RU2795411C2 - Monoclonal antibodies to interleukin-31 for veterinary use - Google Patents

Abstract

FIELD: biotechnology; immunology.

SUBSTANCE: invention relates to monoclonal antibodies against IL-31. Also the compositions containing monoclonal antibodies against IL-31, as well as nucleic acids, vectors, and host cells for producing such antibodies are disclosed.

EFFECT: invention provides novel anti-IL-31 antibodies for the prevention and treatment of IL-31 mediated disorders in mammalian veterinary medicine, in particular for safe and effective alternative treatments for atopic and allergic dermatoses in cats and horses.

29 cl, 26 dwg, 2 tbl, 20 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of recombinant monoclonal antibodies and their use in clinical and scientific techniques, including diagnostic techniques. The present invention also provides isolated anti-IL-31 antibodies in the form of veterinary compositions useful in the treatment of an IL-31 mediated disorder in a mammal such as a cat, dog or horse.

PRIOR ART

Atopic dermatitis is defined by the American College of Veterinary Dermatology Working Group as "an inflammatory, allergic skin disease associated with itching, genetic predisposition, and characteristic clinical features" (Olivry, et at. Veterinary Immunology and Immunopathology 2001; 81: 143 -146). According to this working group, in dogs, the disease is associated with allergen-specific IgE (Olivry, et al. 2001 supra; Marsella & Olivry Clinics in Dermatology 2003; 21: 122-133). The symptoms that are most noticeable and of most concern to pet owners are severe itching accompanied by secondary alopecia and erythema.

The factors that may be involved in the development of allergic dermatitis are numerous and poorly understood. Atopic dermatitis can be provoked by food components (Picco, et al. Vet Dermatol. 2008; 19: 150-155), as well as environmental allergens such as fleas, dust mites, ragweed, plant extracts, and so on. The role of hereditary factors is also important. Despite the absence of a confirmed predisposition in any breed, it is believed that heredity in some way increases the predisposition to develop atopic dermatitis (Sousa & Marsella Veterinary Immunology and Immunopathology 2001; 81: 153-157; Schwartzman, et al. Clin. Exp. Immunol. 1971 ; 9: 549-569).

An estimated 10% of the entire dog population has atopic dermatitis (Marsella & Olivry 2003 supra; Scott, et al. Canadian Veterinary Journal 2002; 43: 601-603; Hillier Veterinary Immunology and Immunopathology 2001; 81: 147-151). Worldwide, approximately 4.5 million dogs are affected by this chronic and lifelong disease. The incidence of atopic dermatitis appears to be on the rise. A predisposition based on breed and sex has been suspected, but this may vary considerably by geographic area (Hillier, 2001 supra; Picco, et al. 2008 supra).

In cats, allergic dermatitis is an inflammatory skin condition accompanied by itching, which is believed to be due to an abnormal immune system response to substances that do not cause a reaction in healthy cats. The most common sign of allergic dermatitis in cats is chronic, recurrent itching. Common clinical manifestations of allergic dermatitis in cats include spontaneous alopecia, miliary dermatitis, eosinophilic granulomatous complexes (including plaques, granulomas, and indolent ulcers), and focal head and neck pruritus characterized by excoriations, erosions, and/or ulcers. Available data do not show a predisposition based on breed and sex, and younger cats seem to be more likely to develop the disease (Hobi et al. Vet Dermatol 2011 22: 406-413; Ravens et al. Vet Dermatol 2014; 25: 95- 102; Buckely In Practice 2017; 39: 242-254).

Currently, treatment options for cats diagnosed with allergic dermatitis depend on the severity of the clinical signs of the disease, its duration, and owner preferences, and include allergen-specific immunotherapy and antipruritic drugs such as glucocorticoids and cyclosporins (Buckley, supra). Immunotherapy is effective in some patients but requires frequent injections and may take 6-9 months to clinical improvement (Buckley, supra). Immunosuppressive drugs, such as glucocorticoids and cyclosporins, are usually effective, however, their long-term use often leads to undesirable effects.

In horses, atopic dermatitis has been considered as a possible cause of pruritus. A growing body of evidence points to the role of environmental allergens in atopic dermatitis in horses. The disease may be seasonal or non-seasonal, depending on the allergen(s) involved. Data on predisposition based on age, breed, and sex are scarce. In a preliminary study from the School of Veterinary Medicine, University of California, Davis (SVM-UCD), the median age of onset was 6.5 years, with Thoroughbred saddle horses being the most common breed, with 25% of the horses studied, and horses (usually geldings) were affected almost twice as often as mares; however, these data were obtained using only 24 horses and have not yet been compared with the general population of sick animals. The most common clinical sign of atopic dermatitis in horses is pruritus, usually localized to the muzzle, distal limbs, or trunk. Alopecia, erythema, urticaria and papules are possible. Urticaria can be very pronounced, but not accompanied by itching. Horses may have a family history of urticarial atopic dermatitis. Horses may suffer from secondary pyoderma, which is typically characterized by excessive scaling, small epidermal "corollas" or crusted papules ("miliary dermatitis"). The diagnosis of atopic dermatitis is based on clinical signs and the exclusion of other diagnoses, especially insect hypersensitivity (Culicoides) (White Clin Tech Equine Pract 2005; 4: 311-313; Fadok Vet Clin Equine 2013; 29 541-550). Currently, the treatment of atopic dermatitis in horses is carried out both symptomatically, suppressing the inflammation and itching provoked by an allergic response, and acting on a specific cause (that is, determining the allergens involved and making an allergen-specific vaccine). A symptomatic approach is usually necessary for short-term therapy in order to ensure patient comfort and minimize self-injury. This approach is based on a combination of topical and systemic therapies, including antihistamines, essential fatty acids, pentoxifylline, and glucocorticoids. The main approach to the control of allergy to environmental elements involves the identification of allergens that trigger the hypersensitivity reaction. Most dermatologists acknowledge that allergen-specific immunotherapy can be beneficial for atopic horses. However, most horses generally do not improve until after the first 6 months of immunotherapy (Marsella Vet Clin Equine 2013; 29: 551-557). In addition, long-term use of immunosuppressive drugs in horses can lead to undesirable effects.

Interleukin-31 (IL-31), a cytokine produced by type 2 T helper cells, has been shown to cause pruritus in humans, mice, and dogs (Bieber N Engl J Med 2008; 358: 1483-1494; Dillon et al Nat Immunol 2004; 5:752-60; U.S. Patent No. 8,790,651 to Bammert et at; Gonzalez et al Vet Dermatl 2013; 24(1): 48-53). IL-31 binds to a co-receptor consisting of the IL-31 receptor A (IL-31RA) and the oncostatin M receptor (OSMR) (Dillon et al. 2004 supra and Bilsborough et al. J Allergy Clin Immunol. 2006 117(2):418 -25). Activation of the receptors results in STAT phosphorylation via the JAK receptor(s). This co-receptor has been shown to be expressed in macrophages, keratinocytes, and dorsal root ganglia.

Recently, IL-31 has been found to be involved in dermatitis, pruritus, allergy, and airway hypersensitivity. Cytopoint®, a canine anti-IL-31 monoclonal antibody manufactured by Zoetis Inc. (Parsippany, NJ), reduces itching and skin lesions in dogs with atopic dermatitis (Gonzalez et al. 2013 supra, Michels et al. Vet Dermatol. 2016; Dec; 27(6): 478-e129). It would be desirable to provide other anti-IL-31 antibodies for the prevention and treatment of IL-31 mediated disorders in veterinary mammals. Given the current unmet need for safe and effective alternative treatment options for atopic and allergic dermatoses in cats and horses, it would be particularly desirable to provide feline and equine anti-IL-31 antibodies to reduce pruritus and skin lesions in cats and horses with atopic dermatitis.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to a region of a mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor, wherein the binding of said antibody to said region is affected by mutations in the binding region of the 15H05 epitope. selected from at least one of the following: (a) a region approximately between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO:157 (Feline_IL31_wildtype); (b) regions approximately between amino acid residues 124 and 135 of the canine IL-31 sequence shown in SEQ ID NO: 155 (Canine_IL31); and (c) regions approximately between amino acid residues 118 and 129 of the equine IL-31 sequence shown in SEQ ID NO: 165 (Equine_IL31).

In one embodiment, the above mutations in the 15H05 epitope binding region are selected from at least one of the following: (a) a mutant where positions 126 and 128 of SEQ ID NO: 157 are replaced with alanine; (b) a mutant where positions 126 and 128 of SEQ ID NO: 155 are replaced by alanine; and (c) a mutant where positions 120 and 122 of SEQ ID NO: 165 are changed to alanine.

In one embodiment, the monoclonal antibody of the present invention binds to the 15H05 epitope region. That is, in one embodiment, the present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to a region of a mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor, wherein the binding region is a 15H05 epitope binding region selected from at least one of the following: (a) regions approximately between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (Feline_IL31_wildtype); (b) regions approximately between amino acid residues 124 and 135 of the canine IL-31 sequence shown in SEQ ID NO: 155 (Canine_IL31); and (c) regions approximately between amino acid residues 118 and 129 of the equine IL-31 sequence shown in SEQ ID NO: 165 (Equine_IL31).

In one embodiment, the mammalian IL-31 to which the antibody or antigen-binding fragment specifically binds is feline IL-31, wherein the antibody binds to a region approximately between amino acid residues 125 and 134 of the feline IL-31 sequence shown in SEQ ID NO: 157 (Feline_IL31_wildtype). In some embodiments, an antibody that binds to a given region of feline IL-31 contains a VL chain containing changes in framework region 2 (FW2) selected from the following: asparagine in place of lysine at position 42, isoleucine in place of valine at position 43, valine in place of leucine at position 43 position 46, asparagine instead of lysine at position 49, and combinations thereof, where the positions correspond to SEQ ID NO: 127 (FEL_15H05_VL1).

In one embodiment, the mammalian IL-31 to which the antibody or antigen-binding fragment specifically binds is canine IL-31, wherein the antibody binds to a region between approximately amino acid residues 125 and 134 of the canine IL-31 sequence shown in SEQ ID NO: 155 (Canine_IL31).

In another embodiment, the mammalian IL-31 to which the antibody or antigen-binding fragment specifically binds is equine IL-31, wherein the antibody binds to a region approximately between amino acid residues 117 and 128 of the equine IL-31 sequence shown in SEQ ID NO: 165 (Equine_IL31).

In one embodiment, the monoclonal antibody, or antigen-binding fragment thereof, contains the following combinations of hypervariable region (CDR) sequences:

1) 15H05 antibody: heavy chain variable region (VH) CDR1 (VH-CDR1) SYTIH (SEQ ID NO: 1), VH-CDR2 NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 WGFKYDGEWSFDV (SEQ ID NO: 3) , light chain variable region (VL) CDR1 (VL-CDR1) RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 KASNLHI (SEQ ID NO: 5) and VL-CDR3 LQSQTYPLT (SEQ ID NO: 6); or

2) variant (1) differing from the original 15H05 antibody by the addition, deletion and/or substitution of one or more amino acid residues in at least one of CDR1, CDR2 or CDR3 VH or VL.

In one embodiment, the 15H05/1505 antibody variant contains a substitution at one or more of the following positions in the CDR: residue 4 (I) of SEQ ID NO: 1; residues 1-3 (NIN), 5-7 (TSG), 9-11 (TEN) and 13 (Q) SEQ ID NO: 2; residues 4 (K), 6(D) and 13 (V) of SEQ ID NO: 3 in CDR1, 2 and 3 of the heavy chain, respectively, and residues 3-7 (SQGIS) of SEQ ID NO: 4, residues 3 (S) and 5 (L) SEQ ID NO: 5 and residues 4(Q), 5 (T) and 9 (T) of SEQ ID NO: 6 in CDRL1, 2 and 3, respectively. In one embodiment, one or more of these substitutions are conservative amino acid substitutions.

In one embodiment, the 15H05 monoclonal antibody above contains at least one of the following:

a) a light chain variable region containing FEL_15H05_VL1_FW2:

b) heavy chain variable region containing FEL_15H05_VH1:

The present invention also provides a monoclonal antibody, or antigen-binding fragment thereof, comprising the following combinations of hypervariable region (CDR) sequences:

1) ZIL1 antibody: heavy chain variable region (VH) CDR1 (VH-CDR1) SYGMS (SEQ ID NO: 13), VH-CDR2 HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 VYTTLAAFWTDNFDY (SEQ ID NO: 15) , light chain variable region (VL) CDR1 (VL-CDR1) SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 SDGNRPS (SEQ ID NO: 17) and VL-CDR3 QSFDTTLDAYV (SEQ ID NO: 18);

2) ZIL8 antibody: VH-CDR1 DYAMS (SEQ ID NO: 19), VH-CDR2 GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 GFPGSFEH (SEQ ID NO: 21), VL-CDR1 TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 YNSDRPS (SEQ ID NO: 23), VL-CDR3 SVYDRTFNAV (SEQ ID NO: 24);

3) ZIL9 antibody: VH-CDR1 SYDMT (SEQ ID NO: 25), VH-CDR2 DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 RDTERPS (SEQ ID NO: 29), VL-CDR3 ESAVDTGTLV (SEQ ID NO: 30);

4) ZIL11 antibody: VH-CDR1 TYVMN (SEQ ID NO: 31), VH-CDR2 SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 SMVGPFDY (SEQ ID NO: 33), VL-CDR1 SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 KDTERPS (SEQ ID NO: 35), VL-CDR3 ESAVSSDTIV (SEQ ID NO: 36);

5) ZIL69 antibody: VH-CDR1 SYAMK (SEQ ID NO: 37), VH-CDR2 TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 GNAESGCTGDHCPPY (SEQ ID NO: 39), VL-CDR1 SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 KDTERPS (SEQ ID NO: 41), VL-CDR3 ESAVSSETNV (SEQ ID NO: 42);

6) ZIL94 antibody: VH-CDR1 TYFMS (SEQ ID NO: 43), VH-CDR2 LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 FWRAFND (SEQ ID NO: 45), VL-CDR1 GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 DTGSRPS (SEQ ID NO: 47), VL-CDR3 SLYTDSDILV (SEQ ID NO: 48);

7) ZIL154 antibody: VH-CDR1 DRGMS (SEQ ID NO: 49), VH-CDR2 YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 WDGSSFFDY (SEQ ID NO: 51), VL-CDR1 KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 KVSNRDP (SEQ ID NO: 53), VL-CDR3 MQAIHFPLT (SEQ ID NO: 54);

8) ZIL159 antibody: VH-CDR1 SYVMT (SEQ ID NO: 55), VH-CDR2 GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 GDIVATGTSY (SEQ ID NO: 57), VL-CDR1 SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 KDTERPS (SEQ ID NO: 59), VL-CDR3 KSAVSIDVGV (SEQ ID NO: 60);

9) ZIL171 antibody: VH-CDR1 TYVMN (SEQ ID NO: 61), VH-CDR2 SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 SMVGPFDY (SEQ ID NO: 63), VL-CDR1 SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 KDTERPS (SEQ ID NO: 65), VL-CDR3 ESAVSSDTIV (SEQ ID NO: 66);

10) 04H07 antibody: VH-CDR1 SYWMN (SEQ ID NO: 200), VH-CDR2 MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), VH-CDR3 QDIVTTVDY (SEQ ID NO: 202), VL-CDR1 KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 WASTRES (SEQ ID NO: 204), VL-CDR3 QQGYTYPFT (SEQ ID NO: 205);

11) Antibody 06A09: VH-CDR1 SYWMN (SEQ ID NO: 206), VH-CDR2 MIDPSDSETHYNQIFRD (SEQ ID NO: 207), VH-CDR3 QDIVTTVDY (SEQ ID NO: 208), VL-CDR1 KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 WASTRES (SEQ ID NO: 210), VL-CDR3 QQHYGYPFT (SEQ ID NO: 211); or

12) variant (1)-(11) that differs from the corresponding parent antibody ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more amino acids residue in at least one of the CDR1, CDR2 or CDR3 VH or VL.

In some embodiments of the present invention:

1) the ZIL1 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL1_VL:

b) heavy chain variable region containing CAN-ZIL1_VH:

2) the ZIL8 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL8_VL:

And

b) heavy chain variable region containing CAN-ZIL8_VH:

contains at least one of the following:

c) a light chain variable region containing ZTS_5864_VL:

d) heavy chain variable region containing ZTS_5864_VH:

contains at least one of the following:

e) light chain variable region containing ZTS 5865 VL:

f) heavy chain variable region containing ZTS 5865 VH:

3) the ZIL9 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL9_VL:

b) heavy chain variable region containing CAN-ZIL9_VH:

4) the ZIL11 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL11_VL:

b) heavy chain variable region containing CAN-ZIL11_VH:

5) the ZIL69 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL69_VL:

b) heavy chain variable region containing CAN-ZIL69_VH:

6) the ZIL94 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL94_VL:

b) heavy chain variable region containing CAN-ZIL94_VH:

7) the ZIL154 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL154_VL:

b) heavy chain variable region containing CAN-ZIL154_VH:

8) the ZIL159 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL159_VL:

b) heavy chain variable region containing CAN-ZIL159_VH:

9) the ZIL171 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL171_VL:

b) heavy chain variable region containing CAN-ZIL171VH:

10) the 04H07 antibody contains at least one of the following:

a) light chain variable region containing Mu_04H07_VL:

b) heavy chain variable region containing Mu_04H07_VH:

11) the 06A09 antibody contains at least one of the following:

a) light chain variable region containing Mu_06A09_VL:

b) heavy chain variable region containing Mu_06A09_VH:

In one embodiment, the monoclonal antibody or antigen-binding fragment thereof of the present invention attenuates, inhibits, or neutralizes an IL-31-mediated pruritic or allergic condition in a mammal. In one embodiment, such a mammal is selected from a dog, cat, or horse.

In some embodiments, the monoclonal antibody is a chimeric antibody. In other embodiments, the antibody is caninized, felinized, equinized, fully canine, fully feline, or fully equine.

The present invention also provides a veterinary composition comprising a therapeutically effective amount of at least one antibody, or antigen-binding portion thereof, as described above.

Also provided is a method for treating an IL-31 mediated disorder in a subject, comprising administering to the subject a therapeutically effective amount of at least one antibody, or antigen-binding portion thereof, described above.

In one embodiment, the IL-31 mediated disorder is an pruritic or allergic condition. In some embodiments, the pruritic or allergic condition is an pruritic condition selected from atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. In other embodiments, the pruritic or allergic condition is an allergic condition selected from the group consisting of allergic dermatitis, summer eczema, urticaria, sunburn, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease and inflammatory processes resulting from autoimmunity.

In other embodiments, the IL-31 mediated disorder is tumor progression. In some embodiments, the IL-31 mediated disorder is an eosinophilic disease or mastocytomas.

Furthermore, a method for inhibiting IL-31 activity in a mammal is provided, comprising administering to the mammal an antibody, or an antigen-binding portion thereof, as described above.

Also provided is an antibody, or an antigen-binding fragment thereof, as described above, for use in the treatment of a mammal with an IL-31 mediated disorder.

Also provided is the use of an antibody or antigen-binding fragment thereof, as described above, for the treatment of a mammal with an IL-31 mediated disorder.

Also provided is a method for detecting IL-31, which includes: incubating a sample containing IL-31 in the presence of an antibody or its antigen-binding portion as described above; and detecting an antibody associated with IL-31 in the sample. In one embodiment, the method further comprises quantifying IL-31 in the sample.

The present invention also provides a host cell that produces a monoclonal antibody, or an antigen-binding portion thereof, comprising at least one of the following combinations of hypervariable region (CDR) sequences:

1) 15H05 antibody: heavy chain variable region (VH) CDR1 (VH-CDR1) SYTIH (SEQ ID NO: 1), VH-CDR2 NINPTSGYTENNQRFKD (SEQ ID NO: 2), VH-CDR3 WGFKYDGEWSFDV (SEQ ID NO: 3) , light chain variable region (VL) CDR1 (VL-CDR1) RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 KASNLHI (SEQ ID NO: 5) and VL-CDR3 LQSQTYPLT (SEQ ID NO: 6);

2) ZIL1 antibody: heavy chain variable region (VH) CDR1 (VH-CDR1) SYGMS (SEQ ID NO: 13), VH-CDR2 HINSGGSSTYYADAVKG (SEQ ID NO: 14), VH-CDR3 VYTTLAAFWTDNFDY (SEQ ID NO: 15) , light chain variable region (VL) CDR1 (VL-CDR1) SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 SDGNRPS (SEQ ID NO: 17) and VL-CDR3 QSFDTTLDAYV (SEQ ID NO: 18);

3) ZIL8 antibody: VH-CDR1 DYAMS (SEQ ID NO: 19), VH-CDR2 GIDSVGSGTSYADAVKG (SEQ ID NO: 20), VH-CDR3 GFPGSFEH (SEQ ID NO: 21), VL-CDR1 TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 YNSDRPS (SEQ ID NO: 23), VL-CDR3 SVYDRTFNAV (SEQ ID NO: 24);

4) ZIL9 antibody: VH-CDR1 SYDMT (SEQ ID NO: 25), VH-CDR2 DVNSGGTGTAYAVAVKG (SEQ ID NO: 26), VH-CDR3 LGVRDGLSV (SEQ ID NO: 27), VL-CDR1 SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 RDTERPS (SEQ ID NO: 29), VL-CDR3 ESAVDTGTLV (SEQ ID NO: 30);

5) ZIL11 antibody: VH-CDR1 TYVMN (SEQ ID NO: 31), VH-CDR2 SINGGGSSPTYADAVRG (SEQ ID NO: 32), VH-CDR3 SMVGPFDY (SEQ ID NO: 33), VL-CDR1 SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 KDTERPS (SEQ ID NO: 35), VL-CDR3 ESAVSSDTIV (SEQ ID NO: 36);

6) ZIL69 antibody: VH-CDR1 SYAMK (SEQ ID NO: 37), VH-CDR2 TINNDGTRTGYADAVRG (SEQ ID NO: 38), VH-CDR3 GNAESGCTGDHCPPY (SEQ ID NO: 39), VL-CDR1 SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 KDTERPS (SEQ ID NO: 41), VL-CDR3 ESAVSSETNV (SEQ ID NO: 42);

7) ZIL94 antibody: VH-CDR1 TYFMS (SEQ ID NO: 43), VH-CDR2 LISSDGSGTYYADAVKG (SEQ ID NO: 44), VH-CDR3 FWRAFND (SEQ ID NO: 45), VL-CDR1 GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 DTGSRPS (SEQ ID NO: 47), VL-CDR3 SLYTDSDILV (SEQ ID NO: 48);

8) ZIL154 antibody: VH-CDR1 DRGMS (SEQ ID NO: 49), VH-CDR2 YIRYDGSRTDYADAVEG (SEQ ID NO: 50), VH-CDR3 WDGSSFFDY (SEQ ID NO: 51), VL-CDR1 KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 KVSNRDP (SEQ ID NO: 53), VL-CDR3 MQAIHFPLT (SEQ ID NO: 54);

9) ZIL159 antibody: VH-CDR1 SYVMT (SEQ ID NO: 55), VH-CDR2 GINSEGSRTAYADAVKG (SEQ ID NO: 56), VH-CDR3 GDIVATGTSY (SEQ ID NO: 57), VL-CDR1 SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 KDTERPS (SEQ ID NO: 59), VL-CDR3 KSAVSIDVGV (SEQ ID NO: 60);

10) ZIL171 antibody: VH-CDR1 TYVMN (SEQ ID NO: 61), VH-CDR2 SINGGGSSPTYADAVRG (SEQ ID NO: 62), VH-CDR3 SMVGPFDY (SEQ ID NO: 63), VL-CDR1 SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 KDTERPS (SEQ ID NO: 65), VL-CDR3 ESAVSSDTIV (SEQ ID NO: 66);

11) 04H07 antibody: VH-CDR1 SYWMN (SEQ ID NO: 200), VH-CDR2 MIDPSDSEIHYNQVFKD (SEQ ID NO: 201), VH-CDR3 QDIVTTVDY (SEQ ID NO: 202), VL-CDR1 KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 WASTRES (SEQ ID NO: 204), VL-CDR3 QQGYTYPFT (SEQ ID NO: 205);

12) Antibody 06A09: VH-CDR1 SYWMN (SEQ ID NO: 206), VH-CDR2 MIDPSDSETHYNQIFRD (SEQ ID NO: 207), VH-CDR3 QDIVTTVDY (SEQ ID NO: 208), VL-CDR1 KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 WASTRES (SEQ ID NO: 210), VL-CDR3 QQHYGYPFT (SEQ ID NO: 211); or

13) variant (1)-(12) that differs from the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, or 06A09 by the addition, deletion, and/or substitution of one or more one amino acid residue in at least one of CDR1, CDR2 or CDR3 VH or VL.

Also provided is a method for producing an antibody comprising culturing a host cell as described above under conditions leading to antibody production and isolating the antibody from the host cell or host cell culture medium.

The isolated nucleic acids of the present invention are described below. Such nucleic acids may comprise a nucleic acid sequence encoding the variable heavy or variable light chain CDR sequences described above. Alternatively, the isolated nucleic acid of the present invention may comprise a nucleic acid sequence encoding both the heavy and light chain variable region CDRs.

In one embodiment, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the following combinations of heavy chain variable region hypervariable region (CDR) sequences:

1) 15H05: heavy chain variable region (VH) CDR1 (VH-CDR1) SYTIH (SEQ ID NO: 1), VH-CDR2 NINPTSGYTENNQRFKD (SEQ ID NO: 2) and VH-CDR3 WGFKYDGEWSFDV (SEQ ID NO: 3);

2) ZIL1: VH-CDR1 SYGMS (SEQ ID NO: 13), VH-CDR2 HINSGGSSTYYADAVKG (SEQ ID NO: 14) and VH-CDR3 VYTTLAAFWTDNFDY (SEQ ID NO: 15);

3) ZIL8: VH-CDR1 DYAMS (SEQ ID NO: 19), VH-CDR2 GIDSVGSGTSYADAVKG (SEQ ID NO: 20) and VH-CDR3 GFPGSFEH (SEQ ID NO: 21);

4) ZIL9: VH-CDR1 SYDMT (SEQ ID NO: 25), VH-CDR2 DVNSGGTGTAYAVAVKG (SEQ ID NO: 26) and VH-CDR3 LGVRDGLSV (SEQ ID NO: 27);

5) ZIL11: VH-CDR1 TYVMN (SEQ ID NO: 31), VH-CDR2 SINGGGSSPTYADAVRG (SEQ ID NO: 32) and VH-CDR3 SMVGPFDY (SEQ ID NO: 33);

6) ZIL69: VH-CDR1 SYAMK (SEQ ID NO: 37), VH-CDR2 TINNDGTRTGYADAVRG (SEQ ID NO: 38) and VH-CDR3 GNAESGCTGDHCPPY (SEQ ID NO: 39);

7) ZIL94: VH-CDR1 TYFMS (SEQ ID NO: 43), VH-CDR2 LISSDGSGTYYADAVKG (SEQ ID NO: 44) and VH-CDR3 FWRAFND (SEQ ID NO: 45);

8) ZIL154: VH-CDR1 DRGMS (SEQ ID NO: 49), VH-CDR2 YIRYDGSRTDYADAVEG (SEQ ID NO: 50) and VH-CDR3 WDGSSFDY (SEQ ID NO: 51);

9) ZIL159: VH-CDR1 SYVMT (SEQ ID NO: 55), VH-CDR2 GINSEGSRTAYADAVKG (SEQ ID NO: 56) and VH-CDR3 GDIVATGTSY (SEQ ID NO: 57);

10) ZIL171: VH-CDR1 TYVMN (SEQ ID NO: 61), VH-CDR2 SINGGGSSPTYADAVRG (SEQ ID NO: 62) and VH-CDR3 SMVGPFDY (SEQ ID NO: 63);

11) 04H07: VH-CDR1 SYWMN (SEQ ID NO: 200), VH-CDR2 MIDPSDSEIHYNQVFKD (SEQ ID NO: 201) and VH-CDR3 QDIVTTVDY (SEQ ID NO: 202);

12) 06A09: VH-CDR1 SYWMN (SEQ ID NO: 206), VH-CDR2 MIDPSDSETHYNQIFRD (SEQ ID NO: 207) and VH-CDR3 QDIVTTVDY (SEQ ID NO: 208); or

13) variant (1)-(12) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more than one amino acid residue in at least one of the CDR1, CDR2 or CDR3 VH.

In one embodiment, the isolated nucleic acid described above may further comprise a nucleic acid sequence encoding at least one of the following combinations of light chain variable region hypervariable region (CDR) sequences:

1) 15H05: light chain variable region (VL) CDR1 (VL-CDR1) RASQGISIWLS (SEQ ID NO: 4), VL-CDR2 KASNLHI (SEQ ID NO: 5) and VL-CDR3 LQSQTYPLT (SEQ ID NO: 6);

2) ZIL1: VL-CDR1 SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 SDGNRPS (SEQ ID NO: 17) and VL-CDR3 QSFDTTLDAYV (SEQ ID NO: 18);

3) ZIL8: VL-CDR1 TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 YNSDRPS (SEQ ID NO: 23) and VL-CDR3 SVYDRTFNAV (SEQ ID NO: 24);

4) ZIL9: VL-CDR1 SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 RDTERPS (SEQ ID NO: 29) and VL-CDR3 ESAVDTGTLV (SEQ ID NO: 30);

5) ZIL11: VL-CDR1 SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 KDTERPS (SEQ ID NO: 35) and VL-CDR3 ESAVSSDTIV (SEQ ID NO: 36);

6) ZIL69: VL-CDR1 SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 KDTERPS (SEQ ID NO: 41) and VL-CDR3 ESAVSSETNV (SEQ ID NO: 42);

7) ZIL94: VL-CDR1 GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 DTGSRPS (SEQ ID NO: 47) and VL-CDR3 SLYTDSDILV (SEQ ID NO: 48);

8) ZIL154: VL-CDR1 KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 KVSNRDP (SEQ ID NO: 53) and VL-CDR3 MQAIHPLT (SEQ ID NO: 54);

9) ZIL159: VL-CDR1 SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 KDTERPS (SEQ ID NO: 59) and VL-CDR3 KSAVSIDVGV (SEQ ID NO: 60);

10) ZIL171: VL-CDR1 SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 KDTERPS (SEQ ID NO: 65) and VL-CDR3 ESAVSSDTIV (SEQ ID NO: 66);

11) 04H07: VL-CDR1 KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 WASTRES (SEQ ID NO: 204), VL-CDR3 QQGYTYPFT (SEQ ID NO: 205);

12) 06A09: VL-CDR1 KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 WASTRES (SEQ ID NO: 210), VL-CDR3 QQHYGYPFT (SEQ ID NO: 211); or

13) variant (1)-(12) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more than one amino acid residue in at least one of the CDR1, CDR2 or CDR3 VH or VL.

In one embodiment, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the following combinations of light chain variable region hypervariable region (CDR) sequences:

1) 15H05: Light chain variable region (VL) CDR1 (VL-CDR1) RASQGISIWLS (SEQIDNO:4), VL-CDR2 KASNLHI (SEQ ID NO: 5) and VL-CDR3 LQSQTYPLT (SEQ ID NO: 6);

2) ZIL1: VL-CDR1 SGSTNNIGILAAT (SEQ ID NO: 16), VL-CDR2 SDGNRPS (SEQ ID NO: 17) and VL-CDR3 QSFDTTLDAYV (SEQ ID NO: 18);

3) ZIL8: VL-CDR1 TGSSSNIGSGYVG (SEQ ID NO: 22), VL-CDR2 YNSDRPS (SEQ ID NO: 23) and VL-CDR3 SVYDRTFNAV (SEQ ID NO: 24);

4) ZIL9: VL-CDR1 SGESLNEYYTQ (SEQ ID NO: 28), VL-CDR2 RDTERPS (SEQ ID NO: 29) and VL-CDR3 ESAVDTGTLV (SEQ ID NO: 30);

5) ZIL11: VL-CDR1 SGESLSNYYAQ (SEQ ID NO: 34), VL-CDR2 KDTERPS (SEQ ID NO: 35) and VL-CDR3 ESAVSSDTIV (SEQ ID NO: 36);

6) ZIL69: VL-CDR1 SGESLNKYYAQ (SEQ ID NO: 40), VL-CDR2 KDTERPS (SEQ ID NO: 41) and VL-CDR3 ESAVSSETNV (SEQ ID NO: 42);

7) ZIL94: VL-CDR1 GLNSGSVSTSNYPG (SEQ ID NO: 46), VL-CDR2 DTGSRPS (SEQ ID NO: 47) and VL-CDR3 SLYTDSDILV (SEQ ID NO: 48);

8) ZIL154: VL-CDR1 KASQSLLHSDGNTYLD (SEQ ID NO: 52), VL-CDR2 KVSNRDP (SEQ ID NO: 53) and VL-CDR3 MQAIHPLT (SEQ ID NO: 54);

9) ZIL159: VL-CDR1 SGETLNRFYTQ (SEQ ID NO: 58), VL-CDR2 KDTERPS (SEQ ID NO: 59) and VL-CDR3 KSAVSIDVGV (SEQ ID NO: 60);

10) ZIL171: VL-CDR1 SGKSLSYYYAQ (SEQ ID NO: 64), VL-CDR2 KDTERPS (SEQ ID NO: 65) and VL-CDR3 ESAVSSDTIV (SEQ ID NO: 66);

11) 04H07: VL-CDR1 KSSQSLLYSINQKNHLA (SEQ ID NO: 203), VL-CDR2 WASTRES (SEQ ID NO: 204) and VL-CDR3 QQGYTYPFT (SEQ ID NO: 205);

12) 06A09: VL-CDR1 KSSQSLLYSINQKNFLA (SEQ ID NO: 209), VL-CDR2 WASTRES (SEQ ID NO: 210) and VL-CDR3 QQHYGYPFT (SEQ ID NO: 211); or

13) variant (1)-(12) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more than one amino acid residue in at least one of CDR1, CDR2 or CDR3 VL.

