isolated and then combined. Alternatively, the host organisms can be co-cultured and the chains allowed to spontaneously associate in the culture medium, followed by recovery of the assembled immunoglobulin or fragment, or both the heavy and light chains can be expressed in the same host cell. Methods for producing chimeric antibodies are well known in the art (see, e.g., U.S. Patent No. 6,284,471; U.S. Patent No. 5,807,715; U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816,397).

[00096] As used herein, "felinized" forms of non-feline (e.g., murine) antibodies (i.e., felinized antibodies) are antibodies that contain little or no sequence derived from a non-feline immunoglobulin. In most cases, felinized antibodies are feline immunoglobulins (the recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-feline species (the donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and binding extent. In some cases, framework region (FR) residues of the feline immunoglobulin are replaced by corresponding residues from a non-feline species. In addition, felinized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. Such modifications are typically performed to further improve the characteristics of the antibody. Typically, a felinized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the complementarity determining loops (CDRs) correspond to those of an immunoglobulin from a species other than a feline, and all or substantially all of the FR residues are those of a feline immunoglobulin sequence. The felinized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically a portion of a feline immunoglobulin constant region.

[00097] As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding domain of a heterologous "adhesin" protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of the amino acid sequence of an adhesin with a desired binding specificity that is distinct from the antigen recognition and binding site (antigen binding site) of an antibody (i.e., is "heterologous"), with an immunoglobulin constant domain sequence.

[00098] As used herein, the term "ligand binding domain" refers to any native receptor or any region or derivative thereof that retains at least the qualitative ligand binding capacity of the corresponding native receptor. In certain embodiments of the invention, the receptor is derived from a cell surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin superfamily. Other receptors that are not members of the immunoglobulin superfamily but are nonetheless specifically encompassed by this definition are cytokine receptors, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules (e.g., E-, L-, and P-selectins).

[00099] As used in the context of this document, the term "receptor binding domain" refers to any native ligand of a receptor, including, for example, cell adhesion molecules, or any region or derivative of said native ligand that retains at least the qualitative receptor binding capacity of the corresponding native ligand.

[000100] As used herein, the term "isolated" polypeptide is a polypeptide that has been identified and separated from and/or isolated from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments of the invention, the isolated polypeptide is purified (1) to a polypeptide content greater than 95% by weight as determined by the Lowry method, and preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated polypeptide includes a polypeptide in situ in recombinant cells, since at least one component of the natural environment of such a polypeptide will not be present. Typically, however, an isolated polypeptide is obtained by at least one purification step.

[000101] When used in the context of this document, the terms "disorder" and "disease" are used interchangeably to refer to any condition that would benefit from treatment with a variant polypeptide (a polypeptide comprising a variant Fc region of the present invention), including chronic and acute disorders or diseases (e.g., pathological conditions that predispose a patient to a particular disorder).

[000102] As used herein, the term "receptor" refers to a polypeptide capable of binding at least one ligand. A preferred receptor is a cell surface receptor or a soluble receptor having an extracellular ligand binding domain and, optionally, other domains (e.g., a transmembrane domain, an intracellular domain, and/or a membrane anchor). The receptor to be assessed in the assay described herein can be an intact receptor or a fragment thereof, or a derivative thereof (e.g., a fusion protein comprising a receptor binding domain fused to one or more heterologous polypeptides). Moreover, the receptor to be assessed for its binding properties can be present in a cell or isolated and optionally coated on an assay plate or some other solid phase, or can be directly labeled and used as a probe.

Feline wild-type IgG

[000103] Feline IgGs are well known in the art and are fully described, for example, in Strietzel et al., 2014, Vet Immunol Immunopathol., vol. 158 (3-4), pages 214-223. In one embodiment of the invention, the feline IgG is IgG1 _a . In another embodiment of the invention, the feline IgG is IgG1 _b . In another embodiment of the invention, the feline IgG is IgG2. In a specific embodiment of the invention, the feline IgG is IgG1 _a .

[000104] The amino acid and nucleotide sequences of IgG1 _a , IgG1 _b and IgG2 are also well known in the art.

[000105] In one example, an IgG of the present invention comprises a constant domain, such as a CH1, CH2, or CH3 domain, or a combination thereof. In another example, a constant domain of the present invention comprises an Fc region, such as a CH2 or CH3 domain, or a combination thereof.

[000106] In a specific example, said wild-type constant domain comprises the amino acid sequence set forth in SEQ ID NO.: 3. In some embodiments of the invention, said wild-type IgG constant domain is a homologue, variant, isomer, or functional fragment of the sequence of SEQ ID NO.: 3, but without any mutation at position 428 or 434. Each possibility represents a separate embodiment of the invention.

[000107] IgG constant domains also include polypeptides with amino acid sequences substantially similar to the amino acid sequence of said heavy and/or light chains. Substantially the same amino acid sequence is defined herein as a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a reference amino acid sequence, as determined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988).

[000108] The invention also includes nucleic acid molecules that encode the IgGs or fragments thereof described herein. In one embodiment of the invention, the nucleic acids may encode an antibody heavy chain comprising, for example, CH1, CH2, CH3 regions, or a combination thereof. In another embodiment of the invention, the nucleic acids may encode an antibody heavy chain comprising, for example, any one of the VH regions or a portion thereof, or any one of the VH CDR regions, including any variants thereof. The invention also includes nucleic acid molecules that encode an antibody light chain comprising, for example, any one of the CL regions or a portion thereof, any one of the VL regions or a portion thereof, or any one of the VL CDR regions, including any variants thereof. In certain embodiments of the invention, the nucleic acid encodes both the heavy and light chains, or portions thereof.

[000109] The amino acid sequence of the wild-type constant domain set forth in SEQ ID NO: 3 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 4.

Modified feline IgG

[000110] The inventors of the present invention have discovered that substitution of the amino acid residue serine (Ser or S) at position 428 or 434 with another amino acid surprisingly and unexpectedly increased the affinity for FcRn and increased the half-life of IgG. The terms "position 428" or "position 434" when used herein refer to a position numbered according to the EU index as per Kabat et al. (1991).

