Modified Fc region

Abstract

The present invention relates to the field of modified constant domains of canine or feline antibodies with altered immune effector functions and uses thereof. More specifically, the present application relates to modified Fc fragments with significantly reduced FcyRI and Clq binding.

Description

Technical Field

[0001] This invention relates to the field of modified constant domains of canine or feline antibodies with altered immune effector functions and their uses. More specifically, this application relates to modified Fc fragments having significantly reduced binding to FcγRI and C1q.

[0002] The accompanying sequence list (in txt format) forms part of the disclosure of this application. Background Technology

[0003] Immunoglobulin G or IgG antibodies are large tetrameric proteins. Each IgG protein consists of two identical light chains and two identical heavy chains linked together by disulfide bonds. There are two types of light chains, called κ and λ chains. Each light chain consists of a variable domain (VL) and a constant domain (CL). The heavy chain also consists of a variable domain (VH) and three constant domains called CH1, CH2, and CH3. A highly flexible amino acid region in the central portion of the heavy chain, known as the "hinge region," connects the CH1 and CH2 domains.

[0004] In principle, antibodies can be divided into two independent subunits: the "Fab" fragment, which consists of the light chain and the VH and CH1 domains of the heavy chain; and the "Fc" fragment, which contains the remaining CH2 and CH3 domains of the heavy chain. The Fab fragment is responsible for antigen recognition and binding, while the Fc fragment interacts with the immune system, mediating effector functions such as antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0005] Human immunoglobulin G antibodies (IgG) have been extensively studied, and four human IgG subclasses have been described based on their biological functions, biochemical properties, and DNA sequences, referred to as IgG1, IgG2, IgG3, and IgG4 (Davies DR, Metzger H., “Structural basis of antibody function”, Annu Rev Immunol. 1983; 1:87–117; Jefferis R, Lund J, Goodall M., “Recognition sites on human IgG for Fc gamma receptors: the role of glycosylation”, Immunol Lett. 1995; 44(2-3):111–117; Shakib F., “The human IgG subclasses”, 1990, Pergamon Press, New York; Kenneth Murphy PT, Walport). Each subclass possesses distinct characteristics and participates in the immune system in different ways, mediated by varying binding affinities of immune effector proteins, including complement protein C1q, the Fcγ receptor (FcγR), and the neonatal Fc receptor (FcRn). These interacting mates play key roles in complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and serum half-life, respectively.

[0006] In humans, complement activation is most effectively triggered by IgG1 and IgG3 (Brüggemann M, Williams GT, Bindon CI, “Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies”, J Exp Med. 1987; 166(5):1351–1361; Michaelsen TE, Aase A, Westby C, Sandlie I., “Enhancement of complement activation and cytolysis of human IgG3 by deletion of hinge exons”, Scand J Immunol. 1990; 32(5):517–528).The binding of the antibody constant domain (Fc) to C1q, the first protein in the complement cascade, initiates complement, which helps activate phagocytes and destroy pathogens (Schifferli JA, Ng YC, Peters DK, “The role of complement and its receptors in the elimination of immune complexes”, N Engl J Med. 1986; 315(8):488–495; Garred P, Michaelsen TE, Aase A, “The IgG subclass pattern of complement activation depends on epitope density and antibody and complement concentration”, Scand J Immunol. 1989; 30(3):379–382; Moore GL, Chen H, Karki S, Lazar GA, “Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector function”). (effector functions), MAbs. 2010; 2(2):181–189). It has been shown that IgG1 and IgG3 are the human IgG subclasses with the strongest ability to trigger ADCC in blood monocytes. The high affinity of these subclasses for FcγRI and FcγRIII is associated with ADCC activity. Conversely, the binding of other subclasses to the inhibitory receptor FcγRIIb helps to reduce ADCC activity. M, “Fc receptor biology”, Annu Rev Immunol. 1997; 15: 203–234; Armour KL, Clark MR, Hadley AG, Williamson LM, “Recombinant human IgG molecules lacking FcγreceptorI binding and monocyte triggering activities”, Eur J Immunol. 1999; 29(8): 2613–2624; 2-J; Clynes RA, Towers TL, Presta LG, Ravetch JV, “Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets”. Nat Med. 2000; 6(4): 443–446.The binding of antibodies to FcRn on epithelial cells is associated with antibody recycling and is also related to serum half-life (Ghetie V, Hubbard JG, Kim JK, Tsen MF, Lee Y, Ward ES, “Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice”, Eur J Immunol. 1996; 26(3):690–696; Wilsker DF, Hayes KC, Schoenfeld D, Simister NE, “Increased clearance of IgG in mice that lack beta 2-microglobulin: possible protective role of FcRn”, Immunology. 1996; 89(4):573–578; Praetor A, Hunziker W, “β(2)-Microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn”, J Cell Sci. 2002; 115(Pt11):2389–2397; Jefferis R, “Antibody therapeutics: isotype and glycoform selection”, Expert Opin Biol Ther. 2007; 7(9):1401–1413.

[0007] Besides human IgG, the immunoglobulin classes of rodents (mice and rats) have been well characterized. In contrast, less is known about the functional properties of IgG subclasses in companion animals such as dogs and cats.

[0008] Canine IgG consists of four subclasses, referred to as caIgG-A (HC-A), caIgG-B (HC-B), caIgG-C (HC-C), and caIgG-D (HC-D) (Tang L, Sampson C, Dreitz MJ, McCall C, “Cloning and characterization of cDNAs encoding fourdifferent canine immunoglobulin gamma chains”, Vet Immunol Immunopathol. 2001; 80(3-4):259–270). Furthermore, canine FcγRs similar to human receptors I, IIA, IIB, and III have been described (Nimmerjahn F, Ravetch JV, “Fc gamma receptors: old friends and new family members”, Immunity. 2006; 24(1):19–28), but an inhibitory canine FcγRIIA could not be confirmed in another study (Bergeron LM, McCandless EE, Dunham S et al., “Comparative functional characterization of canine IgG subclasses”, Vet Immunol Immunopathol. 2014; 157(1-2):31–41). In vitro binding experiments revealed that canine HC-B and HC-C bind strongly to canine FcγRs, while HC-A binds only weakly (Bergeron, 2013). Similarly, HC-B and HC-C have been reported to bind tightly to human C1q proteins, while HC-A and HC-D bind with almost no affinity (Bergeron, 2013). Except for HC-C, all other canine subtypes bind strongly to FcRn (Bergeron, 2013).

[0009] Antibody purification strategies typically involve an affinity chromatography step with Staphylococcus protein A. Of the four canine subclasses, only HC-B has been reported to bind strongly to protein A. HC-A exhibits a weak affinity for Staphylococcus protein A, and neither HC-C nor HC-D subclasses bind. However, using Staphylococcus protein G resin, all four canine subclasses can be purified (Bergeron, 2013).

[0010] Depending on the therapeutic application, the selection of the appropriate antibody subclass is crucial, and one needs to consider whether the involvement of humoral or cellular components of the immune system is beneficial or may even lead to unwanted side effects of the drug. For example, therapeutic antibodies targeting tumor cell growth or pathogens should have potent effector functions. Conversely, soluble mediators or cell surface receptors targeting healthy cells to block receptor-ligand interactions typically require the absence of any CDC or ADCC activity to prevent target cell death or unwanted cytokine secretion. Disease areas requiring silent antibody formats include, but are not limited to, inflammatory diseases (e.g., rheumatoid arthritis, psoriasis, inflammatory bowel disease), allergies (e.g., asthma), pain (e.g., osteoarthritis pain, cancer pain, back pain), and eye diseases (e.g., age-related macular degeneration).

[0011] It is well known that IgG antibodies mediate effector functions such as ADCC through the binding of their Fc moiety to the Fc receptor family, while CDC is mediated through the binding of the Fc moiety to the first component of complement, C1q. Enhancement or elimination of effector function can be achieved through mutations in the Fc moiety of the antibody that alter the affinity for the corresponding interacting molecule. In the prior art, numerous reports describe amino acid substitutions that can be introduced into antibody molecules to modulate their effector function. For example, the substitution of asparagine for alanine (N297A) in human IgG1 produces non-glycosylated antibodies that significantly reduce antibody binding to several Fc receptors (Shields RL, Namenuk AK, Hong K et al., "High-resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and Fc Rn and design of IgG1 variants with improved binding to the Fc gamma R", J Biol Chem. 2001; 276(9):6591–6604). Furthermore, the substitution of asparagine for alanine (D265A) also significantly reduces antibody binding to Fc receptors. Each of the N297A and D265A substitutions also showed significant impairment of CDC (Shields 2001).Other similar reports have identified potential substitutes in antibodies that reduce or eliminate effector function (e.g., Xu D, Alegre ML, Varga SS, “In vitro characterization of five humanized OKT3 effector function variant antibodies”, Cell Immunol. 2000; 200(1):16–26; Alegre ML, Collins AM, Pulito VL, “Effect of a single amino acid mutation on the activating and immunosuppressive properties of a “humanized” OKT3 monoclonal antibody”, J Immunol. 1992; 148(11):3461–3468; Bolt S, Routledge E, Lloyd I, “The generation of humanized, nonmitogenic CD3 monoclonal antibodies that retain in vitro immunosuppressive properties”). A humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties), Eur J Immunol. 1993; 23(2): 403–411; Tao MH, Morrison SL, “Study on deglycosylated chimeric mouse-human IgG”."The role of carbohydrate in the structure and effector functions mediated by the human IgG constant region" (Studies of aglycosylated chimeric mouse-human IgG, J Immunol. 1989; 143(8): 2595–2601); Walker MR, Lund J, Thompson KM, Jefferis R, "Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fcgamma RI and / or Fcgamma RII receptors" (Biochem J. 1989; 259(2): 347–353).

[0012] The N297 residue is conserved not only in humans but also throughout the entire mammalian class, particularly in dogs, cats, cattle, camels, horses, macaques, monkeys, opossums, mice, rabbits, sheep, chimpanzees, rats, and pigs. It is well known that strongly conserved residues across a large number of species are associated with conserved functional phenotypes of the corresponding residues, and the introduction of N297A mutations into any other species can also lead to reduced immune effector function, as demonstrated in humans.

[0013] Therefore, EP 2 705 057A1 discloses the production of non-glycosylated canine antibodies by introducing asparagine into canine HC-B and HC-C as a substitution for alanine (N297A). The practical characteristic of the variant is that the binding to C1q is eliminated or reduced. However, deglycosylation may negatively affect the plasma half-life of the antibody, as shown by Chen et al. (Chen TF, Sazinsky SL, Houde D et al., “Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fcgamma RIIA and Fcgamma RIIIAs”, J Mol Biol. 2017; 429(16):2528–2541), and thus may require higher doses or more frequent dosing of recombinant antibodies. In addition, deglycosylation may reduce thermal stability (Ghirlando R, Lund J, Goodall M, Jefferis R, “Glycosylation of human IgG-Fc: influences on structure revealed by differential scanning micro-calorimetry”, Immunol Lett. 1999; 68(1):47–52) and increase sensitivity to proteolysis (Raju TS, Scallon BJ, “Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain”, Biochem Biophys ResCommun. 2006; 341(3):797–803).