In addition, according to the present invention, a vector containing at least one of the nucleic acids described above is provided.

The present invention also provides a method for improving the uniformity and/or characteristics of a feline antibody, comprising: expressing a nucleotide sequence encoding a feline IgG kappa light chain and a nucleotide sequence encoding a feline IgG heavy chain in a host cell to obtain a feline antibody, wherein the nucleotide sequence a feline IgG kappa light chain coding sequence comprises a kappa light chain constant region nucleotide sequence in which the sequence encoding the C-terminal QRE sequence otherwise present in the wild-type feline IgG kappa light chain constant region has been modified and/or deleted. Such modifications may include modifications to the nucleotide sequence resulting, for example, in deletions, substitutions, or additions of one or more amino acids at the C-terminus.

In one embodiment, a method for improving the uniformity and/or characteristics of a feline antibody comprises:

a) providing a nucleotide sequence encoding a wild-type feline IgG antibody kappa light chain constant region containing the C-terminal amino acid sequence of QRE;

b) deleting and/or modifying the sequence encoding the C-terminal QRE in the nucleotide sequence provided in (a) to obtain an altered nucleotide sequence of the kappa light chain constant region;

c) combining the altered nucleotide sequence of the kappa light chain constant region obtained in (b) with a nucleotide sequence encoding a feline IgG kappa light chain variable region to obtain a nucleotide sequence encoding a complete feline IgG kappa light chain; And

d) expressing the nucleotide sequence encoding the complete feline IgG kappa light chain obtained in (c) and the nucleotide sequence encoding the feline IgG heavy chain in a host cell to produce a feline antibody in which the C-terminal QRE sequence is otherwise wild-type feline IgG kappa present in the light chain constant region has been modified and/or deleted.

In one embodiment, improving the homogeneity and/or characteristics of the feline antibody comprises reducing free IgG kappa light chain levels and thereby increasing the percentage of intact feline IgG antibody monomer.

In one embodiment, the nucleotide sequence encoding the feline IgG kappa light chain and the nucleotide sequence encoding the feline IgG heavy chain are present in the same vector used to transform the host cell. In another embodiment, the nucleotide sequence encoding the feline IgG kappa light chain and the nucleotide sequence encoding the feline IgG heavy chain are present in separate vectors used to transform the host cell.

In one embodiment of a method for improving the uniformity and/or characteristics of a feline antibody, the feline antibody specifically binds to a target involved in a cytokine and/or growth factor mediated disorder. In one specific embodiment, the feline antibody specifically binds to feline IL-31 or feline NGF.

In one embodiment, the feline antibody comprises a kappa light chain constant region having the sequence:

(SEQ ID NO: 186) или ее вариант. Такие варианты могут включать, например, добавление или модификацию одного или более чем одного аминокислотного остатка на С-конце SEQ ID NO: 186.

(SEQ ID NO: 186) or a variant thereof. Such options may include, for example, the addition or modification of one or more amino acid residues at the C-terminus of SEQ ID NO: 186.

In addition, the present invention provides a method for improving the homogeneity and/or characteristics of a canine antibody, comprising: expressing a nucleotide sequence encoding a canine IgG kappa light chain and a nucleotide sequence encoding a canine IgG heavy chain in a host cell to obtain a canine antibody, wherein the nucleotide sequence encoding the canine IgG kappa light chain comprises a kappa light chain constant region nucleotide sequence in which the sequence encoding the C-terminal QRVD sequence otherwise present in the wild-type canine IgG kappa light chain constant region (Canine LC Kappa wt, SEQ ID NO: 194) has been modified and/or removed. Such modifications may include modifications to the nucleotide sequence resulting, for example, in deletions, substitutions, or additions of one or more amino acids at the C-terminus.

In one embodiment, a method for improving the uniformity and/or characteristics of a canine antibody comprises:

a) providing a nucleotide sequence encoding a wild-type canine IgG kappa light chain constant region containing the C-terminal QRVD amino acid sequence;

b) removing and/or modifying the sequence encoding the C-terminal QRVD in the nucleotide sequence provided in (a) to obtain an altered nucleotide sequence of the kappa light chain constant region;

c) combining the altered nucleotide sequence of the kappa light chain constant region obtained in (b) with a nucleotide sequence encoding a canine IgG kappa light chain variable region to obtain a nucleotide sequence encoding a complete canine IgG kappa light chain; And

d) expressing the nucleotide sequence encoding the complete canine IgG kappa light chain obtained in (c) and the nucleotide sequence encoding the canine IgG heavy chain in a host cell to produce a canine antibody in which the C-terminal QRVD sequence is otherwise wild-type canine IgG kappa present in the light chain constant region has been modified and/or removed.

In one embodiment, improving the uniformity and/or characteristics of a canine antibody comprises reducing free IgG kappa light chain levels and thereby increasing the percentage of intact canine IgG antibody monomer.

In one embodiment, the nucleotide sequence encoding the canine IgG kappa light chain and the nucleotide sequence encoding the canine IgG heavy chain are present in the same vector used to transform the host cell. In another embodiment, the nucleotide sequence encoding the canine IgG kappa light chain and the nucleotide sequence encoding the canine IgG heavy chain are present in separate vectors used to transform the host cell.

In one embodiment of a method for improving the uniformity and/or characteristics of a canine antibody, the canine antibody specifically binds to a target involved in a cytokine and/or growth factor mediated disorder. In one specific embodiment, the canine antibody specifically binds to canine IL-31.

In one embodiment of a method for improving the uniformity and/or characteristics of a canine antibody, the canine antibody comprises a kappa light chain constant region having the sequence:

(SEQ ID NO: 179) или ее вариант. Такие варианты могут включать, например, добавление или модификацию одного или более чем одного аминокислотного остатка на С-конце SEQ ID NO: 179.

(SEQ ID NO: 179) or a variant thereof. Such options may include, for example, the addition or modification of one or more amino acid residues at the C-terminus of SEQ ID NO: 179.

The modified kappa light chain constant regions described herein may be used in combination with any number of feline and canine antibodies, including, for example, without limitation, any of the canine or feline antibodies described in the specification and claims of this invention. It is contemplated that canine and feline antibodies having targets other than IL-31 can also be suitably combined with the altered kappa light chain constant regions disclosed herein. The present invention includes any feline or canine antibody containing such altered kappa light chain constant regions disclosed herein because, based on the description and claims of the present invention, such antibodies would be expected to have improved uniformity and/or performance.

BRIEF DESCRIPTION OF GRAPHICS

On FIG. 1 is an alignment showing the conservation of the amino acid sequence of IL-31 from different species. In particular, a comparison of SEQ ID NO: 155 (canine IL-31), SEQ ID NO: 157 (feline IL-31), SEQ ID NO: 165 (equine IL-31) and SEQ ID NO: 181 (human IL-31) is shown. 31). Also indicated are the percentage amino acid sequence identity of canine, feline, equine and human IL-31.

On FIG. 2 details the affinity with which each of the murine CDR candidate antibodies bind to feline and canine IL-31 using surface plasmon resonance (SPR) and the Biacore system (Biacore Life Sciences (GE Healthcare), Uppsala, Sweden).

On FIG. 3 is a table showing the activity (IC ₅₀ (μg/mL)) of mouse CDR candidate antibodies measured in canine and feline cell assays. In particular, the ability of candidate antibodies to inhibit IL-31 mediated STAT phosphorylation in canine (DH-82) or feline (FCWF4) macrophage-like cells was evaluated.

On FIG. 4 shows the results obtained with canine CDR candidate monoclonal antibodies binding to various proteins using both the indirect ELISA method and the Biacore method. In the case of indirect ELISA, binding (OD in ELISA) to wild-type feline IL-31 and feline IL-31 15H05 mutant having mutations in the epitope region of the monoclonal antibody 15H05 was evaluated. To confirm binding, a Biacore assay was performed using surfaces with canine, feline, equine, human IL-31, feline IL-31 mutant 15H05, and feline IL-31 11E12 mutant proteins at a single antibody concentration analyzed. The feline IL-31 mutant 11E 12 had mutations in the epitope region of the monoclonal antibody 11E12.

Fig. 5. In FIG. 5A shows alignment with the VL sequence of the mouse antibody 11E12 (SEQ ID NO: 73) comparing the sequences of the previously disclosed caninized 11E12, designated as Can_11E12_VL_cUn_1 (SEQ ID NO: 182) and CAN_11E12_VL_cUn_FW2 (SEQ ID NO: 184), with the felinized variants, designated as FEL_11E12_VL1 (SEQ ID NO: 113) and FEL_11E12_VL1_FW2 (SEQ ID NO: 117). Under alignment in Fig. 5A dots are marked showing the positions of important changes in Fel_11E12_VL1 that were required to restore the affinity of this antibody for the IL-31 protein. On FIG. 5B shows the alignment of the VL sequence of the mouse antibody 15H05, here designated MU_15H05_VL (SEQ ID NO: 69), with the VL sequences of felinized 15H05, here designated FE1_15H05_VL1 (SEQ ID NO: 127) and FEl_15H05_VL FW2 (SEQ ID NO: 135). Points under alignment in Fig. 5B indicate the necessary changes in the VL of felinized 15H05 (Fel_15H05_VL1) that were needed not only to restore, but also to improve its affinity for canine and feline IL-31 compared to murine and chimeric forms of this antibody.

Fig. 6. In FIG. 6A shows an alignment of wild-type feline IL-31 (SEQ ID NO: 157) with the 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161) mutants, highlighting the positions of the alanine substitutions. On FIG. 6B shows a homologous model of feline IL-31 highlighting the positions of two amino acids involved in binding to antibodies 11E12 (site 1) and 15H05 (site 2). On FIG. 6C is a graph showing the results obtained for the binding of monoclonal antibodies 11E12 and 15H05 to wild-type feline IL-31 and IL-31 mutant proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161) using wild type and these mutants as adsorbed antigens.

On FIG. 7 is a graph showing competitive binding assays of mAbs 15H05 and 11E12 using Biacore. On FIG. 7A shows competition binding of mouse antibodies 15H05 and 11E12 to canine IL-31. On FIG. 7B shows competition binding of 15H05 and 11E12 antibodies to feline IL-31 surface.

On FIG. 8 is a graph showing the results obtained for the binding of individual OSMR and IL-31Ra receptor subunits to wild-type feline IL-31 and mutant IL-31 proteins 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161 ) when using the wild type and these mutants as adsorbed antigens.

On FIG. 9 is a graph showing the preliminary efficacy of chimeric mouse-feline 11E12, chimeric mouse-feline 15H05, and felinized 11E12 (Feline 11E12 1.1) in an IL-31 induced itch model in cats.

On FIG. 10 is a graph showing the in vivo performance evaluation of the felinized anti-IL-31 antibody 15H05, named ZTS-361, in a feline itch provocation model. On FIG. 10A shows baseline behavioral signs of pruritus before challenge in the T01 placebo vehicle and T02 antibody ZTS-361 groups from day -7 to day 28, where day zero is the day of antibody administration in the T02 group. On FIG. 10B shows the efficacy of the ZTS-361 antibody demonstrating a significant reduction in pruritus observed on days 7 (p less than 0.0001), 21 days (p less than 0.0027), and 28 days (p less than 0.0238) after challenge with IL- 31 compared to vehicle control placebo.

Fig. 11. In FIG. 11A is a graph showing plasma levels of IL-31 in client dogs with atopic and allergic dermatitis compared to normal laboratory animals. On FIG. 11B is a graph showing the results of a recent study to determine serum levels of IL-31 in cats with a presumptive diagnosis of allergic dermatitis (AD) from several different geographic areas of the United States. On FIG. 11C is a graph showing the pharmacokinetic profile of canine IL-31 in dogs following subcutaneous administration at a dose of 1.75 μg/kg.

On FIG. 12 shows a non-reducing 4-12% SDS-PAGE comparing lane 1 showing ZTS-361 whose heavy chain is (SEQ ID NO:121; FEL_15H05_VH1) fused to a feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1). The ZTS-361 light chain is (SEQ ID NO:135; FEL-15H05-VL1 FW2) combined with an IgG light chain constant region (SEQ ID NO: 175; Feline LC Kappa G minus). Lane 2 shows mouse 15H05 whose heavy chain is (SEQ ID NO: 67; MU_15H05_VH) fused to a mouse IgG heavy chain constant region (SEQ ID NO: 188; Mouse_HC_IgG1). The mouse 15H05 light chain is (SEQ ID NO: 69; MU_15H05_VL) fused to a mouse IgG light chain constant region (SEQ ID NO: 190; Mouse_LC_Kappa). Intact refers to IgG with two heavy chains and two light chains held together by interchain disulfide bonds and an estimated molecular weight of approximately 150 kDa. HHL refers to "Heavy Heavy Light (heavy heavy light)" and is an IgG without one light chain with an estimated molecular weight of approximately 125 kDa. HH refers to "Heavy Heavy (heavy heavy)" and is an IgG without both light chains with an estimated molecular weight of approximately 100 kDa. HL refers to "Heavy Light (heavy light)" and is an IgG with one heavy chain, one light chain and an estimated molecular weight of approximately 75 kDa. L refers to "Light (light)" and is an IgG with a single light chain and an estimated molecular weight of approximately 25 kDa, also referred to here as a free light chain.

Fig. 13. In FIG. 13A shows the results of non-reducing capillary gel electrophoresis (NR-CGE) comparing IgG from stable cell lines expressing ZTS-361 or mouse 15H05 (each described above). The percentage of monomer and minor species was calculated from experimental readings obtained using NR-CGE and shown as electropherograms in FIG. 13V. The time corrected area (TCA) is defined as the ratio of the area of an individual peak as measured by the instrument to the time at which that peak emerges. Total TCA is defined as the sum of the TCA of all peaks greater than or equal to 0.3%. The percentage of intact IgG monomer (% Monomer) and individual fragments (%HHL and %L) is calculated from their individual TCA as a percentage of the total TCA. The percentage of fragments (% Fragments) is the sum of the areas of all peaks, the molecular weight of which is less than that of intact IgG.

Fig. 14. In FIG. 14A shows a non-reducing 4-12% SDS-PAGE comparing IgG from individual stable ZTS-361 CHO clones (described above and in Section 1.9 of the Examples). Lanes 1 and 8 provide a standard IgG sample for comparison. The percentage of monomer is calculated by densitometric analysis of each band moving according to an estimated molecular weight of approximately 150 kDa using BioRad VersaDoc software. The percentage of fragments (% Fragments) is the sum of the individual bands with lower molecular weight. On FIG. 14B shows the results of non-reducing capillary gel electrophoresis (NR-CGE) comparing IgG from individual stable ZTS-361 CHO clones. The time corrected area (TCA) is defined as the ratio of the area of an individual peak as measured by the instrument to the time at which that peak emerges. Total TCA is defined as the sum of the TCA of all peaks greater than or equal to 0.3%. The percentage of intact IgG monomer (% Monomer) and the percentage of fragments (% Fragments) are calculated from their individual TCA as a percentage of the total TCA. The percentage of fragments (% Fragments) is the sum of the areas of all peaks, the molecular weight of which is less than that of intact IgG.

On FIG. 15 shows the C-terminal amino acids of the Ig kappa light chain constant region protein of the indicated species. In Canine LC kappa wt, the C-terminal amino acid residues shown are positions 103-109 of SEQ ID NO: 194 and the nucleotide residues shown are positions 307-330 of SEQ ID NO: 195; in Feline LC kappa G minus (G-), the C-terminal amino acid residues shown are positions 105-110 of SEQ ID NO: 175 and the nucleotide residues shown are positions 313-330 of SEQ ID NO: 176; in Pig LC kappa, the C-terminal amino acid residues shown are positions 104-108 of SEQ ID NO: 196 and the nucleotide residues shown are positions 310-327 of SEQ ID NO: 197; in Mink LC kappa, the C-terminal amino acid residues shown are positions 105-108 of SEQ ID NO: 198 and the nucleotide residues shown are positions 313-327 of SEQ ID NO: 199; in Human LC kappa, the C-terminal amino acid residues shown are positions 102-106 of SEQ ID NO: 192 and the nucleotide residues shown are positions 304-321 of SEQ ID NO: 193; in Mouse LC kappa, the C-terminal amino acid residues shown are positions 102-106 of SEQ ID NO: 190 and the nucleotide residues shown are positions 304-321 of SEQ ID NO: 191. The gray box shows the position of the C-terminal cysteine forming an interchain disulfide association with an IgG heavy chain constant region required for an intact antibody. The gray triangle indicates an increase in the percentage of kappa light chain utilization in IgG of the indicated species, i.e. dog (Canine), cat (Feline) and pig (Pig) (Aran et al. 1996 Zentralbl Veterinarmed. Nov; 43(9):573-6) , mink (Mink) (Bovkun et al. 1993 Eur J Immunol. Aug; 23(8): 1929-34), mice (Mouse) (Woloschak et al. 1987 Mol Immunol. Jul; 24(7):751-7 ) and Human (Barandun et al. 1976 Blood. Jan; 47(1):79-89). Nucleotides shown in light gray highlight codons in which a single nucleotide substitution results in a stop codon.

Fig. 16. In FIG. 16A is a pictographic representation of feline IgG highlighting the relative positions of putative interchain and intrachain disulfide bonds. The CYS15 amino acid residue shown is position 15 of Feline HC AlleleA wt (SEQ ID NO: 171) and Feline HC AlleleA 1 (SEQ ID NO: 173) and the nucleotide residues shown are positions 43-45 of Feline HC AlleleA wt (SEQ ID NO: 172) and 43-45 Feline HC AlleleA 1 (SEQ ID NO: 174), respectively. The CYS107 amino acid residue shown is position 107 of Feline LC Kappa G minus (SEQ ID NO: 175), and the nucleotide residues shown are positions 319-321 of Feline LC Kappa G minus (SEQ ID NO: 176). On FIG. 16B is a homologous model of ZTS-361 highlighting the positions of CYS15 and CYS107 described above. On FIG. 16C is an enlarged view of the circled area in FIG. 16B, with re-isolation of two cysteine positions allowing for interchain pairing of feline heavy and light chains. Reticulated shells shown are the calculated electrostatic contribution of kappa QRE light chain constant region residues immediately following CYS107 shown in Feline LC kappa G- in FIG. 15.

Fig. 17. In FIG. 17A shows the sequence identification numbers corresponding to the heavy and light chains used to create stable CHO cell lines producing antibodies ZTS-361 and ZTS-1505 (described above and/or in Section 1.9 of the Examples section). Feline LC Kappa G minus QRE minus (SEQ ID NO: 186) was isolated, which corresponds to the nucleotide sequence of Feline LC Kappa G minus QRE minus (SEQ ID NO: 187). On FIG. 17B shows the results of non-reducing capillary gel electrophoresis (NR-CGE) comparing IgG from individual stable ZTS-1505 CHO clones. The time corrected area (TCA) is defined as the ratio of the area of an individual peak as measured by the instrument to the time at which that peak emerges. Total TCA is defined as the sum of the TCA of all peaks greater than or equal to 0.3%. The percentage of intact IgG monomer (% Monomer) and the percentage of fragments (% Fragments) are calculated from their individual TCA as a percentage of the total TCA. The percentage of fragments (% Fragments) is the sum of the areas of all peaks, the molecular weight of which is less than that of intact IgG. On FIG. 17C shows a comparison of one stable CHO clone producing the ZTS-361 antibody with one stable CHO clone producing ZTS-1505. Comparison of two stable clones was performed under 8 independent variants of cultivation conditions, designated as A-G. The percentage of variability of each culture after 14 days of cultivation is indicated. The titer indicates the amount of antibody produced after 14 days of culture by each stable clone under the appropriate culture conditions. The percentage of monomer calculated from the results of NR-CGE for each clone grown using different culture conditions is indicated.

Fig. 18. In FIG. 18A shows percent identity when comparing the variable regions of anti-feline NGF and anti-IL-31 antibodies calculated using ClustalW software. On FIG. 18B and 18C show the alignment of the variable heavy and light chains, respectively, of anti-feline IL-31 and NGF antibodies, where the CDRs are boxed.

On FIG. 19 shows NR-CGE results comparing anti-feline IL-31 and anti-feline NGF antibodies with and without kappa constant chain C-terminal modification.

On FIG. 20 shows sequence alignment of feline and equine IL-31 using ClustalW.

On FIG. 21 shows the alignment of the variable regions of the heavy (FIG. 21A) and light (FIG. 21B) chains of antibodies 04H07 and 06A09 compared to mouse antibody 15H05 using ClustalW. For comparison, the location of each of the six CDRs is framed.

On FIG. 22 is a Biacore sensorgram showing the mean plus/minus 3 SD profile of the anti-IL-31 antibody ZTS-1505 used to determine the threshold response for screening for alanine CDR mutants.

On FIG. 23 shows the results of mutagenesis using alanine substitutions in the CDR of the heavy chain of the ZTS-1505 antibody for binding to IL-31 and inhibition of IL-31 mediated pSTAT signals, compared with the wild-type antibody.

On FIG. 24 shows the results of mutagenesis using alanine substitutions in the CDR of the light chain of the ZTS-1505 antibody for binding to IL-31 and inhibition of IL-31 mediated pSTAT signals compared to the wild-type antibody.

Fig. 25. In FIG. 25A and 25B show the binding affinity of two felinized antibodies, herein designated ZTS-5864 and ZTS-5865, and their activity against cells, respectively.

On FIG. 26 is a graph showing the results of an in vivo evaluation of the efficacy of the felinized anti-IL-31 antibody ZTS-5864 in a feline pruritus model.

The antibodies described in Fig. 12, 13, and 14 were obtained under culture conditions equivalent to culture conditions "A" in FIG. 17C.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is the heavy chain variable region CDR1, referred to herein as MU_15H05_VH_CDR1;

SEQ ID NO: 2 is the heavy chain variable region CDR2, referred to herein as MU_15H05_VH_CDR2;

SEQ ID NO: 3 is the heavy chain variable region CDR3, referred to herein as MU_15H05_VH_CDR3;

SEQ ID NO: 4 is the light chain variable region CDR1, referred to herein as MU_15H05_VL_CDR1;

SEQ ID NO: 5 is the light chain variable region CDR2, referred to herein as MU_15H05_VL_CDR2;

SEQ ID NO: 6 is the light chain variable region CDR3, referred to herein as MU_15H05_VL_CDR3;

SEQ ID NO: 7 is the heavy chain variable region CDR1, referred to herein as 11E12-VH-CDR1;

SEQ ID NO: 8 is the heavy chain variable region CDR2, referred to herein as 11E12-VH-CDR2;

SEQ ID NO: 9 is the heavy chain variable region CDR3, referred to herein as 11E12-VH-CDR3;

SEQ ID NO: 10 is the light chain variable region CDR1, referred to herein as 11E12-VL-CDR1;

SEQ ID NO: 11 is the light chain variable region CDR2, referred to herein as 11E12-VL-CDR2;

SEQ ID NO: 12 is the light chain variable region CDR3, referred to herein as 11E12-VL-CDR3;

SEQ ID NO: 13 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL1_VH_CDR1;

SEQ ID NO: 14 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL1_VH_CDR2;

SEQ ID NO: 15 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL1_VH_CDR3;

SEQ ID NO: 16 is the light chain variable region CDR1, referred to herein as CAN_ZIL1_VL_CDR1;

SEQ ID NO: 17 is the light chain variable region CDR2, referred to herein as CAN_ZIL1_VL_CDR2;

SEQ ID NO: 18 is the light chain variable region CDR3, referred to herein as CAN_ZIL1_VL_CDR3;

SEQ ID NO: 19 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL8_VH_CDR1;

SEQ ID NO: 20 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL8_VH_CDR2;

SEQ ID NO: 21 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL8_VH_CDR3;

SEQ ID NO: 22 is the light chain variable region CDR1, referred to herein as CAN_ZIL8_VL_CDR1;

SEQ ID NO: 23 is the light chain variable region CDR2, referred to herein as CAN_ZIL8_VL_CDR2;

SEQ ID NO: 24 is the light chain variable region CDR3, referred to herein as CAN_ZIL8_VL_CDR3;

SEQ ID NO: 25 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL9_VH_CDR1;

SEQ ID NO: 26 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL9_VH_CDR2;

SEQ ID NO: 27 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL9_VH_CDR3;

SEQ ID NO: 28 is the light chain variable region CDR1, referred to herein as CAN_ZIL9_VL_CDR1;

SEQ ID NO: 29 is the light chain variable region CDR2, referred to herein as CAN_ZIL9_VL_CDR2;

SEQ ID NO: 30 is the light chain variable region CDR3, referred to herein as CAN_ZIL9_VL_CDR3;

SEQ ID NO: 31 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL11_VH_CDR1;

SEQ ID NO: 32 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL11_VH_CDR2;

SEQ ID NO: 33 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL11_VH_CDR3;

SEQ ID NO: 34 is the light chain variable region CDR1, referred to herein as CAN_ZIL11_VL_CDR1;

SEQ ID NO: 35 is the light chain variable region CDR2, referred to herein as CAN_ZIL11_VL_CDR2;

SEQ ID NO: 36 is the light chain variable region CDR3, referred to herein as CAN_ZIL11_VL_CDR3;

SEQ ID NO: 37 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL69_VH_CDR1;

SEQ ID NO: 38 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL69_VH_CDR2;

SEQ ID NO: 39 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL69_VH_CDR3;

SEQ ID NO: 40 is the light chain variable region CDR1, referred to herein as CAN_ZIL69_VL_CDR1;

SEQ ID NO: 41 is the light chain variable region CDR2, referred to herein as CAN_ZIL69_VL_CDR2;

SEQ ID NO: 42 is the light chain variable region CDR3, referred to herein as CAN_ZIL69_VL_CDR3;

SEQ ID NO: 43 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL94_VH_CDR1;

SEQ ID NO: 44 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL94_VH_CDR2;

SEQ ID NO: 45 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL94_VH_CDR3;

SEQ ID NO: 46 is the light chain variable region CDR1, referred to herein as CAN_ZIL94_VL_CDR1;

SEQ ID NO: 47 is the light chain variable region CDR2, referred to herein as CAN_ZIL94_VL_CDR2;

SEQ ID NO: 48 is the light chain variable region CDR3, referred to herein as CAN_ZIL94_VL_CDR3;

SEQ ID NO: 49 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL154_VH_CDR1;

SEQ ID NO: 50 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL154_VH_CDR2;

SEQ ID NO: 51 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL154_VH_CDR3;

SEQ ID NO: 52 is the light chain variable region CDR1, referred to herein as CAN_ZIL154_VL_CDR1;

SEQ ID NO: 53 is the light chain variable region CDR2, referred to herein as CAN_ZIL154_VL_CDR2;

SEQ ID NO: 54 is the light chain variable region CDR3, referred to herein as CAN_ZIL154_VL_CDR3;

SEQ ID NO: 55 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL159_VH_CDR1;

SEQ ID NO: 56 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL159_VH_CDR2;

SEQ ID NO: 57 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL159_VH_CDR3;

SEQ ID NO: 58 is the light chain variable region CDR1, referred to herein as CAN_ZIL159_VL_CDR1;

SEQ ID NO: 59 is the light chain variable region CDR2, referred to herein as CAN_ZIL159_VL_CDR2;

SEQ ID NO: 60 is the light chain variable region CDR3, referred to herein as CAN_ZIL159_VL_CDR3;

SEQ ID NO: 61 is the heavy chain variable region CDR1, referred to herein as CAN_ZIL171_VH_CDR 1;

SEQ ID NO: 62 is the heavy chain variable region CDR2, referred to herein as CAN_ZIL171_VH_CDR2;

SEQ ID NO: 63 is the heavy chain variable region CDR3, referred to herein as CAN_ZIL171_VH_CDR3;

SEQ ID NO: 64 is the light chain variable region CDR1, referred to herein as CAN_ZIL171_VL_CDR 1;

SEQ ID NO: 65 is the light chain variable region CDR2, referred to herein as CAN_ZIL171_VL_CDR2;

SEQ ID NO: 66 is the light chain variable region CDR3, referred to herein as CAN_ZIL171_VL_CDR3;

SEQ ID NO: 67 is the heavy chain variable region, referred to herein as MU_15H05_VH;

SEQ ID NO: 68 is the nucleotide sequence encoding the heavy chain variable region referred to herein as MU_15H05_VH;

SEQ ID NO: 69 is the light chain variable region referred to herein as MU_15H05_VL;

SEQ ID NO: 70 is the nucleotide sequence encoding the light chain variable region referred to herein as MU_15H05_VL;

SEQ ID NO: 71 is the heavy chain variable region referred to herein as MU-11E12-VH;

SEQ ID NO: 72 is the nucleotide sequence encoding the heavy chain variable region, referred to herein as MU-11E12-VH;

SEQ ID NO: 73 is the light chain variable region referred to herein as MU-11E12-VL;

SEQ ID NO: 74 is the nucleotide sequence encoding the light chain variable region referred to herein as MU-11E12-VL;

SEQ ID NO: 75 is the heavy chain variable region referred to herein as CAN-ZIL1_VH;

SEQ ID NO: 76 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL1_VH;

SEQ ID NO: 77 is the light chain variable region referred to herein as CAN-ZIL1_VL;

SEQ ID NO: 78 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL1_VL;

SEQ ID NO:79 is the heavy chain variable region referred to herein as CAN-ZIL8_VH;

SEQ ID NO: 80 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL8_VH;

SEQ ID NO: 81 is the light chain variable region referred to herein as CAN-ZIL8_VL;

SEQ ID NO: 82 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL8_VL;

SEQ ID NO: 83 is the heavy chain variable region referred to herein as CAN-ZIL9_VH;

SEQ ID NO: 84 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL9_VH;

SEQ ID NO: 85 is the light chain variable region referred to herein as CAN-ZIL9_VL;

SEQ ID NO: 86 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL9_VL;

SEQ ID NO: 87 is the heavy chain variable region referred to herein as CAN-ZIL11_VH;

SEQ ID NO: 88 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL11_VH;

SEQ ID NO: 89 is the light chain variable region referred to herein as CAN-ZIL11_VL;

SEQ ID NO: 90 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL11_VL;

SEQ ID NO: 91 is the heavy chain variable region referred to herein as CAN-ZIL69_VH;

SEQ ID NO: 92 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL69_VH;

SEQ ID NO: 93 is the light chain variable region referred to herein as CAN-ZIL69_VL;

SEQ ID NO: 94 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL69_VL;

SEQ ID NO: 95 is the heavy chain variable region referred to herein as CAN-ZIL94_VH;

SEQ ID NO: 96 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL94_VH;

SEQ ID NO: 97 is the light chain variable region referred to herein as CAN-ZIL94_VL;

SEQ ID NO: 98 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL94_VL;

SEQ ID NO: 99 is the heavy chain variable region referred to herein as CAN-ZIL154_H;

SEQ ID NO: 100 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL154_VH;

SEQ ID NO: 101 is the light chain variable region referred to herein as CAN-ZIL154_VL;

SEQ ID NO: 102 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL154_VL;

SEQ ID NO: 103 is the heavy chain variable region referred to herein as CAN-ZIL159_VH;

SEQ ID NO: 104 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL159_VH;

SEQ ID NO: 105 is the light chain variable region referred to herein as CAN-ZIL159_VL;

SEQ ID NO: 106 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL159_VL;

SEQ ID NO: 107 is the heavy chain variable region referred to herein as CAN-ZIL171_VH;

SEQ ID NO: 108 is the nucleotide sequence encoding the heavy chain variable region referred to herein as CAN-ZIL171_VH;