[000111] Accordingly, in one embodiment of the invention there is provided a modified IgG comprising: a constant domain of a feline IgG comprising at least one amino acid substitution compared to a constant domain of a wild-type feline IgG, wherein said substitution is at amino acid residue 428, numbered according to the EU index, as per Kabat. The serine at position 428 may be substituted with any other amino acid. For example, serine at position 428 may be substituted with leucine (i.e., S428L), asparagine (i.e., S428N), alanine (i.e., S428A), phenylalanine (i.e., S428F), glycine (i.e., S428G), isoleucine (i.e., S428I), lysine (i.e., S428K), histidine (i.e., S428H), methionine (i.e., S428M), glutamine (i.e., S428Q), arginine (i.e., S428R), threonine (i.e., S428T), valine (i.e., S428V), tryptophan (i.e., S428W), tyrosine (i.e., S428Y), cysteine (i.e., S428C), aspartic acid (i.e., S428D), glutamic acid (i.e., S428E), or proline (i.e., S428P). In a particular embodiment of the invention, said substitution is a leucine substitution (e.g., S428L).

[000112] In a specific example, a mutant constant domain of the present invention comprises the amino acid sequence set forth in SEQ ID NO.: 1. In some embodiments of the present invention, said mutant IgG constant domain is a homologue, variant, isomer, or functional fragment of the sequence SEQ ID NO.: 1, but with a mutation at position 428. Each possibility represents a separate embodiment of the present invention.

[000113] The amino acid sequence of the mutant constant domain set forth in SEQ ID NO.: 1 is encoded by a corresponding mutant nucleic acid sequence, such as a mutant form of the nucleic acid sequence set forth in SEQ ID NO.: 4.

[000114] In another embodiment of the present invention, there is provided a modified IgG comprising: a constant domain of a feline IgG comprising at least one amino acid substitution compared to a constant domain of a wild-type feline IgG, wherein said substitution is at amino acid residue 434, numbered according to the EU index, as per Kabat. The serine at position 434 may be substituted with any other amino acid. For example, serine at position 434 may be substituted with histidine (i.e., S434H), asparagine (i.e., S434N), alanine (i.e., S434A), phenylalanine (i.e., S434F), glycine (i.e., S434G), isoleucine (i.e., S434I), lysine (i.e., S434K), leucine (i.e., S434L), methionine (i.e., S434M), glutamine (i.e., S434Q), arginine (i.e., S434R), threonine (i.e., S434T), valine (i.e., S434V), tryptophan (i.e., S434W), tyrosine (i.e., S434Y), cysteine (i.e., S434C), aspartic acid (i.e., S434D), glutamic acid (i.e., S434E), or proline (i.e., S434P). In a particular embodiment of the invention, said substitution is a histidine substitution (e.g., S434H).

[000115] In a specific example, a mutant constant domain of the present invention comprises the amino acid sequence set forth in SEQ ID NO.: 2. In some embodiments of the present invention, said mutant IgG constant domain is a homologue, variant, isomer, or functional fragment of the sequence SEQ ID NO.: 2, but with a mutation at position 434. Each possibility represents a separate embodiment of the present invention.

[000116] The amino acid sequence of the mutant constant domain set forth in SEQ ID NO.: 2 is encoded by a corresponding mutant nucleic acid sequence, such as a mutant form of the nucleic acid sequence set forth in SEQ ID NO.: 4.

[000117] In some embodiments of the present invention, said feline IgG constant domain comprises substitutions of serine at both position 428 and position 434 with leucine and histidine, respectively.

[000118] The modified IgG of the present invention provides a half-life ranging from about 8 days to about 26 days. In one embodiment of the invention, the modified IgG of the present invention provides a half-life of about 10, 12, 15, 17, 20, 23, or 26 days. In a particular embodiment, the modified IgG of the present invention provides a half-life of greater than 10 days.

Methods for producing the antibody molecules provided in this invention

[000119] Methods for producing antibody molecules are well known in the art and are fully described in U.S. Patent Nos. 8,394,925; 8,088,376; 8,546,543; 10,336,818; and 9,803,023, and U.S. Patent Application No. 20060067930, which are incorporated herein by reference in their entireties. Any suitable method, process, or technique known to one of skill in the art may be used. An antibody molecule having a variant Fc region of the present invention can be produced according to methods well known in the art. In some embodiments of the present invention, the variant Fc region may be fused to a heterologous polypeptide of choice, such as an antibody variable domain or a receptor or ligand binding domain.

[000120] With the advent of molecular biology techniques and recombinant technologies, a person skilled in the art can obtain antibody and antibody-like molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of antibodies. Such antibodies can be obtained either by cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains with assembly of the synthesized chains to form active tetrameric (H2L2) structures that have affinity for specific epitopes and antigenic determinants. This has made it possible to obtain ready-made antibodies having sequences characteristic of neutralizing antibodies from different species and sources.

[000121] Regardless of the source of the antibodies or the methods by which they are produced recombinantly, or the methods by which they are synthesized, whether in vitro or in vivo, using transgenic animals, large laboratory or commercial cell cultures, using transgenic plants, or by direct chemical synthesis that does not use living organisms at any stage of the process, all antibodies have a similar overall 3-dimensional structure. This structure is often referred to as H2L2, which refers to the fact that antibodies typically contain two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions that are capable of interacting with a structurally complementary antigenic target. The regions that interact with said target are called "variable" regions, or "V" regions, and are characterized by differences in amino acid sequence from antibodies with different antigenic specificities. The variable regions of the H chain and the L chain contain amino acid sequences capable of specifically binding to antigenic targets.

[000122] As used herein, the term "antigen-binding region" refers to that portion of an antibody molecule that contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for that antigen. The antigen-binding region of an antibody includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues. Within the variable regions of the H and L chains that provide the antigen-binding regions are smaller sequences that are termed "hypervariable" because of their extreme variability between antibodies of different specificities. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDRs." The CDRs are responsible for the major specificity of an antibody for a particular antigenic determinant structure.