[0014] Furthermore, it has been reported that the HC-A and HC-D isoforms do not bind C1q and do not induce complement activation and potential other downstream effects such as ADCC and ADCP.

[0015] Although canine antibodies of the HC-A and HC-D isotypes lack complement binding, which is ideal for applications where target neutralization is not required, their weak binding to staphylococcal protein A complicates the development of commercially viable preparation and purification methods. Conversely, deglycosylated canine HC-B antibodies retain binding to staphylococcal protein A, making this variant a more suitable candidate for antibodies lacking effector function. For human antibodies, deglycosylation has proven successful in eliminating binding to low-affinity FcγRs and effector functions such as CDC and ADCC. However, it is also recognized that effector functions can be preserved under affinity-based binding conditions (Lo M, Kim HS, Tong RK et al., “Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice”, J Biol Chem. 2017; 292(9):3900–3908; Nesspor TC, Raju TS, Chin CN, Vafa O, Brezski RJ, “Avidity confers FcγR binding and immune effector function to aglycosylated immunoglobulin G1”, J Mol Recognit. 2012; 25(3):147–154). Furthermore, as disclosed in another study (WO 2015 / 091910 A2), a single substitution at the N297A position of canine HC-B (which is similar to human IgG1 and produces deglycosylated antibodies) does not completely eliminate both ADCC and CDC effector functions in the corresponding canine antibodies.

[0016] To develop “silent” therapeutic antibodies for dogs, naturally occurring canine IgG subclasses and deglycosylated HC-B variants do not meet all the required properties, namely, the lack of effector functions such as antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), a long in vivo half-life, and the possibility of purification using industrial standard techniques such as protein A chromatography.

[0017] In addition to the N297A mutant mentioned above, several other silent mutants are known in the prior art.

[0018] WO 2018 / 073185 A1 discloses that mutations at residues 253, 255, and 257 may enhance the binding of the constant region to FcRn. However, no effect on ADCC or CDC is shown.

[0019] WO 2019 / 035010 A1 hypothesizes that mutations at residues 5, 38, 38, 97, and 98, identified after analyzing the protein sequences and 3D structures of canine IgG-B and IgG-C and comparing them with IgG-A and IgG-D, might affect ADCC activity. However, this hypothesis has not been experimentally verified.

[0020] WO 2015 / 091910 A2 discloses that mutations at residues 4, 31, 63, 93, and 95 reduce binding to C1q and FcγRI; however, it does not show that binding to FcRn is not reduced in the same way.

[0021] Little is known about the functional characteristics of feline IgG. Two allele sequences, referred to as feline IgG1a and 1b, have been described, functioning similarly to human IgG1 and expected to induce potent effector functions in vivo (Strietzel CJ, Bergeron LM, Oliphant T, Mutchler VT, Choromanski LJ, Bainbridge G, “In vitro functional characterization of feline IgGs”, Vet Immunol Immunopathol. 2014; 158(3-4):214–223). The same authors also reported the existence of a rare IgG sequence now known as feline IgG2. This additional IgG does not bind to recombinant fFcγRI or fFcγRIII, and therefore has negligible binding to hC1q, indicating a lack of effector function.

[0022] The fundamental problem of this invention is to provide Fc fragments of canine and feline antibodies that overcome the shortcomings of Fc fragments known in the art and whose binding to C1q and FcγR is enhanced, reduced, or eliminated. Summary of the Invention

[0023] The fundamental problem of this invention is solved by the appended claims and the polypeptides and methods further described herein.

[0024] The present invention solves the aforementioned problem by providing a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc fragment, relative to the wild-type Fc fragment, includes a substitution of at least one amino acid selected from at least one of amino acid positions 235, 239, 270, and / or 331. Preferably, the Fc fragment is derived from isotype B of canine IgG.

[0025] In a preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc fragment comprises a substitution of at least two amino acids selected from at least two of the amino acids at positions 234, 235, 239, 270, and / or 331. More preferably, the two amino acids are 235 and 239; 235 and 270; 235 and 331; 239 and 270; 239 and 331; 270 and 331; 234 and 235; 234 and 239; 234 and 270; or 234 and 331.

[0026] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc fragment comprises substitutions of at least three amino acids selected from at least three of the amino acid positions 234, 235, 239, 270, and / or 331. More preferably, the three amino acid positions are 235, 239, and 270; 239, 270, and 331; 235, 270, and 331; or 235, 239, and 331.

[0027] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc fragment comprises amino acids selected from amino acid positions 234, 235, 239, 270, and 331, more preferably amino acids at amino acid positions 235, 239, 270, and 331, and most preferably at least four substitutions of amino acids L235, S239, D270, and P331.

[0028] The polypeptide according to the invention may comprise SEQ ID NO: 8 to SEQ ID NO: 29, more preferably SEQ ID NO: 18, 19, 26, 27 or 29. Most preferably, the polypeptide comprises SEQ ID NO: 19 or 27.

[0029] Surprisingly, the peptides according to the invention exhibit reduced binding affinity for C1q and / or Fc receptors relative to peptides containing the corresponding wild-type Fc fragments. Under physiological conditions of an unimpaired immune system, reduced or decreased binding to C1q and / or FcγRIs leads to a reduction or complete elimination of complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC)-induced immune effector functions. The inclusion of at least one alternative peptide in the Fc fragment, resulting in reduced or decreased binding to C1q and / or FcγRIs, and / or the resulting reduction or complete elimination of CDC or ADCC, is also referred to herein as “silencing.”

[0030] In an embodiment of the invention, preferably the polypeptide is derived from canine IgG isotype B, and the Fc fragment surprisingly maintains its ability to bind to neonatal Fc receptor (FcRn) and protein A.

[0031] As in Figure 4 As shown, the polypeptides according to the present invention, comprising mutated Fc fragments of the HC-B isotype, maintain their ability to bind to neonatal Fc receptors (FcRn).

[0032] The invention will be described in more detail below.

[0033] Detailed description

[0034] In a first aspect, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises, relative to the wild-type Fc region, a substitution of at least one amino acid selected from at least one of amino acid positions 235, 239, 270, and / or 331. Preferably, the Fc region is derived from isotype B of canine IgG.

[0035] Unless otherwise explicitly described in a specific implementation, the term “amino acid position” as used herein refers to the amino acid position numbered according to the EU numbering system when the canine or feline Fc region is compared with human IgG1 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Woof et al., Molec. Immunol. 23:319-330 (1986); Duncan et al., Nature 332:563 (1988); Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel et al., Proc. Natl. Acad. Sci. USA 88:9036-9040 (1991)). The position number in the Fc region of canines or felines corresponds to the position number in the compared human IgG. For example... Figure 6a and 6bAs shown, the alignment of the canine and feline sequences with the human sequence resulted in gaps in the canine and feline sequences. Therefore, some sequences do not have an amino acid at every consecutive numbered position. Although the EU numbering system was originally applied to human IgG1 antibodies, it is also applicable to antibodies from other species. In antibodies or antibody-derived molecules, the amino acid positions numbered according to the EU system can be readily transferred to an alternative numbering system for antibodies, for example, according to Chothia (Chothia C and Lesk AM (1987), “Canonical structures for the hypervariable regions of immunoglobulins”, J Mol Biol. 196(4):901-17; Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air G, Sheriff S, Padlan EA, Davies D, Tulip WR (1989), “Conformations of immunoglobulin hypervariable regions”, Nature. 342(6252):877-83) or IMGT (Lefranc MP, Giudicelli V, Ginestoux C, Bodmer J, Müller W, Bontrop R, Lemaitre M, Malik A, Barbié V, Chaume D (1999), “IMGT, the international ImMunoGeneTics database” (Nucleic Acids Res. 27(1):209-12). Figure 6 shows the canine and feline Fc wild-type sequences with numbers according to the EU system.

[0036] The amino acid positions according to the present invention can also be numbered according to Tang et al. (Tang L, Sampson C, Dreitz MJ, McCall C (2001), “Cloning and characterization of cDNAs encoding four different canine immunoglobulin γ chains”, Vet Immunol Immunopathol. 80(3-4):259-70), page 266. Figure 2 The positions in the alignment shown are used for assignment. It should be noted that the position numbering according to Tang et al. also includes vacancy positions in the alignment and does not begin with the first amino acid in the Fc region, but rather with the N-terminus of the complete canine antibody, including the CDR and leader sequence. In this optional numbering according to Tang et al., amino acid positions 262, 263, 267, 298, and 359 correspond to EU numbers 234, 235, 239, 270, and 331, respectively.

[0037] The term "Fc fragment" refers to a fragment of an immunoglobulin that contains at least part or all of the heavy chain constant region 2 (C2 or CH2) and heavy chain constant region 3 (C3 or CH3), or a crystallizable fragment of an immunoglobulin obtained by papain digestion. A fragment is understood to be part of a larger polypeptide sequence. Therefore, a "fragment" typically has an amino acid sequence defined by a C-terminus and / or an N-terminus. The terms "C2" or "CH2," and "C3" or "CH3," are used interchangeably. Furthermore, unless explicitly stated otherwise, the terms "Fc region" and "Fc domain" are used interchangeably when referring to the Fc CH2 and CH3 sequences of immunoglobulin. In the context of this invention, the boundaries of the CH2 and CH3 regions of canine immunoglobulin isotypes HC-A, HC-B, HC-C, and HC-D are defined according to Tang et al. (Tang L, Sampson C, Dreitz MJ, McCall C (2001), “Cloning and characterization of cDNAs encoding fourdifferent canine immunoglobulin gamma chains”, Vet Immunol Immunopathol. 80(3-4):259-70), which is incorporated herein by reference.

[0038] The Fc region according to the present invention is derived from the Fc region of a dog, and is therefore a canine Fc region, or derived from a cat, and is therefore a feline Fc region.

[0039] The term "dog" or "canine" refers to all domesticated dogs, the subspecies of Canis lupus familiaris, or domestic dogs (Canis familiaris). Similarly, the term "cat" or "cat" refers to domesticated cats, the domestic cat (Felis catus), the Angora cat (Felis catus domesticus Felus angorensis), and the common cat (Felis vulgaris).

[0040] The Fc region according to the invention contains at least one amino acid substitution relative to the wild-type Fc region. The term "substitution" means that an amino acid in the sequence is replaced by at least one other amino acid, preferably by one amino acid. The polypeptide of the invention may contain 1, 2, 3, 4, 5, 6 or more amino acid substitutions.

[0041] In the context of this invention, a "wild-type" Fc region is an Fc region having a naturally occurring amino acid sequence that has not been artificially modified, for example, by introducing mutations through genetic engineering methods. A wild-type sequence of an Fc region according to the invention that contains at least one amino acid substitution relative to a wild-type Fc region is also referred to herein as the "corresponding wild-type" or "corresponding wild-type sequence." An Fc region containing at least one amino acid substitution relative to the "wild-type" is also referred to in the context of this invention as a "mutant."