SEQ ID NO: 109 is the light chain variable region referred to herein as CAN-ZIL171_VL;

SEQ ID NO: 110 is the nucleotide sequence encoding the light chain variable region referred to herein as CAN-ZIL171_VL;

SEQ ID NO: 111 is the heavy chain variable region referred to herein as FEL_11E12_VH1;

SEQ ID NO: 112 is the nucleotide sequence encoding the heavy chain variable region referred to herein as FEL_11E12_VH1;

SEQ ID NO: 113 is the light chain variable region referred to herein as FEL_11E12_VL1;

SEQ ID NO: 114 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_11E12_VL1;

SEQ ID NO: 115 is the light chain variable region referred to herein as FEL_11E12_VL2;

SEQ ID NO: 116 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_11E12_VL2;

SEQ ID NO: 117 is the light chain variable region referred to herein as FEL_11E12_VL1_FW2;

SEQ ID NO: 118 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_11E12_VL1_FW2;

SEQ ID NO: 119 is the light chain variable region referred to herein as FEL_11E12_VL1_K46Q;

SEQ ID NO: 120 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_11E12_VL1_K46Q;

SEQ ID NO: 121 is the heavy chain variable region referred to herein as FEL_15H05_VH1;

SEQ ID NO: 122 is the nucleotide sequence encoding the heavy chain variable region referred to herein as FEL_15H05_VH1;

SEQ ID NO: 123 is the heavy chain variable region referred to herein as FEL_15H05_VH2;

SEQ ID NO: 124 is the nucleotide sequence encoding the heavy chain variable region referred to herein as FEL_15H05_VH2;

SEQ ID NO: 125 is the heavy chain variable region referred to herein as FEL_15H05_VH3;

SEQ ID NO: 126 is the nucleotide sequence encoding the heavy chain variable region referred to herein as FEL_15H05_VH3;

SEQ ID NO: 127 is the light chain variable region referred to herein as FEL_15H05_VL1;

SEQ ID NO: 128 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1;

SEQ ID NO: 129 is the light chain variable region referred to herein as FEL_15H05_VL2;

SEQ ID NO: 130 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL2;

SEQ ID NO: 131 is the light chain variable region referred to herein as FEL_15H05_VL3;

SEQ ID NO: 132 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL3;

SEQ ID NO: 133 is the light chain variable region referred to herein as FEL_15H05_VL1_FW1;

SEQ ID NO: 134 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW1;

SEQ ID NO: 135 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2;

SEQ ID NO: 136 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2;

SEQ ID NO: 137 is the light chain variable region referred to herein as FEL_15H05_VL1_FW3;

SEQ ID NO: 138 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW3;

SEQ ID NO: 139 is the light chain variable region referred to herein as FEL_15H05_VL1_FW1_FW2;

SEQ ID NO: 140 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW1_FW2;

SEQ ID NO: 141 is the light chain variable region referred to herein as FEL_15H05_VL1_FW1_FW3;

SEQ ID NO: 142 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW1_FW3;

SEQ ID NO: 143 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2_FW3;

SEQ ID NO: 144 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2_FW3;

SEQ ID NO: 145 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2_K42N;

SEQ ID NO: 146 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2_K42N;

SEQ ID NO: 147 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2_V43I;

SEQ ID NO: 148 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2_V43I;

SEQ ID NO: 149 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2_L46V;

SEQ ID NO: 150 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2_L46V;

SEQ ID NO: 151 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2_Y49N;

SEQ ID NO: 152 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2_Y49N;

SEQ ID NO: 153 is the light chain variable region referred to herein as FEL_15H05_VL1_FW2_K42N_V43I;

SEQ ID NO: 154 is the nucleotide sequence encoding the light chain variable region referred to herein as FEL_15H05_VL1_FW2_K42N_V43I;

SEQ ID NO: 155 is the amino acid sequence of canine IL-31 protein, referred to herein as Canine_IL31;

SEQ ID NO: 156 is the nucleotide sequence encoding the canine IL-31 protein, referred to herein as Canine_IL31;

SEQ ID NO: 157 is the amino acid sequence, herein referred to as Feline_IL31_wildtype, which corresponds to the wild-type feline IL-31 protein with a C-terminal His tag;

SEQ ID NO: 158 is the nucleotide sequence encoding the amino acid sequence referred to here as Feline_IL31_wildtype;

SEQ ID NO: 159 is the amino acid sequence here referred to as Feline_IL_31_E_coli, which corresponds to the N-terminal His-tagged feline IL-31 protein;

SEQ ID NO: 160 is the nucleotide sequence encoding the amino acid sequence referred to herein as Feline_IL_31_E_coli;

SEQ ID NO: 161 is the amino acid sequence, herein referred to as Feline_IL31_11E12_mutant, which corresponds to a feline IL-31 HE 12 mutant protein with a C-terminal His tag;

SEQ ID NO: 162 is the nucleotide sequence encoding the amino acid sequence referred to herein as Feline_IL31_11E12_mutant;

SEQ ID NO: 163 is the amino acid sequence referred to herein as Feline_IL31_15H05_mutant, which corresponds to the C-terminal His tag mutant feline IL-31 protein 15H05;

SEQ ID NO: 164 is the nucleotide sequence encoding the amino acid sequence referred to herein as Feline_IL31_15H05_mutant;

SEQ ID NO: 165 is the amino acid sequence of the equine IL-31 protein, referred to here as Equine_IL31;

SEQ ID NO: 166 is the nucleotide sequence encoding the equine IL-31 protein, referred to here as Equine_IL31;

SEQ ID NO: 167 is the amino acid sequence here referred to as Feline_OSMR_hIgG1_Fc, which corresponds to the extracellular domain of feline OSMR fused to human IgG1 Fc;

SEQ ID NO: 168 is the nucleotide sequence encoding the amino acid sequence referred to herein as Feline_OSMR_hIgG1_Fc;

SEQ ID NO: 169 is the amino acid sequence here referred to as Feline_IL31Ra_HIgG1_Fc_X1_Fn3, which corresponds to feline IL-31Ra fused to human IgG1 Fc;

SEQ ID NO: 170 is the nucleotide sequence encoding the amino acid sequence referred to herein as Feline_IL31Ra_HIgG1_Fc_X1_Fn3;

SEQ ID NO: 171 is a feline heavy chain, referred to here as Feline_HC_AlleleA_wt;

SEQ ID NO: 172 is the nucleotide sequence encoding the feline heavy chain referred to herein as Feline_HC_AlleleA_wt;

SEQ ID NO: 173 is a feline heavy chain, referred to herein as Feline_HC_AlleleA_1, which has been modified to replace the M, L, and G at positions 120, 121, and 123, respectively, of the wild-type sequence of SEQ ID NO: 171 with an alanine (A) to elimination of the effector function of the antibody;

SEQ ID NO: 174 is the nucleotide sequence encoding the feline heavy chain referred to herein as Feline_HC_AlleleA_1;

SEQ ID NO: 175 is a feline kappa light chain, referred to herein as Feline_LC_Kappa_G_minus, which has been modified to eliminate the (G-) glycosylation at position 103 such that the N normally present at that position in the wild-type feline kappa light chain has been changed to Q;

SEQ ID NO: 176 is the nucleotide sequence encoding the amino acid sequence of the feline kappa light chain, referred to herein as Feline_LC_Kappa_G_minus;

SEQ ID NO: 177 is the canine heavy chain referred to herein as Canine_HC_65_1;

SEQ ID NO: 178 is the nucleotide sequence encoding the canine heavy chain referred to herein as Canine_HC_65_l;

SEQ ID NO: 179 is canine kappa light chain, referred to herein as Canine_LC_Kappa;

SEQ ID NO: 180 is the nucleotide sequence encoding the canine kappa light chain, referred to herein as Canine_LC_Kappa;

SEQ ID NO: 181 is the amino acid sequence of human IL-31.

SEQ ID NO: 182 is the mAb light chain variable region sequence referred to herein as Can_11E12_VL_cUn_1;

SEQ ID NO: 183 is the nucleotide sequence encoding the mAb light chain variable region sequence referred to herein as Can_11E12_VL_cUn_l;

SEQ ID NO: 184 is the mAb light chain variable region sequence referred to herein as Can_11E12_VL_cUn_FW2;

SEQ ID NO: 185 is the nucleotide sequence encoding the mAb light chain variable region sequence referred to herein as Can_11E12_VL_cUn_FW2;

SEQ ID NO: 186 is a feline kappa light chain, referred to herein as Feline_LC_Kappa_G_minus_QRE_minus, which has been modified to (1) eliminate the (G-) glycosylation at position 103 such that the N normally present at that position in wild-type feline kappa light chain, was changed to Q, and (2) deletion of the C-terminal QRE compared to wild type;

SEQ ID NO: 187 is the nucleotide sequence encoding the feline kappa light chain, referred to herein as Feline_LC_Kappa_G_minus_QRE_minus;

SEQ ID NO: 188 is the mouse heavy chain, referred to here as Mouse_HC_IgG1;

SEQ ID NO: 189 is the nucleotide sequence encoding the mouse heavy chain, referred to here as Mouse_HC_IgG1;

SEQ ID NO: 190 is mouse kappa light chain, referred to herein as Mouse_LC_Kappa;

SEQ ID NO: 191 is the nucleotide sequence encoding mouse kappa light chain, referred to herein as Mouse_LC_Kappa;

SEQ ID NO: 192 is human kappa light chain, referred to herein as Human_LC_Kappa;

SEQ ID NO: 193 is the nucleotide sequence encoding the human kappa light chain, referred to herein as Human_LC_Kappa;

SEQ ID NO: 194 is wild-type canine kappa light chain, referred to herein as Canine_LC_Kappa_wt;

SEQ ID NO: 195 is the nucleotide sequence encoding wild-type canine kappa light chain, referred to herein as Canine_LC_Kappa_wt;

SEQ ID NO: 196 is porcine kappa light chain, referred to herein as Pig_LC_Kappa;

SEQ ID NO: 197 is the nucleotide sequence encoding the porcine kappa light chain, referred to herein as Pig_LC_Kappa;

SEQ ID NO: 198 is mink kappa light chain, referred to herein as Mink_LC_Kappa;

SEQ ID NO: 199 is the nucleotide sequence encoding the mink kappa light chain, referred to herein as Mink_LC_Kappa;

SEQ ID NO: 200 is the heavy chain variable region CDR1, referred to herein as Mu_04H07_VH_CDR1;

SEQ ID NO: 201 is the heavy chain variable region CDR2, herein referred to as Mu_04H07_VH_CDR2;

SEQ ID NO: 202 is the heavy chain variable region CDR3, referred to herein as Mu_04H07_VH_CDR3;

SEQ ID NO: 203 is the light chain variable region CDR1, referred to herein as Mu_04H07_VL_CDR1;

SEQ ID NO: 204 is the light chain variable region CDR2, referred to herein as Mu_04H07_VL_CDR2;

SEQ ID NO: 205 is the light chain variable region CDR3, referred to herein as Mu_04H07_VL_CDR3;

SEQ ID NO: 206 is the heavy chain variable region CDR1, referred to herein as Mu_06A09_VH_CDR1;

SEQ ID NO: 207 is the heavy chain variable region CDR2, referred to herein as Mu_06A0_VH_CDR2;

SEQ ID NO: 208 is the heavy chain variable region CDR3, referred to herein as Mu_06A09_VH_CDR3;

SEQ ID NO: 209 is the light chain variable region CDR1, referred to herein as Mu_06A09_VL_CD1;

SEQ ID NO: 210 is the light chain variable region CDR2, referred to herein as Mu_06A09_VL_CD2;

SEQ ID NO: 211 is the light chain variable region CDR3, referred to herein as Mu_06A09_VL_CDR3;

SEQIDNO:212 is the heavy chain variable region referred to herein as Mu_04H07_VH;

SEQ ID NO: 213 is the nucleotide sequence encoding the heavy chain variable region referred to herein as Mu_04H07_VH;

SEQ ID NO:214 is the light chain variable region referred to herein as Mu 04H07 VL;

SEQ ID NO: 215 is the nucleotide sequence encoding the light chain variable region referred to herein as Mu_04H07_VL;

SEQ ID NO:216 is the heavy chain variable region referred to herein as Mu_06A09_VH;

SEQ ID NO:217 is the nucleotide sequence encoding the heavy chain variable region referred to herein as Mu_06A09_VH;

SEQ ID NO: 218 is the light chain variable region referred to herein as Mu_06A09_VL;

SEQ ID NO:219 is the nucleotide sequence encoding the light chain variable region referred to herein as Mu_06A09_VL;

SEQ ID NO: 220 is the heavy chain variable region, referred to herein as ZTS_768_VH;

SEQ ID NO: 221 is the nucleotide sequence encoding the heavy chain variable region, referred to herein as ZTS_768_VH;

SEQ ID NO: 222 is the light chain variable region, referred to herein as ZTS_768_VL;

SEQ ID NO: 223 is the nucleotide sequence encoding the light chain variable region referred to herein as ZTS_768_VL;

SEQ ID NO: 224 is the heavy chain variable region, referred to herein as ZTS_943_VH;

SEQ ID NO: 225 is the nucleotide sequence encoding the heavy chain variable region referred to herein as ZTS_943_VH;

SEQ ID NO: 226 is the light chain variable region, referred to herein as ZTS_943_VL;

SEQ ID NO: 227 is the nucleotide sequence encoding the light chain variable region referred to herein as ZTS_943_VL;

SEQ ID NO: 228 is the heavy chain variable region, referred to herein as ZTS_5864_VH;

SEQ ID NO: 229 is the nucleotide sequence encoding the heavy chain variable region referred to herein as ZTS_5864_VH;

SEQ ID NO: 230 is the light chain variable region, referred to herein as ZTS_5864_VL;

SEQ ID NO: 231 is the nucleotide sequence encoding the light chain variable region, referred to herein as ZTS_5864_VL;

SEQ ID NO: 232 is the heavy chain variable region, referred to herein as ZTS_5865_VH;

SEQ ID NO: 233 is the nucleotide sequence encoding the heavy chain variable region referred to herein as ZTS_5865_VH;

SEQ ID NO: 234 is the light chain variable region referred to herein as ZTS 5865 VL;

SEQ ID NO: 235 is the nucleotide sequence encoding the light chain variable region referred to herein as ZTS_5865_VL;

SEQ ID NO: 236 is the amino acid sequence of the feline kappa light chain, referred to herein as Feline LC Lambda;

SEQ ID NO: 237 is the nucleotide sequence encoding the feline kappa light chain, referred to herein as Feline_LC_Lambda.

DEFINITIONS

Before a detailed description of the present invention, some terms used in the context of the present invention will be defined. Where necessary, other terms are defined in addition to these terms elsewhere in the application. Unless otherwise expressly defined here, terms related to the area under consideration are used in this description in the meanings accepted in this area.

Unless the context clearly dictates otherwise, when used in this specification and claims, the singular forms include the plural. For example, reference to "antibody" includes many such antibodies.

When used here, the term "comprising/comprising" is intended to mean that the compositions and methods include these elements, without excluding others.

As used herein, "epitope" refers to an antigenic determinant recognized by the CDR of an antibody. In other words, an epitope refers to that portion of any molecule that is capable of being recognized and bound by an antibody. Unless otherwise indicated, as used herein, the term "epitope" refers to the region of IL-31 with which an anti-IL-31 agent interacts.

An "antigen" is a molecule or portion of a molecule capable of being bound by an antibody, which is further capable of being recognized and bound by an antibody (the binding region of the corresponding antibody may be referred to as a paratope). Typically, epitopes are composed of reactive surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charging characteristics. Epitopes are antigenic determinants on a protein that are recognized by the immune system. The components of the immune system that recognize epitopes are antibodies, T cells, and B cells. Presentation of T cell epitopes occurs on the surface of antigen presenting cells (APCs) and is typically 8-11 (MHC class I) or 15 or more (MHC class II) amino acids in length. Recognition of the presented MHC-peptide complex by T cells is critical for their activation. These mechanisms allow proper recognition of self and "foreign" proteins such as bacteria and viruses. Independent amino acid residues, which may not be contiguous, contribute to interaction with the APC binding groove and subsequent recognition by the T-cell receptor (Janeway, Travers, Walport, Immunobiology: The Immune System in Health and Disease. ^5th edition New York: Garland Science ; 2001). Epitopes recognized by soluble antibodies and cell surface-bound B-cell receptors vary greatly in length and degree of continuity (Sivalingam and Shepherd, Immunol. 2012; 51(3-4): 304-309). In addition, even epitopes that are linear or located on a contiguous stretch of protein sequence will often have non-adjacent amino acids that are key points of contact with the paratopes of the antibody or the B-cell receptor. Epitopes recognized by antibodies and B cells may be conformational epitopes with amino acids constituting a common three-dimensional contact area on a protein and depend on the properties of the protein's tertiary and quaternary structure. These residues are often located in spatially separated regions of the primary amino acid sequence.

As used herein, a "mimotope" is a linear or restricted peptide that mimics an epitope of an antigen. The mimotope may have a primary amino acid sequence capable of inducing an effector T cell response and/or a three-dimensional structure necessary for binding to B cells leading to the maturation of an acquired immune response in the animal. An antibody to a given antigen epitope will recognize a mimotope that mimics that epitope.

The term "specifically" in the context of antibody binding refers to high avidity and/or high affinity for binding of an antibody to a specific antigen, ie, polypeptide, or epitope. In many embodiments, a specific antigen is an antigen (or a fragment or subfraction of an antigen) used to immunize a host animal from which antibody-producing cells are isolated. The specific binding of an antibody to an antigen is stronger than the binding of the same antibody to other antigens. Antibodies that specifically bind to a polypeptide may be able to bind to other polypeptides at a low but detectable level (eg, 10% or less of the indicated binding to the polypeptide of interest). Such weak binding or background binding is easily distinguished from specific binding of the antibody to the corresponding polypeptide, for example, by using appropriate controls. Typically, the binding affinity of specific antibodies to an antigen corresponds to a K _D of 10 ^-7 M or less, for example, 10 ^-8 M or less (for example, 10 ^-9 M or less, 10 ^-10 or less, 10 ^-11 or less, 10 ^-12 or less, or 10 ^-13 or less, and so on).

As used herein, the term "antibody" refers to an intact immunoglobulin having two light and two heavy chains. Thus, a single isolated antibody or fragment can be a polyclonal antibody, a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a heterochimeric antibody, a caninized antibody, a felinized antibody, a fully canine antibody, a fully feline antibody, or a fully equine antibody. The term "antibody" preferably refers to monoclonal antibodies, fragments thereof and immunological binding equivalents thereof, which can bind to the IL-31 protein and fragments thereof. The term antibody is used to refer to both homogeneous molecules and a mixture, such as a serum product, containing many different molecules.

"Native antibodies" and "native immunoglobulins" are typically heterotetrameric glycoproteins of approximately 150,000 Da, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds between immunoglobulin heavy chains of different isotypes varies. Each heavy and light chain also has regularly repeated intrachain disulfide bonds. Each heavy chain has at one end a variable domain (V _H ) followed by several constant domains. Each light chain has a variable domain at one end (V _L ) and a constant domain at its other end; the light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. I believe that certain amino acid residues form the surface of the variable domains of the light and heavy chains facing each other.

The term "antibody fragment" refers to a structure smaller than an intact antibody, including, without limitation, an isolated single antibody chain, Fv construct, Fab construct, Fc construct, light chain variable region or hypervariable region (CDR) sequence, etc. Further.

The term "variable" region includes framework regions and CDRs (also known as hypervariable regions) and refers to the fact that certain positions of the variable domains of different antibodies differ significantly in sequence and are involved in the specific binding of each particular antibody to its particular antigen. However, the distribution of variability in the variable domains of antibodies is uneven. In the variable domains of both the light chain and the heavy chain, it is concentrated in three segments called hypervariable regions. The more conserved portions of the variable domains are called framework regions (FRs). Each native heavy and light chain variable domain contains a number of FRs, most of which have a β-sheet configuration and are connected by three hypervariable regions that form loops connecting these β-sheet structures and, in some cases, are part of them. Within each chain, the FRs keep the hypervariable regions in close proximity to each other, and these, together with the hypervariable regions of the other chain, are involved in the formation of the antigen-binding site of an antibody (see Kabat, et al., Sequences of Proteins of Immunological Interest, ^5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669). The constant domains are not directly involved in the binding of an antibody to an antigen, but perform various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that allow binding to an antigen. The hypervariable region contains "hypervariable region" or "CDR" amino acid residues (Kabat et al. (1991) supra) and/or "hypervariable loop" residues (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)) . Variable domain residues that are not hypervariable region residues as defined herein are "framework regions" or "FR" residues.

Cleavage of antibodies with papain results in two identical antigen-binding fragments, called "Fab fragments", each of which has one antigen-binding site, and a residual "Fc fragment", the name of which reflects its ability to crystallize rapidly. Upon treatment with pepsin, an F(ab') ₂ fragment is formed having two antigen-binding sites and the ability to cross-link antigen.

"Fv" is the minimum antibody fragment containing a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain linked by tight non-covalent interactions. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer. Together, these six hypervariable regions provide the binding specificity of the antibody to the antigen. However, even a single variable domain (or half of the Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind an antigen, but with less affinity than the entire binding site.

The Fab fragment also contains a light chain constant domain and a heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the C-terminus of the heavy chain CH1 domain, including one or more antibody hinge cysteines. Fab'-SH refers here to Fab' in which the cysteine residue(s) of the constant domain have a free thiol group. F(ab') ₂ antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines in between. Other chemical combinations of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are currently five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 (as they are commonly used in mice and humans). The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known in many species. The prevalence of individual isotypes and the functional activity associated with these constant domains are species-specific and require experimental determination.

"Monoclonal antibody", as defined here, is an antibody produced by a single clone of cells (specifically, a single clone of cells such as hybridoma cells), and therefore is a single pure homogeneous type of antibody. All monoclonal antibodies derived from the same clone are identical and have the same antigenic specificity. The term "monoclonal" refers to one cell clone, one cell and the progeny of that cell.

A "totally canine antibody", as defined herein, is a monoclonal antibody produced by a clone of cells (usually a CHO cell line) and is therefore one pure, homogeneous type of antibody. Identified antibodies from individual B cells of immunized mammals, such as dogs, are obtained in the form of recombinant IgG proteins after their variable domain sequences have been determined. Transfer of these variable domains to canine constant domains (heavy chain constant region and kappa or lambda light chain constant region) results in recombinant fully canine antibodies. All fully canine monoclonal antibodies derived from the same clone are identical and have the same antigenic specificity. The term "monoclonal" refers to one cell clone, one cell and the progeny of that cell.

A "totally feline antibody", as defined herein, is a monoclonal antibody produced by a clone of cells (usually a CHO cell line) and is therefore one pure, homogeneous type of antibody. Identified antibodies from individual B cells of immunized mammals, such as dogs, are obtained in the form of recombinant IgG proteins after their variable domain sequences have been determined. Transfer of these variable domains to feline constant domains (heavy chain constant and kappa or lambda light chain constant region) results in recombinant all-feline antibodies. All fully feline monoclonal antibodies derived from the same clone are identical and have the same antigenic specificity. The term "monoclonal" refers to one cell clone, one cell and the progeny of that cell.

A "fully equine antibody", as defined herein, is a monoclonal antibody produced by a clone of cells (usually a CHO cell line) and is therefore one pure, homogeneous type of antibody. Identified antibodies from individual B cells of immunized mammals, such as dogs, are obtained in the form of recombinant IgG proteins after their variable domain sequences have been determined. Transfer of these variable domains to equine constant domains (heavy chain constant region and kappa or lambda light chain constant region) results in recombinant all-equine antibodies. All fully equine monoclonal antibodies derived from the same clone are identical and have the same antigenic specificity. The term "monoclonal" refers to one cell clone, one cell and the progeny of that cell.

Here monoclonal antibodies include, in particular, "chimeric" antibodies (immunoglobulins), in which part of the heavy and/or light chain is identical or homologous to the corresponding sequences of antibodies derived from a certain species, while the rest of the chain (s) is identical or homologous to the corresponding sequences of antibodies originating from another species, as well as fragments of such antibodies, provided that they exhibit the desired biological activity. Typically, chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, usually by genetic engineering, from the genes of the variable and constant regions of antibodies belonging to different species. For example, variable segments of mouse monoclonal antibody genes can be combined with canine constant segments. In one embodiment of the chimeric mouse-canine IgG, the antigen-binding site is mouse-derived while the Fc portion is canine.

"Caninized" forms of non-canine (eg, murine) antibodies are genetically engineered antibodies containing a minimal sequence derived from a non-canine immunoglobulin. Canine antibodies are canine immunoglobulin sequences (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues of a non-canine species (donor antibody), such as a mouse, having the desired specificity, affinity and activity. In some cases, framework region (FR) residues of canine immunoglobulin sequences are replaced by corresponding residues of a non-canine species. In addition, caninized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve the properties of the antibody. In general, a caninized antibody will contain substantially all of at least one, and usually two, variable domains wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of a non-canine immunoglobulin sequence and all or substantially all of the FRs are canine FR sequences. immunoglobulin. Optionally, the caninized antibody will also comprise the entire immunoglobulin constant region (Fc), typically a canine immunoglobulin constant region (Fc), or at least a portion thereof. In one embodiment of species adaptation or caninization of mouse IgG, mouse CDRs are transferred to canine framework regions.

"Felinized" forms of non-feline (eg, murine) antibodies are genetically engineered antibodies containing a minimal sequence derived from a non-feline immunoglobulin. Felinized antibodies are feline immunoglobulin sequences (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues of a non-feline species (donor antibody), such as a mouse, having the desired specificity, affinity and activity. In some instances, framework region (FR) residues of feline immunoglobulin sequences are replaced by corresponding residues of a non-feline species. In addition, felinized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve the properties of the antibody. In general, a felinized antibody will contain substantially all of at least one, and usually two, variable domains wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of a non-feline immunoglobulin sequence and all or substantially all of the FRs are feline FR sequences. immunoglobulin. Optionally, the felinized antibody will also comprise the entire immunoglobulin constant region (Fc), typically a feline immunoglobulin constant region (Fc), or at least a portion thereof.

"Equinized" forms of non-equine (eg, murine) antibodies are genetically engineered antibodies containing a minimal sequence derived from a non-equine immunoglobulin. Equinized antibodies are equine immunoglobulin (recipient antibody) sequences in which the hypervariable region residues of the recipient are replaced by hypervariable region residues of a non-equine species (donor antibody), such as a mouse, having the desired specificity, affinity and activity. In some cases, framework region (FR) residues of equine immunoglobulin sequences are replaced by corresponding residues of a non-equine species. In addition, equinized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve the properties of the antibody. In general, an equinized antibody will contain substantially all of at least one, and usually two, variable domains wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of a non-equine immunoglobulin sequence and all or substantially all of the FRs are equine FR sequences. immunoglobulin. Optionally, the equinized antibody will also comprise the entire immunoglobulin constant region (Fc), typically an equine immunoglobulin constant region (Fc), or at least a portion thereof.

"All-canine" antibodies are genetically engineered antibodies that do not contain sequences derived from non-canine immunoglobulins. Fully canine antibodies are canine immunoglobulin (recipient antibody) sequences in which hypervariable region residues are derived from a naturally occurring canine antibody (donor antibody) having the desired specificity, affinity and activity. In some cases, framework region (FR) residues of canine immunoglobulin sequences are replaced by corresponding residues of a non-canine species. In addition, fully canine antibodies may contain residues that are not present in the recipient or donor antibody, for example, including but not limited to changes in the CDR to modify affinity. These modifications are made to further improve the properties of the antibody. In general, a fully canine antibody will comprise substantially all of at least one, and usually two, variable domains wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of a canine immunoglobulin sequence and all or substantially all of the FRs are canine immunoglobulin FR sequences. Optionally, a fully canine antibody will also contain the entire constant region (Fc) of an immunoglobulin, typically a constant region (Fc) of a canine immunoglobulin sequence, or at least a portion thereof.

"Full feline" antibodies are genetically engineered antibodies that do not contain sequences derived from non-feline immunoglobulins. Fully feline antibodies are feline immunoglobulin (recipient antibody) sequences in which hypervariable region residues are derived from a naturally occurring feline antibody (donor antibody) having the desired specificity, affinity and activity. In some instances, framework region (FR) residues of feline immunoglobulin sequences are replaced by corresponding residues of a non-feline species. In addition, fully feline antibodies may contain residues that are not present in the recipient antibody or in the donor antibody, for example, including, without limitation, changes in the CDR to modify affinity. These modifications are made to further improve the properties of the antibody. In general, a fully feline antibody will comprise substantially all of at least one, and usually two, variable domains wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of the feline immunoglobulin sequence and all or substantially all of the FRs are FRs of the feline immunoglobulin sequence. Optionally, a fully feline antibody will also comprise the entire constant region (Fc) of an immunoglobulin, typically a constant region (Fc) of a feline immunoglobulin sequence, or at least a portion thereof.

"Full equine" antibodies are genetically engineered antibodies that do not contain sequences derived from non-equine immunoglobulins. Fully equine antibodies are equine immunoglobulin (recipient antibody) sequences in which hypervariable region residues are derived from a naturally occurring equine antibody (donor antibody) having the desired specificity, affinity and activity. In some cases, framework region (FR) residues of equine immunoglobulin sequences are replaced by corresponding residues of a non-equine species. In addition, fully equine antibodies may contain residues that are not present in the recipient or donor antibody, for example, including but not limited to changes in the CDR to modify affinity. These modifications are made to further improve the properties of the antibody. In general, a fully equine antibody will comprise substantially all of at least one, and usually two, variable domains wherein all or substantially all of the hypervariable regions correspond to hypervariable regions of an equine immunoglobulin sequence and all or substantially all of the FRs are FRs of an equine immunoglobulin sequence. Optionally, a fully equine antibody will also comprise a complete immunoglobulin constant region (Fc), typically a constant region (Fc) of an equine immunoglobulin sequence, or at least a portion thereof.

The term "heterochimeric", as defined herein, refers to an antibody in which one of the antibody chains (heavy or light) is caninized, felinized, or equinized, while the other is chimeric. In one embodiment, a felinized heavy chain variable region (where all CDRs are murine and all FRs are feline) is paired with a chimeric light chain variable region (where all CDRs are murine and all FRs are murine). In this embodiment, the heavy chain variable region and the light chain variable region are fused to a feline constant region.

A "variant" of an anti-IL-31 antibody refers here to a molecule that differs in amino acid sequence from the amino acid sequence of the "parent" anti-IL-31 antibody by the addition, deletion, and/or substitution of one or more amino acid residues in the sequence of the parent antibody and retains at least at least one desired activity of the parent anti-IL-31 antibody. Desired activities may include the ability to specifically bind to an antigen, the ability to reduce, inhibit or neutralize IL-31 activity in an animal, and the ability to inhibit IL-31 mediated pSTAT signals in a cellular assay. In one embodiment, the variant contains one or more amino acid substitutions in one or more hypervariable and/or framework regions of the parent antibody. For example, a variant may contain at least one, eg, from about one to about ten, and preferably from about two to about five substitutions in one or more hypervariable and/or framework regions of the parent antibody. Typically, a variant will have an amino acid sequence that is at least 50% identical, more preferably at least 65%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85% identical. , more preferably at least 90% and most preferably at least 95% identical to the amino acid sequences of the heavy or light chain variable domains of the parent antibody. Identity or homology in relation to a given sequence is defined here as the percentage of amino acid residues in a candidate sequence that are identical to those of the parent antibody, after sequence alignment and, if necessary, the introduction of gaps to achieve the maximum percentage of sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions in an antibody sequence should not be considered as affecting sequence identity or homology. The variant retains the ability to bind to IL-31 and preferably has the desired activities that are superior to those of the parent antibody. For example, a variant may have higher binding affinity, enhanced ability to reduce, inhibit, or neutralize IL-31 activity in an animal, and/or enhanced ability to inhibit IL-31 mediated pSTAT signals in a cellular assay.

A "variant" nucleic acid refers here to a molecule whose sequence is different from the "original" nucleic acid. The divergence of polynucleotide sequences may be the result of mutational changes such as deletions, substitutions or additions of one or more nucleotides. Each of these changes may be present in a given sequence alone or in combination, one or more than one time.

Here, the "original" antibody is the antibody encoded by the amino acid sequence used to generate the variant. In one embodiment, the parent antibody has a canine framework region and, if present(s), canine antibody constant region(s). For example, the parent antibody may be a canine or canine antibody. As another example, the parent antibody may be a feline or feline antibody. As another example, the parent antibody may be an equine or equine antibody. As another example, the parent antibody is a mouse monoclonal antibody.