[000123] CDRs are non-contiguous stretches of amino acids in variable regions, but regardless of species, the positional locations of these critical amino acid sequences in the variable regions of the heavy and light chains have been found to have similar locations in the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies have three CDRs, each of which is non-contiguous with the others. In all mammalian species, antibody peptides contain constant (i.e., highly conserved) and variable regions, and within the latter are CDRs and so-called "framework regions" consisting of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

[000124] The present invention further provides a vector comprising at least one of the nucleic acids described above. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be identified, each of which would be capable of encoding a given amino acid. The likelihood that a particular oligonucleotide will actually constitute the actual coding sequence can be estimated by considering the anomalous base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing the antibody or portion thereof. Such "codon usage rules" are described in Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). By applying the "codon usage rules" of Lathe, a single nucleotide sequence or set of nucleotide sequences can be identified that contain a theoretical "most probable" nucleotide sequence capable of encoding feline IgG sequences. It is also contemplated that antibody coding regions for use in the present invention can also be obtained by altering existing antibody genes using standard molecular biology techniques that result in variants of the antibodies and peptides described herein. Such variants include, but are not limited to, deletions, additions, and substitutions in the amino acid sequence of the antibodies or peptides.

[000125] For example, one class of substitutions are conservative amino acid substitutions. Such substitutions are those that replace a given amino acid in a feline antibody peptide with another amino acid with similar characteristics. Typically, conservative substitutions are considered to be substitutions between the aliphatic amino acids Ala, Val, Leu, and Ike; substitutions between the hydroxyl residues Ser and Thr, substitutions between the acidic residues Asp and Glu, substitutions between the amide residues Asn and Gln, substitutions between the basic residues Lys and Arg, substitutions between the aromatic residues Phe, Tyr, and the like. Guidance on which amino acid changes are likely to be phenotypically inconspicuous is provided by Bowie et al., 247 Science 1306-10 (1990).

[000126] Variants of feline antibodies and peptides may be fully functional or may lack one or more functions. Fully functional variants typically contain only conservative variations or variations at non-critical residues or in non-critical regions. Functional variants may also contain substitutions of similar amino acids that result in little or no change in function. Alternatively, such substitutions may have some positive or negative effect on function. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or a substitution, insertion, inversion, or deletion at a critical residue or in a critical region.

[000127] Amino acids that are critical for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as epitope binding or ADCC activity in vitro. Sites that are critical for ligand binding to the receptor can also be identified by structural analysis, such as crystallography, nuclear magnetic resonance, or photoaffinity labeling Smith et al., 224 J. Mol. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[000128] Moreover, polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids. In addition, many amino acids, including terminal amino acids, can be modified either by natural processes such as processing or other post-translational modifications, or by chemical modification techniques that are well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition transfer RNA-mediated modifications of amino acids to proteins, such as arginylation and ubiquitination. Such modifications are well known to those skilled in the art and have been described in great detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, have been described in major texts such as Proteins Structure and Molecular Properties (2nd ed., T. E. Creighton, W. H. Freeman & Co., N. Y., 1993). Many detailed reviews are available on the subject, such as Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, N. Y., 1983); Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663 Arm. NY Acad. Sci. 48-62 (1992).

[000129] In another aspect of the invention, derivatives of antibodies are provided. A "derivative" of an antibody contains additional chemical moieties that are not normally part of the protein. Covalent modifications of the protein are included within the scope of the invention. Such modifications can be introduced into the molecule by reacting target amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents well known in the art is useful for cross-linking an antibody or fragment to a water-insoluble carrier matrix or to other macromolecular carriers.

[000130] Derivatives also include radioactively labeled monoclonal antibodies. For example, those labeled with radioactive iodine (251,1311), carbon (4C), sulfur (35S), indium, tritium ( ^H3 ), or the like; conjugates of monoclonal antibodies with biotin or avidin, with enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase; as well as conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters) or fluorescent agents (such as phycobiliproteins).

[000131] Another derivative of the bifunctional antibody of the present invention is a bispecific antibody prepared by combining portions of two separate antibodies that recognize two different antigen groups. This can be achieved by cross-linking or recombination techniques. In addition, fragments can be added to the antibody or portion thereof to increase half-life in vivo (e.g., by increasing clearance time from the bloodstream). Such techniques include, for example, the addition of PEG moieties (also referred to as "PEGylation"), and are well known in the art. See U.S. Patent Application Publication No. 20030031671.

[000132] In some embodiments of the invention, nucleic acids encoding said antibody are introduced directly into a host cell, and said cell is incubated under conditions sufficient to induce expression of said encoded antibody. After said nucleic acids have been introduced into the cell, said cell is typically incubated, typically at a temperature of 37°C, sometimes with selection, for a period of about 1-24 hours to allow expression of said antibody. In one embodiment of the invention, said antibody is secreted into the supernatant of the medium in which said cell grows. Traditionally, monoclonal antibodies have been produced as native molecules in mouse hybridoma lines. In addition to said technology, the present invention provides for expression of antibodies from recombinant DNA. This allows for the production of antibodies, as well as a repertoire of antibody derivatives and fusion proteins, in a selected host species.

[000133] A nucleic acid sequence encoding at least one antibody, portion, or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques, including blunt or sticky ends for ligation, restriction enzyme digestion to obtain appropriate ends, filling in cohesive ends as necessary, alkaline phosphatase treatment to avoid unwanted joining, and ligation with appropriate ligases. Techniques for such manipulations, as described, for example, in Maniatis et al., MOLECULAR CLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and 1989); and Ausubel et al. 1993, as noted above, can be used to construct nucleic acid sequences that encode an antibody molecule or an antigen-binding region thereof.

[000134] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences that contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences that encode the polypeptide. A functional linkage is one in which the regulatory DNA sequences and the DNA sequence desired to be expressed are linked in such a way as to provide for the expression of genes as peptides or antibody portions in recoverable amounts. The precise nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in the art. See, for example, Sambrook et al., 2001, supra; Ausubel et al., 1993, supra.