[0042] The Fc region according to the present invention can be selected from canine immunoglobulin G isotype A (also known as HC-A, HCA, caIgG-A), immunoglobulin G isotype B (also known as HC-B, HCB, caIgG-B), immunoglobulin G isotype C (also known as HC-C, HCC, caIgG-C), or immunoglobulin G isotype D (also known as HC-D, HCD, calgG-D). Preferably, the Fc region is selected from isotype B. The feline Fc region can be derived from immunoglobulin G isotype 1a (also known as IgG1a), isotype 1b (also known as IgG1b), and isotype 2 (also known as IgG2).

[0043] In certain embodiments, the canine wild-type sequence mentioned herein is disclosed in Figure 6a and 6b And the sequences in Table 1a according to SEQ ID NO: 1 to 4.

[0044] Table 1a Canine wild-type sequence

[0045]

[0046] The sequence from the 234th to the 331st underscores describes the range of sequences containing the substitutions as described above.

[0047] Optionally, the canine wild-type sequence according to the invention is published elsewhere:

[0048] Table 1b Canine wild-type sequence

[0049]

[0050]

[0051] In a particular implementation, the wild-type feline sequences mentioned herein are those in Table 2, and Figure 6a and 6b The sequences shown are based on SEQ ID NO: 5 to 7.

[0052] Table 2: Wild-type sequence of felines

[0053]

[0054] Optionally, the wild-type feline sequence according to the present invention is disclosed in Striezel et al. (Strietzel CJ, Bergeron LM, Oliphant T, Mutchler VT, Choromanski LJ, Bainbridge G (2014), “In vitro functional characterization of feline IgGs”, VetImmunol Immunopathol, 158(3-4):214-23, p. 220).

[0055] In a preferred embodiment, the polypeptide according to the invention comprises at least a sequence corresponding to amino acids 234 to 331 according to Kabat numbering, which has a substitution of at least one amino acid selected from at least one of amino acid positions 235, 239, 270, and / or 331 relative to the wild-type Fc region. The underlined sequences in Tables 1a and 2 correspond to amino acids 234 to 331 according to Kabat numbering in the canine or feline wild-type Fc region sequence.

[0056] In summary, the wild-type sequence according to the present invention can preferably be selected from any of the sequences of amino acids 234 to 331 (according to the Kabat number) of the wild-type sequence disclosed on page 220 of GeneBank accessions AF354264, AF354265, AF354266, AF354267, or Striezel, etc. Therefore, the (amino acid) sequences (according to the Kabat number) of the wild-type sequences disclosed on page 220 of GeneBank accessions AF354264, AF354265, AF354266, AF354267, and Striezel, etc., are expressly incorporated herein by reference and thus constitute a part of the disclosure of this application.

[0057] As further described herein, the substitution of an amino acid at least one of the amino acid positions selected from the 235th, 239th, 270th, and / or 331st amino acid positions relative to the wild-type Fc fragment may, in various different embodiments, be described as an amino acid substitution at at least one of the (amino acid) positions corresponding to the 235th, 239th, 270th, and / or 331st amino acid positions in the (amino acid) sequence corresponding to the wild-type Fc fragment. Therefore, the designation of a substitution of at least one amino acid at at least one of the amino acid positions selected from the 235th, 239th, 270th, and / or 331st amino acid positions relative to the wild-type Fc fragment may, in various different embodiments, be described as an amino acid substitution at at least one of the (amino acid) positions corresponding to the 235th, 239th, 270th, and / or 331st amino acid positions in the (amino acid) sequence corresponding to the wild-type Fc fragment.

[0058] Therefore, the designation of a substitution of at least one amino acid at positions 235, 239, 270, and / or 331 relative to at least one of the wild-type Fc fragments selected from amino acid positions 235, 239, 270, and / or 331 may, in various different embodiments, be described as a substitution of at least one amino acid at position 235, 239, 270, and / or 331 in the (amino acid) sequence of the wild-type Fc fragment disclosed in any of SeqID NOs: 1 to 7, or in GeneBank accessions AF354264, AF354265, AF354266, AF354267, or Striezel et al., page 220, according to Kabat number.

[0059] As further described herein, the terms “relative to wild-type Fc fragment (region)” and “amino acid sequence relative to wild-type Fc fragment (region)” are used interchangeably herein.

[0060] According to the present invention, a polypeptide sequence having an "amino acid substitution relative to the wild-type Fc region" at a designated position is a polypeptide characterized by having an amino acid sequence that, apart from the designated substitution, has at least 96%, preferably 98%, more preferably 99%, and most preferably 100% identity with the alleged wild-type sequence. Therefore, the polypeptide according to the present invention may also contain other mutations besides the "substitution of at least one amino acid" described herein, such as insertions, deletions, or substitutions.

[0061] In the context of this invention, "percentage (%) identity" relative to a given amino acid sequence is defined as the percentage of amino acid residues in a reference sequence that are identical to amino acid residues in the compared amino acid sequence, after sequence alignment and, if necessary, the introduction of vacancies to achieve maximum percentage sequence identity, and without regard to any conservative substitutions as part of sequence identity. The aim is that alignments for determining percentage sequence identity in this invention can be performed in various ways known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilled in the art are generally able to determine suitable parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0062] Amino acid substitutions can be conserved or non-conserved. Amino acids can be grouped according to common side chain properties: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In non-conservative substitutions, an amino acid from one group is exchanged with an amino acid from a different group. In conserved substitutions, an amino acid from one group is exchanged with another amino acid from the same group.

[0063] As an alternative to replacing a natural amino acid contained in a wild-type polypeptide sequence with another natural amino acid, the term amino acid substitution also covers replacing a natural amino acid with an amino acid derivative. When used herein, "amino acid derivative" refers to any non-natural amino acid, modified amino acid, and / or amino acid analogue not found in mammals. Exemplary amino acid derivatives include natural amino acids not found in humans (e.g., selenocysteine ​​and pyrrolidone, which can be found in certain microorganisms) or chemically modified amino acids.

[0064] In a preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises, relative to the wild-type Fc region, at least one amino acid substitution selected from at least one of L235, S239, D270 and / or P331.

[0065] As in Figure 2 As disclosed herein, the inventors have surprisingly discovered that any single mutation in L235, S239, D270 and / or P331 affects the binding of the Fc fragment to both C1q and FcγRI. These results are particularly surprising because mutations in S239, D270, or P331 in human Fc do not impair binding to human FcγRI, as disclosed by Shields et al. (Shields RL, Namenuk AK, Hong K et al., “High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R”, J Biol Chem. 2001; 276(9):6591–6604). Therefore, in a preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises at least one amino acid substitution selected from amino acids at positions 239, 270, and / or 331.

[0066] The observed difference between the effects of mutations in the canine Fc region and the corresponding effects in the human Fc region was also confirmed in Experiment 1 using the mutation M234A / L235A. For this mutation, a significant reduction in effector function has been described in human systems (Xu D, Alegre ML, Varga SS, et al., “In vitro characterization of five humanized OKT3 effector function variant antibodies”, Cell Immunol. 2000; 200(1):16–26). In contrast, the same mutation in the canine construct eliminates binding to FcγRI, but the variant can still bind C1q.

[0067] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises a substitution of at least two amino acids selected from at least two of the amino acids at positions 234, 235, 239, 270, and / or 331. Preferably, the amino acid substitution is selected from at least two of the amino acids M234, L235, S239, D270, and / or P331, particularly at least two amino acids selected from the amino acids S239, D270, or P331. More preferably, the two amino acids are amino acids at the following positions: 235 and 239; 235 and 270; 235 and 331; 239 and 270; 239 and 331; 270 and 331; 234 and 235; 234 and 239; 234 and 270; 234 and 331; 234 and 331. More preferably, the two amino acids are: L235 and S239; L235 and D270; L235 and P331; S239 and D270; S239 and P331; D270 and P331; M234 and L235; M234 and S239; M234 and D270; M234 and P331. Most preferably, the two amino acid positions are 235 and 331, and the corresponding amino acids are L235 and P331. Specifically, the substitution can be: M234A and L235A; L235A and S239A; L235A and D270A; L235A and P331G; S239A and D270A; S239A and P331G; or D270A and P331G.

[0068] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises substitutions of at least two amino acids selected from amino acid positions 234, 235, 239, 270 and / or 331, wherein at least one of the two amino acid positions is selected from 239, 270 and / or 331.

[0069] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises substitutions of at least three amino acids selected from at least three of amino acid positions 234, 235, 239, 270, and / or 331. Preferably, the amino acid substitutions are at least selected from M234, L235, S239, D270, and / or P331. More preferably, the three amino acid positions are: 235, 239, and 270; 239, 270, and 331; 235, 270, and 331; or 235, 239, and 331. More preferably, the three amino acids are: L235, S239, and D270; S239, D270, and P331; L235, D270, and P331; or L235, S239, and P331. Most preferably, the three amino acid positions are 235, 239, and 270; or 235, 239, and 331, with the corresponding amino acids being L235, S239, and D270; or L235, S239, and P331. Specifically, the substitutions can be: L235A, S239A, and D270A; S239A, D270A, and P33G1; L235A, D270A, and P331G; or L235A, S239A, and P331G.

[0070] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc region comprises at least four substitutions selected from amino acid positions 234, 235, 239, 270, and 331, preferably amino acids at positions 235, 239, 270, and 331, more preferably L235, S239, D270, and P331. Specifically, the substitutions may be L235A, S239A, D270A, and P331G.

[0071] In the aforementioned polypeptides, the one or more substitutions are preferably replacements of wild-type amino acids with alanine, glycine, glutamine, valine, or serine. More preferably, leucine, preferably L235, is replaced by alanine, glutamine, or valine, most preferably alanine. Serine, preferably S239, is preferably replaced by alanine or valine, most preferably alanine. Aspartic acid, preferably D270, is preferably replaced by alanine or valine, most preferably alanine. Proline, preferably P331, is preferably replaced by glycine, alanine, or serine, most preferably glycine.

[0072] Therefore, one or more of the replacements described above are selected from L235A, S239A, D270A and / or P331G.

[0073] In a preferred embodiment of the invention, the polypeptide according to the invention comprises at least a canine Fc fragment derived from immunoglobulin isotype B.

[0074] As further described herein, the terms “Fc region” and “Fc domain” and “Fc segment” are used interchangeably. Specifically, the terms “Fc region” and “Fc segment” are used interchangeably herein. More specifically, the terms “canine or feline Fc region” and “canine or feline Fc segment” are used interchangeably herein. The same applies to the terms “wild-type Fc region” and “wild-type Fc segment”, which are also used interchangeably herein.

[0075] The polypeptide according to the invention may comprise a sequence selected from SEQ ID Nos: 8 to 29 disclosed in Table 3.

[0076] Table 3: Canine HC-B variants:

[0077] The following sequences correspond to AA 234 to 331 of SEQ ID NO: 2. The mutation location is indicated based on the AA position of SEQ ID NO: 2. The mutated AA is indicated in bold.

[0078]

[0079]

[0080] Preferably, the polypeptide according to the invention comprises a sequence selected from SEQ ID NO: 18, 19, 26, 27 or 29. Most preferably, the polypeptide comprises SEQ ID NO: 19 or 27.