The terms "antigen-binding region", "antigen-binding fragment" and the like, as used in this description and claims, refer to that part of the antibody molecule that contains amino acid residues that interact with the antigen and give the antibody its specificity and affinity for the antigen. The binding region of an antibody contains "framework" amino acid residues necessary to maintain the correct conformation of the antigen-binding residues. An antigen-binding fragment of an antibody of the present invention may alternatively be referred to herein, for example, as an IL-31 specific peptide or polypeptide or an anti-IL-31 peptide or polypeptide.

The term "isolated" means that a substance (eg, antibody or nucleic acid) is separated and/or isolated from a component of its natural environment. Impurity components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the substance, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. With respect to a nucleic acid, an isolated nucleic acid may include a nucleic acid separated from the 5'-3' sequences to which it is normally associated on a chromosome. In preferred embodiments, the substance will be purified to greater than 95% by weight of the substance, and most preferably to greater than 99% by weight. An isolated substance includes the substance in situ in recombinant cells, since at least one component of the natural environment of the substance will be absent. Typically, however, the recovered material will be obtained by at least one purification step.

As used herein, the word "label" refers to a detectable compound or composition directly or indirectly conjugated to an antibody or nucleic acid. The label itself may be detectable per se (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a detectable chemical change in the substrate compound or substrate composition.

The terms "nucleic acid", "polynucleotide", "nucleic acid molecule" and the like may be used interchangeably herein to refer to the series of nucleotide bases (also referred to as "nucleotides") in DNA and RNA. The nucleic acid may contain deoxyribonucleotides, ribonucleotides and/or their analogs. The term "nucleic acid" includes, for example, single-stranded and double-stranded molecules. A nucleic acid can be, for example, a gene or gene fragment, exons, introns, a DNA molecule (eg cDNA), an RNA molecule (eg mRNA), recombinant nucleic acids, plasmids and other vectors, primers and probes. Both 5'-3' polynucleotides (sense) and 3'-5' polynucleotides (antisense) are included.

"Subject" or "patient" refers to an animal in need of treatment, which can be carried out by the molecules of the invention. Animals that can be treated according to the invention include vertebrates, with particularly preferred examples being mammals such as dogs, cats and horses.

A "therapeutically effective amount" (or "effective amount") refers to an amount of an active ingredient, such as an agent of the invention, sufficient to produce beneficial or desired results when administered to a subject or patient. An effective amount may be administered using one or more administrations, applications, or doses. A therapeutically effective amount of a composition of the invention can be readily determined by one of skill in the art. In the context of this invention, a "therapeutically effective amount" is an amount that results in an objectively measurable change in one or more parameters associated with the treatment of an pruritic or allergic condition, including clinical improvement in symptoms. Of course, the therapeutically effective amount will vary depending on the particular subject and the condition being treated, the body weight and age of the subject, the severity of the disease state, the particular compound chosen, the administration regimen used, the time of administration, the route of administration, and the like, and all. this can be easily determined by a person skilled in the art.

As used herein, the term "therapeutic" encompasses the full range of treatment options for a disease or disorder. The "therapeutic" agent of the invention may act in a prophylactic or preventative manner, including techniques designed to affect animals that may be identified as at risk (pharmacogenetics), or in an alleviating or curative manner, or may act to reduce the rate or extent of progression at least one symptom of the disease or disorder being treated.

"Treatment", "treat" and the like refers to both therapeutic treatment and prophylactic or preventive measures. Animals in need of treatment include both those that already have the disorder and those that should be prevented from having the disorder. The term "treatment" of a disease or disorder includes preventing or protecting against the disease or disorder (i.e., ensuring that clinical symptoms do not develop), suppressing the disease or disorder (i.e., arresting or containing the development of clinical symptoms), and/or ameliorating the disease or disorder (i.e., providing regression of clinical symptoms). It should be understood that the distinction between "prevention" and "containment" of a disease or disorder is not always possible because the last inducing event or events may be unknown or latent. Accordingly, the term prevention should be understood as a type of "treatment" encompassing both "prevention" and "containment". Thus, the term "treatment" includes "prophylaxis".

The term "allergic condition" is defined here as a disorder or disease caused by the interaction of the immune system with a substance that is foreign to the body. This foreign substance is called an "allergen". Common allergens include airborne allergens such as pollen, dust, mold, dust mite proteins, saliva from insect bites, and so on. Examples of allergic conditions include, without limitation, the following: allergic dermatitis, summer eczema, urticaria, equine sunburn, inflammatory airway disease, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, and inflammatory processes resulting from autoimmunity such as irritable bowel (IBS).

The term "pruritic condition" is defined here as a disease or disorder characterized by an intense sensation of itching that prompts rubbing or scratching of the skin to obtain relief. Examples of conditions accompanied by itching include, without limitation, the following: atopic dermatitis, allergic dermatitis, eczema, psoriasis, scleroderma and pruritus.

When used here, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All of these terms also include their offspring, representing all and any subsequent generations. It should be understood that all progeny may not be identical due to intentional or unintentional mutations. In the context of expression of a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell (eg, bacterial cells, yeast cells, mammalian cells, and insect cells), whether in vitro or in vivo. For example, the host cells may be in a transgenic animal. The host cell may be used as a recipient of the vectors and may include any transformable organism capable of replicating the vector and/or expressing the heterologous nucleic acid encoded by the vector.

The term "composition" is intended to mean a combination of an active agent and another compound or other composition, which may be inert (eg, a label) or active, such as an adjuvant.

As defined herein, pharmaceutically acceptable carriers suitable for use in the invention are well known to those skilled in the art. Such carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other commonly used diluents, adjuvants and excipients may be added according to conventional techniques. Such carriers may include ethanol, polyols and suitable mixtures thereof, vegetable oils and injectable organic esters. Buffers and pH adjusting agents may also be used. Buffers include, without limitation, salts derived from an organic acid or base. Exemplary buffers include, without limitation, organic acid salts such as citric acid salts, e.g. citrates, ascorbic acid, gluconic acid, histidine-HCl, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, tris, tromethamine hydrochloride or phosphate buffers. Carriers for parenteral administration may include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose and sodium chloride, Ringer's lactate, or fixed oils. Carriers for intravenous administration may include fluid and nutrient replenishers such as Ringer's dextrose based replenishers and the like. Preservatives and other additives may also be present in pharmaceutical carriers, such as, for example, antimicrobial agents, antioxidants, chelating agents (eg, EDTA), inert gases, and the like. The present invention is not limited to the choice of carrier. The preparation of these pharmaceutically acceptable compositions from the components described above, having suitable pH, isotonicity, stability and other conventional characteristics, is within the skill of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000, and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Rowe et al., APhA Publications, 2003.

The term "conservative amino acid substitution" refers to any amino acid substitution of a given amino acid residue, where the substituting residue is chemically so similar to the given residue that the substitution does not significantly reduce the function (eg, enzymatic activity) of the polypeptide. Conservative amino acid substitutions are well known in the art and examples are described in, for example, US Pat. In a preferred embodiment, a conservative amino acid substitution will be any substitution occurring within one of the following six groups:

• 1. small aliphatic almost non-polar residues: Ala, Gly, Pro, Ser and Thr;

• 2. large aliphatic non-polar residues: Ile, Leu and Val; met;

• 3. polar negatively charged residues and their amides: Asp and Glu;

• 4. amides of polar negatively charged residues: Asn and Gln; His;

• 5. polar positively charged residues: Arg and Lys; His; And

• 6. large aromatic residues: Trp and Tyr; Phe.

In a preferred embodiment, the conservative amino acid substitution will be any of the following substitutions, listed as native residue (conservative substitution) pairs: Ala (Ser); Arg(lys); Asn (Gln; His); Asp (Glu); Gln(Asn); Glu (Asp); Gly (Pro); His(Asn;Gln); Ile (Leu; Val); Leu (Ile; Val); Lys (Arg; Gln; Glu); Met (Leu; Ile); Phe (Met; Leu; Tyr); Ser(Thr); Thr(Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (Ile; Leu).

Just as a polypeptide may contain a conservative amino acid substitution(s), its corresponding polynucleotide may contain a conservative codon substitution(s). A codon substitution is considered conservative if, when expressed, it results in a conservative amino acid substitution as described above. In the polynucleotides of the present invention, a degenerate codon change that does not result in an amino acid change may also be useful. Thus, for example, it is possible to mutate a polynucleotide encoding a selected polypeptide useful in an embodiment of the present invention by degenerate codon substitution to approximate the codon usage frequency to that exhibited by the expression host cell to be transformed with said polynucleotide, or to otherwise improve its expression.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to the specific methods, protocols, reagents, etc. described herein, and may, as such, vary. The terms used here are only intended to describe specific embodiments and do not limit the scope of the present invention, defined solely by the claims.

Unless otherwise defined, scientific and technical terms used in connection with the antibodies described herein have the meaning in which they are usually understood by experts in this field. In addition, unless the context otherwise requires, the singular includes the plural, and the plural includes the singular. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligo- or polynucleotides, and the corresponding techniques described herein, are well known and widely used in the art.

For recombinant DNA, oligonucleotide synthesis, tissue culture, and transfection, standard techniques (eg, electroporation, lipofection) are used. Enzymatic reactions and purification procedures are carried out in accordance with the manufacturer's descriptions or as they are usually carried out in this field or as described here. The techniques described above are generally carried out following conventional methods well known in the art and described in various general and more specialized sources cited and discussed herein. See, for example, Sambrook et al. MOLECULAR CLONING: LAB. MANUAL (3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y., 2001) and Ausubel et al. Current Protocols in Molecular Biology (New York: Greene Publishing Association/Wiley Interscience), 1993. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, and the associated laboratory procedures described here, are well known and widely used. in this area. For chemical synthesis, chemical analysis, manufacture of pharmaceuticals, compositions, their delivery and treatment of patients, standard techniques are used.

Except in the working examples or otherwise where indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should always be understood as modified by the term "approximately".

All of these patents and other publications are expressly incorporated here by reference for the purpose of describing and disclosing, for example, the methods described in such publications that can be applied in connection with the present invention. These publications are cited solely by virtue of their disclosure prior to the filing date of this application.

According to the present invention proposed recombinant monoclonal antibodies and peptides and their use in clinical and scientific techniques, including diagnostic techniques.

The advent of molecular biological methods and recombinant technologies has led to the possibility of recombinant production of antibodies and antibody-like molecules and, thus, the creation of gene sequences encoding certain amino acid sequences present in the polypeptide structure of antibodies. Such antibodies can be obtained by cloning gene sequences encoding the polypeptide chains of these antibodies, or direct synthesis of these polypeptide chains and assembly of the synthesized chains with the formation of active tetrameric (H ₂ L ₂ ) structures with affinity for certain epitopes and antigenic determinants. This made it possible to quickly obtain antibodies having sequences characteristic of neutralizing antibodies from various species and sources.

Regardless of the source of the antibodies, or how they are recombinantly constructed, or how they are synthesized, in vitro or in vivo, using transgenic animals, using large laboratory or commercial size cell cultures, using transgenic plants, or using direct chemical synthesis without the use of living organisms at any stage of the process , all antibodies have a similar overall 3-dimensional structure. This structure is often referred to as H ₂ L ₂ and this indicates the fact that antibodies usually contain two light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are called "variable" or "V" regions, and are characterized by differences in the amino acid sequence of antibodies with different antigenic specificities. The variable regions of the H or L chains contain amino acid sequences capable of specifically binding to antigenic targets.

As indicated above, the terms "antigen-binding region", "antigen-binding fragment" and the like, as used in this description and claims, refer to that part of the antibody molecule that contains amino acid residues that interact with the antigen and give the antibody its specificity and affinity for antigen. The binding region of an antibody contains "framework" amino acid residues necessary to maintain the correct conformation of the antigen-binding residues. The antigen-binding fragment of an antibody of the present invention may be referred to herein, for example, as an IL-31 specific peptide or polypeptide or an anti-IL-31 peptide or polypeptide.

Within the variable regions of the H or L chains that provide antigen binding regions, there are smaller sequences called "hypervariable" because of their extreme variability between antibodies of different specificities. Such hypervariable regions are also referred to as "hypervariable regions" or "CDRs". These CDRs provide the basic specificity of the antibody for the structure of a certain antigenic determinant.

The CDRs are non-contiguous stretches of amino acids within the variable regions, but it has been found that, regardless of species, these critical amino acid sequences are located in the heavy and light chain variable regions in a similar manner. The heavy and light chain variable regions of all antibodies have three CDR regions that are not adjacent to each other.

In all mammalian species, antibody peptides contain constant (i.e., highly conserved) and variable regions, and, within the variable regions, there are CDRs and so-called "framework regions" formed by amino acid sequences located within the heavy or light chain variable region, but outside the CDR.

As for the antigenic determinant recognized by the CDR regions of an antibody, it is also referred to as an "epitope". In other words, an epitope refers to that part of any molecule that is capable of being recognized and bound by an antibody (the binding region of the corresponding antibody may be called a paratope).

An "antigen" is a molecule or portion of a molecule capable of being bound by an antibody, which is further capable of inducing in an animal the production of an antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. It is assumed that the specific interaction mentioned above indicates that the antigen will interact in a highly selective manner with its corresponding antibody, and not with a variety of other antibodies that can be induced by other antigens.

It is assumed that the antibodies of the present invention include both intact immunoglobulin molecules and their parts, fragments, peptides and derivatives, such as, for example, Fab, Fab', F(ab') ₂ , Fv, Fse, CDR regions, paratopes or any portion (eg, polypeptide) or peptide sequence of an antibody capable of binding to an antigen or epitope. An antibody is considered "able to bind" or "able to bind" to a molecule if it is capable of specifically interacting with the molecule and thereby causing the molecule to bind to the antibody.

The antibodies of the present invention also include chimeric antibodies, heterochimeric antibodies, caninized antibodies, felinized antibodies, or equinized antibodies, as well as fragments, portions, peptides, or derivatives thereof, obtained by any known technique, such as, without limitation, enzymatic cleavage, peptide synthesis, or recombinant methods. Such antibodies of the present invention are capable of specifically binding to at least one of canine IL-31 or feline IL-31. Fragments or portions of antibodies may lack the Fc fragment of an intact antibody and are subject to faster clearance from circulation and may have less non-specific tissue binding compared to intact antibody. Examples of antibody fragments can be fragments prepared from intact antibodies using methods well known in the art, such as proteolytic cleavage with enzymes such as papain (to give Fab fragments) or pepsin (to give P(ab') ₂ fragments). ). See, for example, Wahl et al., 24 J. Nucl. Med. 316-25 (1983). Parts of antibodies can be obtained by any of the above methods or can be obtained by expression of a part of the recombinant molecule. For example, the CDR region(s) of a recombinant antibody can be isolated and subcloned into a suitable expression vector. See, for example, US Pat. No. 6,680,053.

The terms "antigen-binding region", "antigen-binding fragment" and the like, as used in this description and claims, refer to that part of the antibody molecule that contains amino acid residues that interact with the antigen and give the antibody its specificity and affinity for the antigen. The binding region of an antibody contains "framework" amino acid residues necessary to maintain the correct conformation of the antigen-binding residues. An antigen-binding fragment of an antibody of the present invention may alternatively be referred to herein, for example, as an IL-31 specific peptide or polypeptide or an anti-IL-31 peptide or polypeptide.

Nucleotide and amino acid sequences of clones 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 and 06A09

In some embodiments, the present invention provides novel monoclonal antibodies that specifically bind to at least one of canine IL-31, feline IL-31, or equine IL-31. In one embodiment, the monoclonal antibody of the invention binds to canine IL-31, feline IL-31, or equine IL-31 and prevents them from binding to their co-receptor complexes containing the IL-31 receptor A (IL-31Ra) and the oncostatin-specific receptor. M (OsmR or IL-31Rb), and their activation of these complexes. The monoclonal antibodies of the present invention are referred to herein as "15H05", "ZIL1", "ZIL8", "ZIL9", "ZIL11", "ZIL69", "ZIL94", "ZIL154", "ZIL159", "ZIL171", "04H07 " and "06A09", which indicates the numbers assigned to their clones. Here "15H05", "ZIL1", "ZIL8", "ZIL9", "ZIL11", "ZIL69", "ZIL94", "ZIL154", "ZIL159", "ZIL171", "04H07" and "06A09" also refer to to the monoclonal antibody portion, paratope, or CDR that specifically binds to IL-31 epitopes designated 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, and 06A09 due to their ability to bind to 15H05 antibodies , ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 and 06A09, respectively. The same names can be used to refer to a number of recombinant, chimeric, heterochimeric, caninized, felinized, equinized, fully canine, fully feline and/or fully equine forms of 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 and 06A09 described here. In some embodiments, 15H05 may alternatively be referred to here as 1505, at least in view of the fact that they have the same CDR.

In one embodiment, the present invention provides a monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to a region of a mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor, wherein the binding of said antibody to said region is affected by mutations in the binding region of the 15H05 epitope. selected from at least one of the following: (a) a region approximately between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (Feline_IL31_wildtype); (b) regions approximately between amino acid residues 124 and 135 of the canine IL-31 sequence shown in SEQ ID NO: 155 (Canine_IL31); and (c) regions approximately between amino acid residues 118 and 129 of the equine IL-31 sequence shown in SEQ ID NO: 165 (Equine_IL31). In one embodiment, mutations in the binding region of the 15H05 epitope are selected from at least one of the following: (a) a mutant where positions 126 and 128 of SEQ ID NO: 157 are replaced by alanine; (b) a mutant where positions 126 and 128 of SEQ ID NO: 155 are replaced by alanine; and (c) a mutant where positions 120 and 122 of SEQ ID NO: 165 are changed to alanine.

In one specific embodiment, an antibody of the present invention binds to the 15H05 epitope binding region described above. That is, in one embodiment, the present invention provides a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to a region of a mammalian IL-31 protein involved in the interaction of the IL-31 protein with its co-receptor, wherein the binding region is selected from at least one of the following: (a) regions approximately between amino acid residues 124 and 135 of the feline IL-31 sequence shown in SEQ ID NO: 157 (Feline_IL31_wildtype); (b) regions approximately between amino acid residues 124 and 135 of the canine IL-31 sequence shown in SEQ ID NO: 155 (Canine_IL31); and (c) regions approximately between amino acid residues 118 and 129 of the equine IL-31 sequence shown in SEQ ID NO: 165 (Equine_IL31).

In one embodiment, the mammalian IL-31 to which the antibody or antigen-binding fragment specifically binds is feline IL-31, wherein the antibody binds to a region approximately between amino acid residues 125 and 134 of the feline IL-31 sequence shown in SEQ ID NO: 157 (Feline_IL3_wildtype). In some embodiments, the antibody that binds to feline IL-31 contains a VL chain containing changes in the frame region 2 (FW2) selected from the following: asparagine instead of lysine at position 42, isoleucine instead of valine at position 43, valine instead of leucine at position 46 , asparagine instead of lysine at position 49, and combinations thereof, where the positions correspond to the numbering of SEQ ID NO: 127 (FEL_15H05V_L1).

In one embodiment, the monoclonal antibody, or antigen-binding fragment thereof, contains the following combinations of hypervariable region (CDR) sequences:

1) 15H05 antibody:

2) variant (1) differing from the original 15H05 antibody by the addition, deletion and/or substitution of one or more amino acid residues in at least one of CDR1, CDR2 or CDR3 VH or VL.

In one embodiment, the 15H05 antibody described above contains at least one of the following heavy and/or light chain variable regions:

a) a light chain variable region containing FEL_15H05_VL1_FW2:

b) heavy chain variable region containing FEL_15H05_VH1:

In another embodiment, the monoclonal antibody, or antigen-binding fragment thereof, contains the following combinations of hypervariable region (CDR) sequences:

1) ZIL1 antibody: Heavy chain variable region (VH) CDR1 (VH-CDR1)

2) ZIL8 antibody:

3) ZIL9 antibody:

4) ZIL11 antibody:

5) ZIL69 antibody:

6) ZIL94 antibody:

7) ZIL15 4 antibody:

8) ZIL159 antibody:

9) ZIL171 antibody:

10) 04H07 antibody:

11) antibody 06A09:

12) variant (1)-(11) that differs from the corresponding parent antibody ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more amino acids residue in at least one of the CDR1, CDR2 or CDR3 VH or VL.

In some embodiments of the present invention:

1) the ZIL1 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL_1VL:

b) heavy chain variable region containing CAN-ZIL1_VH:

2) the ZIL8 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL8_VL:

b) heavy chain variable region containing CAN-ZIL8-VH:

a ZIL8 antibody contains at least one of the following:

c) a light chain variable region containing ZTS_5864_VL:

d) heavy chain variable region containing ZTS 5864 VH:

a ZIL8 antibody contains at least one of the following:

e) light chain variable region containing ZTS_5865_VL:

f) heavy chain variable region containing ZTS_5865_VH:

3) the ZIL9 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL9_VL:

b) heavy chain variable region containing CAN-ZIL9_VH:

4) the ZIL11 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL11_VL:

b) heavy chain variable region containing CAN-ZIL11_VH:

5) the ZIL69 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL69_VL:

b) heavy chain variable region containing CAN-ZIL69_VH:

6) the ZIL94 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL94_VL:

b) heavy chain variable region containing CAN-ZIL94_VH:

7) the ZIL154 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL154_VL:

b) heavy chain variable region containing CAN-ZIL154_VH:

8) the ZIL159 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL159_VL:

b) heavy chain variable region containing CAN-ZIL159_VH:

9) the ZIL171 antibody contains at least one of the following:

a) light chain variable region containing CAN-ZIL171VL:

b) heavy chain variable region containing CAN-ZIL_171_VH:

10) the 04H07 antibody contains at least one of the following:

a) light chain variable region containing Mu_04H07_VL:

b) heavy chain variable region containing Mu_04H07_VH:

11) the 06A09 antibody contains at least one of the following:

a) light chain variable region containing Mu_06A09_VL:

b) heavy chain variable region containing Mu_06A09_VH:

In other embodiments, the invention provides a host cell that produces an antibody as described above.

The scope of the present invention also includes nucleotide sequences encoding the variable regions of the light and heavy chains of the anti-IL-31 antibody of the present invention. The scope of the invention also includes any nucleotide sequence encoding the amino acid sequence 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07, 06A09 or their polypeptides or peptides specific for IL-31.

In some embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the following combinations of heavy chain variable region hypervariable region (CDR) sequences:

1) 15H05: Heavy chain variable region CDR1

2)

3) ZIL8:

4) ZIL9:

5) ZIL11:

6) ZIL69:

7) ZIL94:

8) ZIL154:

9) ZIL159:

10) ZIL171:

11) 04H07:

или12) 06A09:

or

13) variant (1)-(12) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more than one amino acid residue in at least one of CDR1, CDR2 or CDR3_VH.

In one embodiment, the isolated nucleic acid described above may further comprise a nucleic acid sequence encoding at least one of the following combinations of light chain variable region hypervariable region (CDR) sequences:

1) 15H05:

2) ZIL1:

3) ZIL8:

4) ZIL9:

5) ZIL11:

6) ZIL69:

7) ZIL94:

8) ZIL154:

9) ZIL159:

10) ZIL171:

11) 04H07:

или12) 06A09:

or

13) variant (1)-(12) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more than one amino acid residue in at least one of CDR1, CDR2 or CDR3 VH or VL.

In one embodiment, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding at least one of the following combinations of light chain variable region hypervariable region (CDR) sequences:

1) 15H05:

2) ZIL1:

3) ZIL8:

4) ZIL9:

5) ZIL11:

6) ZIL69:

7) ZIL94:

8) ZIL154:

9) ZIL159:

10) ZIL171:

11) 04H07:

или12) 06A09:

or

13) variant (1)-(12) differing from the CDR of the corresponding parent antibody 15H05, ZIL1, ZIL8, ZIL9, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, ZIL171, 04H07 or 06A09 by the addition, deletion and/or substitution of one or more than one amino acid residue in at least one of CDR1, CDR2 or CDR3 VL.

In addition, according to the present invention, a vector containing at least one of the nucleic acids described above is provided.

As will be described in more detail below, a nucleic acid sequence encoding at least one of the above-described combinations of heavy chain variable region hypervariable region (CDR) sequences may be present in the same vector as a nucleic acid sequence encoding at least one of the above-described combinations of light chain variable region CDR sequences. Alternatively, a nucleic acid sequence encoding at least one of the above-described combinations of light chain variable CDR sequences and a nucleic acid sequence encoding at least one of the above-described combinations of heavy chain variable region CDR sequences may be present in different vectors.

Due to the degeneracy of the genetic code, more than one codon may be used to code for a particular amino acid. Using the genetic code, one or more different nucleotide sequences can be determined, each of which will be able to encode a specified amino acid. The likelihood that a particular oligonucleotide will indeed constitute the actual coding sequence for XXX can be assessed by considering patterns of abnormal base pairing and the frequency of actual use of a particular codon (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing anti-IL-31 antibody or a portion thereof. specific for IL-31. Such "codon rules" are disclosed by Lathe, et al., 183 J. Molec. Biol. 1-12 (1985). Using Lathe's "codon usage rules", a single nucleotide sequence or group of nucleotide sequences can be determined containing a theoretical "most likely" nucleotide sequence capable of encoding anti-IL-31 sequences. Furthermore, it is contemplated that antibody coding regions for use in the present invention may also be provided by modifying existing antibody genes using standard molecular biology techniques resulting in the production of antibody variants (agonists) and peptides described herein. Such variants include, without limitation, deletions, additions, and substitutions in the amino acid sequence of anti-IL-31 antibodies or polypeptides or peptides specific for IL-31, including CDR regions of an antibody. For example, residues that are not critical for antigen binding located in the CDRs or other regions of the antibody can be replaced. Examples of the types of experiments used to assess whether particular residues are critical for antigen binding are described in Section 1.21 of the Examples section below. In one embodiment, one or more of the substitutions are conservative amino acid substitutions, as described in more detail herein. However, the antibody variants of the present invention, including CDR variants, are not limited to conservative amino acid substitutions.

For example, one class of substitutions are conservative amino acid substitutions. Such substitutions are substitutions of a given amino acid in an anti-IL-31 antibody peptide with another amino acid with similar characteristics. Substitutions of one amino acid (residue) for another amino acid (residue) within the following groups are usually considered conservative: aliphatic amino acids Ala, Val, Leu and Ile; hydroxyl residues Ser and Thr; acid residues Asp and Glu; amide residues Asn and Gln; basic residues Lys and Arg, aromatic residues Phe, Tyr; etc. For information on which amino acid changes are not likely to result in phenotypic changes, see Bowie et al., 247 Science 1306-10 (1990).

A variant or agonist of anti-IL-31 antibodies or polypeptides or peptides specific for IL-31 may be fully functional or may lack one or more functional activities. Fully functional variants usually contain only conservative changes or changes in residues or areas that are not critical. Functional variants may also contain similar amino acid substitutions resulting in little or no functional change. Alternatively, such substitutions may affect functions in a positive or negative way to some extent. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions or truncations, or substitutions, insertions, inversions or deletions of a critical residue or critical region.

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

Moreover, polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, formation disulfide bonds, demethylation, covalent cross-linking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids to proteins, mediated by transfer RNAs such as arginylation, and ubiquitination.

Such modifications are well known to those skilled in the art and are described in detail in the scientific literature. Some particularly common modifications, such as, for example, glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in most seminal publications such as Proteins-Structure and Molecular Properties (2nd ed., T E. Creighton, W.H. Freeman & Co., NY, 1993). There are many detailed reviews on this topic, such as Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. 182 Meth. Enzymol. 626-46 (1990) and Rattan et al. 663 Ann. NY Acad. sci. 48-62 (1992).

Accordingly, the antibodies, polypeptides and peptides specific for IL-31 of the present invention also encompass derivatives or analogs in which the substituting amino acid residue is different from the residues encoded by the genetic code.

Similarly, the additions and substitutions in the amino acid sequence, as well as the variations and modifications described above, may be equally applicable to the amino acid sequence of an IL-31 antigen and/or epitope or peptides thereof and are thus included in the present invention. As indicated above, the genes encoding the monoclonal antibody of the present invention are particularly effective in recognizing IL-31.

Antibody derivatives

The scope of this invention includes antibody derivatives. A "derivative" of an antibody contains additional chemical moieties that are not normally part of the protein. The scope of this invention includes covalent modifications of proteins. Such modifications can be introduced into the molecule through the interaction of the target amino acid residues of the antibody with an organic derivatizing agent capable of interacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents, well known in the art, is useful for crosslinking an antibody or fragment to a water insoluble support matrix or other macromolecular carriers.

Derivatives also include: radiolabeled monoclonal antibodies labeled, for example, with radioactive iodine ( ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), indium ( ¹¹¹ In, tritium ( ³ H) or the like; conjugates monoclonal antibodies with biotin or avidin, with enzymes such as horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase; and conjugates of monoclonal antibodies with bioluminescent agents (such as luciferase), chemiluminescent agents (such as acridine esters), or fluorescent agents (such as phycobiliproteins).

Another bifunctional antibody derivative of the present invention is a bispecific antibody obtained by combining parts of two separate antibodies recognizing two different antigenic groups. This can be achieved by cross-linking or recombinant techniques. In addition, moieties may be added to the antibody or portion thereof to increase the in vivo half-life (eg, increasing clearance time from the circulation). Such techniques include, for example, the addition of PEG moieties (also referred to as PEGylation) and are well known in the art. See US Patent Application Publication No. 20030031671.

Recombinant expression of antibodies

In some embodiments, nucleic acids encoding the corresponding monoclonal antibody are introduced directly into the host cell and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. Typically, after the appropriate nucleic acids have been introduced into the cell, the cell is incubated, typically at 37° C., with occasional selection, for a period of approximately 1-24 hours to allow expression of the antibody. In one embodiment, the antibody is secreted into the supernatant of the medium in which the cell is grown.

Traditionally, monoclonal antibodies are obtained in the form of native molecules in mouse hybridoma lines. In addition to this technology, according to the present invention proposed recombinant expression of DNA monoclonal antibodies. This allows caninized, felinized, equinized, all-canine, all-feline, and all-equine antibodies, as well as a range of antibody derivatives and fusion proteins, to be obtained from a selected host species.

A nucleic acid sequence encoding at least one anti-IL-31 antibody, portion or polypeptide of the present invention can be recombined with vector DNA according to conventional techniques, including blunt ends or stepped ends for ligation, digestion with restriction enzymes to obtain suitable ends, appropriate filling of sticky finally, treatment with alkaline phosphatase to avoid undesirable coupling and ligation with suitable ligases. Techniques for performing such manipulations are disclosed, for example, in Maniatis et al., MOLECULAR CLONING, LAB. MANUAL (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al. 1993 supra and can be used to construct nucleic acid sequences encoding a monoclonal antibody molecule or its antigen-binding region.

A nucleic acid molecule, such as DNA, is considered "capable of expressing" a polypeptide if it contains nucleotide sequences containing information necessary for the regulation of transcription and translation, and such sequences are "operably linked" to the nucleotide sequences encoding the polypeptide. A functional linkage is a linkage in which regulatory DNA sequences and a DNA sequence to be expressed are linked in a manner that allows the gene to be expressed as anti-IL-31 peptides or antibody portions in recoverable amounts. The specific nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in the art. See, for example, Sambrook et al., 2001 supra; Ausubel et al., 1993 supra.

Accordingly, the present invention encompasses the expression of an anti-IL-31 antibody or a peptide or polypeptide specific for IL-31 in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including bacterial, yeast, insect, fungal, avian, and mammalian cells in vivo or in situ, or host cells derived from mammalian, insect, avian, or yeast origin. The mammalian cell or tissue may be of human, primate, hamster, rabbit, rodent, bovine, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used.

In one embodiment, the nucleotide sequence to be introduced will be incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. For this purpose, any of a wide range of vectors can be used. See, for example, Ausubel et al., 1993 supra. Factors important for the selection of a particular plasmid or viral vector include: ease of recognition and selection of recipient cells containing the vector from those recipient cells that do not contain the vector; the desired number of copies of the vector in a particular host; and the desirability of being able to transfer the vector between host cells of different species.

Examples of prokaryotic vectors known in the art include plasmids such as plasmids capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, .pi.VX). Such plasmids are disclosed, for example, in Maniatis et al., 1989 supra; Ausubel et al., 1993 supra. Bacillus plasmids include pC194, pC221, pT127 and so on. Such plasmids are disclosed in Gryczan, in THE MOLEC. bio. OF THE BACILLI 307-329 (Academic Press, NY, 1982). Suitable Streptomyces plasmids include pIJ101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)) and Streptomyces bacteriophages such as phi.C31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45 -54 (Akademiai Kaido, Budapest, Hungary 1986)). An overview of Pseudomonas plasmids is provided in John et at, 8 Rev. Infect. Dis. 693-704 (1986), Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978), and Ausubel et al., 1993 supra.