[000135] Accordingly, the present invention encompasses the expression of an antibody or peptide in either prokaryotic or eukaryotic cells. Suitable host organisms include bacterial or eukaryotic host organisms, including bacterial, yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or host cells derived from mammals, insects, birds, or yeast. The mammalian cell or tissue may be from a human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat. Any other suitable mammalian cell known in the art may also be used.

[000136] In one embodiment of the invention, the nucleotide sequence of the invention will be included in a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a wide variety of vectors can be used for this purpose. See, for example, Ausubel et al., 1993, supra. Factors that are important in selecting a particular plasmid or viral vector include: the ease with which recipient cells containing the vector can be recognized and selected from those recipient cells that do not contain the vector; the number of copies of the vector that are desired in a particular host organism; and whether the ability to "move" the vector between host cells of different species is desirable.

[000137] Examples of prokaryotic vectors known in the art include plasmids, such as those that are capable of replication in E. coli (such as, for example, pBR322, CoIE1, pSC101, pACYC 184, .pi.vX). Such plasmids are described, for example, in Maniatis et al, 1989, as cited above; Ausubel et al, 1993, as cited above. Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are described in Gryczan, THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include p1J101 (Kendall et al., 169 J. Bacteriol. 4177–83 (1987) and Streptomyces bacteriophages such as phLC31 (Chater et al., SIXTH INTL SYMPOSIUM ON ACTINOMYCETALES BIO. 45–54 (Akademiai Kaido, Budapest, Hungary 1986). For reviews of Pseudomonas plasmids, see John et al., 8 Rev. Infect. Dis. 693–704 (1986); lzaki, 33 Jpn. J. Bacteriol. 729–42 (1978); and Ausubel et al., 1993, as noted above.

[000138] Alternatively, gene expression elements suitable for expressing cDNAs encoding antibodies or peptides include, but are not limited to, the following: (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983), the Rous sarcoma virus LTR (Gorman et al, 79 Proc. Natl. Acad. Sci., USA 6777 (1982), and the Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985); (b) splice regions and polyadenylation sites, such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites, such as in SV40 (Okayama et al., 1983).

[000139] Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51 Gene 21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, the SV40 late region mRNA splicing, the rabbit S-globin intermediate sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation elements. For immunoglobulin genes, which consist partly of cDNA and partly of genomic DNA (Whittle et al., 1 Protein Engin. 499 (1987)), the transcriptional promoter may be human cytomegalovirus, the transcriptional enhancers may be cytomegalovirus and mouse/human immunoglobulin, and the mRNA splicing and polyadenylation regions may be native chromosomal immunoglobulin sequences.

[000140] In one embodiment of the invention, for expression of cDNA genes in rodent cells, the transcription promoter is a viral LTR sequence, the transcription promoter enhancers are one or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, the splice region contains an intron greater than 31 bp, and the polyadenylation and transcription termination regions are derived from a native chromosomal sequence corresponding to the immunoglobulin chain being synthesized. In other embodiments of the invention, cDNA sequences encoding other proteins are combined with the above expression elements to achieve expression of said proteins in mammalian cells.

[000141] Each fusion gene can be assembled into or inserted into an expression vector. Recipient cells capable of expressing the immunoglobulin chain gene product are then transfected separately with a peptide or gene encoding an H or L chain, or co-transfected with an H and L chain gene. The transfected recipient cells are cultured under conditions that allow expression of the inserted genes, and the expressed immunoglobulin chains or intact antibodies or fragments thereof are recovered from the culture.

[000142] In one embodiment of the invention, the fusion genes encoding the peptide or the H and L chains, or portions thereof, are assembled into separate expression vectors, which are then used to co-transfect a recipient cell. Alternatively, the fusion genes encoding the H and L chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the antibody, the recipient cell line can be myeloma cells. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by the transfected immunoglobulin genes and have the machinery for glycosylation of said immunoglobulin. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, and the secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as feline or non-feline B lymphocytes, feline or non-feline hybridoma cells, or interspecies heterohybridoma cells.

[000143] An expression vector carrying an antibody construct or polypeptide of the present invention can be introduced into an appropriate host cell by any of a variety of suitable methods, including biochemical methods such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical methods such as electroporation, direct microinjection, and microparticle bombardment Johnston et al, 240 Science 1538 (1988).

[000144] Yeast may provide significant advantages over bacteria in the production of immunoglobulin H and L chains. Yeast perform post-translational peptide modifications, including glycosylation. There are now a number of recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing the leader sequences (i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[000145] Yeast gene expression systems can be routinely assessed for levels of production, secretion, and stability of peptides, antibodies, and fragments and regions thereof. Any of a number of yeast gene expression systems can be used that contain promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large quantities when yeast is grown in glucose-rich media. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and termination signals of the phosphoglycerate kinase (PGK) gene can be used. A number of approaches can be used to evaluate optimal expression plasmids for expression of cloned immunoglobulin cDNAs in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985.

[000146] Bacterial strains can also be used as host organisms for the production of the antibody molecules or peptides described in this invention. In connection with said bacterial hosts, plasmid vectors are used that contain a replicon and control sequences that are obtained from species compatible with the host cell. Said vector carries a replication site, as well as specific genes that are capable of providing phenotypic selection in transformed cells. A number of approaches can be used to assess the expression of antibody plasmids, fragments and regions thereof, or antibody chains encoded by cloned immunoglobulin cDNAs in bacteria (see Glover, 1985, as cited above; Ausubel, 1993, as cited above; Sambrook, 2001, as cited above; Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, N.Y. (1994-2001); Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2001).