[0081] The polypeptide according to the invention can be a binding molecule. The term "binding molecule" according to the invention emphasizes that the polypeptide contains at least one domain that specifically binds to a ligand, preferably a polypeptide, more preferably an epitope. Most preferably, the at least one ligand-specifically binding domain is a complementarity-determining region (CDR) of an antibody or antibody fragment.

[0082] Therefore, in a preferred embodiment of the invention, the polypeptide according to the invention may be an antibody, an antibody fragment, or a polypeptide containing an antibody fragment. Preferably, the antibody, antibody fragment, or polypeptide containing an antibody fragment binds to an epitope disclosed below.

[0083] When used in this document, the term “antibody” refers to any form of antibody, such as monoclonal antibodies including full-length monoclonal antibodies, polyclonal antibodies, and multispecific antibodies such as bispecific antibodies.

[0084] When used herein, the terms "antibody fragment" or "antigen-binding fragment" refer to all segments of an antibody that exhibit antigen-binding properties, i.e., antibody fragments that retain the ability to specifically bind to antigens bound by the corresponding full-length antibody. Thus, "antibody fragment" includes at least one, but preferably all, CDR regions of the full-length antibody from which they are derived. Examples of antigen-binding fragments or antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

[0085] The polypeptides according to the invention can be canine or feline antibodies. The terms "canine antibody" or "feline antibody" refer to an antibody containing an Fc region sequence (canine or feline sequence) having at least 96%, preferably 98%, more preferably 99%, and most preferably 100% identity with a complete canine or feline antibody, except for the mutations according to the invention. The term "complete canine or feline antibody" refers to an antibody that completely comprises a sequence derived from canine or feline genes. In some cases, this can be a canine or feline antibody having an antibody gene sequence derived from a canine or feline chromosome and the modifications outlined herein. "Canine antibodies" or "feline antibodies" can also be recombinantly produced in cells of different species, such as mouse, human, or hybridoma cells. The antibodies can also be derived from synthetic or semi-synthetic antibody sequence libraries. These sequences can comprise sequences encoded by canine and feline genes, as well as artificial sequences, such as artificial CDRs. Therefore, the canine or feline antibodies can contain modifications not typically present in antibodies produced in canine or feline cells, such as sugar attachments.

[0086] The polypeptides according to the invention can also be canine or feline antibodies. A "canine antibody" or "feline antibody" is a form of antibody that correspondingly contains sequences derived from both canine and non-canine (e.g., mouse) antibodies, or sequences derived from both feline and non-feline (e.g., mouse) antibodies. Typically, canine or feline antibodies will contain at least one, usually two or all, CDRs derived from a non-canine or non-feline organism, and substantially canine or feline sequences other than said CDRs.

[0087] The polypeptide according to the invention can also be a chimeric antibody. A "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species. A chimeric antibody may, for example, contain a variable domain from an antibody derived from rodents (e.g., mouse or rat antibodies) and a constant domain from canines or cats.

[0088] The polypeptides according to the present invention can also be fusion proteins. Fusion proteins may be formed by the complete or partial fusion of the constant domain of a canine immunoglobulin heavy chain into the extracellular domain of a cytokine or chemokine receptor or other transmembrane protein.

[0089] The binding properties and immune effector functions of Fc of different canine immunoglobulin isotypes were determined by Bergeron et al. (Bergeron LM, McCandless EE, Dunham S et al., “Comparative functional characterization of canine IgG subclasses”, VetImmunol Immunopathol. 2014; 157(1-2):31–41), as shown in Table 4.

[0090] Table 4 Binding and effector properties of canine IgG isotypes

[0091]

[0092]

[0093] "+++" indicates very tight binding or high reactivity, "++" indicates good binding, "+" indicates some binding observed, "-- / +" indicates almost no activation / binding, and "--" indicates no binding (in Table 4 above).

[0094] Fcγ receptor I (FcγRI) is also commonly referred to as CD64. Fcγ receptor III (FcγRIII) is also commonly referred to as CD16.

[0095] In one key aspect, the peptide according to the invention exhibits reduced binding affinity for C1q and / or Fc receptors relative to peptides containing the corresponding wild-type Fc regions. Preferably, the Fc receptors with reduced binding affinity are FcγRI and FcγRIII. The reduced binding according to the invention can be characterized by an increase in KD of the peptide with the corresponding receptor by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 100-fold, at least 1000-fold, at least 10000-fold, or at least 100000-fold.

[0096] The binding of the polypeptide according to the invention to C1q and / or Fc receptors can be determined by in vitro binding assays known in the art. For example, binding to C1q and / or Fc, particularly FcγRI receptors, can be determined as disclosed in Example 1, or in Sections 2.5 and 2.6 of Bergeron et al. 2014, or WO 2015 / 091910, all of which are incorporated herein by reference. Therefore, the assays for determining binding to C1q and / or Fc, particularly FcγRI receptors, as disclosed in Example 1, or in Sections 2.5 and 2.6 of Bergeron et al. 2014, or WO 2015 / 091910, constitute a part of the disclosure of this application.

[0097] Antibodies, especially in large-scale commercialization, are typically isolated and purified via protein A binding. However, HC-B, which exhibits the strongest protein A binding, also shows strong binding to C1q and Fcγ receptor I (FcγRI), thereby activating the immune effector function (CDC) of the complement system and inducing cytolytic activity (ADCC), as discussed above, which is unacceptable for many indications treated with therapeutic antibodies.

[0098] Surprisingly, as Figure 2 As shown, peptides containing a single substitution of L235, D270, or P331, comprising SEQ IDs 9, 13, and 15, have exhibited weak or significantly reduced binding to FcγRI or C1q. Notably, binding to FcγRI is substantially absent for the peptide comprising SEQ IDs 9, and binding to C1q is substantially absent for the peptide comprising SEQ ID 15.

[0099] Even more surprisingly, for the polypeptide containing only the two substitutions L235 and P331 in SEQ ID 19, the binding with FcγRI and C1q is virtually non-existent.

[0100] Compared to the prior art mutation disclosed in EP 2 705 057A1 (which includes a mutation at position 297 of Kabat, resulting in antibody deglycosylation), the peptide according to the invention achieves silencing of the constant region of the antibody, particularly the highly active isotype HC-B, without deglycosylation. Since deglycosylation can negatively impact antibody clearance from circulation, the present invention moderately offers an advantage over the prior art.

[0101] In addition, such as Figure 4As shown, the mutated Fc fragments of the HC-B isotype according to the present invention maintain their ability to bind to the neonatal Fc receptor (FcRn). It is generally known that binding to FcRn increases the half-life of IgG by reducing lysosomal degradation in endothelial and bone marrow-derived cells. Therefore, maintaining binding to FcRn in peptides with significantly reduced or absent binding to FcγRI and C1q is highly advantageous for recombinant therapeutic antibodies.

[0102] Therefore, the polypeptide according to the invention is characterized in that its binding to FcRn is unimpaired or substantially unimpaired relative to the corresponding wild-type polypeptide. The binding of the polypeptide to FcγRI and / or C1q is preferably significantly reduced or decreased. The substantially unimpaired binding of the polypeptide to FcRn according to the invention can be characterized by a KD increase of less than 2, 3, 5, 10, 25, or 50 times with FcRn. The binding to FcRn can be determined as disclosed in Example 1 of this application.

[0103] Advantageously, the polypeptide provided by the present invention comprises at least a canine or feline Fc fragment derived from immunoglobulin subtype HC-B, which, relative to the wild-type Fc region, includes a substitution of at least one amino acid selected from at least one of amino acid positions 235, 239, 270 and / or 331, and has reduced binding to the FcγRI receptor and / or C1q relative to the corresponding wild-type polypeptide, but has substantially unimpaired binding to protein A.

[0104] As shown in Section 1.3 of Example 1, the polypeptides of the present invention can be purified by binding to protein A. Therefore, the polypeptides of the present invention are characterized in that the binding to protein A is substantially unimpaired relative to the corresponding wild-type polypeptide. The binding of the polypeptide to FcγRI and / or C1q is preferably significantly reduced or diminished. The binding of the polypeptide to protein A according to the present invention can be characterized by a change in the KD of the polypeptide to protein A of less than 2, 3, 5, or 10 times when the mutated polypeptide is compared to the corresponding wild-type polypeptide.

[0105] In a preferred embodiment, the polypeptide is a glycosylated polypeptide that, compared to the corresponding wild-type polypeptide, exhibits significantly reduced or absent binding to the FcγRI receptor and / or C1q, and exhibits binding to the neonatal Fc receptor (FcRn) and protein A as described above. The binding to FcRn and protein A can be characterized by the change in KD of the mutant polypeptide according to the invention compared to the wild-type, as described above.

[0106] As demonstrated by a significant reduction in binding to the FcγRI receptor and / or C1q, the present invention provides peptides as described above that, compared to peptides containing the corresponding wild-type Fc region, induce significantly reduced immune effector function upon administration to a subject. Figure 4 As shown, the peptides according to the invention may not bind substantially to the FcγRI receptor and / or C1q. Therefore, after administration of said peptide to a subject, there may be substantially no immune effector function compared to peptides containing the corresponding wild-type Fc region.

[0107] The subject receiving the peptide may be a subject with an undamaged immune system. Preferably, the subject receiving the peptide is a canine or feline subject, more preferably a canine or feline patient (or a canine or feline animal).