Alternatively, gene expression elements useful for the expression of cDNA encoding anti-IL-31 antibodies or peptides include, without limitation: (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl. Acad. Sci., USA 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites, such as regions and sites derived from the late region of SV40 (Okayarea et al., MCB, 3: 280 (1983); and (c) polyadenylation sites, such as in SV40 (Okayama et al., 1983, supra).

Immunoglobulin cDNA genes can be expressed as described by Weidle et al., 51(1) Gene 21-29 (1987), using, as expression elements, the SV40 early promoter and its enhancer, mouse immunoglobulin H chain promoter enhancers, late splicing region of SV40 mRNA, rabbit S-globin intercalation sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements.

For immunoglobulin genes that are part cDNA, part genomic DNA (Whittle et al., 1 Protein Engin. 499-505 (1987)), the transcriptional promoter can be the human cytomegalovirus promoter, the promoter enhancers can be the cytomegalovirus and murine/human immunoglobulin enhancers , and mRNA splicing and polyadenylation regions may be native immunoglobulin chromosomal sequences.

In one embodiment, for expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are a mouse immunoglobulin heavy chain enhancer and/or a viral LTR enhancer, the splicing region contains an intron larger than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the synthesized immunoglobulin chain. In other embodiments, cDNA sequences encoding other proteins are combined with the above expression elements to allow expression of the proteins in mammalian cells.

Each fused gene can be assembled into an expression vector or introduced into an expression vector. Recipient cells capable of expressing the chimeric immunoglobulin chain gene are then transfected with a single gene encoding an anti-IL-31 peptide or chimeric H chain or chimeric L chain, or co-transfected with chimeric H chain and chimeric L chain genes. The transfected recipient cells are cultured under conditions allowing the introduced genes to be expressed, and the expressed immunoglobulin chains, intact antibodies or fragments are recovered from the culture.

In one embodiment, fusion genes encoding an anti-IL-31 peptide, or chimeric H and L chains, or portions thereof, are assembled into separate expression vectors, which are then used to co-transfect a recipient cell. Alternatively, the fusion genes encoding the chimeric H and L chains can be assembled on the same expression vector.

The recipient cell line for transfection with expression vectors and production of the chimeric antibody may be myeloma cells. Myeloma cells can provide the synthesis, assembly and secretion of immunoglobulins encoded by the immunoglobulin genes used in transfection, and have a mechanism for immunoglobulin glycosylation. Myeloma cells can be grown in culture or in the mouse peritoneum, in which case the secreted immunoglobulin can be obtained from ascitic fluid. Other suitable recipient cells include lymphoid cells such as B-lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells.

An expression vector containing a chimeric, caninized, felinized, equinized, fully canine, fully feline, or fully equine anti-IL-31 antibody construct, or a polypeptide or peptide specific for IL-31 (e.g., the antigen-binding portion of an antibody described herein), according to the present of the invention may be introduced into an appropriate host cell by any of a variety of suitable methods, including biochemical methods such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and the use of polycations such as diethylaminoethyldextran (DEAE-dextran), and such mechanical methods such as electroporation, direct microinjection and microparticle bombardment. Johnston et al., 240 Science 1538-1541 (1988).

Yeast can provide significant advantages over bacteria in the production of H- and L-chains of immunoglobulins. In yeast, post-translational modifications of peptides occur, including glycosylation. There are now a number of recombinant DNA techniques that use strong promoter sequences and high copy number plasmids that can be used to generate desired proteins in yeast. Yeasts recognize the leader sequences of cloned mammalian gene products and secrete peptides containing the leader sequences (ie, prepeptides). Hitzman et al., 11th Int'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

Yeast gene expression systems can be routinely assessed for levels of production, secretion, and stability of anti-IL-31 peptides, antibody, and assembled murine and chimeric, heterochimeric, caninized, felinized, equinized, wholly canine, wholly feline, or wholly equine antibodies, fragments and regions thereof. . Any of a number of yeast gene expression systems can be used, including promoter and termination elements of actively expressed genes encoding glycolytic enzymes produced in large quantities when the yeast is grown in high glucose media. Known glycolytic genes may also provide very effective transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be used. Several techniques can be used to evaluate the optimal expression of plasmids for expression of cloned immunoglobulin cDNA in yeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK (1985).

Bacterial strains can also be used as hosts for obtaining the antibody molecules or peptides described in this invention. Together with these bacterial hosts, plasmid vectors containing replicon and control sequences derived from a species compatible with the host cell are used. The vector contains a replication site, as well as certain genes capable of providing phenotypic selection of transformed cells. Several techniques can be used to evaluate the expression of plasmids to generate murine, chimeric, heterochimeric, caninized, felinized, equinized, fully canine, fully feline, or fully equine antibodies, antibody fragments and regions or chains encoded by cloned immunoglobulin cDNA, in bacteria, several techniques can be applied (see below). Glover 1985 supra Ausubel 1993 supra Sambrook 2001 supra Colligan et al., eds. Current Protocols in Immunology, John Wiley & Sons, NY, NY (1994-2001) Colligan et al., eds. Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001)).

Mammalian host cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including removal of leader peptides, folding and assembly of H and L chains, glycosylation of antibody molecules, and secretion of functional antibody proteins.

Mammalian cells that may be useful as hosts for the production of antibody proteins, in addition to cells of lymphoid origin described above, include cells derived from fibroblasts such as Vero cells (ATCC CRL 81) or CHO-K1 (ATCC CRL 61).

A variety of vector systems are available for expression of cloned anti-IL-31 H and L chain peptide genes in mammalian cells (see Glover, 1985 supra). Various techniques can be used to obtain complete H ₂ L ₂ antibodies. Simultaneous expression of H and L chains in the same cells is possible, allowing intracellular association and binding of H and L chains to be achieved with the formation of complete tetrameric H ₂ L ₂ antibodies and/or peptides against IL-31. Simultaneous expression is possible using the same or different plasmids in the same host. The genes for the H and L chains and/or anti-IL-31 peptides can be placed on the same plasmid, which is then transfected into cells, thus directly selecting cells expressing both chains. Alternatively, cells can first be transfected with a single chain coding plasmid, eg an L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker. Cell lines producing anti-IL-31 peptides and/or H ₂ L ₂ molecules by any of the above methods can be transfected with plasmids encoding additional copies of the peptides, H-, L- or H- and L-chains in combination with additional selectable markers , to obtain cell lines with improved properties, such as increased production of assembled H ₂ L ₂ antibody molecules or increased stability of transfected cell lines.

For long-term production of high yield recombinant antibodies, stable expression can be used. For example, cell lines can be obtained that stably express an antibody molecule. Instead of using expression vectors containing viral replicators, host cells can be transformed using immunoglobulin expression cassettes and a selectable marker. Cells obtained after the introduction of foreign DNA can be grown for 1-2 days in an enriched medium and then transferred to a selective medium. The selectable marker in the recombinant plasmid confers selection resistance and allows cells to stably integrate the plasmid into the chromosome and grow to form foci, which in turn can be cloned and propagated into cell lines. Cell lines thus obtained may be particularly useful in screening and evaluating compounds/components that interact directly or indirectly with an antibody molecule.

Once an antibody of the invention has been produced, it can be purified by any method of purification of immunoglobulin molecules known in the art, such as chromatography (e.g., ion exchange, affinity, especially affinity chromatography using a specific antigen after protein A, and gel filtration on columns), centrifugation , by differential solubility, or by any other standard protein purification technique. Many embodiments involve the secretion of antibodies into the culture medium and their isolation from the culture medium.

Pharmaceutical applications

Anti-IL-31 antibodies or IL-31 specific polypeptides or peptides of the present invention can be used, for example, in the treatment of pruritic and/or allergic conditions in domestic animals such as dogs, cats and horses. In one embodiment, such polypeptides or peptides comprise an antigen-binding fragment of the anti-IL-31 antibodies described herein. More specifically, the invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, an antibody or polypeptide or peptide of the invention. An antibody may be a chimeric, heterochimeric, caninized, felinized, equinized, fully equine, fully feline, or fully equine antibody of the present invention. Intact immunoglobulins or binding fragments thereof, such as Fab, are also contemplated. The antibody and its pharmaceutical compositions according to this invention can be administered parenterally, for example, subcutaneously, intramuscularly or intravenously.

Anti-IL-31 antibodies and/or IL-31 specific polypeptides and/or IL-31 specific peptides of the present invention may be administered as single therapeutic agents or in combination with other therapeutic agents. They may be administered alone, but are usually administered with a pharmaceutical carrier selected based on the route of administration chosen and standard pharmaceutical practice.

Administration of the antibodies disclosed herein may be by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous or intramuscular injection), oral or topical administration of antibodies (usually in a pharmaceutical composition) to the surface of the respiratory tract. Topical administration to the surface of the respiratory tract may be by intranasal administration (eg, using a dropper, swab, or inhaler). Topical administration of antibodies to the surface of the respiratory tract can also be accomplished by inhalation administration, such as preparing respirable particles of a pharmaceutical composition (including both solid and liquid particles) containing antibodies in the form of an aerosol suspension, followed by inhalation of these respirable particles. subject. Methods and apparatus for administering respirable particles of pharmaceutical compositions are well known and any conventional technique may be employed. Oral administration may be, for example, in the form of a liquid or solid composition suitable for oral administration.

In some desirable embodiments, the antibodies are administered by parenteral injection. For parenteral administration, anti-IL-31 antibodies or polypeptides or peptides may be formulated as a solution, suspension, emulsion, or lyophilized powder, together with a pharmaceutically acceptable parenteral vehicle. For example, the excipient may be an antibody solution or mixture dissolved in a suitable carrier such as an aqueous carrier; such excipients are water, saline, Ringer's solution, dextrose solution, trehalose or sucrose solution, or 5% serum albumin, 0.4% saline, 0.3% glycine, and the like. Liposomes and non-aqueous vehicles such as fixed oils can also be used. These solutions are sterile and usually free of particulate matter. These compositions can be sterilized by conventional and well known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients necessary to create conditions close to physiological, such as agents for adjusting pH and buffering agents, agents for correcting tonicity and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, lactate sodium and so on. The concentration of antibody in these compositions can vary widely, for example, from less than about 0.5%, typically from 1% or at least about 1%, to 15% or 20% by weight, and will be selected based on the first a queue of fluid volumes, viscosities, and so on, according to the particular mode of administration chosen. The filler or lyophilized powder may contain additives that maintain isotonicity (eg sodium chloride, mannitol) and chemical stability (eg buffers and preservatives). The composition is sterilized by commonly used techniques.

Existing methods for making parenterally administered compositions will be known or apparent to those skilled in the art and are described in more detail, for example, in REMINGTON'S PHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

The antibodies of this invention may be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques may be used. It will be appreciated by those skilled in the art that lyophilization and reconstitution may result in varying degrees of loss of antibody activity and that this may necessitate adjustments in the applied levels.

Compositions containing the present antibodies, or a mixture thereof, may be administered to prevent recurrence and/or therapeutic treatment of an existing disease. Suitable pharmaceutical carriers are described in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES, the standard guide in the art.

In therapeutic use, the compositions are administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially stop the development or alleviate the disease and its complications. An amount adequate to achieve this is defined as a "therapeutically effective dose" or "therapeutically effective amount". Amounts effective for such use will depend on the severity of the disease and the general state of the subject's own immune system, but will typically range from about 0.1 mg of antibody per kg of body weight to about 15 mg of antibody per kg of body weight, preferably from about 0 .3 mg of antibody per kg of body weight to about 12 mg of antibody per kg of body weight. In one embodiment, a therapeutically effective amount will provide efficacy for at least one month at a dose of up to 12 mg/kg of body weight. The minimal presence of foreign substances and the reduction in the likelihood of "foreign substance" rejection achieved by the presence of true antibodies, such as canine, feline and equine antibodies, can allow these antibodies to be administered in substantial excess.

Of course, the dose administered will vary depending on known factors such as the pharmacodynamic characteristics of the particular agent, as well as the mode and route of its administration, the age, health status and body weight of the recipient, the nature and severity of symptoms, concomitant treatment, frequency of administration and the desired effect.

As a non-limiting example, treatment of IL-31 mediated pathology in dogs, cats, or horses may be in the form of biweekly or once monthly administration of the anti-IL-31 antibodies of the present invention in the dosage range described above.

Examples of antibodies for therapeutic use in dogs, cats or horses are high affinity (they may also be high avid) antibodies and fragments, regions and derivatives thereof having high activity against IL-31 in vivo of the present invention. Antibody fragments and regions may alternatively be referred to herein as polypeptides or peptides of the present invention containing the antigen-binding portion of an anti-IL-31 antibody.

Single or multiple administration of the compositions may be at dose levels and type selected by the treating veterinarian. In any case, pharmaceutical compositions should provide an amount of the antibody or antibodies of this invention sufficient to effectively treat the subject.

Diagnostic applications

The present invention also provides the above antibodies, polypeptides and/or peptides against IL-31 for use in diagnostic methods for the detection of IL-31 in pets with or suspected to have an pruritic and/or allergic condition.

Anti-IL-31 antibodies, polypeptides and/or peptides of the present invention are useful for immunoassays that detect or quantify IL-31 or anti-IL-31 antibodies in a sample. An immunoassay for IL-31 typically involves incubating a clinical or biological specimen in the presence of a high affinity (or high avidity) anti-IL-31 antibody, polypeptide or peptide of the present invention having a detectable label that can selectively bind to IL-31, and detection of the labeled polypeptide, peptide or antibody bound in the sample. Various clinical assay techniques are well known in the art. See, for example, IMMUNOASSAYS FOR THE 80'S (Voller et al., eds., Univ. Park, 1981). Such samples include tissue biopsy samples, blood, serum and feces or fluids obtained from animal subjects and analyzed by ELISA as described below.

In some embodiments, antigen-antibody binding is detected without the use of a solid support. For example, the binding of an antigen to an antibody can be detected in a liquid format.

In other embodiments, the anti-IL-31 antibody, polypeptide, or peptide may, for example, be fixed to nitrocellulose or other solid support capable of immobilizing cells, cell particles, or soluble proteins. The support can then be washed with suitable buffers followed by treatment with a detectably labeled polypeptide, peptide or antibody specific for IL-31. The solid support can then be washed with buffer a second time to remove unbound polypeptide, peptide or antibody. Then, at known method steps, the amount of bound label on the solid support can be determined.

"Solid support" or "carrier" refers to any support capable of binding a polypeptide, peptide, antigen, or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene fluoride (PVDF), dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. For purposes of the present invention, the carrier must be somewhat soluble or insoluble. The support material may have virtually any possible structural configuration, provided that the associated molecule retains the ability to bind to IL-31 or anti-IL-31 antibody. Thus, the substrate configuration can be spherical, like a bead, or cylindrical, like the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface may be flat, such as a sheet, a culture dish, a test strip, and so on. For example, the substrates may contain polystyrene beads. Many other suitable carriers for binding an antibody, polypeptide, peptide, or antigen will be known to those skilled in the art, or they will be able to determine such carriers through routine experimentation.

In well known method steps, the binding activity of a given batch of polypeptide, peptide and/or anti-IL-31 antibody can be determined. Those skilled in the art can determine the optimal operating conditions for analysis through routine experimentation.

Labeling of a polypeptide, peptide and/or antibody specific for IL-31 with a detectable label can be accomplished by binding them to an enzyme for use in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The bound enzyme interacts with the available substrate, leading to the formation of a chemical group, which can be detected, for example, spectrophotometric, fluorimetric or visual methods. Enzymes that can be used to label antibodies specific for IL-31 of the present invention with a detectable label include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

Radiolabeling of antibodies specific for IL-31 allows the detection of IL-31 using radioimmunoassay (RIA). See Work et al., LAB. TECHNIQUES & BIOCHEM. IN MOLEC. Bio. (No. Holland Pub. Co., NY, 1978). The radioactive isotope can be detected by methods such as using a gamma counter or scintillation counter, or by autoradiography. Isotopes particularly useful for the purposes of the present invention include ³ H, ¹²⁵ I, ¹³¹ I, ³⁵ S, ¹⁴ C, and ¹²⁵ I.

It is also possible to label antibodies specific for IL-31 with a fluorescent compound. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. The compounds most commonly used for fluorescent labeling are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthalaldehyde, and fluorescamine.

Labeling of antibodies specific for IL-31 with a detectable label can also be done using fluorescent metals such as ¹²⁵ Eu or other lanthanide metals. These metals can be attached to an IL-31 specific antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In addition, labeling of antibodies specific for IL-31 with a detectable label can be carried out in combination with a chemiluminescent compound. The presence of a chemiluminescently labeled antibody is then determined by detecting the luminescence resulting from the chemical interaction. Examples of compounds useful for chemiluminescent labeling are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalic acid ester.

Similarly, a bioluminescent compound can be used to label an antibody, portion, fragment, polypeptide or derivative specific for IL-31 of the present invention. Bioluminescence is a type of chemiluminescence in biological systems where the efficiency of the chemiluminescent interaction is enhanced by a catalytic protein. The presence of a bioluminescent protein is determined by detecting luminescence. Important bioluminescent compounds used for labeling are luciferin, luciferase and aequorin.

Detection of an antibody, portion, fragment, polypeptide, or derivative specific for IL-31 can be performed with a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or with a fluorometer, for example, if the label is a fluorescent substance. In the case of an enzyme label, detection can be carried out by colorimetric methods using the substrate of the respective enzyme. Detection can also be carried out by visual comparison of the degree of enzymatic interaction of the substrate with standards prepared in a similar way.

For purposes of the present invention, IL-31 as detected by the above assays may be present in a biological sample. Any sample containing IL-31 can be used. For example, the sample is a biological fluid such as, for example, blood, serum, lymph, urine, feces, inflammatory exudate, cerebrospinal fluid, amniotic fluid, tissue extract or homogenate, and the like. The invention is not limited to analyzes using only these samples, however, in light of the present description, a person skilled in the art will be able to determine the appropriate conditions to allow the use of other samples.

In situ detection can be performed by obtaining a histological sample from an animal subject and delivering a combination of labeled antibodies of the present invention to such a sample. An antibody (or portion thereof) can be delivered by applying or overlaying a labeled antibody (or portion thereof) onto a biological sample. Using this technique, it is possible to determine not only the presence of IL-31, but also the distribution of IL-31 in the analyzed tissue. In the practice of the present invention, one skilled in the art will recognize that any of a wide variety of histological methods (such as staining techniques) can be modified to perform such in situ detection.

An antibody, fragment or derivative of the present invention may be adapted for use in an immunometric assay, also known as a "two site" or "sandwich" assay. A typical immunometric assay involves binding a specified amount of unlabeled antibody (or antibody fragment) to a solid support that is insoluble in the fluid being analyzed, adding a specified amount of soluble antibody having a detectable label, and/or determining the amount of the ternary complex formed by the antibody fixed on the solid phase , antigen and labeled antibody.

The antibodies can be used to detect IL-31 quantitatively or qualitatively in a sample, or to detect cells expressing IL-31. This can be done by immunofluorescent techniques using a fluorescently labeled antibody (see below) in combination with fluorescence microscopy, flow cytometric or fluorimetric detection. For diagnostic purposes, antibodies may be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (secondary antibodies) capable of interacting with the antibody, such as antibodies specific for canine or feline immunoglobulin constant regions. Alternatively, direct labeling of antibodies is possible. A wide range of labels can be used, such as radionuclides, fluorescent labels, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), and so on. Many types of immunological assays are available, such as those discussed previously, and are well known to those skilled in the art.

In one embodiment, the diagnostic method for detecting IL-31 is a lateral flow immunoassay. It is also known as an immunochromatographic assay, Rapid ImmunoMigration (RIM™) or test strip assay. In essence, lateral flow immunoassays are immunoassays adapted to run along a single axis so that they can be used in a test strip format. A number of variations of this technology have been developed and used in commercial products, but they all operate on the same basic principle. A typical test strip consists of the following components: (1) sample area - an absorbent area onto which the sample to be analyzed is applied; (2) conjugate or reagent zone - this zone contains antibodies specific for the target analyte conjugated to colored particles (usually colloidal gold particles or latex microspheres); (3) reaction membrane - usually a membrane of hydrophobic nitrocellulose or cellulose acetate, on which antibodies against the target analyte are immobilized, located along a line crossing the membrane, forming a capture zone or test line (a control zone may also be present containing antibodies specific to to conjugated antibodies). and (4) a swab or waste reservoir - another absorbent zone designed to move the sample across the membrane for capillary reaction and collection. Typically, the components of the strip are fixed to an inert support material and can be presented in simple test strip format or in a plastic case with a sample port and a reaction window with a capture zone and a control zone.

There are two main types of lateral flow immunoassays used in microbiological studies: dual antibody sandwich assays and competitive assays. In the dual antibody sandwich assay format, the sample moves from the sample zone through the conjugate zone, where any target analyte present in the sample will bind to the conjugate. The sample then continues to move across the membrane until it reaches the capture zone, where the target analyte/conjugate complex will bind to the immobilized antibodies, forming a visible line on the membrane. The sample is then moved further down the strip until it reaches the control zone where excess conjugate will bind to form a second visible line on the membrane. This control line indicates that the sample has traveled across the membrane properly. Two distinct lines on the membrane indicate a positive result. A single line in the control zone indicates a negative result. Competitive assays differ from the two-antibody sandwich assay format in that the conjugate zone contains antibodies that are already bound to the target analyte or its analog. If the target analyte is present in the sample, it will therefore not bind to the conjugate and remain unlabeled. Once the sample has moved across the membrane and reached the capture zone, the excess unlabeled analyte will bind to the immobilized antibody and block the capture of the conjugate, so no visible line will form. The unbound conjugate will then bind to the antibodies in the control zone, forming a visible control line. A single control line on the membrane indicates a positive result. Two visible lines in the capture zone and control zone indicate a negative result. However, if there is no excess of unlabeled target analyte, a faint line may form in the capture zone, indicating an indeterminate result. There are a number of options for lateral flow technology. Depending on the target analyte, the capture zone on the membrane may contain immobilized antigens or enzymes rather than antibodies. It is also possible to use multiple capture zones for multiple analysis. For example, commercial test strips have been developed to detect both E. coli Shiga toxins, ST1 and ST2, separately in the same sample.

Importantly, the antibodies of the present invention may be useful in diagnosing pruritus and/or allergies in dogs, cats, or horses. More specifically, the antibody of the present invention allows to determine the overexpression of IL-31 in pets. Thus, the antibody of the present invention can be an important tool for immunohistochemistry. In one embodiment, provided herein is an assay procedure wherein an IL-31 mimotope (peptide) is used to capture an antibody of the present invention labeled for detection in the assay. The affinity of this captured antibody for the attached mimotope will be lower than its affinity for native circulating IL-31 in the host species. In this embodiment, the host species-derived fluid is incubated with a labeled antibody-mimotope complex bound to a solid surface. IL-31 present in the assay fluid derived from the host species will have a higher affinity for the antibody, thus resulting in the release of the labeled antibody from the solid surface, after which it can be removed during the washing steps. Therefore, the level of IL-31 in the analyzed fluid can be correlated with the signal attenuation that occurs on the surface associated with the mimotope. Such an assay is expected to be useful in measuring IL-31 in research or clinical settings for use as a diagnostic assay.

The antibodies of the present invention can be used in antibody matrices particularly suitable for assessing gene expression profiles.

Sets

The scope of the present invention also includes kits for the practical application of the respective methods. The kits contain at least one or more than one of the antibodies of the present invention, a nucleic acid encoding said antibody/antibodies, or a cell containing said nucleic acid. In one embodiment, an antibody of the present invention may be presented in a container, usually in lyophilized form. Antibodies, which can be conjugated to a label or toxin or be unconjugated, are usually included in kits with buffers such as tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, such as serum albumin, or the like. In most cases, these substances will be present in an amount of less than 5 wt. %, based on the amount of active antibody, and are usually present in a total amount of at least about 0.001 wt. %, again based on antibody concentration. In many cases, it will be desirable to include an inert filler or excipient to dilute the active ingredients, where the excipient may be present in an amount of from about 1% to 99 wt. %. the whole composition. If a secondary antibody capable of binding to the primary antibody is used in the assay, it will usually be provided in a separate vial. The secondary antibody is usually conjugated to a label and included in a composition similar to the antibody compositions described above. In most cases, the kit will also contain instructions for use.

In one embodiment, the kit of the present invention is a test strip kit (lateral flow immunoassay kit) useful for detecting canine, feline or equine IL-31 protein in a sample. Such a test strip typically includes: a sample area onto which the sample to be analyzed is applied; a conjugate or reagent zone containing an antibody specific for canine, feline, or equine IL-31 conjugated to colored particles (usually colloidal gold particles); a membrane for carrying out the reaction, on which antibodies against IL-31 are immobilized, located along a line crossing the membrane, forming a capture zone or test line (a control zone containing antibodies specific for conjugated antibodies may also be present); and another absorbent zone, designed to move the sample across the membrane for reaction under the action of capillary forces and its collection. In most cases, the test strip kit will also contain instructions for use.

Methods for improving the uniformity and/or characteristics of a feline or canine antibody

Such methods are described above in the "Summary of the Invention" section, as well as in the Examples and Drawings of the present application.

The present inventors surprisingly found that deletion or modification of the C-terminus of the kappa light chain constant region of those animal species in which native germline sequences encode additional residues located beyond the terminal light chain cysteine beneficially affects both the production of homogeneous recombinant antibodies of these species. , and on the amount of antibody produced by a stable cell line (for example, increasing the output). The results described here confirm that additional amino acid residues beyond the terminal cysteine of the feline (and canine) kappa light chain reduce the efficiency of pairing with the heavy chain, leading to mismatch and poor antibody production.

In one embodiment, the present invention provides a method for improving the uniformity and/or characteristics of a feline antibody. The method comprises expressing a nucleotide sequence encoding a feline IgG kappa light chain and a nucleotide sequence encoding a feline IgG heavy chain in a host cell to produce a feline antibody, wherein the nucleotide sequence encoding the feline IgG kappa light chain comprises a nucleotide sequence of a light a kappa chain in which the sequence encoding the C-terminal QRE sequence, otherwise present in the wild-type feline IgG kappa light chain constant region, has been modified and/or deleted. Considering the C-terminal amino acid residues of the feline immunoglobulin kappa light chain constant domain shown in FIG. 15, in one embodiment, the present invention proposes the deletion of the C-terminal "QRE" located immediately after the CYS107 feline kappa light chain sequence of SEQ ID NO: 175. This modification was found to improve production of monomeric recombinant feline IgG. However, the present invention is not limited to this modification. For example, even adding three additional consecutive amino acids to the C-terminus of the cysteine at position 107 instead of the native QRE may be acceptable if these amino acids have minimal effect on electrostatic charge.

In addition, the present invention provides a method for improving the uniformity and/or characteristics of a canine antibody. The method comprises expressing a nucleotide sequence encoding a canine IgG kappa light chain and a nucleotide sequence encoding a canine IgG heavy chain in a host cell to produce a canine antibody, wherein the nucleotide sequence encoding the canine IgG kappa light chain comprises a nucleotide sequence of a canine IgG kappa constant region. a kappa chain in which the sequence encoding the C-terminal QRVD sequence, otherwise present in the wild-type canine IgG kappa light chain constant region, has been modified and/or deleted. Considering the C-terminal amino acid residues of the canine immunoglobulin kappa light chain constant domain shown in FIG. 15, in one embodiment, the present invention provides for deletion of the C-terminal "QRVD" located just after the CYS105 sequence of the canine kappa light chain of SEQ ID NO: 194. However, the present invention is not limited to this modification. For example, even adding three additional consecutive amino acids to the C-terminus of the cysteine at position 105 instead of the native QRVD may be acceptable if these amino acids have minimal effect on electrostatic charge.

The results presented here clearly demonstrate that the methods described above are applicable to structurally different antibodies recognizing completely different targets, and therefore it is likely that these modifications will be applicable to a wide range of feline antibodies, including, without limitation, anti-IL-31 and anti-IL-31 antibodies. NGF, as well as other mammalian antibodies having additional C-terminal amino acids in the kappa light chain constant region. Without wishing to be bound by theory, this light chain modification appears to result in more precise pairing of immunoglobulin chains when induced to be produced by stable CHO cell lines, resulting in an increase in monomeric IgG and possibly an increase in overall antibody yield. Both of these results are highly desirable from the point of view of the manufacture of drugs containing antibodies for commercial use.

In one embodiment, any of the anti-IL-31 antibodies disclosed herein may contain deletions and/or modifications to the kappa light chain constant region disclosed herein. For example, in one embodiment, a feline antibody of the present invention comprises a kappa light chain constant region wherein the "QRE" normally present at the C-terminus of the kappa light chain constant region has been removed and optionally replaced with three or less than three additional amino acids that provide minimal effect on electrostatic charge. In one specific embodiment, the feline antibody of the present invention comprises a feline kappa light chain having the sequence RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFQRSEC (SEQ ID NO: 186) or a variant thereof.

The invention will now be described further by way of the non-limiting examples below. In the "Examples" section below and in the drawings, any data presented for antibodies whose designation contains "11E12" is intended for comparison with the antibodies of the present invention.

EXAMPLES

1.1. Production of canine interleukin-31 (cIL-31) from Chinese hamster ovary (CHO) cells

The amino acid sequence conservation of the interleukin-31 protein varies between homologous species (Fig. 1), but its structural architecture is believed to be similar to that of other members of the type I cytokine family (Boulay et al. 2003, Immunity. Aug; 19(2):159 -632003; Dillon et al. 2004 Nat Immunol. Jul; 5(7):752-60). This ascending and descending bundle topology is important for the receptor recognition mechanism shared by these cytokines (Dillon et al. supra, Comelissen et al. 2012 Eur J Cell Biol. Jun-Jul; 91(6-7):552-66). Sequence variability of IL-31 proteins from different species makes it impossible to predict whether antibodies raised against one species will cross-react with other species due to differences in epitope patterns and local amino acid composition. Therefore, in this work, many forms of the IL-31 protein were considered, representing many types and expression systems. Canine IL-31 protein (cIL-31) was prepared for use as an immunogen and reagent for assaying the affinity and activity of selected antibodies. Recombinant cIL-31 was generated in CHO cells using the CHROMOS ACE (Artificial Chromosome Expression) system (Chromos Molecular Systems, Inc., Burnaby, British Columbia) to produce a secreted canine IL-31 protein having the sequence (SEQ ID NO: 155; Canine_IL31 ) to which the nucleotide sequence corresponds (SEQ ID NO: 156; Canine_IL31). A conditioned medium was prepared from 400 ml of cell culture (CHO cell line) and dialyzed against 10 volumes of QA buffer (20 mM Tris, pH 8.0, 20 mM NaCl) for 4.5 hours. After dialysis, the medium was filtered through a 0.2 µm filter and loaded onto a SOURCE™ Q column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with QA buffer at a rate of 1 ml/min. The protein was eluted using a multi-stage linear gradient. Most of the cIL-31 remained in the flow through (FT) fraction, a small amount of cIL-31 was eluted at the beginning of the gradient. The identity of the protein was previously confirmed by Western blotting and mass spectrometry (MS) analysis of trypsin digest products. Protein in the FT fraction was concentrated 4-5 times and dialyzed overnight against phosphate buffered saline (PBS) at 4°C. The stability of the protein was checked after dialysis in PBS. There was no precipitation and proteolysis after several days at 4°C. Deglycosylation experiments using N-glycosidase F resulted in a protein that formed a single band of approximately 15 kDa on SDS-PAGE. Protein concentration was determined using the bicinchonin assay (BCA assay) with bovine serum albumin (BSA) as standard (ThermoFisher Scientific, Inc., Rockford, IL). The resulting protein solution was divided into aliquots, quickly frozen (liquid N ₂ ) and stored at -80°C.

1.2. Transient expression of wild-type feline interleukin-31 (fIL-31) and mutant fIL-31 in CHO cells

To facilitate the identification of antibodies with suitable epitope-binding properties, wild-type feline IL-31 and mutant feline IL-31 proteins were expressed in a mammalian cell expression system for their preparation, purification and evaluation in affinity and cellular assays. The IL-31 antibody 11E12 binding site has been described previously (US Pat. No. 8,790,651 to Bammert, et al.). Characterization of the new binding site on IL-31 recognized by the 15H05 antibody is described here. The designation "wild type" corresponds to the full-length protein of feline IL-31 without changes in native amino acid residues. Mutant proteins were designated by the names of their respective antibodies (11E12 and 15H05), indicating mutations in the amino acids of the IL-31 protein, which (if changed) affect its binding to the corresponding antibody. The determination of suitable mutations required for the 15H05 feline IL-31 protein is described below in Section 1.10. The task was to change the amino acids in the IL-31 epitope and confirm the reduction in binding to the corresponding antibody. Screening can then compare and determine if new candidate antibodies will bind to the wild-type protein but not to the mutant. The selected novel antibodies can then be grouped by binding to the same epitope as the 11E12 or 15H05 antibody or a similar epitope.