[000147] Host cells of mammalian origin can be grown in vitro or in vivo. Mammalian cells provide for post-translational modifications of the immunoglobulin protein molecules, including removal of the leader peptide, folding and assembly of the H and L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein. Mammalian cells that may be useful as host organisms for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin such as Vero cells (ATCC CRL 81) or CHO-K1 cells (ATCC CRL 61). A variety of vector systems are available for the expression of cloned peptides from the H and L chain genes in mammalian cells (see Glover, 1985, cited above). A variety of approaches can be used to produce complete H2L2 antibodies. Co-expression of the H and L chains in the same cells may be possible to achieve intracellular association and linkage of the H and L chains into complete tetrameric H2L2 antibodies and/or peptides. Said co-expression may occur using either the same or different plasmids in the same host. The genes for both H and L chains and/or peptides may be placed on the same plasmid, which may then be transfected into cells, and then cells expressing both chains may be selected directly. Alternatively, cells may first be transfected with a plasmid encoding one chain, such as the L chain, followed by transfection of the resulting cell line with a plasmid encoding the H chain containing a second selection marker. Cell lines producing H2L2 peptides and/or molecules by either pathway can be transfected with plasmids encoding additional copies of the H, L, or H plus L chain peptides, in combination with additional selection markers, to generate cell lines with improved properties, such as higher production of assembled H2L2 antibody molecules or increased stability of transfected cell lines.

[000148] Stable expression can be used to produce recombinant antibodies in high yields over a long period of time. For example, cell lines can be engineered that stably express the antibody molecule. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selection marker. Following the introduction of foreign DNA, the engineered cells can be grown for 1-2 days in an enriched nutrient medium and then transferred to a selection medium. The selection marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into the chromosome and grow to form foci, which in turn can be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful in screening and evaluating compounds/components that directly or indirectly interact with the antibody molecule.

[000149] Once an antibody of the present invention has been obtained, it can be purified by any method known in the art for purifying an immunoglobulin molecule, such as chromatography (e.g., ion exchange, affinity, particularly post-protein A specific antigen affinity, and size cutoff column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. In many embodiments of the present invention, the antibodies are secreted from the cell into the culture medium and collected from the culture medium.

[000150] In another aspect of the invention, there is provided an antibody comprising: a feline IgG constant domain comprising at least one amino acid substitution compared to a wild-type feline IgG constant domain, wherein said substitution is at amino acid residue 428, 434, or a combination thereof. In one embodiment of the invention, said substitution is a substitution of serine at position 428 with leucine (S428L). In another embodiment of the invention, said substitution is a substitution of serine at position 434 with histidine (S434H).

[000151] The antibody having said substitution may be any suitable antibody known to one skilled in the art. In one example, said antibody is an anti-IL-31 antibody. In another example, said antibody is an anti-NGF antibody.

[000152] An anti-IL-31 antibody without the substitution described herein is well known in the art and is fully described, for example, in U.S. Patent Nos. 10,526,405; 10,421,807; 9,206,253; and 8,790,651. An anti-NGF antibody without the substitution described herein is also well known in the art and is fully described, for example, in U.S. Patent Nos. 10,125,192; 10,093,725; 9,951,128; 9,617,334; and 9,505,829.

[000153] In one embodiment of the invention, an anti-IL-31 antibody of the invention (i.e., an antibody having a substitution) reduces, inhibits, or neutralizes an IL-31-mediated pruritic or allergic condition. In another embodiment of the invention, an anti-IL-31 antibody of the invention reduces, inhibits, or neutralizes IL-31 activity in a cat.

[000154] The VL, VH, and CDR sequences of anti-IL-31 antibodies are well known in the art and are fully described, for example, in U.S. Patents Nos. 10,526,405; 10,421,807; 9,206,253; and 8,790,651. In one example, an anti-IL-31 antibody of the invention may comprise at least one of the following complementarity determining region (CDR) sequences: variable heavy (VH)-CDR1 of SEQ ID NO: 15, VH-CDR2 of SEQ ID NO: 16, VH-CDR3 of SEQ ID NO: 17, variable light (VL)-CDR1 of SEQ ID NO: 20, VL-CDR2 of SEQ ID NO: 21, and VL-CDR3 of SEQ ID NO: 22. In some embodiments, an anti-IL-31 antibody of the invention may comprise at least one CDR described herein.

[000155] In one embodiment of the invention, an anti-IL-31 antibody of the invention may comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19. In another embodiment of the invention, an anti-IL-31 antibody of the invention may comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[000156] In one embodiment of the invention, a mutant anti-NGF antibody of the invention (i.e., an antibody having a substitution) reduces, inhibits, or neutralizes NGF activity in an animal and/or enhances the ability to inhibit NGF binding to Trk A and p75 to treat an NGF-mediated pain or condition.

[000157] The VL, VH, and CDR sequences of anti-NGF antibodies are also well known in the art and are fully described in, for example, U.S. Patent Nos. 10,125,192; 10,093,725; 9,951,128; 9,617,334; and 9,505,829. In one example, an anti-NGF antibody of the present invention may include at least one of the following complementarity determining region (CDR) sequences: variable heavy (VH)-CDR1 according to SEQ ID NO: 24, VH-CDR2 according to SEQ ID NO: 25, VH-CDR3 according to SEQ ID NO: 26, variable light (VL)-CDR1 according to SEQ ID NO: 28, VL-CDR2 according to SEQ ID NO: 29, and VL-CDR3 according to SEQ ID NO: 30.

[000158] In another example, an anti-NGF antibody of the present invention may include at least one of the following complementarity determining region (CDR) sequences: variable heavy (VH)-CDR1 according to SEQ ID NO: 32, VH-CDR2 according to SEQ ID NO: 33, VH-CDR3 according to SEQ ID NO: 34, variable light (VL)-CDR1 according to SEQ ID NO: 36, VL-CDR2 according to SEQ ID NO: 37, and VL-CDR3 according to SEQ ID NO: 38.

[000159] In one embodiment of the invention, an anti-NGF antibody of the invention may comprise a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27 or 35.

[000160] In another embodiment of the invention, an anti-NGF antibody of the invention may comprise a heavy chain variable region comprising a variable region with an amino acid sequence set forth in SEQ ID NO: 23 or 31.

Pharmaceutical and veterinary use

[000161] The present invention also provides a pharmaceutical composition comprising the molecules of the present invention and one or more pharmaceutically acceptable carriers. More particularly, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, an antibody or peptide of the present invention.