[0108] As described above, the ligand-specific binding domain can bind to epitopes derived from proteins selected from 17-IA, 4-1BB, 4Dc, 6-keto-PGF1α, 8-iso-PGF2α, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, activin RIBALK-4, activin RIIA, activin RUB, ADAM, ADAM 10, ADAM 12, ADAM 15. ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonists, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic peptide AV / B3 integrin, Axl, B2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, β-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP BMP-3 osteogenic protein, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB(ALK-6), BRK-2, RPK-1, BMPR-II(BRK-3), BMPs, β-NGF, BOK, bufotalin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC Complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / ZIP, CBL, CCI, CCK2, CCL, CCL1, CCL11. CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL 23. CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CC R1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD2 1. CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD50, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80, CD89, CD95, CD123, CD133, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD154, CD163, CD164, CEACAM5, CEACAM6, CFTR, CGRP, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CX3CL1, CXCL11. CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerator, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxigenin, DNAM-1, Dnase (deoxyribonuclease), Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot Eosinophil activation chemokine 1, EpCAM, Ephrin (liver glycoprotein) B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor 1a, Factor VII, Factor V11c, Factor IX, Factor XI, Fibroblast activation protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-3 ligand, Flt-4, Follicle-stimulating hormone, Fractal chemokine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6. GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myogenic inhibitory protein), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, glycoprotein llb / llla (GP llb / llla), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV), gH envelope glycoprotein, HCMVUL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her1 (Erb-B1, EGFR), Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSVgD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MM), HIV gp120, HIV 1MB gp120 V3 ring, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-13R IL-15, IL-18, IL-18R, IL-22, IL-23, IL-31, IL-31R, IL-33, IL-33R, Interferon (INF)-α, INF-β, INF-γ, Inhibin, iNOS, Insulin A-chain, Insulin B-chain, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, IP-10, l-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 1 1. Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, Lipoproteins LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surfactant, luteinizing hormone, lymphotoxin B receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11. MMP-12, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, MMP-3, MMP-1, MPIF, Mpo, MSK, MSP, mucin (Mud), MUC18, Müllerian inhibitor, Mug, Musk, Nav1.3, Nav1.5, Nav1.7, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, enkephalin, neurotrophic factor-3,-4 or-6, neurotrophic factor-β, NGF, NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX4 0L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PD-1, PD-L1, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, Placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Prochastin, Protein C, PS, PSA, PSCA, Prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, Renin, Respiratory syncytial virus (RSV)F, RSVFgp, Ret, Rheumatoid Factor, RLIP76, RPA2, RSK, 5100, SCF / KL, SDF-1, Serine, Serum Albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, ST2, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (Tumor-associated Glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT Testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β panspecific, TGF-βR1 (ALK-5), TGF-βR11, TGF-βR11b, TGF-βR11, TGF-β1, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-β, TNF-β2, TNF-α, TNFR1, TNFc, TNF-RII, TNFRSF10A (TRAIL) R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF1 1A (RANK ODF R, TRANCE R), TNFRSF1 1B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF Rl CD120a, p55-60), TNFRSF1 B (TNF RllCD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIM, TNFC R), TNFRSF4 (OX40ACT35, TXGP1R), TNFRSF5 (CD40p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF) BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α Conectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb) TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand, CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TSLP, TSLPR, TARC, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, tumor-associated antigen CA 125, expression LewisTumor-associated antigens of Y-related carbohydrates, TWEAK, TXB2, Ung, uPA, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (fit-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT1OA, WNT1OB, WNT1 1. Receptors for WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD and / or hormones and growth factors, toxins, parasitic epitopes, bacterial epitopes and / or viral epitopes.

[0109] Preferably, the epitope is derived from a protein selected from CTLA-4, EGF, Her1 (Erb-B1, EGFR), IgE, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-10, IL-12, IL-17, IL-17R, IL-18, IL-18R, IL-23, IL-31, IL-31R, IL-33, IL-33R, integrin α4 / β7, NGF, TNF-α, PD-1, PD-L1, and / or VEGF.

[0110] Epitopes derived from molecules, especially proteins, can be peptide epitopes contained within the corresponding target sequence, or conformational epitopes established by the corresponding target structure.

[0111] On the other hand, the present invention relates to a pharmaceutical composition comprising the polypeptide described herein and optionally a pharmaceutically acceptable carrier. A “pharmaceutical composition” is a composition comprising the polypeptide according to the invention and other compounds that are toxicologically acceptable and capable of storing and administering the polypeptide according to the invention to a treated subject, allowing the polypeptide to exert its target pharmacological and biological activities.

[0112] The pharmaceutically acceptable carrier may include agents that are non-toxic to cells or mammals exposed to it at the doses and concentrations used, such as diluents, stabilizers, adjuvants, or other types of excipients. Examples of pharmaceutically acceptable carriers include alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, canine or other animal albumin, buffers such as phosphates, citrates, tromethorphan, or HEPES buffers, glycine, sorbic acid, potassium sorbate, mixtures of metaglycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica or magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sucrose, mannitol, or amino acids including but not limited to arginine.

[0113] This invention also relates to methods of using the polypeptide or pharmaceutical compositions described herein for treating diseases. Similarly, this invention relates to the use of the polypeptide or pharmaceutical compositions described herein in methods of treating diseases. The methods of treating diseases include the step of administering the polypeptide or pharmaceutical composition described herein to a patient requiring treatment, preferably to a canine or feline subject.

[0114] In a preferred embodiment, the disease is an inflammatory disease, allergy, pain, (auto)immune disease, neurological disorder, eye disease, cardiovascular dysfunction, or infectious disease.

[0115] Preferably, the inflammatory disease can be selected from rheumatoid arthritis, osteoarthritis, psoriasis, atopic dermatitis, and inflammatory bowel disease; the allergy can be asthma; the cancer can be selected from lymphoma, melanoma, angiosarcoma, mast cell tumor, osteosarcoma, brain cancer, breast cancer, and colon cancer; the pain can be selected from osteoarthritis pain, cancer pain, back pain, postoperative pain, and neuropathic or inflammatory pain; the (auto)immune disease can be selected from systemic lupus erythematosus; the neurological disorder can be selected from epilepsy; the eye disease can be selected from age-related macular degeneration; the cardiovascular dysfunction can be selected from hypertension and congestive heart failure; and the infectious disease can be selected from hepatitis, canine distemper, canine infectious respiratory disease, and feline immunodeficiency virus.

[0116] The Fc domain, antibody, or Fc fusion protein of the present invention can be used to treat infectious or parasitic diseases, including those caused by external and internal parasites in dogs, as well as respiratory infections, urinary tract infections, and skin infections, particularly skin infections, soft tissue infections, and otitis media.

[0117] As mentioned above, the antibodies and Fc fusion proteins of the present invention can be used for therapeutic, diagnostic, or research purposes or methods.

[0118] On the other hand, the present invention relates to polynucleotides encoding polypeptides according to the present invention. The polynucleotide may be an isolated polynucleotide. The polynucleotide may be contained in a vector such as a plasmid or an artificial chromosome. The polynucleotide may be operably linked to transcription and translation control sequences. In this case, the term "operably linked" means that the transcription and translation control sequences function to functionally transcribe and translate the polynucleotide to express the encoded polypeptide.

[0119] The vector may be contained within a cell. The cell is preferably a host cell suitable for recombinant expression of an antibody or antibody fragment. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate cells, yeast cells, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 cells, and CHO cells, and cell lines derived from them such as 293-6E, DG44, CHO-S, and CHO-K cells.

[0120] On the other hand, the present invention relates to a method for generating a polypeptide comprising an Fc fragment, wherein the method comprises at least the following steps:

[0121] Introducing at least one mutation into a polynucleotide encoding at least one Fc fragment, wherein the mutation in the polynucleotide results in a substitution of at least one amino acid as disclosed above within the polypeptide encoded by the polynucleotide.

[0122] Express a polypeptide containing at least one of the replaced Fc fragments in a host cell.

[0123] Preferably, the mutated polynucleotide encodes at least one of the Seq ID NOs: 1 to 29 as described above.

[0124] Furthermore, the present invention relates to a method for reducing the immune effector function of a peptide containing an Fc fragment, wherein the method comprises the following steps:

[0125] At least one mutation is introduced into a polypeptide containing an Fc fragment, wherein the mutation in the polynucleotide results in a substitution of at least one amino acid as disclosed above in the polypeptide containing the Fc fragment encoded by the polynucleotide.

[0126] Express a polypeptide containing at least one of the replaced Fc fragments in a host cell.

[0127] The present invention will be explained in more detail with reference to the following drawings and embodiments. Attached Figure Description

[0128] Figure 1 A schematic overview of the variants tested in this study is shown.

[0129] Figure 2 The binding of wild-type and mutant variants to C1q(A) or FcγRI(B) is shown. All IgG variants were generated as cell culture supernatants (ccSups) and tested at different dilutions. IgG derived from the supernatant was captured by binding to the target antigen and used to detect binding to C1q or FcγRI. The signals were plotted as bars and normalized to the HC-B wild-type variant showing the greatest binding to C1q or FcγRI.

[0130] Figure 3 The binding of wild-type and mutant variants to C1q (A) or FcγRI (B and C) is shown. All IgG variants were generated as cell culture supernatants (ccSups) and tested at different dilutions. IgG derived from the supernatant was captured by binding to the target antigen and used to detect binding to C1q (A), while IgG was captured by antigen (B) and Fab anti-canine IgG (C) and used to detect binding to FcγRI. The signals were plotted as bars and normalized to the HC-B wild-type variant showing the maximum binding to C1q or FcRI. Combining two or more mutations produced Fc variants with completely disrupted binding to C1q or FcRI.

[0131] Figure 4 The binding of selected variants to C1q (A), FcγRI (B), and FcRn (C) is shown. All IgG variants were tested as purified IgG at different concentrations. Only the wild-type HC-B variant showed binding to C1q and FcγRI, while other isotypes did not bind to the corresponding molecules even at high concentrations. Comparable binding to the FcRn receptor was observed for HC-B-LP and HC-B_LSDP, interestingly, HC-A wt showed only minimal binding. Mutations in the Fc portion of the antibody variants had no effect on antigen recognition (D).

[0132] Figure 5 The binding of selected variants to C1q (A), FcγRI (B), and FcRn (C) is shown. All IgG variants were tested as purified IgG at different concentrations. The wild-type HC-B variant showed strong binding to C1q and FcγRI, while HC-A wt did not bind to the corresponding molecules even at high concentrations. The Fc-engineered HC-B variant HC-B_ML showed slightly reduced binding to C1q but completely lacked FcγRI binding. Comparable binding to the FcRn receptor was observed for HC-B wt and HC-B_ML, interestingly, HC-A wt showed only minimal binding. Mutations in the Fc portion of the antibody variants had no effect on antigen recognition (D).

[0133] Figure 6 shows a comparison of the wild-type sequences of the constant regions of canine IgG isotypes HC-A (SEQ ID NO: 1), HC-B (SEQ ID NO: 2), HC-C (SEQ ID NO: 3), HC-D (SEQ ID NO: 4), feline IgG 1a (SEQ ID NO: 5), feline IgG 1b (SEQ ID NO: 6), and feline IgG 2 (SEQ ID NO: 7) with the wild-type sequences of the constant region of human IgG1. Detailed Implementation

[0134] Aspects and embodiments of the present invention

[0135] 1. A polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc fragment comprises, relative to a wild-type Fc fragment, at least one amino acid selected from amino acid positions 235, 239, 270 and / or 331, preferably selected from at least one of L235, S239, D270 and / or P331.

[0136] 2. The polypeptide according to aspect 1, wherein the wild-type sequence (the sequence of the wild-type Fc fragment) is selected from any of the following: Seq ID NOs: 1 to 7, the sequences of GeneBank accession numbers AF354264, AF354265, AF354266, and AF354267 (according to Kabat numbers), particularly the sequences of amino acids 234 to 331 (according to Kabat numbers) of GeneBank accession numbers AF354264, AF354265, AF354266, and AF354267, or the wild-type sequence disclosed in Striezel et al., page 220.

[0137] 3. The polypeptide according to aspect 1 or 2, wherein the Fc fragment comprises at least two amino acid substitutions selected from positions 234, 235, 239, 270 and / or 331, preferably selected from at least two amino acid substitutions selected from L235 and S239, L235 and D270, L235 and P331, L235 and P331, S239 and D270, S239 and P331, D270 and P331, M234 and L235, M234 and S239, M234 and D270, M234 and P331.

[0138] 4. The polypeptide according to any of the foregoing aspects, wherein the Fc fragment comprises a substitution of at least two amino acids selected from positions 234, 235, 239, 270 and / or 331, wherein at least one of the two amino acid positions is selected from positions 239, 270 and / or 331.