We performed codon optimization and synthesis of expression constructs for expression in Chinese hamster ovary (CHO) cells. The synthesized genes were cloned into pD2529 (ATUM vector) for transient expression. The wild-type feline IL-31 protein is shown in (SEQ ID NO: 157; Feline_IL31_wildtype), which corresponds to the nucleotide sequence (SEQ ID NO: 158; Feline_IL31_wildtype). The mutant feline IL-31 11E12 protein is shown in (SEQ ID NO: 161; Feline_IL31_11E12_mutant), which corresponds to the nucleotide sequence (SEQ ID NO: 162; Feline_IL31_11E12_mutant). The 15H05 mutant feline IL-31 protein is shown in (SEQ ID NO: 163; Feline_IL31_15H05_mutant), which corresponds to the nucleotide sequence (SEQ ID NO: 164; Feline_IL31_15H05_mutant). Recombinant feline IL-31 proteins were expressed in ExpiCHO-S™ cells (ThermoFisher Scientific, Inc., Rockford, IL) following the manufacturer's protocol with maximum titer for transient expression in CHO. Twelve days after transfection, cells were centrifuged and filtered to obtain secreted protein in conditioned medium. For each construct (wild-type and mutants), 120 ml of conditioned medium (from CHO cell culture filtered through a 0.2 μm filter) was adjusted to 30 mS/cm with NaCl, 5 mM imidazole and pH 7.4. Each medium sample was combined with 5 ml of HisPur Cobalt resin (ThermoFisher Scientific, Inc., Rockford, IL) pre-equilibrated with 5 mM imidazole, 20 mM sodium phosphate, 300 mM NaCl, pH 7.4. Each sample and resin was allowed to stir at 4° C. overnight. Resins were collected (and separated from the unbound fraction) by passing through BioRad Econcolumns (Bio-Rad, Hercules, CA). The resins were washed using 5×5 ml of buffer (as described above) and then eluted using 5×5 ml of 500 mM imidazole in the same buffer. Fractions were analyzed by SDS-PAGE. The concentration was measured by BCA protein analysis using standard methods.

1.3 Production of feline interleukin-31 (fIL-31) from E. coli

Recombinant feline IL-31 protein was generated in an E. coli expression host for use as an assay reagent and for in vivo challenge studies to induce pruritus in cats. A gene representing feline IL-31 was synthesized for optimal expression in E. coli. Received expression constructs with the gene of the full-length feline IL-31 containing N-terminal 6-His-tag for detection and purification. This feline IL-31 protein is shown in (SEQ ID NO: 159; Feline_IL-31_E_coli), which corresponds to the nucleotide sequence (SEQ ID NO: 160; Feline_IL-31_E_coli). Sequence verified plasmids were used to transform E. coli BL21 DE3 (Invitrogen Corp., Carlsbad, CA) and subsequent protein expression was performed.

Cell paste (262.3 g) from E. coli was lysed as follows. The cell paste was resuspended in 500 ml of 50 mM Tris, pH 8, filtered through a stainless mesh filter to remove particles, and then lysed by double passing through a microfluidizer at 1300 psi (8.963 MPa). The resulting lysate (volume approximately 1200 ml) was divided into four bottles and centrifuged at 12000g for 20 minutes at 10°C. The supernatant was removed by decantation. Each pellet was washed by suspending in 300 ml of 5 mM EDTA, 0.5% Triton X-100, pH 9.0, followed by centrifugation at 12000g for 50 minutes at 10°C. The supernatant was removed by decantation. The washed precipitates were stored at -20° C. until folding and isolation.

Before isolation, one of the precipitates was washed with water to remove residual detergent and then centrifuged at 10,000 g for 20 minutes at 4°C. The supernatant was again decanted. Finally, the washed precipitate was solubilized in 60 ml 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, 5 mM imidazole, pH 7.4. The precipitate was allowed to stir at room temperature for approximately 25 minutes before another centrifugation at 10000g for 20 minutes at 4°C. This time, the supernatant was decanted and stored for further processing. The pellet (resuspended in water to original volume) was left for SDS-PAGE only. Prior to folding, IMAC (Immobilized Metal Affinity Chromatography) was performed on the crude supernatant to increase the purity. In this case, 15 ml of Ni-NTA Superflow (Qiagen Ele, Germantown, MD, catalog # 30450, pre-equilibrated in the same buffer) was added to the clarified supernatant and allowed to stir at room temperature for approximately 90 minutes. The unbound fraction was decanted and left for SDS-PAGE. The IMAC resin was washed with 5 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, pH 7.4 (identical to solubilization buffer). Elution (first with 7.5 ml and then several times with 15 ml with monitoring of protein elution by Bradford analysis) was performed using 200 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine-HCl, pH 7.4. Eluted fractions containing protein (according to Bradford analysis) were pooled (125 ml) for further processing.

IL-31 protein folding was performed as follows. IL-31 was reduced by adding dithiothreitol to a final concentration of 10 mm and allowed to mix at room temperature for 2 hours. Then the diluted sample was added dropwise to 2500 ml (20-fold volume) PBS with 1 M NaCl with rapid stirring. This theoretical concentration of urea should be approximately 0.4 M. The remainder of the urea was removed by slow dialysis against 3 changes of PBS (4 l each) at 4°C during the night. After dialysis, the sample was filtered through a 0.2 μm filter to remove any unfolded or precipitated protein.

The sample was further purified by a second round of IMAC, this time eluting with a linear gradient. Fifteen ml of Ni-NTA Superflow resin was added to each batch of sample and allowed to bind with stirring (submersible magnetic stirrer) overnight at 4°C. The unbound fraction was again decanted and left. Ni-NTA Superflow resin was loaded into an XK16 column (GE Healthcare Lifesciences, Marlborough, MA) and coupled to a proprietary AKTA chromatography system (GE Healthcare Lifesciences, Marlborough, MA). The column was then washed with 50 mM Tris, 300 mM NaCl, pH 8.2, followed by elution with 150 ml linear gradient from 0 to 500 mM imidazole in wash buffer. Fractions were analyzed by SDS-PAGE. Fractions with sufficient purity of IL-31 were pooled and another buffer exchange was performed by dialysis against 3 changes of PBS (2 L each) at 4° C. overnight. Finally, after dialysis, a folded and purified sample was obtained, sterilized by filtration, concentration measured, aliquoted, flash frozen in a dry ice/isopropanol bath and stored at -80°C.

1.4. Method for determining the affinity of antibodies against IL-31 against IL-31 using surface plasmon resonance

The affinity with which mAb candidates bind to feline and canine IL-31 was determined using surface plasmon resonance (SPR) and the Biacore system (Biacore Life Sciences (GE Healthcare), Uppsala, Sweden). To avoid differences in affinity associated with differences in surface preparation, possible when antibodies are immobilized on surfaces, a technique with direct conjugation of IL-31 on the surface was used. Immobilization was performed with an amine coupling of 5 μg/ml IL-31 using N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. The chips were quenched with ethanolamine and the affinity with which all candidate mAbs bound to immobilized IL-31 was assessed. All curves were adapted to the model 1:1. Affinity constants (K _D ) less than 1×10 ^-11 M (1E-11 M) do not reach the lower limit of quantitative determination of the device. The results of the affinity measurement are described here.

1.5. Method for determining the activity of antibodies against IL-31, assessed by the inhibition of pSTAT3 signals induced by canine and feline IL-31, in canine and feline macrophages

To identify candidates with inhibitory activity, antibodies were evaluated for their ability to influence IL-31 mediated STAT3 phosphorylation in a canine and feline cell assay. STAT3 phosphorylation was determined in canine DH-82 macrophage-like cells ( ^ATCC® CRL-10389™) or feline FCWF4 macrophage-like cells (ATCC CRL-2787). DH82 and FCWF4 cells were primed with canine interferon-gamma (R&D Systems, Minneapolis, MN), 10 ng/mL for 24 hours, or feline interferon-gamma (R&D Systems, Minneapolis, MN), 125 ng/mL for 96 hours, respectively, to increase receptor expression. Both types of cells were subjected to serum starvation for 2 hours before treatment with IL-31 and mAb. All mAb candidates were evaluated for their ability to inhibit STAT3 phosphorylation induced by 1 μg/ml canine or 0.2 μg/ml feline IL-31 using two independent methods. Analyzes were also performed to demonstrate the cross-reactivity of canine and feline cytokines and the cross-functionality of the ability of antibodies to inhibit signals in both species. To ensure the formation of the complex before cell stimulation, a one-hour co-incubation of mAb and cytokine IL-31 was performed. Stimulation of IL-31 cells was performed for five minutes. Phosphorylation of STAT3 was measured using AlphaLISA SureFire ULTRA™ technology (Perkin Elmer, Waltham, MA). In cases where antibody concentration and purity were unknown, hybridoma supernatants were quantified for their ability to inhibit STAT3 phosphorylation after 1 hour co-incubation with 1 μg/mL canine or 0.2 μg/mL feline IL-31. The activity of individual monoclonal antibodies, as determined by their ability to inhibit IL-31 mediated STAT3 phosphorylation, in these assays was considered as a key selection criterion for further refinement of selected antibodies. The term "activity" refers to the IC ₅₀ value calculated from the results of these assays, which is the concentration of antibody at which the signals induced by IL-31 are reduced to half of their maximum value. The increased activity described here correlates with a lower IC ₅₀ value.

1.6. Identification of mouse and canine monoclinal antibodies recognizing canine and feline interleukin-31 (IL-31)

To identify antibodies, mice and dogs were immunized with recombinant canine IL-31 (SEQ ID NO: 155). The titers of antibodies in the serum of immunized animals were determined using enzyme-linked immunosorbent assay (ELISA). Canine or feline IL-31 (50 ng per well) was immobilized on polystyrene microplates and used as the capture antigen. Serum from immunized animals was diluted in phosphate buffered saline with 0.05% Tween-20 (PBST). The presence of antibodies against IL-31 was detected using an appropriate secondary antibody labeled with HRP. After addition of chromogenic substrate (SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL, Inc., Gaithersburg, Maryland) and ten minutes incubation at room temperature (RT), the reaction was stopped by adding 100 μl of 0.1 N HCl. The absorbance of each well was determined as optical density (OD) at 450 nm. The selection of antibodies for their ability to bind to canine and feline IL-31 was performed using ELISA. In some cases, additional antibody characterization was determined during screening using an ELISA with a mutant form of feline IL-31 protein as the capture antigen. Cells producing antibodies with the desired binding and inhibitory properties were selected for sequence analysis of RNA transcripts corresponding to IgG variable heavy (VH) and variable light (VL) chains.

In the case of mouse antibodies, donor splenocytes from a single responding CF-1 mouse were used for fusion and hybridoma supernatants were screened for antibodies binding to canine or feline IL-31 proteins by ELISA. This led to the identification of one mouse antibody, Mu-15H05, which had subnanomolar affinity for both species of IL-31 (FIG. 2A). Next, the mouse anti-IL-31 antibody 15H05 was subcloned to obtain a hybridoma producing a homogeneous antibody and to sequence the variable region of the heavy and variable region of the light chains. The variable sequences of the mouse anti-IL-31 antibody defined for the 15H05 antibody are as follows: 15H05 heavy chain variable region (SEQ ID NO: 67; MU-15H05-VH) to which the nucleotide sequence (SEQ ID NO: 68; MU- 15H05-VH), the 15H05 light chain variable region (SEQ ID NO: 69; MU-15H05-VL) to which the nucleotide sequence corresponds (SEQ ID NO: 70; MU-15H05-VL).

In addition to the murine 15H05 antibody, the mouse-derived 11E12 antibody previously described in US Pat. No. 8,790,651 to Bammert, et al. Described here is data showing the ability of the 11E12 antibody to bind both canine and feline IL-31 proteins with high affinity. The ability of 11E12 to bind to feline IL-31 made this antibody a suitable candidate for felinization and possible therapeutic use in cats. The variable sequences of the mouse anti-IL-31 antibody previously defined for the 11E12 antibody are as follows: 11E12 heavy chain variable region (SEQ ID NO: 71; MU-11E12-VH) to which the nucleotide sequence (SEQ ID NO: 72; MU -11E12-VH), the 15H05 light chain variable region (SEQ ID NO: 73; MU-11E12-VL), which corresponds to the nucleotide sequence (SEQ ID NO: 74; MU-11E12-VL).

Dogs with elevated titers of antibodies against IL-31 after vaccination were selected for analysis of B-cell populations producing antibodies with the desired phenotypes. B cells for further analysis were obtained from PBMC, bone marrow, spleen, or lymph nodes. Single B cells were expanded into individual wells and analyzed for secreted IgG capable of binding to wild-type canine IL-31 and canine IL-31 mutants 11E12 and 15H05 (AbCellera, Vancouver, British Columbia) using the methods described in US 2012/0009671 A1, US 2016/0252495 A1, US 9,188,593, WO 2015/176162 A9 and WO 2016/123692 A1.

This screening technique is based on known regions of the IL-31 protein critical for signal binding and transduction through its co-receptor complex. The selection of these mutant proteins for screening is described in Section 1.2 of this application. Sequencing of the variable domains of the IgG heavy and light chains was performed after the RT-PCR reaction from individual candidate B cells. This screening resulted in the identification of nine canine antibodies selected for further analysis. The variable sequences of these canine anti-IL-31 antibodies are as follows: ZIL1 heavy chain variable region (SEQ ID NO: 75; CAN-ZIL1_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 76; CAN-ZIL1_VH), light chain variable region ZIL1 (SEQ ID NO: 77; CAN-ZIL1_VL), which corresponds to the nucleotide sequence of (SEQ ID NO: 78; CAN-ZIL1_)VL); ZIL8 heavy chain variable region (SEQ ID NO: 79; CAN-ZIL8_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 80; CAN-ZIL8_VH), ZIL8 light chain variable region (SEQ ID NO: 81; CAN-ZIL8_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 82; CAN-ZIL8_VL); the ZIL9 heavy chain variable region (SEQ ID NO: 83; CAN-ZIL9_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 84; CAN-ZIL9_VH), the ZIL9 light chain variable region (SEQ ID NO: 85; CAN-ZIL9_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 86; CAN-ZIL9_VL); the ZIL11 heavy chain variable region (SEQ ID NO: 87; CAN-ZIL11_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 88; CAN-ZIL11_VH), the ZIL11 light chain variable region (SEQ ID NO: 89; CAN-ZIL11_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 90; CAN-ZIL11_VL); the ZIL69 heavy chain variable region (SEQ ID NO: 91; CAN-ZIL69_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 92; CAN-ZIL69_VH), the ZIL69 light chain variable region (SEQ ID NO: 93; CAN-ZIL69_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 94; CAN-ZIL69_VL); the ZIL94 heavy chain variable region (SEQ ID NO: 95; CAN-ZIL94_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 96; CAN-ZIL94_VH), the ZIL94 light chain variable region (SEQ ID NO: 97; CAN-ZIL94_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 98; CAN-ZIL94_VL); ZIL154 heavy chain variable region (SEQ ID NO: 99; CAN-ZIL154_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 100; CAN-ZIL154_VH), ZIL154 light chain variable region (SEQ ID NO: 101; CAN-ZIL154_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 102; CAN-ZIL154_VL); heavy chain variable region of ZIL159 (SEQ ID NO: 103; CAN-ZIL159_VH), which corresponds to the nucleotide sequence (SEQ ID NO: 104; CAN-ZIL159_VH), light chain variable region of ZIL159 (SEQ ID NO: 105; CAN-ZIL159_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 106; CAN-ZIL159_VL); the ZIL171 heavy chain variable region (SEQ ID NO: 107; CAN-ZIL171_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 108; CAN-ZIL171_VH), the ZIL171 light chain variable region (SEQ ID NO: 109; CAN-ZIL171_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 110; CAN-ZIL171_VL).

The above nine monoclonal antibodies that have been selected for further study may be referred to elsewhere in this specification, drawings or claims as ZIL1, ZIL8, ZIL8, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159 and ZIL171.

1.7. Construction of recombinant chimeric and fully canine antibodies

The variable domains of an antibody enable it to bind to an antigen. The transfer of the full-length variable domain to the appropriate constant region is expected to have little or no effect on the ability of the antibody to bind to the IL-31 immunogen. In order to simultaneously validate the sequencing of heavy and light chain variable regions and obtain homogeneous material, expression vectors have been developed to generate recombinant chimeric and fully canine antibodies in mammalian cell expression systems. The chimeric antibodies described herein consist of the variable sequence (both CDRs and framework regions) of an antibody of a host species transferred onto the respective heavy and light chain constant regions of a feline or canine IgG molecule (e.g., a mouse variable region with a canine constant region is termed a chimeric mouse-canine antibody). The fully canine antibodies described herein consist of the variable sequence (both CDRs and framework regions) of an antibody of the host species (canine) transferred to the respective heavy and light chain constant regions of the canine IgG molecule. Synthetic DNA sequences were constructed for the heavy chain variable region (VH) and light chain variable region (VL) sequences of the selected antibodies. These sequences contain unique restriction sites, a Kozak consensus sequence, and an N-terminal secretory leader sequence to facilitate expression and secretion of the recombinant antibody from a mammalian cell line.

For mouse-feline chimeric antibodies, each respective variable region was cloned into a mammalian expression plasmid containing a feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1) corresponding to the nucleotide sequence (SEQ ID NO: 174; Feline_HC_AlleleA_1), or a feline IgG light chain constant region (SEQ ID NO: 175; Feline_LC_Kappa_G_minus) to which the nucleotide sequence (SEQ ID NO: 176; Feline_LC_Kappa_G_minus) corresponds. For mouse-canine chimeric antibodies or fully canine antibodies, each mouse or canine variable region was cloned into a mammalian expression plasmid containing a canine IgG heavy chain constant region (SEQ ID NO: 177; Canine_HC_65_1) corresponding to the nucleotide sequence (SEQ ID NO : 178; Canine_HC_65_1), or a canine IgG light chain constant region (SEQ ID NO: 179; Canine_LC_Kappa) to which the nucleotide sequence corresponds (SEQ ID NO: 180; Canine_LC_Kappa). Plasmids encoding each heavy and light chain under the control of the CMV promoter were co-transfected into HEK 293 cells using standard methods. After six days of expression, chimeric mAbs were purified from 50 ml supernatants of transiently transfected HEK293FS cells using MabSelect Sure protein A resin (GE Healthcare, Uppsala, Sweden) according to standard protein purification methods. The eluted fractions were pooled, concentrated to approximately 500 μl using a Nanosep Omega centrifuge (Pall Corp., Port Washington, NY) with a nominal molecular weight cutoff of 10,000, dialyzed overnight at 4°C in 1x PBS, pH 7.2, and storage at 4°C. The affinity and activity of selected recombinant antibodies in cell assays are described below.

On FIG. 2 shows in detail the affinity of antibodies to murine CDRs using Biacore. On FIG. 2a shows the affinity of mouse anti-IL-31 antibodies 11E12 and 15H05 and the corresponding affinities of the feline and canine chimeric forms for feline and canine IL-31 surfaces. These data confirm the correct sequence of both mouse antibodies and indicate that conversion to chimeric forms results in antibodies with equivalent or higher affinity than parental mouse antibodies, with the exception of the mouse-feline 15H05 chimeric antibody, which has an affinity for IL- 31 of both species was somewhat reduced as a result of the transformation into a chimeric form.

The fully murine and chimeric forms of the 11E12 and 15H05 antibodies were also analyzed for activity in the canine and feline cell assays described in Section 1.5. The results of these analyzes are shown in FIG. 3. Mouse antibodies 11E12 and 15H05 were analyzed for activity against canine and feline cell types using both canine and feline IL-31 to stimulate signals. When using the feline cytokine, the activity of both mouse antibodies against canine and feline cells was comparable, with the exception of 15H05 against feline IL-31 in feline FCWF4 cells, which showed a slight increase in IC ₅₀ . Murine 15H05 was able to block canine IL-31 signals in both feline and canine cells, with somewhat higher activity in the canine cell assay. These results indicate that the respective epitopes recognized by these antibodies are present on both canine and feline IL-31 and that binding of these antibodies is able to neutralize receptor-mediated cell signals in the respective cell lines of both species.

On FIG. 3 also describes the activity of selected chimeric antibodies in both cell assays. Conversion of mouse antibodies to feline and canine chimeric antibodies had minimal effect on anti-feline IL-31 activity in a feline cell activity assay (IC ₅₀ range 1.15-3.45 μg/mL). Similar results were noted when these chimeric antibodies against feline IL-31 signals were assayed in canine DH82 cells, with a slight increase in activity (IC ₅₀ 0.71 μg/ml) of mouse-canine chimeric 15H05 antibody observed. In general, an increase in IC ₅₀ values against canine IL-31 was observed in both canine and feline cell types. In this format assay, the activity of the mouse-feline 15H05 chimeric antibody was slightly lower than that of the mouse-dog form (IC ₅₀ 28.61 vs 12.49 μg/ml). Similar to mouse antibodies, conversion to canine and feline chimeric forms resulted in minimal changes in activity.

The antibodies described above, which were identified from individual B cells of immunized dogs, were constructed as recombinant IgG proteins after their variable domain sequences were determined. Transfer of these variable domains to canine heavy chain Fc (isotype 65 1) resulted in recombinant all canine antibodies. It was of interest to identify additional canine antibodies that bind to wild-type feline IL-31 and whose binding to the 15H05 feline IL-31 mutant would be reduced (ie, those that would target the 15H05 epitope). Such antibodies derived from this alternative source (dog vs. mouse) provide paratopes (the IL-31 protein-recognizing CDR portion of the antibody) recognizing the 15H05 epitope, thus increasing the diversity of antibodies with different physical properties for selection.

On FIG. 4 shows the results obtained with the binding of these recombinant canine antibodies to various proteins using both the ELISA method and the Biacore method. In an indirect ELISA, antibody binding to wild type feline IL-31 and 15H05 mutant feline IL-31 proteins was evaluated. All nine canine monoclonal antibodies (ZIL1, ZIL8, ZIL8, ZIL11, ZIL69, ZIL94, ZIL154, ZIL159, and ZIL171) were able to bind to wild-type feline IL-31, and this binding was affected by mutations in the mAb 15H05 epitope region, confirming the correct determination of the binding phenotype in the initial screen used to identify them. In comparison, the 11E12 antibody bound wild-type feline IL-31, and this binding was not affected by mutations in the 15H05 epitope region, as indicated by the data presented in FIG. 4. To confirm binding, a Biacore assay was performed using surfaces with canine, feline, equine, human IL-31, feline IL-31 mutant 15H05, and feline IL-31 mutant 11E12 proteins at a single antibody concentration assayed. Similar to ELISA data, all antibodies bound to wild-type feline IL-31. As described above in this section, both mouse 11E12 antibody and mouse 15H05 antibody bound to canine and feline IL-31 surfaces. This double binding property has been shown in three other antibodies: ZIL69 (partial binding to canine protein), ZIL94 and ZIL159. Of this group of nine all-canine antibodies, only ZIL1 and ZIL9 were cross-reactive with equine IL-31. Of note, of all the antibodies analyzed here, the 15H05 antibody was the only one that bound to canine, feline, and equine IL-31, indicating some degree of epitope conservation in these three species. In contrast, none of the antibodies described here bind to human IL-31. Additional Biacore surfaces were used to verify the ELISA results indicating differential binding of antibodies to wild-type feline IL-31 and two proteins with mutations in the 15H05 (15H05 mutant) or 11E12 (11E12 mutant) epitopes. As expected, the murine 11E12 antibody bound to the 15H05 IL-31 mutant and did not bind to the 11E12 IL-31 mutant due to mutations in the corresponding epitope. Similarly, 15H05 did not bind to the 15H05 mutant and retained binding to the 11E12 IL-31 mutant, further confirming that the two antibodies bind to separate epitopes. In support of the ELISA results, all fully canine antibodies were sensitive to mutations in 15H05, with the exception of ZIL94, ZIL154 and ZIL171 (partially sensitive). Differences in results may be due to differences between the two methods of analysis. In addition, it was shown that mutations in 11E12 led to reduced binding of three antibodies: ZIL1 (partial reduction), ZIL8 and ZIL159. These results indicate that the epitope recognized by these antibodies is affected by changes in both regions of the IL-31 protein. Taken together, these results contribute to the characterization of nine antibodies derived from canine B cells that bind to the region of the feline IL-31 protein recognized by the 15H05 antibody.

1.8. Felinization of mouse 11E12 and 15H05 antibodies and optimization of binding affinity

The production of anti-drug antibodies (ADA) can lead to a decrease in the effectiveness of any biotherapeutic proteins, including monoclonal antibodies. A comprehensive review of the literature has shown that species adaptation of monoclonal antibodies reduces the immunogenicity propensity of mAbs, although examples of immunogenic fully human mAbs and non-immunogenic chimeric mAbs can be found. To reduce the risks associated with the production of ADA, when using the monoclonal antibodies against IL-31 proposed here, the technique of felinization was applied. This felinization technique is based on the identification of the most suitable feline germline antibody sequence for CDR transfer. After careful analysis of all available feline germline sequences, both heavy chain and light chain, germline candidates were selected based on their homology to the mouse mAb, and the mouse mAb progenitor CDRs were used to replace the native feline CDRs. The challenge was to maintain high affinity and activity against cells while using feline antibody frameworks to minimize immunogenic potential in vivo. Felinized mAbs were expressed and characterized for their affinity for feline IL-31 and their activity in cell assays. In the event that the felinized antibody lost its ability to bind to IL-31, a systematic analysis was performed to determine (1) the chain that caused the loss of function, (2) the framework region that led to the loss of function, and (3) the amino acid(s) leading to loss of function.

Received synthetic nucleotide constructs corresponding to the felinized variable heavy and light chains of mAb 11E12 and 15H05. After subcloning each variable chain into plasmids containing the respective kappa heavy chain or light chain constant regions, HEK 293 cells were co-transfected with the resulting antibody expression plasmids. Initial attempts at felinization of the 11E12 antibody focused on using single feline VH frameworks (SEQ ID NO: 111; FEL_11E12_VH1) to which the nucleotide sequence (SEQ ID NO: 112; FEL_11E12_VH1) corresponds, when independently combined with VL framework regions (SEQ ID NO : 113; FEL_11E12_VL1) to which the nucleotide sequence corresponds (SEQ ID NO:114; FEL_11E12_VL1), and (SEQ ID NO: 115; FEL_11E12_VL2) to which the nucleotide sequence corresponds (SEQ ID NO: 116; FEL_11E12_VL2), to give Feline 11E12 1.1 and Feline 11E12 1.2, respectively. This species adaptation attempt resulted in a decrease in affinity of Feline 11E12 1.1 for both feline and canine IL-31 proteins and a complete loss of binding in Feline 11E12 1.2 mAb compared to the murine form of this antibody (Fig. 2b). The activity of these antibodies after species adaptation was assessed in assays with canine DH82 cells and feline FCWF4 cells using the feline IL-31 cytokine. The activity of felinized 11E12 1.1 against feline IL-31 in the assay with feline FCWF cells was approximately two-fold reduced compared to the murine form of this antibody. Similar to the complete loss of affinity for felinized 11E12 1.2, this antibody showed a complete loss of activity against cells (FIG. 3). Based on previous experience with caninization of the 11E12 mAb orthologue, a similar technique was used to recover the affinity reduced by felinization (US Pat. No. 8,790,651 to Bammert, et al.). The felinized framework region 2 (FW2) of Feline 11E12 VL1 was replaced with the mouse FW2 (SEQ ID NO: 73; Mu_11E12_VL) corresponding to the nucleotide sequence (SEQ ID NO: 74; Mu_11E12_VL) to give Feline 11E12 VL1 FW2. In addition, a single substitution was made at position 46 of the feline VL (K46Q) to obtain (SEQ ID NO: 119; FEL_11E12_VL1_K46Q), which corresponds to the nucleotide sequence (SEQ ID NO: 120; FEL_11E12_VL1_K46Q). The combination of the above VL with Fel_11E12_VH1 resulted in Feline 11E12 1.1 FW2 and Feline 11E12 1.1 K46Q, respectively. The change in FW2 resulted in the restoration of Feline 11E12 1.1 FW2 affinity for _feline IL-31 protein with a K _D equivalent to that of the mouse and chimeric form (FIGS. 2A and 2B). However, these changes negatively affected the affinity of Feline 11E12 1.1 FW2 for canine IL-31 protein, indicating a clear difference in the ability of the 11E12 antibody to bind to this epitope on feline and canine cytokine. A single amino acid substitution in Feline 11E12 1.1 K46Q did not affect the affinity of this antibody. The increased affinity of the 11E12 1.1 FW2 antibody for feline IL-31 protein resulted in an increase in its activity against the feline cytokine in the canine DH82 cell assay (FIG. 3).

Felinization attempts of the mouse 15H05 antibody focused on combinations of three feline VH frameworks with three feline VL frameworks to produce a total of 9 felinized mAbs. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1), which corresponds to the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1), was combined with (SEQ ID NO: 127; FEL_15H05_VL1), which corresponds to the nucleotide sequence (SEQ ID NO: 128; FEL_15H05_ VL1) c obtaining Feline 15H05 1.1, Feline 15H05 1.2 and Feline 15H05 1.3, respectively. FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2), which corresponds to the nucleotide sequence (SEQ ID NO: 124; FEL_15H05_VH2), was combined with (SEQ ID NO: 127; FEL_15H05_VL1), which corresponds to the nucleotide sequence (SEQ ID NO: 128; FEL_15H05_ VL1) c obtaining Feline 15H05 2.1, Feline 15H05 2.2 and Feline 15H05 2.3, respectively. FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3), which corresponds to the nucleotide sequence (SEQ ID NO: 126; FEL_15H05_VH3), was combined with (SEQ ID NO: 127; FEL_15H05_VL1), which corresponds to the nucleotide sequence (SEQ ID NO: 128; FEL_15H05_ VL1) c obtaining Feline 15H05 3.1, Feline 15H05 3.2 and Feline 15H05 3.3, respectively. Similar to the 11E12 antibody, the first attempt at felinization of the 15H05 antibody resulted in a reduced affinity for the feline IL-31 protein compared to the murine 15H05 and a neutral effect compared to the chimeric mouse-feline 15H05 (FIGS. 2A and 2C). Similar to the binding of feline 11E12 antibody to canine IL-31, certain combinations of feline VH and VL frameworks in 15H05 had a neutral or positive effect on affinity for canine IL-31 (see Figure 2C: Feline 15H05 1.1, 2.2 and 3.2).

To restore the affinity of the felinized 15H05 antibody, the VH of each felinized 15H05 was combined with the VL of the mouse 15H05 to generate heterochimeric antibodies. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1), which corresponds to the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1), was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 70; MU_15H05_VL ), resulting in Feline 15H05 VH1 mouse VL. FEL_15H05_VH2 (SEQ ID NO: 123; FEL_15H05_VH2), which corresponds to the nucleotide sequence (SEQ ID NO: 124; FEL_15H05_VH2), was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 70; MU_15H05_VL ), resulting in Feline 15H05 VH2 mouse VL. FEL_15H05_VH3 (SEQ ID NO: 125; FEL_15H05_VH3), which corresponds to the nucleotide sequence (SEQ ID NO: 126; FEL_15H05_VH3), was combined with MU_15H05_VL (SEQ ID NO: 69; MU_15H05_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 70; MU_15H05_VL ), resulting in Feline 15H05 VH3 mouse VL. These heterochimeric antibodies with felinized VH and mouse VL were analyzed for their affinity for canine and feline IL-31. The combination of VH1 and VH3 of felineized 15H05 with VL of mouse 15H05 resulted in restoration of affinity for feline IL-31 to a level equivalent to or greater than that of the murine and chimeric forms. This trend towards improved affinity was also noted with canine IL-31 protein (FIGS. 2A and 2C).