[000162] "Pharmaceutically acceptable carriers" include any excipient that is nontoxic to the cell or animal exposed to it at the dosages and concentrations employed. The pharmaceutical composition may include one or more additional therapeutic agents.

[000163] “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms that are medically suitable for contact with animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications, consistent with a reasonable benefit/risk ratio.

[000164] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buffers, chelating agents, antioxidants, isotonic agents, and absorption delaying agents.

[000165] Pharmaceutically acceptable carriers include water; saline; phosphate-saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphate, citrate and other organic acids; ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; EDTA; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, polyethyleneglycol (PEG) and poloxamers; isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride; and combinations thereof.

[000166] The pharmaceutical compositions of the present invention can be prepared in various forms, including, for example, liquid, semi-solid or solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, suppositories, tablets, pills or powders. In some embodiments of the present invention, said compositions are formulated in the form of injection or infusion solutions. Said composition can be in a form suitable for intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, oral, topical or transdermal administration. Said composition can be formulated in the form of an immediate, controlled, extended or delayed release pharmaceutical composition.

[000167] The compositions of the present invention can be administered either as single therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are typically administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. Administration of the antibodies described herein can be by any suitable route, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), oral administration, or topical administration of the antibodies (typically contained in a pharmaceutical composition) to the surface of the respiratory tract. Topical administration to the surface of the respiratory tract can be accomplished by intranasal administration (e.g., via a dropper, swab, or inhaler). Local administration of antibodies to the surface of the respiratory tract can also be accomplished by inhalation administration, for example, by creating respirable particles of a pharmaceutical composition (including both solid and liquid particles) containing the antibodies in the form of an aerosol suspension, and then causing the subject to inhale said respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical compositions are well known, and any conventional technique can be used.

[000168] In some desirable embodiments of the invention, the antibodies are administered by parenteral injection. For parenteral administration, the antibodies or molecules can be formulated in the form of a solution, suspension, emulsion, or lyophilized powder in combination with a pharmaceutically acceptable carrier for parenteral administration. For example, the carrier can be a solution of the antibody or mixture thereof dissolved in an acceptable carrier such as an aqueous carrier, such carriers being water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, and the like. Liposomes and non-aqueous carriers such as fixed oils can also be used. The solutions are sterile and generally free of solid particles. The compositions can be sterilized by conventional, well-known sterilization techniques. Said compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibodies in said pharmaceutical formulations may vary widely, for example, from less than about 0.5% by weight, usually at or at least about 1% by weight, up to 15% or 20% by weight, and is selected mainly on the basis of liquid volumes, viscosity values, etc., in accordance with the specific route of administration selected. The carrier or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). Said pharmaceutical formulation is sterilized using conventional techniques. Actual methods for preparing compositions for parenteral administration are known or obvious to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

[000169] The antibodies or molecules of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques can be used. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of loss of antibody activity and that use levels may need to be adjusted to compensate. Compositions containing the antibodies or mixtures thereof can be administered to prevent relapse and/or for the therapeutic treatment of an existing disorder. Suitable pharmaceutical carriers are described in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, a standard reference in the art. In therapeutic use, the compositions are administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially arrest or alleviate said disease and its complications.

[000170] Effective doses of the compositions of the present invention for treating the conditions or diseases described herein vary depending on a variety of factors, including, for example, but not limited to, the pharmacodynamic characteristics of the particular agent, the route and route of administration; the target site; the physiological state of the animal; other drugs administered; the nature of the treatment - prophylactic or therapeutic; the age, health and body weight of the recipient; the nature and severity of the symptoms, the nature of concomitant treatment, the frequency of treatment, and the desired effect.

[000171] Single or multiple administrations of said compositions can be carried out at dosage levels and in a schedule selected by the attending veterinarian. In any case, said pharmaceutical compositions should provide an amount of the antibody(ies) of the present invention sufficient to effectively treat the subject. In some embodiments of the present invention, said composition is administered once every two months, once every three months, once every four months, once every five months, once every six months, or once every seven months.

[000172] Treatment doses can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[000173] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount." A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the required doses and for the required periods of time. A therapeutically effective amount of a molecule may vary depending on factors such as the stage of the disease, the age, sex, and body weight of the subject, as well as the ability of the molecule to elicit the desired response in the subject. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the molecule.

[000174] In another aspect of the invention, the compositions of the invention can be used, for example, in the treatment of various diseases and disorders in cats. As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, including prophylactic or preventive measures, the purpose of which is to prevent or slow down (reduce) an undesirable physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, the following: relief of symptoms, reduction in the severity of the disease or condition, stabilization of the disease or condition (i.e., when the disease or condition does not worsen), delay or slowing of the progression of the disease or condition, alleviation or alleviation of the disease or condition, and remission (partial or complete) of the disease or condition, whether detectable or not. Those in need of treatment include those who already have the disease or condition, as well as those who are prone to have the disease or condition, or those in whom the disease or condition is to be prevented.

[000175] A composition comprising a mutant molecule of the present invention can be used to treat any suitable disease or disorder. For example, a mutant anti-IL-31 antibody of the present invention can be used to treat an IL-31-mediated pruritic condition or an allergic condition. Examples of an IL-31-mediated pruritic condition include, for example, but are not limited to, atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. Examples of an IL-31-mediated allergic condition include, for example, but are not limited to, allergic dermatitis, summer eczema, urticaria, edema, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity.

[000176] The anti-NGF mutant antibody of the present invention can be used to treat an NGF-mediated pain or condition. Examples of pain include, but are not limited to, chronic pain, inflammatory pain, pain following a surgical incision, neuropathic pain, fracture pain, osteoporosis-associated fracture pain, postherpetic neuralgia, cancer pain, pain resulting from burns, pain associated with wounds, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal disorder, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthropathies, extra-articular rheumatism, periarticular disorder, or peripheral neuropathy. In a specific embodiment of the present invention, said pain is osteoarthritic pain.

[000177] All patents and literature references cited in this document are hereby incorporated by reference in their entirety.