[0139] 5. The polypeptide according to any of the foregoing aspects, wherein the Fc fragment comprises at least three amino acid positions selected from amino acid positions 234, 235, 239, 270 and / or 331, preferably L235, S239 and D270; S239, D270 and P331; L235, D270 and P331; or substitutions of at least three amino acids selected from L235, S239 and P331.

[0140] 6. The polypeptide according to any of the foregoing aspects, wherein the Fc fragment comprises at least four selected from amino acid positions 234, 235, 239, 270 and 331, preferably at least four substitutions of amino acids 235, 239, 270 and 331.

[0141] 7. The polypeptide according to any of the foregoing aspects, wherein the one or more substitutions are alanine or glycine, preferably wherein the one or more substitutions are L235A, S239A, D270A and / or P331G.

[0142] 8. The polypeptide according to any of the foregoing aspects, wherein the canine Fc fragment is derived from IgG isotypes IgG-A, IgG-B, IgG-C or IgG-D, most preferably from the Fc fragment of IgG isotype IgG-B.

[0143] 9. The polypeptide according to any of the foregoing aspects, comprising a sequence selected from SEQ ID NOs 8 to 29, preferably SEQ ID NOs 18, 19, 26, 27 or 29.

[0144] 10. The polypeptide according to any of the foregoing aspects, wherein the polypeptide is a binding molecule, preferably an antibody, an antibody fragment, or a polypeptide containing an antibody fragment.

[0145] 11. The polypeptide according to any of the foregoing aspects, wherein the polypeptide has a reduced binding affinity for C1q and / or Fc receptors relative to a polypeptide comprising the corresponding wild-type Fc fragment.

[0146] 12. The polypeptide according to aspect 11, wherein the Fc receptor is FcγRI or FcγRIII.

[0147] 13. The polypeptide according to any of the foregoing aspects, characterized in that the binding of the polypeptide to FcRn and / or protein A is substantially unimpaired relative to the corresponding wild-type polypeptide.

[0148] 14. The polypeptide according to any of the foregoing aspects, wherein the polypeptide, relative to a polypeptide comprising the corresponding wild-type Fc fragment, induces reduced immune effector function upon administration to a subject, wherein preferably the subject has an undamaged immune system.

[0149] 15. The polypeptide according to any of the foregoing aspects, wherein the polypeptide, relative to a polypeptide comprising the corresponding wild-type Fc fragment, induces reduced ADCC or CDC upon administration to a subject.

[0150] 16. The polypeptide according to any of the foregoing aspects, wherein the polypeptide comprises a domain that specifically binds to an epitope, said epitope being derived from a protein selected from CTLA-4, EGF, Her1 (Erb-B1, EGFR), IgE, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-10, IL-12, IL-17, IL-17R, IL-18, IL-18R, IL-23, IL-31, IL-31R, IL-33, IL-33R, integrin α4 / β7, NGF, TNF-α, PD-1, PD-L1, VEGF.

[0151] 17. A pharmaceutical composition comprising a polypeptide according to any of the foregoing aspects.

[0152] 18. A method for treating a disease in canine or feline subjects with a polypeptide or composition according to any of the foregoing aspects, preferably wherein the disease is an inflammatory disease, allergy, cancer, pain, (auto)immune disease, neurological disorder, eye disease, cardiovascular dysfunction, or infectious disease.

[0153] 19. A polynucleotide encoding a polypeptide as described in the foregoing aspects.

[0154] 20. A carrier or cell comprising the polynucleotides described in aspect 19.

[0155] 21. A method for generating a polypeptide comprising Fc C2 and C3 fragments, wherein the method comprises at least the following steps:

[0156] Introducing at least one mutation into a polynucleotide encoding at least one Fc C2 and C3 segment, wherein the mutation in the polynucleotide results in the substitution of at least one amino acid as disclosed above within the polypeptide encoded by the polynucleotide;

[0157] In the host cell, a polypeptide containing at least one of the replaced Fc C2 and C3 fragments is expressed.

[0158] 22. A method for reducing the immune effector function of a peptide comprising Fc C2 and C3 fragments, wherein the method comprises the following steps:

[0159] At least one mutation is introduced into a polypeptide containing Fc C2 and C3 segments, wherein the mutation in the polynucleotide results in the substitution of at least one amino acid as disclosed above within the polypeptide containing the Fc C2 and C3 segments encoded by the polynucleotide.

[0160] In the host cell, a polypeptide containing at least one of the replaced Fc C2 and C3 fragments is expressed.

[0161] 23. A method of treating a disease in a canine or feline subject, the method comprising administering (therapeuticly effective amount) of a polypeptide or (pharmaceutical) composition according to any of the foregoing aspects to a canine or feline subject in need, preferably wherein the disease is an inflammatory disease, allergy, cancer, pain, (auto)immune disease, neurological disorder, eye disease, cardiovascular dysfunction, or infectious disease.

[0162] Example

[0163] Example 1

[0164] 1. Materials and Methods:

[0165] 1.1 Construction of Variants

[0166] A complete canine anti-GFP antibody was used as a model antibody to study the interaction between C1q and FcγRI in Fc mutants containing different antibody constant regions. The antibody was derived from a complete canine phage display library described in WO 2018 / 234438.

[0167] A total of 13 constructs were generated, including constructs containing Fc fragments modified according to Seq ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29. Thus, there were 4 HC-B variants with one amino acid mutation and 6 HC-B variants with combinations of these single mutations, as well as wild-type HC-B as a positive control and wild-type HC-A described as lacking effector function as a negative control in binding assays.

[0168] The construct is schematically depicted in Figure 1 middle.

[0169] The mutant Fc construct was synthesized via PCR mutagenesis and cloned into a proprietary mammalian expression vector that encodes both the heavy and light chain sequences of the anti-GFP IgG antibody.

[0170] 1.2 Production of IgG-containing cell culture supernatant for screening

[0171] use Transfection reagent HEK293-EBNA cells were transfected with mammalian expression vector DNA. Cell culture supernatant was harvested on day 3 post-transfection, and IgG concentration was determined by ELISA (data not shown). The supernatant was used for binding assays.

[0172] 1.3 Production and purification of IgG

[0173] HEK293F suspension cells were grown to the logarithmic growth phase and then used FectoPRO. Transfection was performed using mammalian expression vector DNA. Cell culture supernatant was harvested on day 8 post-transfection and subjected to standard protein A affinity chromatography (MabSelect SURE, GE Healthcare). The buffer was replaced with 1x Dulbcecco's PBS (pH 7.2), and the samples were aseptically filtered (0.2 μm pore size). Protein concentration was determined by UV spectrophotometry, and IgG purity was analyzed by SDS-PAGE under denaturing and reducing conditions, followed by size exclusion chromatography (SEC). Purification was performed on a GE Healthcare Europe GmbH, Freiburg, Germany. Separation was performed using a Superdex 75 HR 10 / 30 column (GE Healthcare Europe GmbH, Freiburg, Germany). For each sample, 10 μl of protein was loaded onto the column, separated at a flow rate of 0.05 mL / min, and UV absorbance at 260 and 280 nm was recorded. The run buffer consisted of Gibco D-PBS, pH 7.4 (Invitrogen, Paisley, USA).

[0174] Quality control following transfection and purification showed that the antibody variant used in the binding assays was of high purity and monomeric, ruling out the possibility that variations in C1q or FcγRI binding were due to aggregates, for example, in the protein preparation (data not shown). Interestingly, the HC-A antibody could also be readily purified using protein A, as evidenced by the lack of detectable antibody in the flow buffer or washing buffer, indicating that all proteins were captured on protein A (data not shown).

[0175] Furthermore, all antibody constructs exhibited the same binding efficiency to their target protein GFP. Figure 4 and Figure 5 1.4 C1q and FcγRI ELISA were performed using cell culture supernatant.

[0176] The binding of the antibody to complement protein C1q was assessed using ELISA. In simple terms, the Maxisorp plate (Nunc) was used to assess the binding. TM The plate was coated with GFP (3 μg / ml) at room temperature (RT) for 1 h. The plate was blocked with PBS containing 5% skim milk. The supernatant containing the antibody was titrated in PBS and incubated with the fixed GFP at room temperature with shaking for 1 h. The bound antibody was then added to purified recombinant human C1q protein (Quidel Corporation, San Diego, CA, USA) at a concentration of 10 μg / ml in M-PBST (PBS supplemented with 0.5% skim milk and 0.05% Tween-20), and the plate was gently shaken and incubated at room temperature for 1 h. After washing with PBS-T (PBS supplemented with 0.05% Tween-20), binding was detected using sheep anti-human C1q antibody conjugated to HRP (Bio-Rad). Using the QuantaBlu Fluorescent Peroxidase Substrate Kit (Thermo), the plate was developed according to the manufacturer's instructions, and fluorescence was measured on a Genios Reader Pro (Tecan) using excitation at 320 nm and emission at 430 nm.

[0177] The binding of the antibody to human FcγRI was assessed using ELISA. In simple terms, the Maxisorp plate (Nunc) was used to assess the binding. TM Incubate the plate at room temperature (RT) for 1 h with GFP (3 μg / ml) or Fab anti-canine IgG (H+L) antibody (5 μg / ml). Block the plate with ChemiBLOCKER (Millipore). Titrate the supernatant containing the antibody in PBS and incubate with the plate on the fixed GFP at room temperature with shaking for 1 h. Add biotinylated recombinant human FcγRI protein at a concentration of 1 μg / ml to the bound antibody in PBS supplemented with 10% ChemiBLOCKER and 0.05% Tween-20 (Sino Biological), and incubate the plate with gentle shaking at room temperature for 1 h. After washing with PBS-T (PBS supplemented with 0.05% Tween-20), detect binding using streptavidin-HRP (Jackson ImmunoResearch). Using the QuantaBlu Fluorescent Peroxidase Substrate Kit (Thermo), the plate was developed according to the manufacturer's instructions, and fluorescence was measured on a Genios Reader Pro (Tecan) using excitation at 320 nm and emission at 430 nm.

[0178] like Figure 2 As shown, the wild-type HC-B variants bind well to both C1q (1A, left panel) and FcγRI protein (1B, right panel). Furthermore, as expected, the wild-type HC-A antibody does not bind to C1q or FcγRI or binds very weakly. Compared to wild-type HC-B, all single mutations showed lower binding to either C1q or FcγRI. However, significant differences exist among the variants. The L235A mutation significantly reduces binding to C1q and weakens binding to FcγRI, while P331G retains some binding to FcγRI but does not recognize C1q. The S239A and D270 variants show reduced binding to both proteins, but the effect is more pronounced for D270A.

[0179] Since no single mutant showed a complete loss of binding to both C1q and FcγRI, different combinations were tested. Results were shown in... Figure 3 The study demonstrated that a combination of two or more mutations could eliminate binding to C1q and FcγRI. Except for the variants HC-B_LS and HC-B_DP, which showed little residual binding to either C1q or FcγRI, all other variants did not bind under the experimental settings.

[0180] To validate the results from the screening assay, several variants were purified and their binding to C1q, FcγRI, and FcRn was tested in a concentration-dependent manner compared to HC-B wt and HC-Awt.