To further refine the positions of the 15H05 frameworks resulting in reduced affinity, one felinized 15H05 VH (FEL_15H05_VH1) was used to couple with separate substitutions in the murine 15H05 VL frameworks. FEL_15H05_VH1 (SEQ ID NO: 122; FEL_15H05_VH1) to which the nucleotide sequence corresponds (SEQ ID NO: 123; FEL_15H05_VH1) was independently combined with FEL_15H05_VL1-FW1 (SEQ ID NO: 133; FEL_15H05_VL1_FW1) to which the nucleotide corresponds sequence (SEQ ID NO : 134; FEL_15H05_VL1_FW1), FEL_15H05_VL1_FW2 (SEQ ID NO: 135; FEL_15H05_VL1_FW2), which corresponds to the nucleotide sequence (SEQ ID NO: 136; FEL_15H05_VL1_FW2), and FEL_15H05_VL1_FW3 (SE Q ID NO: 137; FEL_15H05_VL1_FW3), which corresponds to the nucleotide sequence (SEQ ID NO: 138; FEL_15H05_VL1_FW3) to give Feline 15H05 1.1 FW1, Feline 15H05 1.1 FW2 and Feline 15H05 1.1 FW3, respectively. FW1 mouse 15H05 substitution in Feline 15H05 1.1 reduced affinity for both feline and canine IL-31, however, excellent affinity for both canine and canine IL-31 was obtained when FW2 or FW3 was introduced into Feline 15H05 1.1. feline IL-31, with the FW2 substitution resulting in a greater increase in affinity for both species (FIG. 2C). Additional pairwise framework substitutions were made to determine the degree of affinity modulation using this technique. FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1), which corresponds to the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1), was independently combined with FEL_15H05_VL1_FW1_2 (SEQ ID NO: 139; FEL_15H05_VL1_FW1_FW 2) to which the nucleotide sequence corresponds (SEQ ID NO: 140 ; FEL_15H05_VL1_FW1_FW2), FEL_15H05_VL1_FW2_3 (SEQ ID NO: 143; FEL_15H05_VL1_FW2_FW3), which corresponds to the nucleotide sequence (SEQ ID NO: 144; FEL_15H05_VL1_FW2_FW3), and FEL_15H05_ VL1_FW1_3 (SEQ ID NO: 141; FEL_15H05_VL1_FW1_FW3), which corresponds to the nucleotide sequence (SEQ ID NO: 142; FEL_15H05_VL1_FW1_FW3) to give Feline 15H05 1.1 FW1_2, Feline 15H05 1.1 FW2_3 and Feline 15H05 1.1 FW1_3. Interestingly, replacement of mouse FW1 alone resulted in reduced affinity, while combinations of FW1 with FW2 or FW3 resulted in good affinity for both feline and canine IL-31 (FIG. 2C).

Finally, an attempt was made to minimize the number of backmutations in the feline framework regions, starting with the most promising combinations of feline VH and VL sequences. For this, FEL_15H05_VH1 (SEQ ID NO: 121; FEL_15H05_VH1), which corresponds to the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1) was independently combined with FEL_15H05_VL1_FW2_K42N (SEQ ID NO: 145; FEL_15H05_VL1_F W2_K42N) to which the nucleotide sequence corresponds (SEQ ID NO: 146; FEL_15H05_VL1_FW2_K42N), FEL_15H05_VL1_FW2_V43I (SEQ ID NO: 147; FEL_15H05_VL1_FW2_V43I), which corresponds to the nucleotide sequence (SEQ ID NO: 148; FEL_15H05_VL1_FW2_V43I), F EL_15H05_VL1_FW2_L46V (SEQ ID NO: 149; FEL_15H05_VL1_FW2_L46V), which corresponds to the nucleotide sequence (SEQ ID NO: 150; FEL_15H05_VL1_FW2_L46V), FEL_15H05_VL1_FW2_Y49N (SEQ ID NO: 151; FEL_15H05_VL1_FW2_Y49N) to which the nucleotide sequence corresponds (SEQ ID NO: 152; FEL_15H05_VL1_FW2_Y49N), and FEL_15H05_VL1_FW2_K42N_V43I (SEQ ID NO: 153; FEL_15H05_VL1_FW2_K42N_V43I), which corresponds to the nucleotide sequence (SEQ ID NO : 154; FEL_15H05_VL1_FW2_K42N_V43I) to give Feline 15H05 1.1 K42N, Feline 15H05 1.1 V43I, Feline 15H05 1.1 L46V, Feline 15H05 1.1 Y49N and Feline 15H05 1.1 K42N_ V43I, respectively. While replacement with the introduction of the entire murine FW2 framework region into Felinized 15H05 VL1 resulted in an antibody with excellent affinity for canine and feline IL-31 (Fig. or a negative effect, indicating the need for all 4 substitutions to maintain the optimal tertiary structure, ensuring the location of the CDR on the IL-31 epitope. The increased affinity of felinized 15H05 1.1 FW2 for feline and canine IL-31 led to the choice of this antibody for further work.

On FIG. 5A shows the alignment with the VL sequence of the mouse antibody 11E12 when comparing the sequence of the previously mentioned caninized 11E12 with the felinized variants. Dots are marked below the alignment showing the positions of important changes in Fel_11E12_VL1 that were required to restore the affinity of this antibody for the IL-31 protein. Similarly, in FIG. 5B shows the necessary changes in the VL of felinized 15H05 (Fel_15H05_VL1) that were needed not only to restore, but also to improve its affinity for canine and feline IL-31 compared to the mouse and chimeric form of this antibody.

1.9. Generation of Cell Lines Expressing Felinized Antibodies from Glutamine Synthetase (GS) Plasmids

Felinized 15H05 1.1 FW2 (Felinized 15H05 1.1 FW2) was chosen as a candidate for obtaining stable cell lines that will produce a homogeneous antibody for further characterization. Genes encoding felinized heavy and light chains were cloned into GS plasmids pEE 6.4 and pEE 12.4, respectively (Lonza, Basel, Switzerland) to obtain cell lines. The resulting plasmids were digested according to the manufacturer's protocol and ligated with each other, obtaining a single plasmid for expression in mammalian cells. For ZTS-927, the heavy chain is (SEQ ID NO: 121; FEL_15H05_VH1) to which the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1) corresponds, in combination with the feline IgG heavy chain constant region (SEQ ID NO: 171; Feline_HC_AlleleA_wt) , which corresponds to the nucleotide sequence (SEQ ID NO: 172; Feline_HC_AlleleA_wt). For ZTS-927, the light chain is (SEQ ID NO: 135; FEL-15H05-VL1_FW2) to which the nucleotide sequence (SEQ ID NO: 136; FEL-15H05-VL1_FW2) corresponds, in combination with the feline IgG light chain constant region ( SEQ ID NO: 175; Feline_LC_Kappa_Gminus), which corresponds to the nucleotide sequence (SEQ ID NO: 176; Feline_LC_Kappa_G_minus). For ZTS-361, the heavy chain is (SEQ ID NO: 121; FEL_15H05_VH1), which corresponds to the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1), in combination with the feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1) , which corresponds to the nucleotide sequence (SEQ ID NO: 174; Feline_HC_AlleleA_1). For ZTS-361, the light chain is (SEQ ID NO: 135; FEL-15H05-VL1_FW2) to which the nucleotide sequence (SEQ ID NO: 136; FEL-15H05-VL1_FW2) corresponds, in combination with the feline IgG light chain constant region ( SEQ ID NO: 175; Feline_LC_Kappa_G_minus), which corresponds to the nucleotide sequence (SEQ ID NO: 176; Feline_LC_Kappa_G_minus). For ZTS-1505, the heavy chain is (SEQ ID NO: 121; FEL_15H05_VH1) to which the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1) corresponds, in combination with the feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1) , which corresponds to the nucleotide sequence (SEQ ID NO: 174; Feline_HC_AlleleA_1). For ZTS-1505, the light chain is (SEQ ID NO: 135; FEL-15H05-VL1_FW2) to which the nucleotide sequence (SEQ ID NO: 136; FEL-15H05-VL1_FW2) corresponds, in combination with the feline IgG light chain constant region ( SEQ ID NO: 186; Feline_LC_Kappa_G_minus_QRE_minus), which corresponds to the nucleotide sequence (SEQ ID NO: 187; Feline_LC_Kappa_G_minus_QRE_minus). Affinity and activity data for ZTS-1505 are described below in Section 1.18 of this Example section.

To demonstrate transient antibody production, each plasmid was used to transfect HEK 293 cells and expressed in cultures of various sizes. The protein was isolated from the conditioned HEK medium using white A chromatography according to standard protein purification methods. The medium was loaded onto a chromatographic resin and eluted with a pH shift. Before using the eluted protein, it was subjected to pH adjustment, dialysis and sterilizing filtration. ZTS-361 was subsequently used in a feline pruritus model to evaluate in vivo efficacy.

Antibodies derived from one GS plasmid, ZTS-927 and ZTS-361, were analyzed for affinity and activity. On FIG. 2D shows the results of the affinity assessment of these antibodies using Biacore. The affinity of ZTS-927 and ZTS-361 for feline IL-31 is highly consistent with the affinity of the murine and chimeric forms of their murine 15H05 precursor mAb. The activity of these two antibodies was determined against canine and feline IL-31 using both canine and feline cell assays (FIG. 3). Similar to previous observations, IC ₅₀ values were proportionally higher when using the canine form of IL-31 with both cell types. The IC ₅₀ values of ZTS-927 and ZTS-361 against feline IL-31 were also highly consistent with the IC ₅₀ values of the chimeric and murine forms of the antibody, indicating that the final felineized mAb 15H05 variant generated from a single GS plasmid was suitable for cell line development.

To obtain a stable cell line producing candidate antibodies, the GS plasmid was linearized prior to transfection using the PvuI restriction enzyme, which cleaves the plasmid backbone at a single site. GS-CHOK1SV cells (clone 144E12) were transfected with linearized plasmid DNA by electroporation. After transfection, cells were seeded in 48-well plates (48WP) to obtain stable pools. At at least 50% confluence in 48-well plates, 100 μl of supernatant was analyzed for IgG expression using ForteBio Octet and Protein A biosensors (Pall ForteBio, Fremont, CA). Clones with the best expression were scaled up in 6-well plates (6WP) and then in 125 ml shake flasks (SF). After adaptation of the cells to suspension culture in 125 ml flasks, 2 flasks of the pool of each cell line were stored for long-term storage. Because industrial cell lines must be clonal, the 3 highest expression pools were subcloned by serial dilution in 96-well culture plates. To confirm clonality and to avoid a second round of serial dilution, 96-well plates were imaged using a Molecular Devices Clone-Select Imager (CSI) (Molecular Devices LLC, San Jose, CA) to image single cells and their subsequent growth. Clone selection was based on successful CSI imaging, growth, and antibody production in 96-well plates.

To assess the growth and productivity of cell cultures, the pools with the highest expression in fed-batch culture in 125 ml shake flasks were further evaluated for 14 days. Cell seeding was performed using platform media and a feed consisting of Life Technologies 4 amino acid CHO CD, CDF v6.2 proprietary feed, and 10% glucose. After 14 days in fed-batch culture, the pools were centrifuged and the resulting mAb in CD CHO was isolated by filtering the supernatant through a 0.20 μm polyethersulfone membrane (PES) before purification.

A typical purification consists of two liters of conditioned medium (from CHO cell culture filtered through a 0.2 µm filter) loaded onto a 235 ml MabSelect column (GE healthcare, p/n 17-5199-02). The column was pre-equilibrated using PBS. The sample was loaded with a retention time greater than 2.5 minutes. After loading the column was washed again using PBS and then 25 mm sodium acetate with a pH close to neutral. Elution was carried out using 25 mm acetic acid, pH 3.6, and then desorption using 250 mm acetic acid, 250 mm sodium chloride, pH approximately 2.2. Fractions (50 ml) were collected at the stages of elution and desorption. Conducted continuous monitoring of UV absorption at A280. The peak fractions were pooled, the pH adjusted to approximately 5.5 with 20 mM sodium acetate, and then dialyzed against three buffer changes. The dialysate was collected, sterilized by filtration and stored at 4°C.

1.10. Identification of the IL-31 epitope recognized by the 15H05 antibody

Knowledge of the IL-31 epitope recognized by an antibody is critical to understanding the mechanism by which it neutralizes a cytokine from binding to the IL-31Ra:OSMR co-receptor. In addition, knowledge of the epitope allows (without limitation) to optimize the binding affinity of the antibody and to design epitope peptide mimetics (mimotopes) that can be very useful as capture assays and as subunit vaccines to induce an appropriately targeted immune response. To identify and optimize the peptide capable of binding to the mAb 15H05 paratope, a multi-step technique was used using the CLIPS (Chemical Linkage of Peptides onto Scaffolds) technology (Timmerman et al. J Mol Recognit. 2007; 20(5): 283-299) (Pepscan, Lelystad, The Netherlands). The affinity of mAb 15H05 for both canine and feline IL-31 proteins is high (FIG. 2, MU-15H05), so the primary IL-31 sequence of both species was considered important for the above task. A microarray peptide library corresponding to canine IL-31 protein was generated and used to identify peptides capable of binding to mAb 15H05 using indirect ELISA. After identifying peptides whose primary amino acid sequences corresponded to the mAb 15H05 binding region on IL-31, a focused complete replacement analysis was performed using peptides corresponding to the IL-31 segment, and replacing each of the 12 amino acids in this mAb 15H05 binding region with 19 other possible amino acids at each position. This analysis was necessary to identify key IL-31 amino acid residues involved in binding to mAb 15H05 and also demonstrated where substitutions in the canine primary sequence lead to increased binding to the antibody.

Canine IL-31 protein amino acids recognized by the 11E12 antibody have been previously described (US Pat. No. 8,790,651 to Bammert, et al.). It describes a mutational analysis of canine IL-31 protein showing the positions of canine IL-31 protein that affect binding to mAb 11E12 when changed to alanine. Based on the total substitution assay described for mAb 15H05 above and the previously known epitope bound by 11E12, mutant forms of feline IL-31 were generated where two key epitope residues recognized by each antibody were replaced with alanine (mutants are described in Section 1.2 above ). Mutations of each epitope were named according to the antibody recognizing the site of the mutation (11E12 and 15H05 mutants against native wild-type protein sequence).

On FIG. 6A shows an alignment of wild-type feline IL-31 (SEQ ID NO: 157) with the 15H05 (SEQ ID NO: 163) and 11E12 (SEQ ID NO: 161) mutants, highlighting the positions of the alanine substitutions. IL-31 belongs to the IL-6 family of cytokines with four helical bundles with ascending and descending architecture (CATH database, Dawson et al. 2017 Nucleic Acids Res. 2017 Jan 4; 45 (Database issue): D289 D295). Its homologous model was derived from the structure of human IL-6 1P9M (Boulanger et al. 2003 Science. Jun 27; 300(5628):2101-4) using MOE software (Chemical Computing Group, Montreal, Quebec, Canada). On FIG. 6B shows a homologous model of feline IL-31 highlighting amino acid positions involved in binding to antibodies 11E12 (site 1) and 15H05 (site 2). The binding sites for each antibody appear to be located in different regions of the IL-31 protein molecule.

To determine the effect on the ability of mAbs 11E12 and 15H05 to bind to these feline IL-31 mutants, an indirect ELISA was performed using these mutants adsorbed directly onto an immunoassay plate. On FIG. 6C shows the results of this ELISA demonstrating that mAbs 11E12 and 15H05 are able to bind wild-type feline IL-31 in this assay format. When using the 11E12 mutant as the capture protein, the binding of mAb 11E12 is significantly weakened, and the binding of mAb 15H05 is partially weakened. Previous analysis of the 11E12 epitope in canine IL-31 (described in U.S. Patent No. 8,790,651 to Bammert, et al.) showed that 4 amino acid residues affect mAb binding with analine mutations, so in this case, 2 residue mutations may not be sufficient for complete elimination of high affinity binding of mAb 11E12 using this ELISA format. A slight weakening of mAb 15H05 binding to the 11E12 mutant is probably due to the translational effects of mutations from the displacement of the two forward helices affecting the 15H05 binding site. Mutations designed to affect the binding of mAb 15H05 (15H05 mutant) demonstrate complete loss of the ability of mAb 1505 to bind to this IL-31 mutant in ELISA. In contrast to the 11E12 mutant, the random helix changes recognized by the 15H05 mAb (15H05 mutant) do not affect the binding of the 11E12 mAb, further confirming that the two epitopes are separate from each other (FIG. 6C).

1.11. Competitive Binding Assay of mAbs 15H05 and 11E12 Using Biacore

To further characterize the IL-31 epitopes bound by mAb 15H05 and 11E12, blocking experiments were performed using Biacore, where a surface containing IL-31 protein was obtained, followed by sequential addition of antibodies. On FIG. 7 shows the relative binding of each antibody to IL-31 after 11E12 or 15H05 capture. Columns labeled HBS-EP (assay buffer) indicate the maximum signal obtained by self-binding of each antibody to an IL-31 surface without competition. On FIG. 7A shows competition binding of mouse antibodies 15H05 and 11E12 to canine IL-31. These results clearly show that the 15H05 and 11E12 antibodies are able to bind to canine IL-31 in the presence of each other, indicating that they recognize different epitopes of this protein. The sensorgrams associated with FIG. 7A show the very slow dissociation kinetics of both antibodies on this newly formed Biacore surface and hence the inability to occupy new binding sites when the same antibody is added (data not shown).

On FIG. 7B shows competitive binding of 15H05 and 11E12 antibodies to feline IL-31 surfaces, again indicating no overlap in the epitopes they recognize. Binding of additional antibody in the presence of the same antibody is the result of an increased rate of dissociation due to the poor quality of the surface used. Elevated dissociation rates are seen which can be compared to the Kd values obtained using the newly formed feline IL-31 surfaces shown in FIG. 2.

These results further support the epitope mapping data presented in Section 1.10 and indicate that the CDRs present in MU-15H05 and MU-11E12 recognize separate epitopes. The epitope recognized by the 15H05 antibody differs from that of the 11E12 antibody described in US Pat. These data highlight the spatial separation of binding sites described in the feline IL-31 homologous model (FIG. 6B) and support the hypothesis that this cytokine surface is critical for interaction with the IL-31Ra:OSMR receptor complex.

1.12. Synthesis and characterization of soluble feline IL-31 co-receptor (IL-31RA and OSMR)

The heterochimeric human IL-31 co-receptor complex, consisting of IL31Ra and OSMR subunits, has been shown to be required for IL-31-mediated intracellular activation of the JAK-STAT pathway and has been implicated in atopic skin diseases (Dillon et al. 2004 Nat Immunol. Jul; 5 (7):752-60, Dreuw et al 2004 J Biol Chem 279:36112-36120 and Diveu et al 2004 Eur Cytokine Netw 15:291-302). More recently, the human subunit of IL-31 Ra has been described as the primary binding event occurring upon contact of IL-31 with cell surface receptors, and this event is a necessary condition for attracting OSMR with subsequent formation of a high affinity receptor complex (Le Saux et al. 2010 J Biol Chem. Jan 29;285(5):3470-7). Here, the inventors describe data according to which feline IL-31 is capable of independent binding to both OSMR and IL-31Ra. This is a new observation of great importance for understanding how the IL-31 protein interacts with the IL-31Ra:OSMR co-receptor and the biological role of IL-31 in its independent interaction with individual subunits.

To understand how IL-31 binds to its co-receptor and to characterize the inhibitory properties of the identified antibodies, two forms of the receptor were synthesized. Both separate subunits of the IL-31 receptor IL-31Ra (SEQ ID NO: 169; Feline_IL31Ra_HIgG1_Fc_X1_Fn3) to which the nucleotide sequence corresponds (SEQ ID NO: 170; Feline_IL31Ra_HIgG1_Fc_X1 Fn3) and OSMR (SEQ ID NO: 167; Feline_OSMR_hIgG1_Fc ) to which the nucleotide sequence corresponds (SEQ ID NO: 168; Feline_OSMR_hIgG1_Fc) were constructed as fusion proteins with human IgG1 Fc. Based on homology with human homologues, a cytokine-binding domain, a fibronectin III domain, and an immunoglobulin-like domain were identified. To assess individual receptor subunits, the extracellular domains of OSMR and IL-31Ra (with its putative N-terminal proximal fibronectin III domain) were generated as fusion proteins with human IgG1 Fc, in both cases using their native signal peptides. All synthetic cassettes were cloned into pcDNA3.1, expressed in the ExpiCHO system and purified as described above.

To analyze the ability of these forms of receptors to bind to wild-type IL-31 proteins and mutant IL-31 proteins, an indirect ELISA was performed by adsorbing 100 μl of each respective protein onto an Immulon 2HB plate (1 μg/ml) overnight in carbonate/bicarbonate buffer (Sigma C3041-100CAP) at 4°C. The ELISA plates were then blocked with blocking buffer with 5% NFDM in PBST for 1 hour at room temperature, followed by binding to each receptor construct at several concentrations at room temperature for 1 hour. After washing with PBST, the presence of the bound receptor (fused to Fc) was determined using a mouse anti-human IgG1 antibody (Lifetech A10684, 1:500 dilution) for 1 hour at room temperature. The wells were washed again with PBST and developed with 3,3',5,5'-tetramethylbenzidine (TMB) KPL Sureblue microwell substrate. On FIG. 8 shows the results of this indirect ELISA using wild-type feline IL-31 protein and capture mutant forms of feline IL-31 protein. These data demonstrate the ability of wild-type feline IL-31 to independently bind to IL-31Ra receptor subunits and OSMR. These observations differ from previous reports of the primary binding of the IL-31 protein to the IL-31Ra subunit, followed by recruitment of OSMR to the appropriate site. Since the biological role of IL-31 has not yet been determined, it is very important to understand the dynamics of its binding to receptors and the possible consequences of a weakening of its role in diseases such as atopic dermatitis. For this reason, these data were taken into account when characterizing antibodies that bind to epitopes that can reduce the ability of IL-31 to recognize IL-31Ra and OSMR.

In Section 1.2, the inventors describe the weakened binding of antibodies 11E 12 and 15H05 to mutants in which key amino acids of the binding sites are replaced by alanine (mutants 11E12 and 15H05, respectively). Therefore, it was of great interest to understand the effect of these mutations on the ability to bind to individual IL-31Ra and OSMR receptor subunits. On FIG. 8 shows that mutations at either the 11E12 or 15H05 binding sites result in a complete loss of the ability to bind to IL-31Ra and OSMR, indicating that both antibodies bind to the epitopes required for IL-31 to interact with both receptor subunits. The lack of binding could also be due to changes in the conformation of IL-31 as a result of mutations, however, these mutants are still able to bind to the antibody, indicating that the lack of binding is due to other reasons. These key findings support the ability of the 11E12 and 15H05 antibodies (and their derivatives) to recognize epitopes on IL-31 that neutralize the ability of this cytokine to signal through its co-receptor, as well as block the binding of this cytokine to cells through any of the receptors under consideration. These data confirm the identification of antibodies capable of removing IL-31 from the circulation and depriving it of its ability to bind to surface cellular or soluble forms of receptors.

1.13. In vivo evaluation of chimeric antibodies in a feline itch provocation model using IL-31

The ability of an antibody to effectively neutralize its target can be assessed in vitro by examining binding to the appropriate epitope on the target protein with suitable affinity and activity in cell assays, allowing extrapolation to in vivo activity. The above describes the steps taken to characterize two series of antibodies derived from mouse mAb precursors 11E12 and 15H05. Section 1.7 describes the production of mouse-feline chimeric forms of mAbs 11E12 and 15H05, with the resulting affinity for canine and feline IL-31 comparable to the parent mouse monoclonal antibody (FIG. 2A). The mouse-feline chimeric forms of 11E12 and 15H05 also had comparable IC ₅₀ values, demonstrating inhibition of feline IL-31-induced pSTAT3 signals in canine and feline macrophages (FIG. 3). During the felinization process described in Section 1.8, the murine mAb 11E 12 was converted to a felinized variant (Feline 11E12 1.1) with a subsequent decrease in affinity for canine and feline IL-31 (Fig. 3) and a decrease in activity against feline IL-31 signaling in dog and cat cages (Fig. 3). Prior to the optimization of the felinized 11E12 and 15H05 antibodies described in Section 1.8, it was of interest to understand the ability of these prefelinized and chimeric forms to neutralize the pruritus inducing activity of feline IL-31 in a feline itch provocation model. Of interest were the pharmacodynamic effects of these various antibodies in neutralizing pruritus and understanding any correlation thereof with affinity, cell activity, or epitope recognition that might influence efficacy. Analysis of the range of activity on cells correlated with in vivo efficacy in the pruritus provocation model may allow prediction of further optimization with necessary in vitro assays 1.1), a model of IL-31-induced pruritus was developed in cats. Following intravenous administration of 0.5 μg/mL of feline IL-31 (SEQ ID NO: 159; Feline_IL-31_E_coli), which corresponds to the nucleotide sequence (SEQ ID NO: 160; Feline_IL-31_E_coli), cats will experience transient behavioral signs of pruritus, including (but not limited to) licking, biting, scratching, and shaking the head or body. Friction against the cage was not considered as a manifestation of itching. Itching is monitored by a qualified examiner for 30 minutes prior to and 1 hour after IL-31 protein administration. For this study, challenge with feline IL-31 was performed no earlier than 1 month prior to antibody administration. On day zero, a dose of 0.5 mg/kg of antibody was combined with 0.5 μg/kg of feline IL-31 at room temperature for 60 minutes prior to injection of the pre-coupled complex into each animal. For the control, a "no mAb" control was included. The mAb dose used corresponds to a large molar excess of the antibody relative to the cytokine. The described monitoring of manifestations of itching was carried out on days 0, 7 and 21. The results shown in FIG. 9 show a significant improvement (p < 0.05) in pruritus scores with the 15H05 chimeric mouse-feline mAb at days 0, 7 and 21 compared to placebo controls. Although chimeric mouse-feline 11E12 showed an initial trend toward efficacy at day zero, it did not provide a significant reduction in pruritus compared to placebo vehicle at any time point. Feline 11E12 1.1 (Feline 11E12 1.1) did not improve pruritus at day 0 and did not show a trend towards efficacy when compared to vehicle placebo, so no further challenge with IL-31 was performed on days 7 and 21.

Taken together, these results show a clear difference between the activity of these antibodies and the lack of efficacy of feline 11E12 1.1 in preventing behavioral signs of IL-31-induced pruritus in cats. Decreased affinity and activity of feline 11E12 1.1 likely led to its ineffectiveness in vivo. When comparing the results on the effectiveness of chimeric mouse-feline 11E12 and chimeric mouse-feline 15H05, the differences are less pronounced. The _Kd values of the chimeric forms of both mAbs are comparable to their murine progenitors, with slightly higher affinity for mouse-feline 11E12 for both feline and canine IL-31 (FIG. 2A). However, this increase in affinity does not translate directly into an increase in activity, as the chimeric mouse-feline 15H05 IC ₅₀ against pSTAT3 signals induced by feline IL-31 in feline FCWF4 cells is approximately 2-fold higher than that of the chimeric mouse-feline 11E12 (Fig. 3). These data indicate that recognition of feline IL-31 by the hypervariable regions of the 15H05 antibody leads to greater neutralization of the signaling ability of this cytokine through its co-receptor, which in turn makes this antibody more effective in blocking itch in cats. The differences in IC ₅₀ observed in these cell-based assays make them a promising tool for predicting in vivo performance and detecting subtle differences in epitope recognition between antibodies from the same lot and from different lots.

1.14. Evaluation of the Efficacy of Felinized Anti-IL-31 15H05 Antibodies in Vivo in a Cat Pruritus Model

Based on the positive efficacy results of chimeric mouse-feline 15H05 described above, further work was done to increase the affinity and activity of felinized 15H05 (described above in Section 1.8). Systematic substitutions of the light chain variable region frameworks of the feline 15H05 1.1 antibody (Feline 15H05 1.1) resulted in the identification of Feline 15H05 1.1 FW2 with increased affinity for both feline and canine IL-31 compared to murine 15H05 (Figure 2) . The incorporation of heavy and light chains of Feline 15H05 1.1 FW2 into one plasmid resulted, after their production in the HEK and CHO expression systems, in the production of antibodies ZTS-927 and ZTS-361. The affinity and activity of both antibodies obtained when expressed from the same plasmid are also described in FIG. 2 and 3, respectively.

The fully feline anti-feline IL-31 mAb ZTS-361 was evaluated for its ability to neutralize behavioral manifestations of pruritus in an in vivo IL-31-induced pruritus model. On FIG. 10A shows baseline behavioral signs of pruritus before challenge in the T01 placebo vehicle and T02 antibody ZTS-361 groups from day -7 to day 28, where day zero is the day of antibody administration in the T02 group. As shown in this graph, the behavioral signs of pruritus assessed in the T01 and T02 groups prior to IL-31 administration did not vary significantly, with the number of pruritic manifestations noted during the 30-minute follow-up period ranging from 0 to 10. This study differed from the preliminary model with the feline challenge described in Section 1.13 above in that, on day zero, 4 mg/kg ZTS-361 was administered subcutaneously to cats without combining it with feline IL-31 to form a pre-coupled complex. This allows for a more accurate assessment of efficacy, since by the time of the first administration of IL-31, the ZTS-361 antibody will have been circulating for seven days and will only be able to bind and neutralize circulating IL-31 if the antibody is sufficiently exposed.

For this study, behavioral signs of pruritus were assessed on days 7, 21, and 28 for 1 hour after intravenous administration of IL-31 protein at a dose of 0.5 μg/kg. FIG. 10B shows the efficacy of the ZTS-361 antibody showing a significant reduction in pruritus observed on days 7 (p less than 0.0001), 21 days (p less than 0.0027) and 28 days (p less than 0.0238) after IL challenge. -31, compared to placebo vehicle control. The data obtained in this challenge model confirm previous observations demonstrating the efficacy of chimeric mouse-feline 15H05 and confirm its activity against cells and the significance of the feline IL-31 epitope recognized by CDR 15H05. In addition, these data support the ability of the ZTS-361 antibody to neutralize itch induced by feline IL-31 in vivo and show that this antibody can be used as a therapeutic agent in the treatment of IL-31 mediated disease in cats, including atopic dermatitis. .

Recent data on plasma levels of IL-31 in client animals indicate an increase in the amount of this cytokine in the circulating blood of dogs with atopic and allergic dermatitis compared to normal laboratory beagles (Fig. 11A). A study was recently conducted to determine serum levels of IL-31 in cats with a presumptive diagnosis of allergic dermatitis (AD) from several different geographic areas in the United States. On FIG. 11B shows the results of this study showing that, similar to dogs with atopic and allergic dermatitis, the 73 cats with this presumptive diagnosis included in the study had mean circulating IL-31 levels of 8799 fg/mL, compared to 205 fg/mL. in 17 control cats of the same age. To understand the levels of canine IL-31 in a development study using a prior model, the pharmacokinetic profile of canine IL-31 in dogs was analyzed after its subcutaneous administration at a dose of 1.75 μg/kg. On FIG. 11C shows peak plasma levels during the first hour, reaching a maximum level of approximately 30 ng/mL and maintaining a level of approximately 400 pg/mL after three hours. Based on these data, it is logical to assume that the amount of feline IL-31 in circulating blood after its intravenous administration at a dose of 0.5 μg/ml, used in this model in cats, will significantly exceed its amount observed in natural disease in dogs and cats.

1.15. Analytical methods used to improve leading anti-IL-31 feline antibodies

When developing cell lines, a variety of analytical methods are used to ensure that a therapeutic antibody can be obtained in a stable manner. Careful attention to analytical methods that ensure (without limitation) the identity, purity and activity of the product is critical to the consistent production of leading monoclonal antibodies and a close correlation with its activity and safety in animals of the target species. Because antibodies are homodimers of heterodimeric units held adjacent to each other by interchain disulfide bonds, any disruption of their pairing can result in heterogeneity of the protein drug. Two analytical methods well suited for monitoring antibody dissociation are non-reducing (NR) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and non-reducing (NR) capillary gel electrophoresis (CGE).

NR SDS-PAGE is a convenient method for qualitative determination of the mass of individual protein species in the analyzed sample. SDS enters into a hydrophobic interaction with proteins, uniformly imparting to them a total negative charge, which allows the separation of individual components by their mass. After electrophoretic separation on a polyacrylamide gel, the proteins are stained with a dye such as Coomassie blue to allow their detection. The non-linearity of staining precludes the determination of the absolute amount of protein in individual bands, but allows it to be assessed using densitometric analysis software (VersaDoc, Bio-Rad). Capillary gel electrophoresis, commonly known as CGE, also involves the processing of proteins using SDS, which results in uniform negative charge and dissociation of non-covalent protein complexes. In the presence of an electric field, the proteins associated with the SDS move to the anode and are detected using ultraviolet absorption at a fixed wavelength of 220 nm. Separation is based on the size of the sample components in a capillary filled with a replaceable SDS polymer filter gel matrix. In non-reducing CGE, the alkylating agent iodoacetamide (IAM) is added to minimize disulfide bond shuffling during sample preparation. Intact IgG is separated from any fragmented species to allow quantification of purity. CGE analysis software (of which Empower or 32 Karat are examples) uses a time corrected area (TCA), which is defined as the ratio of the area of an individual peak to the time at which that peak emerges. Total TCA is defined as the sum of the TCA of all peaks greater than or equal to 0.3%. Thus, the percentage of monomeric intact IgG and minor varieties can be calculated from their individual TCA as a percentage of the total TCA.