[000178] The following examples are presented in addition to the preceding description and for a better understanding of the invention described herein. These examples should not be considered as limiting the invention described. It should be understood that the examples and embodiments of the invention described herein are intended for illustrative purposes only, and that various modifications or changes thereof will be understood by those skilled in the art and are included herein, and can be made without departing from the scope of the invention.

EXAMPLES EXAMPLE 1

Construction of Fc mutants of feline IgG

[000179] Construction of all feline IgGs (Fig. 1) was performed as described by Strietzel et al. (Strietzel et al., 2014, Vet Immunol Immunopathol., vol. 158 (3-4), pages 214-223), using plasmids containing the sequence encoding the feline constant regions for the IgG subclass 1 a allele (IgG1a) and the VH/VL sequences for each mAb tested in this example were inserted 5' and in-frame with the nucleotides encoding the indicated constant domains. Mutations were made at positions S434 and S428 of the CH3 domain (Fig. 2) of each plasmid using Agilent's QuikChange II mutagenesis technology and associated Agilent Single Site Mutagenesis Primer Design Tools (https://www.agilent.com/store/primerDesignProgram.jsp).

[000180] Antibody constructs were transiently expressed in either HEK 293 cells using a standard Lipofectamine transfection protocol (Invitrogen Life Technologies, Carlsbad, CA, USA) or CHO cells using protocols from the ExpiCHO transient system (ThermoFisher Scientific). Expression of ExpiCHO followed protocols outlined by ThermoFisher for co-transfection of a plasmid containing the gene sequence encoding the IgG light chain and IgG heavy chain. For expression, equal weight amounts of the heavy chain plasmid and the light chain plasmid were co-transfected in HEK293. Cells were grown for 7 days, after which supernatants were collected for antibody purification. Antibodies were screened for binding to protein A sensors using the Octet QKe assay (Pall ForteBio Corp, Menlo Park, CA, USA). Constructs that bound protein A were purified and quantified as described by Strietzel et al. for protein quality assessment.

EXAMPLE 2

Analysis of binding affinity and activity to the target molecule

[000181] The affinity of each mAb was assessed using Biacore assay technology and the IC50 was determined using a suitable cell-based activity assay. Surface plasmon resonance was performed on a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA, USA) to measure the binding affinities of each antibody to its target molecule. 2.5 μg/mL of each target protein was immobilized by amine coupling using EDC/NHS reagent to give a final density of ~250 RU in flow cells 2-4 of the CM5 sensor chip, respectively.

[000182] Flow cell 1 was used as an internal reference to correct for the effect of the running buffer. Antibody binding was measured at 15°C with a contact time of 250 s and a flow rate of 30 μl/min. The dissociation period was 300 s. Reconstitution was performed using reconstitution buffers (10 mM glycine, pH 1.5, and 10 mM NaOH) and a flow rate of 20 μl/min for 60 s each. Running buffer/diluent (1X HBS-EP, GE Healthcare, BR-1006-69, 10X containing 100 mM HEPES, 150 mM NaCl, 30 mM EDTA and 0.5% v/v surfactant P20, pH 7.4, 1:10 in filtered MQ H2O) was used as a negative control in the same assay format.

[000183] Data were analyzed using Biacore T200 Evaluation software using a dual reference method. The resulting curves were fitted to a 1:1 binding model. No differences in binding affinities or IC50 were detected between WT IgG and mutant IgG S434 and S428 (Table 1).

EXAMPLE 3 In vitro FcRn binding assay

[000184] Feline FcRn was isolated and prepared, and IgG Fc mutants were analyzed against FcRn according to Strietzel et al. A standard rapid amplification of cDNA ends PCR (RACE PCR) method was used to amplify the feline FcRn α subunit and β-microglobulin. FcRn-α subunit and β-microglobulin were co-transfected into HEK 293 cells, and the FcRn complex was affinity purified by immobilized metal affinity chromatography (IMAC) using a C-terminal histidine tag. KD values were measured using the Biacore 3000 or Biacore T200 (GE Healthcare, Pittsburgh, PA, USA) using the CM5 sensor chip.

[000185] FcRn was immobilized onto the sensor surface using a standard amine immobilization method to achieve the desired surface density. HBS-EP was used as the immobilization running buffer, and 10 mM MES; 150 mM NaCl; 0.005% Tween-20; 0.5 mg/mL BSA; pH 6 and pH7.2, and PBS; 0.005% Tween-20; 0.5 mg/mL BSA; pH 7.4 were used as assay running and titration buffers. Fc-mutant IgGs were passed over the receptor surface and affinity was determined using Scrubber2 software (BioLogic Software Pty, Ltd., Campbell, Australia) or T200 Evaluation software (Table 2). Values from blank control wells containing buffer only were subtracted from values for all other wells. Flow cells were reconstituted with 50 mM Tris pH 8. Runs were performed at 15°C.

[000186] Mutations made at positions 434 and 428 had a marked effect on the affinity of these IgGs for FcRn at pH 6. This study demonstrates that the increase in IgG affinity for FcRn is independent of the VHVL domains and is universal for all feline IgG1a.

EXAMPLE 4

Pharmacokinetic studies of Fc mutant IgG in cats

[000187] Pharmacokinetic (PK) studies were conducted to demonstrate the effect of the S434H and S428L mutations on prolonging the half-life of the IgG1a subclass with mAbs generated against multiple target molecules. Study designs varied, but essentially two types of studies were conducted:

[000188] Study Design 1: mAb was administered to groups of 4 male and 4 female domestic shorthair cats at a dose of 2 mg/kg. The first and second doses were administered subcutaneously 28 days apart. The third dose was administered intravenously 28 days later. Serum samples were collected weekly for 14 weeks.

[000189] Study design 2: mAb was administered as a single dose of 2 mg/kg subcutaneously or intravenously to groups of 3 or 4 domestic shorthair cats. Serum samples were collected weekly.