[0181] 1.5 Characterization of Fc variants using purified antibodies

[0182] 1.5.1 C1q ELISA using purified IgG

[0183] Essentially as described above, the binding of purified IgG to complement protein C1q was assessed by ELISA. In short, the purified antibody was titrated in PBS and plated on Maxisorp plates (Nunc... TMFix at room temperature for 1 h. Block the plate with PBS containing 5% skim milk. Add purified recombinant human C1q protein (Quidel Corporation, San Diego, CA, USA) at a concentration of 10 μg / ml in M-PBST (PBS supplemented with 0.5% skim milk and 0.05% Tween-20) to the bound antibody, and gently shake the plate to incubate at room temperature for 1 h. After washing with PBS-T (PBS supplemented with 0.05% Tween-20), detect binding using sheep anti-human C1q antibody conjugated to HRP (Bio-Rad). Develop the plate using the QuantaBlu fluorescent peroxidase substrate kit (Thermo) according to the manufacturer's instructions, and measure fluorescence on a Genios Reader Pro (Tecan) using excitation at 320 nm and emission at 430 nm.

[0184] 1.5.2 FcγRI ELISA using purified IgG

[0185] The binding of the antibody to human FcγRI was assessed by ELISA. In short, the antibody was titrated in PBS and fixed in a Maxisorp plate (Nunc) at room temperature for 1 h. The plate was then blocked with ChemiBLOCKER. To the bound antibody, biotinylated recombinant human FcγRI protein (Sino Biological) was added at a concentration of 1 μg / ml in PBS supplemented with 10% ChemiBLOCKER and 0.05% Tween-20, and the plate was gently shaken and incubated at room temperature for 1 h. After washing with PBS-T (PBS supplemented with 0.05% Tween-20), binding was detected using streptavidin-HRP (Jackson ImmunoResearch). The plate was developed using the QuantaBlu Fluorescent Peroxidase Substrate Kit (Thermo) according to the manufacturer's instructions, and fluorescence was measured on a Genios Reader Pro (Tecan) using excitation at 320 nm and emission at 430 nm.

[0186] 1.5.3 FcRn-ELISA using purified IgG

[0187] The binding of the antibody to canine FcRn was assessed by ELISA. In short, the antibody was titrated in PBS and fixed in a Maxisorp plate (Nunc) at room temperature for 1 h. The plate was then blocked with ChemiBLOCKER. To the bound antibody, biotinylated recombinant canine FcRn protein (Immunitrack) at a concentration of 10 μg / ml was added to pH 6 PBS supplemented with 10% ChemiBLOCKER and 0.05% Tween-20, and the plate was gently shaken and incubated at room temperature for 1 h. After washing with PBS-T (PBS supplemented with 0.05% Tween-20), binding was detected using streptavidin-HRP (Jackson ImmunoResearch). The plate was developed using the QuantaBlu Fluorescent Peroxidase Substrate Kit (Thermo) according to the manufacturer's instructions, and fluorescence was measured on a Genios Reader Pro (Tecan) using excitation at 320 nm and emission at 430 nm.

[0188] 1.5.4 Antigen Binding ELISA

[0189] The binding of the antibody to the model antigen GFP was assessed by ELISA. 3 μg / mL GFP diluted in PBS was fixed in Maxisorp plates (Nunc) at room temperature for 1 h. The plates were blocked with PBS containing 5% skim milk. The antibody was titrated in M-PBST (PBS supplemented with 0.5% skim milk and 0.05% Tween-20), added to the bound antigen, and the plates were gently shaken and incubated at room temperature for 1 h. After washing with PBS-T (PBS supplemented with 0.05% Tween-20), binding was detected using rabbit anti-dog (Fab)2 antibody conjugated to HRP (Sigma). The plates were developed using the QuantaBlu Fluorescent Peroxidase Substrate Kit (Thermo) according to the manufacturer's instructions, and fluorescence was measured on a Genios Reader Pro (Tecan) using excitation at 320 nm and emission at 430 nm.

[0190] 2. Results

[0191] HC-B wt is known to efficiently induce effector functions mediated by binding to C1q and FcγRI proteins, and was used as a positive control in subsequent experiments. In vitro binding experiments from previous studies revealed that canine HC-A binds to C1q and FcγRI with near-zero affinity, resulting in loss of effector function. The selected candidate was purified and tested against wild-type HC-B and HC-A. The variant HC-B_ML contains the double mutation M234A / L235A, which has been described as significantly reducing effector function for human antibodies (Xu D, Alegre ML, Varga SS, et al., “In vitro characterization of five humanized OKT3 effector function variant antibodies”, Cell Immunol. 2000; 200(1):16–26). Surprisingly, the same mutation in the canine construct eliminated binding to FcγRI, but the variant was still able to bind to C1q (see Figure 4 A and B). These results show that the characteristics of mutations in the human Fc region are generally not transferable to the canine Fc region. (This is in contrast to the neonatal receptor FcRn). Figure 4 C) and the antigens targeted by the antibodies ( Figure 4 The binding of D) was comparable. The HC-A wild-type yielded the expected results, but surprisingly, the binding to FcRn was also reduced compared to the HC-B variant.

[0192] As seen in experiments using cell culture supernatant, it was confirmed that variants HC-B_LP and HC-B_LSDP lost their binding to C1q and FcγRI even at high antibody concentrations. Figure 5 A and 5B), while binding to FcRn can be preserved, and recognition of the target antigen is not affected. sequence list <110> Idivo LLC <120> Modified Fc area <130> PCT127675-RPpau <140> Not yet allocated <141> Same as above <150> EP20158132.9 <151> 2020-02-19 <160> 29 <170> BiSSAP 1.3.6 <210> 1 <211> 330 <212> PRT <213> Domestic dog (Canis lupus) <220> <223> HC-A WT <400> 1 Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser 1 5 10 15 Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe 20 25 30 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly 35 40 45 Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu His Ser Leu<000,0461>50 55 60 Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe 65 70 75 80 Thr Cys Asn Val Val His Pro Ala Ser Asn Thr Lys Val Asp Lys Pro 85 90 95 Val Phe Asn Glu Cys Arg Cys Thr Asp Thr Pro Pro Cys Pro Val Pro 100 105 110 Glu Pro Leu Gly Gly Pro Ser Val Leu Ile Phe Pro Pro Lys Pro Lys 115 120 125 Asp Ile Leu Arg Ile Thr Arg Thr Pro Glu Val Thr Cys Val Val Leu 130 135 140 Asp Leu Gly Arg Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp 145 150 155 160 Gly Lys Glu Val His Thr Ala Lys Thr Gln Ser Arg Glu Gln Gln Phe 165 170 175 Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His Gln Asp 180 185 190 Trp Leu Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His Ile Asp Leu 195 200 205 Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Arg Ala His 210 215 220 Lys Pro Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser Ser 225 230 235 240 Ser Asp Thr Val Ser Ile Thr Cys Leu Ile Lys Asp Phe Tyr Pro Pro 245 250 255 Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Arg 260 265 270 Lys His Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 275 280 285 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly Asp 290 295 300 Pro Phe Thr Cys Ala Val Met His Glu Thr Leu Gln Asn His Tyr Thr 305 310 315 320 Asp Leu Ser Leu Ser His Ser Pro Gly Lys 325 330 <210> 2 <211> 335 <212> PRT <213> Domestic dog (Canis lupus) <220> <223> HC-B WT <400> 2 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr Lys Val Asp Lys 85 90 95 Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys 100 105 110 Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu 130 135 140 Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp Pro Glu Val Gln 145 150 155 160 Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln 165 170 175 Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys 195 200 205 Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210 215 220 Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser 225 230 235 240 Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys 245 250 255 Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln 260 265 270 Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu 275 280 285 Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg 290 295 300 Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu 305 310 315 320 His Asn His Tyr Thr Gln Glu Ser Leu Ser His Ser Pro Gly Lys 325 330 335 <210> 3 <211> 333 <212> PRT <213> Canis lupus <220> <223> HC‑C WT <400> 3 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Ser Gln Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr 20 25 30 Ile Pro Glu Pro Val Thr Val Ser Trp Asn Ser Val Ser Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys 85 90 95 Pro Val Ala Lys Glu Cys Glu Cys Lys Cys Asn Cys Asn Asn Cys Pro 100 105 110 Cys Pro Gly Cys Gly Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 115 120 125 Pro Lys Pro Lys Asp Ile Leu Val Thr Ala Arg Thr Pro Thr Val Thr 130 135 140 Cys Val Val Val Asp Leu Asp Pro Glu Asn Pro Glu Val Gln Ile Ser 145 150 155 160 Trp Phe Val Asp Ser Lys Gln Val Gln Thr Ala Asn Thr Gln Pro Arg 165 170 175 Glu Glu Gln Ser Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile 180 185 190 Gly His Gln Asp Trp Leu Ser Gly Lys Gln Phe Lys Cys Lys Val Asn 195 200 205 Asn Lys Ala Leu Pro Ser Pro Ile Glu Glu Ile Ile Ser Lys Thr Pro 210 215 220 Gly Gln Ala His Gln Pro Asn Val Tyr Val Leu Pro Pro Ser Arg Asp 225 230 235 240 Glu Met Ser Lys Asn Thr Val Thr Leu Thr Cys Leu Val Lys Asp Phe 245 250 255 Phe Pro Pro Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu 260 265 270 Pro Glu Ser Lys Tyr Arg Met Thr Pro Pro Gln Leu Asp Glu Asp Gly 275 280 285 Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln 290 295 300 Arg Gly Asp Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn 305 310 315 320 His Tyr Thr Gln Ile Ser Leu Ser His Ser Pro Gly Lys 325 330 <210> 4 <211> 331 <212> PRT <213> Domestic dog (Canis lupus) <220> <223> HC‑D WT <400> 4 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Thr Val Thr Val Pro Ser Ser Arg Trp Pro Ser Glu Thr 65 70 75 80 Phe Thr Cys Asn Val Val His Pro Ala Ser Asn Thr Lys Val Asp Lys 85 90 95 Pro Val Pro Lys Glu Ser Thr Cys Lys Cys Ile Ser Pro Cys Pro Val 100 105 110 Pro Glu Ser Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 115 120 125 Lys Asp Ile Leu Arg Ile Thr Arg Thr Pro Glu Ile Thr Cys Val Val 130 135 140 Leu Asp Leu Gly Arg Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val 145 150 155 160 Asp Gly Lys Glu Val His Thr Ala Lys Thr Gln Pro Arg Glu Gln Gln 165 170 175 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His Gln 180 185 190 Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His Ile Gly 195 200 205 Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala 210 215 220 His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Pro Lys Glu Leu Ser 225 230 235 240 Ser Ser Asp Thr Val Thr Leu Thr Cys Leu Ile Lys Asp Phe Tyr Pro 245 250 255 Pro Glu Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Pro Glu Pro Glu 260 265 270 Ser Lys Tyr His Thr Thr Ala Pro Gln Leu Asp Glu Asp Gly Ser Tyr 275 280 285 Phe Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295 300 Asp Thr Phe Thr Cys Ala Val Met His Glu Ala Leu Gln Asn His Tyr 305 310 315 320 Thr Asp Leu Ser Leu Ser His Ser Pro Gly Lys 325 330 <210> 5 <211> 335 <212> PRT <213> Domestic cat (Felis catus) <220> <223> IgG1a <400> 5 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Thr Thr Ser Gly Ala Thr Val Ala Leu Ala Cys Leu Val Leu Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ala Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Leu Ser Asp Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Arg Lys Thr Asp His Pro Pro Gly Pro Lys Pro Cys Asp Cys 100 105 110 Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr Pro Glu 130 135 140 Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asp Val Gln 145 150 155 160 Ile Thr Trp Phe Val Asp Asn Thr Gln Val Tyr Thr Ala Lys Thr Ser 165 170 175 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys 195 200 205 Val Asn Ser Lys Ser Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210 215 220 Ala Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro Pro Ala 225 230 235 240 Gln Glu Glu Leu Ser Glu Asn Lys Val Ser Val Thr Cys Leu Ile Lys 245 250 255 Ser Phe His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr Gly Gln 260 265 270 Pro Glu Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro Gln Leu Asp Ser 275 280 285 Asp Gly Thr Tyr Phe Val Tyr Ser Lys Leu Ser Val Asp Arg Ser His 290 295 300 Trp Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu Ala Leu 305 310 315 320 His Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly Lys 325 330 335 [[ID=二十]]<210> 6 <211> 335 <212> PRT <213> Domestic cat (Felis catus) <220> <223> IgG1b <400> 6 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Thr Thr Ser Gly Ala Thr Val Ala Leu Ala Cys Leu Val Leu Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ala Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Leu Ser Asp Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Arg Lys Thr Asp His Pro Pro Gly Pro Lys Pro Cys Asp Cys 100 105 110 Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr Pro Glu 130 135 140 Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asp Val Gln 145 150 155 160 Ile Thr Trp Phe Val Asp Asn Thr Gln Val Tyr Thr Ala Lys Thr Ser 165 170 175 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys 195 200 205 Val Asn Ser Lys Ser Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys 210 215 220 Asp Lys Gly Gln Pro His Glu Pro Gln Val Tyr Val Leu Pro Pro Ala 225 230 235 240 Gln Glu Glu Leu Ser Glu Asn Lys Val Ser Val Thr Cys Leu Ile Glu 245 250 255 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ile Thr Gly Gln 260 265 270 Pro Glu Pro Glu Asn Asn Tyr Arg Thr Thr Pro Pro Gln Leu Asp Ser 275 280 285 Asp Gly Thr Tyr Phe Leu Tyr Ser Arg Leu Ser Val Asp Arg Ser Arg 290 295 300 Trp Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu Ala Leu 305 310 315 320 His Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly Lys 325 330 335 <210> 7[[ID=']] <211> 334 <212> PRT <213> Domestic cat (Felis catus) <220> <223> IgG2 <400> 7 Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly 1 5 10 15 Thr Thr Ser Gly Ala Thr Val Ala Leu Ala Cys Leu Val Leu Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ala Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp Leu Ser Asp Thr 65 70 75 80 Phe Thr Cys Asn Val Ala His Pro Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Pro Lys Thr Ala Ser Thr Ile Glu Ser Lys Thr Gly Glu Gly 100 105 110 Pro Lys Cys Pro Val Pro Glu Ile Pro Gly Ala Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr Pro Glu 130 135 140 Val Thr Cys Leu Val Val Asp Leu Gly Pro Asp Asp Ser Asn Val Gln 145 150 155 160 Ile Thr Trp Phe Val Asp Asn Thr Glu Met His Thr Ala Lys Thr Arg 165 170 175 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 180 185 190 Pro Ile Leu His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys 195 200 205 Val Asn Ser Lys Ser Leu Pro Ser Ala Met Glu Arg Thr Ile Ser Lys 210 215 220 Ala Lys Gly Gln Pro His Glu Pro Val Tyr Val Leu Pro Pro Thr Gln 225 230 235 240 Glu Glu Leu Ser Glu Asn Lys Val Ser Val Thr Cys Leu Ile Lys Gly 245 250 255 Phe His Pro Pro Asp Ile Ala Val Glu Trp Glu Ile Thr Gly Gln Pro 260 265 270 Glu Pro Glu Asn Asn Tyr Gln Thr Thr Pro Pro Gln Leu Asp Ser Asp 275 280 285 Gly Thr Tyr Phe Leu Tyr Ser Arg Leu Ser Val Asp Arg Ser His Trp 290 295 300 Gln Arg Gly Asn Thr Tyr Thr Cys Ser Val Ser His Glu Ala Leu His 305 310 315 320 Ser His His Thr Gln Lys Ser Leu Thr Gln Ser Pro Gly Lys 325 330 <210> 8 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_L235(A / Q / V) <220> <221> Variant <222> 2 <223> X = Ala, Gln, Val <400> 8 Met Xaa Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 9 <211> 98 <212> PRT <213> Artificial sequence <220> <223> HC-B_L235A <400> 9 Met Ala Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 10 <211> 98 <212> PRT <213> Artificial sequence <220> <223> HC-B_S239(A / V) <220> <221> variants <222> 6 <223> X = Ala, Val <400> 10 Met Leu Gly Gly Pro Xaa Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 11 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_S239A <400> 11 Met Leu Gly Gly Pro Ala Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 12 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_D270(A / N) <220> <221> Variant <222> 37 <223> X = Ala, Asn <400> 12 Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Xaa Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 13 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_D270A <400> 13 Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Ala Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 14 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_P331(G / A / S) <220> <221> Variant <222> 98 <223> X = Gly, Ala, Ser <400> 14 Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 ​​​​​​​​​​​​​​​​​​​​<211> 98 <212> PRT <213> Artificial sequence <220> <223> HC-B_P331G <400> 15 Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Gly <210> 16 <211> 98 <212> PRT <213> Artificial sequence <220> <223> HC-B_L235(A / Q / V)_S239(A / V) <220> <221> variants <222> 2 <223> X = Ala, Gln, Val <220> <221> Variant <222> 6 <223> X = Ala, Val <400> 16 Met Xaa Gly Gly Pro Xaa Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 17 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_L235A_S239A <400> 17 Met Ala Gly Gly Pro Ala Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95<00,01013>Ser Pro <210> 18 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_L235(A / Q / V)_P331(G / A / S) <220> <221> Variant <222> 2 <223> X = Ala, Gln, Val <220> <221> Variant <222> 98 <223> X = Gly, Ala, Ser <400> 18 Met Xaa Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Xaa <210> 19 <211> 98 <212> PRT <213> Synthetic Sequence <220> <223> HC‑B_L235A_P331G <400> 19 Met Ala Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Gly <210> 20 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_D270(A / N)_P331(G / A / S) <220> <221> Variant <222> 37 <223> X = Ala, Asn <220> <221> Variant <222> 98 <223> X = Gly, Ala, Ser <400> 20 Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Xaa Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Xaa <210> 21 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_D270A_P331G <400> 21 Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Ala Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Gly <210> twenty two <211> 98 <212> PRT <213> Artificial sequence <220> <223> HC‑B HC‑B_L235(A / Q / V)_S239(A / V)_D270(A / N) <220> <221> variants <222> 2 <223> X = Ala, Gln, Val <220> <221> variants <222> 6 <223> X = Ala, Val <220> <221> variants <222> 37 <223> X = Ala, Asn <400> twenty two Met Xaa Gly Gly Pro Xaa Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Xaa Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 23 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> HC‑B_L235A_S239A_D270A <400> 23 Met Ala Gly Gly Pro Ala Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Ala Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 [[ID=�6]]Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 24 <211> 98 <212> PRT <213> Artificial sequence <220> <223> HC‑B_L235(A / Q / V)_S239(A / V)_P331(G / A / S) <220> <221> variants <222> 2 <223> X = Ala, Gln, Val <220> <221> variants <222> 6 <223> X = Ala, Val <220> <221> variants <222> 98 <223> X = Gly, Ala, Ser <400> twenty four Met Xaa Gly Gly Pro Xaa Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Xaa <210> 25 <211> 98 <212> PRT <213> Synthetic sequence <220> <223> HC‑B_L235A_S239A_P331G <400> 25 Met Ala Gly Gly Pro Ala Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Gly <210> 26 <211> 98 <212> PRT <213> Synthetic sequence <220> <223> HC‑B HC‑B_L235(A / Q / V)_S239(A / V)_D270(A / N)_P331(G / A / S) <220> <221> variants <222> 2 <223> X = Ala, Gln, Val <220> <221> variants <222> 6 <223> X = Ala, Val <220> <221> variants <222> 37 <223> X = Ala, Asn <220> <221> variants <222> 98 <223> X = Gly, Ala, Ser <400> 26 Met Xaa Gly Gly Pro Xaa Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Xaa Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Xaa <210> 27 <211> 98 <212> PRT <213> Synthetic sequence <220> <223> HC‑B_L235A_S239A_D270A_P331G <400> 27 Met Ala Gly Gly Pro Ala Val Phe Ile Phe Pro Pro Lys Pro Lys Asp **1** **5** **10** **15** Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp **20** **25** **30** Leu Asp Pro Glu Ala Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly **35** **40** **45** Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn **50** **55** **60** Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp **65** **70** **75** **80** Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Gly <210> 28 <211> 98 <212> PRT <213> Synthetic sequence <220> <223> HC‑B_M234(A / Q / V) _L235(A / Q / V) <220> <221> variants <222> 1 <223> X = Ala, Gln, Val <220> <221> variants <222> 2 <223> X = Ala, Gln, Val <400> 28 Xaa Xaa Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro <210> 29 <211> 98 <212> PRT <213> Artificial sequence <220> <223> HC-B_M234A_L235A <400> 29 Ala Ala Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 1 5 10 15 Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 20 25 30 Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly 35 40 45 Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn 50 55 60 Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp Trp 65 70 75 80 Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro 85 90 95 Ser Pro

Claims

1. An antibody, wherein the Fc fragment of the antibody is a canine or feline Fc fragment, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27.

2. The antibody according to claim 1, wherein the canine Fc fragment is an Fc fragment derived from IgG isotypes IgG-A, IgG-B, IgG-C, or IgG-D.

3. The antibody according to claim 2, wherein the canine Fc fragment is an Fc fragment derived from the IgG isotype IgG-B.

4. The antibody according to claim 1, wherein the antibody has a reduced binding affinity for C1q and / or Fc receptor relative to an antibody comprising the corresponding HC-B wild-type Fc fragment.

5. The antibody according to claim 4, wherein the Fc receptor is FcγRI or FcγRIII.

6. The antibody according to claim 1, characterized in that, The binding of the antibody to FcRn and / or protein A is substantially unimpaired compared to antibodies containing the corresponding HC-B wild-type Fc fragment.

7. The antibody of claim 1, wherein the antibody, relative to an antibody comprising the corresponding HC-B wild-type Fc fragment, induces reduced immune effector function upon administration to a subject.

8. The antibody of claim 7, wherein the subject has an undamaged immune system.

9. The antibody of claim 1, wherein the antibody, relative to an antibody comprising the corresponding HC-B wild-type Fc fragment, induces reduced ADCC or CDC upon administration to a subject.

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