[000190] Pharmacokinetic calculations were performed using a non-compartmental approach (linear trapezoidal rule for AUC calculation) using Watson™. Additional calculations were performed using Excel™, including an AUC correction to account for the overlap of concentration-time profiles after the 2nd and 3rd injections of the drug. Concentration-time summary data and pharmacokinetic data with simple statistics (mean, standard deviation, coefficient of variation) were calculated using Excel™ or Watson™. No other statistical analyses were performed.

[000191] The S428L point mutation in feline IgG1a was shown to increase the half-life of three different feline IgGs in domestic shorthair cats. For mAb 1, the half-life increased from 11 days to 26 days, and for mAb2, from 7.9 days to 23 days. The S434H point mutation in feline IgGla was shown to increase the half-life of one mAb from 10.1 days to 13.2 days.

[000192] The mechanism of action is through increased affinity for feline FcRn at pH 6, and this has been demonstrated in a variety of feline IgGs that bind very different and specific soluble target molecules. Therefore, it has been demonstrated that the half-life extension by the S428L and N434 mutations in feline IgGla is independent of the VHVL domains.

EXAMPLE 5 FcRn Binding Assay

[000193] Feline FcRn was isolated and prepared, and IgG Fc mutants were analyzed against feline FcRn according to the work of Strietzel et al., as discussed above. A standard PCR method was used to amplify the feline FcRn a subunit and β-microglobulin. The FcRn-α subunit and β-microglobulin were co-transfected into HEK 293 cells, and the FcRn complex was purified by affinity purification with AChIM using a C-terminal histidine tag. The FcRn complex was labeled with biotin using the BirA biotinylation enzymatic reaction. KD values were measured using the Biacore T200 (GE Healthcare, Pittsburgh, PA, USA) or Biacore 8K (Cytiva, Marlborough, MA, USA) using the SA sensor chip.

[000194] FcRn was captured on the surface of the indicated sensor using a modified SA capture method. 10 mM MES; 150 mM NaCl; 0.005% Tween-20; 0.5 mg/mL BSA; pH 6 were used as the capture, assay, and titration buffer. 1x HBS-P, 0.5 mg/mL BSA; pH 7.4 were also used as the assay and titration buffer. Fc mutant IgGs were passed over the receptor surface and affinity was determined using T200 Evaluation software or Biacore Insight Evaluation software. Values from blank control wells containing buffer only were subtracted from the values for all other wells. Flow cells were reconstituted with 50 mM Tris pH 8 or pH 9. Runs were performed at 15 °C.

[000195] Mutations made at the corresponding positions had a marked effect on the affinity of these IgGs for FcRn at pH 6. Binding of wild-type (WT) and mutant IgGs to feline FcRn was measured by surface plasmon resonance (Biacore).

[000196] Marked effects on affinity were observed across distinct and structurally distinct antibodies that bind different target molecules (i.e., anti-IL-31 and anti-NGF antibodies) as well as across different versions of antibodies that bind the same target molecule (i.e., different versions of anti-IL-31 and anti-NGF antibodies) (Tables 1-5). Therefore, the enhancement of IgG affinity for FcRn is independent of the VHVL domains or CDR regions. Additionally, marked effects on affinity were observed across multiple IgG subclasses. Overall, these results indicate that the enhancement of IgG affinity for FcRn is independent of feline IgG subclass.

[000197] Preferred embodiments of the present invention have been described herein, but it should be understood that the present invention is not limited to the precise embodiments of the present invention disclosed, and that various changes and modifications can be made thereto by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

Claims (17)

1. A modified IgG having properties of increased binding affinity to the neonatal Fc receptor (FcRn), comprising: a constant domain of feline IgG comprising at least one amino acid substitution compared to the constant domain of wild-type feline IgG, wherein said substitution is at amino acid residue 428 or 434, numbered in accordance with the EU index, as per Kabat. 2. A modified IgG according to claim 1, characterized in that said substitution is a substitution of serine at position 428 with leucine (S428L). 3. A modified IgG according to claim 1, characterized in that said substitution is a substitution of serine at position 434 with histidine (S434H). 4. The modified IgG according to claim 1, characterized in that said constant domain of feline IgG contains two amino acid substitutions, where the first substitution is at amino acid residue 428, and the second substitution is at amino acid residue 434. 5. The modified IgG according to claim 4, characterized in that the first substitution is a substitution of serine at position 428 with leucine (S428L), and the second substitution is a substitution of serine at position 434 with histidine (S434H). 6. The modified IgG according to claim 1, characterized in that said modified IgG has a higher affinity for FcRn than said IgG containing the constant domain of wild-type feline IgG. 7. The modified IgG according to claim 1, characterized in that said modified IgG is feline or feline IgG. 8. The modified IgG according to claim 1, characterized in that said IgG constant domain contains an Fc constant region containing CH2 or CH3 domains, or a combination thereof. 9. The modified IgG according to claim 1, characterized in that said constant domain of IgG contains the amino acid sequence indicated in SEQ ID NO: 1 or 2. 10. A pharmaceutical composition for increasing the half-life of IgG in cat blood serum, comprising a modified IgG according to any one of claims 1-9 and a pharmaceutically acceptable carrier. 11. A kit designed to increase the half-life of IgG in cat blood serum, containing modified IgG according to any of paragraphs 1-9 in a container and instructions for use. 12. An expression vector comprising a nucleic acid sequence encoding a constant domain of a feline IgG modified IgG according to claim 1, wherein said domain has the amino acid sequence indicated in SEQ ID NO: 1 or 2. 13. An isolated host cell containing the vector of claim 12, which produces a modified IgG having properties of increased binding affinity to FcRn. 14. A method for producing an antibody, comprising providing a cell according to item 13 and culturing said cell. 15. Use of an immunoglobulin to increase its half-life in the blood serum of a cat, wherein said immunoglobulin comprises a modified IgG according to any one of claims 1-9. 16. A fusion protein having properties of increased binding affinity to FcRn, comprising a modified IgG according to any one of claims 1-9, fused with a heterologous protein. 17. The use according to claim 15, wherein said immunoglobulin is a molecule against interleukin-31 (IL-31) or against nerve growth factor (NGF).