In light of the promising in vivo performance data for ZTS-361 described in the feline pruritus model (Section 1.14), further characterization of different lots of this antibody was performed using these described analytical methods. It was found that stable pools expressing felinized anti-IL-31 antibody (ZTS-361) had increased levels of lower molecular weight species (including free light chains) visible by Coomassie staining on SDS-PAGE compared to mouse hybridoma precursor 15H05 (Fig. 12). Intact or intact monomer refers here to IgG with two heavy chains and two light chains held together by interchain disulfide bonds and an estimated molecular weight of approximately 150 kDa. HHL refers here to "Heavy Heavy Light (heavy heavy light)", IgG without one light chain with an estimated molecular weight of approximately 125 kDa. HH refers here to "Heavy Heavy (heavy heavy)", IgG without both light chains with an estimated molecular weight of approximately 100 kDa. HL refers here to "Heavy Light (heavy light)", IgG with one heavy chain, one light chain and an estimated molecular weight of approximately 75 kDa. L refers here to "Light (light)", IgG with one light chain and an estimated molecular weight of approximately 25 kDa, also referred to here as a free light chain. Quantification of the same material using NR-CGE revealed a significant decrease in the proportion of intact monomeric IgG (83%) ZTS-361 compared to the murine progenitor 15H05 (94.7%) (Fig. 13a). On FIG. 13B shows electropherograms after separation of samples by NR-CGE. These peaks at different retention times were used to quantify the percent of total TCA for sample 1 (ZTS-361) and sample 2 (mouse 15H05). The sum of minor peaks with a molecular weight less than the major peak of intact IgG was used to calculate the percentage of fragments indicated in FIG. 13A (% Fragments). Stable CHO pools producing ZTS-361 resulted in 17% of the final antibody produced as a fragmented form of felinized IgG compared to 5.3% for murine 15H05 hybridoma.

To facilitate understanding of this phenomenon, individual clonal isolates of CHO cells derived from the ZTS-361 clonal pool were prepared to determine whether the percentage of intact IgG monomer varied between individual clones. On FIG. 14A shows NR SDS-PAGE of purified antibody originating from cultures of 8 individual clones stably expressing ZTS-361 with Coomassie staining. For comparison, lanes labeled 1 and 8 show a control antibody with a known high percentage of intact IgG monomer (approximately 97%). On the right is a quantitative densitometric analysis of the intensity of the bands. In different clones, the percentage of intact monomer varied from 80.2% to 86% with an average value of approximately 82%. Similarly, the percentage of fragments determined from individual clones ranged from 14.0% to 19.5% with an average of 17.5%. On FIG. 14B shows the quantification of these individual clones using NR-CGE. Less variability was noted using this method with an average percentage of intact monomer in the 8 clones analyzed of 86.3% and a percentage of lower molecular weight species of 13.7%. Despite the noted high homogeneity of the percentage of intact IgG monomer for ZTS-361 from individual clones, it was of interest to understand why the overall level of intact IgG monomer was less than that of the murine version of this antibody. It is important to note that the production of antibodies, including but not limited to felinized antibodies, in transient expression systems (examples of which are HEK and CHO cells) resulted in the production of IgG with a high percentage of the monomeric form (from about 88% to about 92%) (data not shown). At the same time, the amount of antibodies obtained from these transient cultures is significantly less than the amount obtained from a stable CHO line. Without wishing to be bound by theory, fragmented antibody species observed with felinized antibodies and antibodies of other species may be due to conditions under which the host cell produces an exceptionally large amount of antibody and inherent limitations in culture conditions and/or molecular composition of the antibody. .

1.16. Consideration of primary amino acid sequences of the IgG kappa light chain constant domain of several mammalian species

Analysis of the potential limitations of the felinized ZTS-361 antibody began by looking at the primary sequence of the antibody. ZTS-361 consists of a heavy chain containing a variable region (SEQ ID NO: 121; FEL_15H05_VH1) to which the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1) corresponds, in combination with a feline IgG heavy chain constant region (SEQ ID NO: 173 ; Feline_HC_AlleleA_1) to which the nucleotide sequence corresponds (SEQ ID NO: 174; Feline_HC_AlleleA_1) and a light chain containing the variable region (SEQ ID NO: 135; FEL-15H05-VL1_FW2) to which the nucleotide sequence corresponds (SEQ ID NO: 136; FEL-15H05-VL1_FW2), in combination with the feline IgG light chain constant region (SEQ ID NO: 175; Feline LC Kappa G minus) to which the nucleotide sequence corresponds (SEQ ID NO: 176; Feline_LC_Kappa_G_minus). The functional properties of naturally occurring feline antibody heavy chain constant regions have been previously described by Strietzel et al. (2014 Veterinary Immunology and Immunopathology April 15; 158(3-4): 214-223). Here, the inventors describe the cloning and expression of the feline ZTS-361 antibody using the heavy chain constant region (SEQ ID NO: 171; Feline_HC_AlleleA_l). SEQ ID NO: 171 Feline HC AlleleA 1 corresponds to feline IgG1a in Strietzel et al. 2014 supra and appears to be functionally equivalent to human IgG1. Comparison of these functional properties and alignment of this heavy chain constant region with other constant regions of a wide range of species revealed no apparent regions of concern resulting in insufficient production of intact IgG monomer (data not shown).

A similar analysis of the kappa constant region used in ZTS-361 (SEQ ID NO: 175; Feline_LC_Kappa_G_minus) reveals a unique aspect of the diversity of kappa light chain constant regions seen in at least feline and canine sequences. Different species use the kappa light chain in their immunoglobulin repertoire at different frequencies. On FIG. 15 shows exemplary C-terminal amino acids and corresponding nucleotides of several kappa light chain constant regions from the indicated species. The percentage of kappa light chain utilization in IgG of the indicated species is given from the following sources: dog (Canine), cat (Feline) and pig (Pig) (Aran et al. 1996 Zentralbl Veterinarmed. Nov; 43(9):573-6), mink (Mink) (Bovkun et al. 1993 Eur J Immunol. Aug; 23(8): 1929-34), mouse (Mouse) (Woloschak et al. 1987 Mol Immunol. Jul; 24(7): 751-7) and Human (Barandun et al. 1976 Blood. Jan; 47(l):79-89). Typically, monoclonal antibodies are obtained using mice. As shown in FIG. 15, mice use the kappa light chain approximately 95% of the time compared to the lambda light chain. In contrast, dogs and cats use predominantly the lambda light chain in their immunoglobulin repertoire (9% and 8%, respectively). By comparison, two non-human mammals (pig and mink) and a human demonstrate balanced use of the kappa and lambda light chains (50%, 46% and approximately 50%, respectively). The image below shows and aligned the position of most of the C-terminal cysteines in different species. This cysteine is critical for quaternary complex formation and structure formation of intact IgG, as it is involved in the formation of an interchain covalent disulfide bond with the constant heavy chain. Without being bound by any theory, it is of great interest to note that at least canine and feline kappa light chains contain a few amino acids after the terminal cysteine. These additional amino acid residues have both polarity (glutamine) and charge (arginine, aspartic and glutamic acids). These residues are usually located where they participate in interactions through hydrogen bonds, including, without limitation, interactions with the external aqueous environment. The nature and location of these additional residues beyond the terminal cysteine may interfere with the formation of the interchain disulfide bond required for the formation of the IgG heterodimer. Two mammals (pig and mink) have fewer extra residues after the C-terminal cysteine and use the kappa and lambda light chains in approximately equivalent ratios (50% and 46%, respectively). Using transient expression, a series of recombinant forms of ZTS-361 were obtained from HEK cells with changes and deletions of amino acids at the C-terminus of the kappa light chain constant region located immediately after cysteine at amino acid position 107 of SEQ ID NO: 175, and the percentage of monomeric IgG in them was analyzed by NR-CGE (data not shown). As stated previously, unpaired light chain and lower molecular weight IgG complexes are more likely to be generated under conditions of antibody overproduction by stable clonal cells. However, NR-CGE allows for differences in the amount of intact IgG monomer versus lower molecular weight contaminants from cultures obtained by transient transfection of cells (eg, HEK and CHO). This allows a qualitative assessment of the percentage of monomeric IgG obtained from transient cultures. This qualitative use of NR-CGE in transient cultures allowed us to analyze the many modifications and deletions of the C-terminus of the ZTS-361 kappa constant chain for the percentage of monomeric IgG (data not shown). Without wishing to be bound by theory, the removal of QRE residues from the C-terminus of the feline kappa chain constant region appears to be the most optimal for obtaining monomeric recombinant feline IgG. It is possible to attach other residues to the C-terminus. In general, the addition of one or two additional amino acids after the cysteine at position 107 of Feline LC Kappa G minus (SEQ ID NO: 175) appears to be acceptable based on a qualitative assessment of percent monomer after transient IgG production. Using the same qualitative analyzes, even adding three additional consecutive amino acids to the C-terminus of the cysteine at position 107 instead of native QRE amino acid residues can be acceptable if these amino acids have minimal effect on electrostatic charge. It should be noted here that the number and chemical properties of additional amino acids after the cysteine at position 107 of Feline LC Kappa G minus (SEQ ID NO: 175) will affect the formation of an interchain disulfide bond between the kappa light chain constant region and the corresponding immunoglobulin heavy chain constant region. It can be assumed that modifications and/or deletions in this region may favorably influence the production of a homogeneous intact form of IgG from a stable recombinant cell line.

The C-terminal amino acid of human and murine kappa light chains is a terminal cysteine, so there are no additional residues that could interact with the environment when forming a disulfide bond with the heavy chain constant region. Therefore, it is hypothesized here that these additional C-terminal residues present in at least the feline and canine kappa light chain constant regions limit the possibility of this disulfide bond formation, which may not normally be seen in vivo due to the small number of these antibody species, but may be of great importance in the overproduction of these forms, which have undergone species adaptation, in laboratory conditions. Such limitations may include the absence of disulfide bond formation between the C-terminus of the kappa light chain and the heavy chain constant region, resulting in the presence of the HHL, HH, HL and L variants described previously when generating a recombinant antibody from a stable cell line. These lower molecular weight species are undesirable in the preparation of a homogeneous drug product and their presence can be a problem in terms of quality and safety.

Also included in this observation are the codons encoding the amino acid residues of this region, shown below the letter of each amino acid in FIG. 15. Mammals have three stop codons that signal the termination of polypeptide translation in the ribosome (TAA, TGA, and TAG). Without wishing to be bound by any particular theory, it has been noted that most of the codons encoding the amino acids after the C-terminal cysteine (and the C-terminal cysteine itself) differ from the stop codons by only one nucleotide (Fig. 15, light gray letters in codons). The inventors conjecture that somatic mutations at these various nucleotide positions could provide the optimal length and amino acid composition of the immunoglobulin kappa light chain constant region for efficient expression and proper pairing of kappa IgG antibody chains. When evaluating the possibility of the presence of the corresponding amino acid, which may be located at the C-terminus next to the terminal cysteine, subtle differences cannot be taken into account. Of note here is the observation that the addition of EA residues to the porcine light chain or Q residues to the mink light chain may not significantly adversely affect interchain disulfide bond formation and expression of the intact IgG molecule. These species show equivalent use of the kappa and lambda light chains and these attachments are acceptable to them. With regard to, without limitation, C-terminal residues of canine and feline kappa light chains, the distance from the C-terminal cysteine and the charged nature of arginine and aspartic and glutamic acid residues are believed to have a significant effect on the ability of feline and canine antibodies to efficiently and correctly form a covalent disulfide bond with the corresponding heavy chain.

1.17. Analysis of the quaternary structure of the facing surfaces of the feline heavy chain and kappa light chain

On FIG. 16A is a pictographic representation of the putative structure of feline IgG whose heavy and light chains are equivalent to ZTS-361 (confirming analytical data not shown). To simplify the positions of intrachain and interchain disulfide bonds with the release of heavy chain cysteine (CYS15) and light chain cysteine (CYS107) are shown in only one branch of the structure. The CYS15 amino acid residue shown is position 15 of Feline HC AlleleA wt (SEQ ID NO: 171) and Feline HC AlleleA 1 (SEQ ID NO: 173) and the nucleotide residues shown are positions 43-45 of Feline HC AlleleA wt (SEQ ID NO : 172) and 43-45 Feline HC AlleleA 1 (SEQ ID NO: 174), respectively. The CYS107 amino acid residue shown is position 107 of Feline LC Kappa G minus (SEQ ID NO: 175) and Feline LC Kappa G minus QRE minus (SEQ ID NO: 186) and the nucleotide residues shown are positions 319-321 of Feline LC Kappa G minus (SEQ ID NO: 176) and 319-321 Feline LC Kappa G minus QRE minus (SEQ ID NO: 187). As stated previously, feline ZTS-361 is functionally equivalent to human IgG1; however, its disulfide bond pattern is more similar to human IgG2 (data not shown). On FIG. 16B shows a homologous pattern corresponding to the two P(ab') ₂ branches of ZTS-361, with the approximate locations of CYS15 and CYS107 highlighted using MOE software (Chemical Computing Group, Montreal, Quebec, Canada) for clarity. On FIG. 16C shows the contribution of additional electrostatic charges to the local environment due to QRE constant kappa light chain residues located immediately after CYS107. The charge of these three amino acids is shown as shells with a mesh surface. In homologous models, regions with random helical structure are shown less accurately than regions with ordered secondary structure elements, such as alpha helices or regions of ordered beta folding. However, this is not significant for heavy chain CYS15 as this region of the IgG constant domain is well defined by the large amount of structural data and the ordered nature of this region with conserved IgG structure. Similarly, the position of CYS107 is determined in the structures of many antibodies, which allows to assess its proximity to the heavy chain CYS15. In this model, the addition of residues after the terminal cysteine of the light chain can only be determined based on the geometry of neighboring residues and the calculation of local energy minima. This mapping of the ZTS-361 kappa feline light chain is the most optimal working model for developing an experimental hypothesis. Taken together, these results indicate that additional amino acid residues beyond the terminal cysteine of the feline kappa light chain (and probably other kappa light chain species) reduce the efficiency of pairing with the heavy chain, leading to a high mismatch rate and poor antibody production. .

1.18. Production of felinized anti-IL-31 antibody ZTS-1505 with a modified C-terminus of the kappa light chain constant region

Due to possible limitations in the stable preparation of a homogeneous preparation of the ZTS-361 antibody, it was considered necessary to eliminate the decrease in the percentage of the resulting monomer. Including, but not limited to, for this purpose, ZTS-1505 was obtained, the heavy chain of which contains the variable region (SEQ ID NO: 121; FEL_15H05_VH1), which corresponds to the nucleotide sequence (SEQ ID NO: 122; FEL_15H05_VH1), in combination with a feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1) to which the nucleotide sequence (SEQ ID NO: 174; Feline_HC_AlleleA_1) corresponds. In the case of ZTS-1505, the light chain contains the variable region (SEQ ID NO:135; FEL-15H05-VL1_FW2) to which the nucleotide sequence (SEQ ID NO:136; FEL-15H05-VL1_FW2) corresponds, in combination with the feline light chain constant region. IgG (SEQ ID NO: 186; Feline_LC_Kappa_G_minus_QRE_minus), which corresponds to the nucleotide sequence (SEQ ID NO: 187; Feline_LC_Kappa_G_minus_QRE_minus). ZTS-1505 is identical to ZTS-361, except for the removal of three additional QRE residues from the very C-terminus of the kappa light chain constant region to avoid the undesirable effects of producing antibodies with lower molecular weight species due to inefficient pairing with the kappa light chain. It is assumed that the removal of these residues is useful for obtaining monomeric IgG. However, the present invention is not necessarily limited to this one modification of the kappa light chain, as other modifications to the kappa light chain have been made, which, according to preliminary experimental data, had a neutral or even positive effect on transient expression (data not shown).

On FIG. 17A describes the variable and constant sequences used to construct the ZTS-361 and ZTS-1505 antibodies, highlighting the differences in the light chain used for ZTS-1505. The production of a stable CHO cell line expressing ZTS-1505 is described in Section 1.9 of this application. Assessing the quality of the antibody was of great interest, so individual clones of stably transfected CHOs were studied for their ability to produce monomeric antibody. On FIG. 17B shows the results of NR-CGE quantification of these individual clones. Twenty-three individual stable CHO clones were analyzed and a high degree of uniformity in the percentage of monomer obtained was noted, with an average value of 90.3%. This corresponds to a 4% increase in average percent monomer yield compared to individual ZTS-361 clonal isolates (FIG. 14B) and 7.3% compared to the stable pool (FIG. 13A). On FIG. 17C shows a direct comparison of individual stable CHO cell lines expressing ZTS-361 and ZTS-1505 antibodies under various culture conditions. It should be noted that the antibodies described in FIG. 12, 13, and 14 were obtained under culture conditions equivalent to culture conditions "A" in FIG. 17C, and these conditions are described in Section 1.9 of this application. Both cell lines were grown for 14 days and viability, titer (antibody yield in g/l) and monomer percentage were determined by NR-CGE. Under all conditions, both cell lines showed a similar percentage of viability. In the case of ZTS-1505, the percentage of monomer by NR-CGE was 4-5% higher than that of ZTS-361, further indicating that the kappa light chain constant region of ZTS-1505 without additional QRE residues had a positive effect on the compound. kappa light chain feline heavy chain constant region. Even more pronounced was the increase in the amount of obtained antibody ZTS-1505 compared to ZTS-361. For ZTS-1505, stable CHO cells produced an average of 3.5 times more antibody than for ZTS-361, which has a wild-type kappa light chain constant region.

To verify that modification of the kappa light chain constant region did not affect the affinity and activity of ZTS-1505, a comparison was made using Biacore and inhibition of IL-31 mediated pSTAT signaling in canine and feline cells. Table 1 below describes the results of the analysis using several types of IL-31 protein as the capture surface. These results show almost identical affinity for ZTS-1505, which has a modification of the kappa light chain constant region, and ZTS-361, which has the C-terminus of the wild-type kappa chain constant region, for canine, feline, and equine IL-31 proteins. Also described herein is the putative binding of ZTS-1505 to the 15H05 and 11E12 feline mutants described in Section 1.2 of this application. Modification of the C-terminus of the kappa chain constant region did not alter epitope selectivity, as demonstrated by the lack of binding of ZTS-1505 to the feline mutant 15H05 protein and retained binding to the feline mutant HE 12, similar to ZTS-361.

140

Table 2 below shows the cell activity data of ZTS-1505 versus ZTS-361. Modification of the ZTS-1505 kappa light chain constant region did not affect the ability of this antibody to inhibit cellular pSTAT signals induced by canine and feline IL-31 in canine DH82 cells and feline FCWF-4 cells, respectively, as indicated by comparable IC ₅₀ values.

Taken together, these results indicate that deletion or modification of the C-terminus of the kappa light chain of those species whose native germline sequences encode these additional residues beneficially affects both the production of uniform recombinant antibodies of these species and the amount of antibody produced. stable cell line (eg, increasing yield). In addition, such modifications, while increasing the quality and quantity of the resulting antibody, do not adversely affect the affinity and activity of the antibody for its IL-31 target protein. It should be noted here that these factors may be useful in generating recombinant antibodies for therapeutic use in many species.

1.19. Confirmation of NR-CGE results using anti-IL-31 mAb 1505 and further demonstration of utility of modified kappa chain constant region C-terminus using anti-NGF antibody

It was of interest to determine whether the increase in the percentage of monomeric IgG is applicable to the production of other antibodies whose target proteins are other than IL-31. To do this, an additional set of pools of stable CHO cell lines with felinized antibodies recognizing feline nerve growth factor beta (NGF) (ZTS-768 and ZTS-943) was obtained. In the sequence of the ZTS-768 antibody, the heavy chain is (SEQ ID NO: 220; ZTS 768 VH) to which the nucleotide sequence (SEQ ID NO: 221; ZTS 768 VH) corresponds, in combination with the feline IgG heavy chain constant region (SEQ ID NO: 171; Feline HC AlleleA wt), which corresponds to the nucleotide sequence (SEQ ID NO: 172; Feline HC AlleleA wt). In the sequence of the ZTS-943 antibody, the heavy chain is (SEQ ID NO: 224; ZTS 943 VH) to which the nucleotide sequence (SEQ ID NO: 225; ZTS 943 VH) corresponds, in combination with the feline IgG heavy chain constant region (SEQ ID NO: 171; Feline HC AlleleAwt), which corresponds to the nucleotide sequence (SEQ ID NO: 172; FelineHCAlleleAwt). In the case of ZTS-768, the light chain variable region is (SEQ ID NO: 222; ZTS 768 VL) to which the nucleotide sequence (SEQ ID NO: 223; ZTS 768 VL) corresponds. In the case of ZTS-768, this light chain variable region is presented in combination with a feline IgG light chain constant region (SEQ ID NO: 175; FelineLCKappaGminus) to which the nucleotide sequence corresponds (SEQ ID NO: 176; Feline LC Kappa G minus). In the case of ZTS-943, the light chain variable region is (SEQ ID NO: 226; ZTS 943 VL) to which the nucleotide sequence corresponds (SEQ ID NO: 227; ZTS 943 VL) in combination with the feline IgG light chain constant region (SEQ ID NO: 186; Feline LC Kappa G minus QRE minus) to which the nucleotide sequence corresponds (SEQ ID NO: 187; FelineLCKappaGminusQREminus). For direct comparison with anti-IL-31 antibodies, stable pools of ZTS-361 and ZTS-1505 were also prepared using identical conditions for culturing and purifying anti-NGF antibodies.

After obtaining and purifying the four antibodies described above, NR-CGE was used to determine the percentage of monomeric intact IgG and minor varieties. Comparison of these antibodies, prepared and purified using identical methods, assessed the usefulness of incorporating a modified C-terminus of the kappa chain constant region into structurally different antibodies that recognize different target proteins. On FIG. 18A shows percent identity when comparing the variable regions of anti-feline NGF and anti-IL-31 antibodies calculated using ClustalW software. On FIG. 18B and 18C show the alignment of the heavy and light chain variable regions, respectively, of anti-feline IL-31 and NGF antibodies, with CDRs highlighted in boxes. Clearly, anti-IL-31 and anti-NGF antibodies differ from each other without showing a common identity, especially in the antigen-binding regions identified as CDRs.

On FIG. 19 shows NR-CGE results comparing anti-feline IL-31 and anti-feline NGF antibodies with and without C-terminus modification of the kappa chain constant region. Similar to previous data, the wild-type kappa light chain-containing anti-IL-31 antibody (ZTS-361) had 80.74% monomeric IgG, with the most abundant minor variant, HHL, accounting for 8.71%. After removing the three C-terminal residues to give ZTS-1505, a clear improvement in monomer percentage was again observed (89.17% monomer at 5.9% HHL). NR-CGE analysis of purified IgG from anti-feline NGF mAb containing wild-type kappa light chain (ZTS-768) showed similar levels of isolated monomer and minor species (80.81% monomer at 12.33% HHL) compared to anti-IL-31 antibody ZTS-361. Of note, after removal of the C-terminal residues from the anti-feline NGF antibody, the same pattern was observed with 88.59% monomer yield, with the most abundant minor species, HHL, accounting for 5.98%.

These results indicate a structural difference between a feline IgG protein containing wild-type amino acid residues (QRE) at the C-terminus of the kappa light chain and an IgG from which these residues have been removed. The results of the experiments described here show that the use of this kappa light chain modification results in monomeric feline IgG with fewer contaminating minor species. In addition, the results described here clearly demonstrate that this method is applicable to structurally different antibodies recognizing completely different targets, and therefore it is likely that this modification will be applicable to a wide range of feline antibodies, as well as to other mammalian antibodies having additional C-terminal amino acids in the kappa light chain constant region. Without wishing to be bound by any theory, this light chain modification appears to result in more precise pairing of immunoglobulin chains when induced to be produced by stable CHO cell lines, resulting in increased monomeric IgG and possibly increased overall antibody yield. Both of these results are highly desirable from the point of view of the manufacture of drugs containing antibodies for commercial use.

1.20. Identification of anti-equine IL-31 antibodies binding to the equivalent region of the equine IL-31 protein compared to mAb 15H05 binding to feline IL-31

Given the promising in vivo efficacy in a feline pruritus model using antibodies from the 15H05 mouse strain described here, it was desirable to identify new antibody substrates that bind to a similar region of the equine IL-31 protein orthologue. To this end, in order to identify antibodies binding to equine IL-31, mice were immunized with recombinant equine IL-31 (SEQ ID NO: 165).

Antibody titers in the serum of immunized animals were determined using ELISA as described previously. For fusion, donor splenocytes from a single responding mouse were used and hybridoma supernatants were screened for antibodies binding to the equine IL-31 protein by ELISA. This led to the identification of two mouse antibodies that bind to a region of the equine IL-31 protein comparable to the 15H05 binding site on feline IL-31. Section 1.10 of this application describes the characterization of the binding site of the 15H05 antibody, illustrating the approximate binding site in the feline IL-31 homologous model shown in FIG. 6B. On FIG. 20 shows alignment of wild-type feline IL-31 (SEQ ID NO: 157) with equine IL-31 (SEQ ID NO: 165) using ClustalW software. The arrows above the alignment indicate the P126 and D128 residues described in Section 1.10 as being within the binding region of the 15H05 antibody to feline IL-31 (site 2) (FIG. 6B). The two anti-equine IL-31 antibodies that share this common binding site in the equine IL-31 protein are 04H07 and 06A09.

Next, 04H07 and 06A09 against equine IL-31 were subcloned to obtain a homogeneous antibody producing hybridoma and to sequence their heavy and light chain variable regions. The sequences of the variable regions of the murine anti-IL-31 antibody defined for the 04H07 antibody are as follows: heavy chain variable region 04H07 (SEQ ID NO: 212; Mu_04H07_VH) to which the nucleotide sequence (SEQ ID NO: 213; Mu_04H07_VH) corresponds, variable region light chain 15H05 (SEQ ID NO: 214; Mu_04H07_VL), which corresponds to the nucleotide sequence (SEQ ID NO: 215; Mu_04H07_VL). Heavy chain variable region 06A09 (SEQ ID NO: 216; Mu_06A09_VH) to which the nucleotide sequence corresponds (SEQ ID NO: 217; Mu_06A09_VH), light chain variable region 06A09 (SEQ ID NO: 218; Mu_06A09_VL) to which the nucleotide sequence corresponds (SEQ ID NO: 219; Mu_06A09_VL). On FIG. 21 shows the alignment of the variable heavy (FIG. 21A) and light (FIG. 21B) chains 04H07 and 06A09 compared to the mouse antibody 15H05 using Clusta1W. For comparison, the location of each of the six CDRs is framed. Anti-equine IL-31 antibodies 04H07 and 06A09 are very similar to each other and probably come from a common clonal lineage. Anti-feline IL-31 antibody 15H05 clearly differs in CDR amino acid sequence and length of CDRH3 and CDRL1 from 04H07 and 06A09 against equine IL-31. Interestingly, the binding site of the 15H05 antibody to feline IL-31 is also preserved in the horse protein (arrows in Fig. 20). These results are another example that structurally different CDRs can recognize a common epitope region on two IL-31 orthologues.

1.21. Alanine scanning of the CDR of anti-IL-31 antibody ZTS-1505

The region of an antibody that provides antigen recognition is the paratope. The paratope is formed by a combination of amino acids of the hypervariable regions (CDRs) of the heavy and light chain variable regions. The binding of an antibody to an antigen is often mediated by the side chains of CDR residues and the side chains or carbohydrate moieties of the antigen. To help identify critical side chains involved in antibody recognition of an antigen, alanine scan mutagenesis was performed on each CDR residue of both the heavy and light chains. These mutants were then analyzed separately for their ability to bind to feline IL-31 using Biacore and evaluated for inhibition of IL-31 mediated signals in an assay with FCWF-4 cells.

To determine the relative affinity of the alanine scan mutant mAbs versus the parent mAb, binding profiles were determined for feline IL-31 coated chips at 100 nM using Biacore T200. The average number of response units after four repetitions obtained for the original mAb, plus/minus 3 standard deviations, was used to calculate the parameters that determine the threshold number of response units, including association and dissociation rates for antibody binding (Fig. 22). The percentage of data points for each mutant that met this threshold was then used to determine the "% similarity score" (FIG. 23). The score of similarity resulting from alanine substitutions at each position of the heavy and light chain CDRs of the ZTS-1505 antibody is shown in FIG. 23 and 24, respectively.

To determine the relative potency of ZTS-1505 mutant antibodies in a cell assay, each antibody was evaluated for its ability to inhibit IL-31 mediated STAT phosphorylation in FCWF-4 cells at a concentration of 15 μg/mL. Relative inhibition of STAT phosphorylation was determined by assessing the % inhibition of STAT phosphorylation by each mutant compared to the parent antibody. The results of replacing each position in the heavy and light chain CDRs of ZTS-1505 with alanine are shown as "percent inhibition versus parental antibody" in FIG. 23 and 24, respectively. By evaluating the effect of individual alanine substitutions on binding affinity and cell activity, the role of the side chains of each CDR amino acid residue in antigen recognition can be assessed. CDR residues that can be exchanged for alanine while maintaining activity are likely to be subject to multiple amino acid substitutions and are not critical to antigen recognition. Based on these data, at least residue 4(1) of SEQ ID NO: 1, residues 1-3 (NIN), 5-7 (TSG), 9-11 (TEN), and 13 (Q) of SEQ ID NO: 2 and residues 4 (K), 6 (D) and 13 (V) of SEQ ID NO: 3 in CDR1, 2 and 3 of the heavy chain, respectively, are not critical for antigen binding. Light chain CDR residues not critical for binding include residues 3-7 (SQGIS) of SEQ ID NO: 4, residues 3 (S) and 5 (L) of SEQ ID NO: 5, and residues 4 (Q), 5 ( T) and 9 (T) SEQ ID NO: 6 of CDRL1, 2 and 3, respectively.

1.22. Binding affinity of two felinized variants of the ZIL8 antibody and their activity against cells

Above, Section 1.6 of this Example section describes the identification of a canine antibody that recognizes feline IL-31, called ZIL8. Initial screening showed that this antibody is able to bind to the feline IL-31 protein, but its binding is affected by the 15H05 mutation. Therefore, it was of interest to obtain a felinized form of this antibody for use as a therapeutic agent in cats. For this, the felinization technique described earlier in Section 1.8 of this application was used. By transferring the ZIL8 CDRs to suitable feline framework regions, antibodies ZTS-5864 and ZTS-5865 were generated. The heavy chain sequence of the ZTS-5864 antibody is (SEQ ID NO: 228; ZTS_5864_VH) to which the nucleotide sequence (SEQ ID NO: 229; ZTS_5864_VH) corresponds, in combination with the feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1 ) to which the nucleotide sequence corresponds (SEQ ID NO: 174; Feline_HC_AlleleA_1). In the case of ZTS-5864, the light chain variable region is (SEQ ID NO: 230; ZTS_5864_VL) to which the nucleotide sequence (SEQ ID NO: 231; ZTS_5864_VL) corresponds. In the case of ZTS-5864, this light chain variable region is presented in combination with a feline IgG light chain constant region (SEQ ID NO: 236; Feline_LC_Lambda) to which the nucleotide sequence (SEQ ID NO: 237; Feline_LC_Lambda) corresponds. The heavy chain sequence of the ZTS-5865 antibody is (SEQ ID NO: 232; ZTS_5865_VH) to which the nucleotide sequence (SEQ ID NO: 233; ZTS_5865_VH) corresponds, in combination with the feline IgG heavy chain constant region (SEQ ID NO: 173; Feline_HC_AlleleA_1 ) to which the nucleotide sequence corresponds (SEQ ID NO: 174; Feline_HC_AlleleA_1). In the case of ZTS-5865, the light chain variable region is (SEQ ID NO: 234; ZTS_5865_VL) to which the nucleotide sequence (SEQ ID NO: 235; ZTS_5865_VL) corresponds. In the case of ZTS-5865, this light chain variable region is presented in combination with a feline IgG light chain constant region (SEQ ID NO: 236; Feline LC Lambda) to which the nucleotide sequence corresponds (SEQ ID NO: 237; Feline_LC_Lambda).

On FIG. 25A and 25B show the affinity of two felinized antibodies ZTS-5864 and ZTS-5865 and their activity against cells. Both antibodies have high affinity for feline IL-31, with the (K _d (M)) affinity of ZTS-5864 approximately 4-fold higher than that of ZTS-5865. Cell activity was assessed using feline IL-31 to stimulate pSTAT3 signals in FCFW4 cells. IC ₅₀ values were calculated for each antibody as described earlier in this application. The activity of ZTS-5864 is approximately 3 times higher when comparing its IC ₅₀ value with the IC ₅₀ of ZTS-5865. It should be noted that both antibodies are considered to be active, with their IC _50s falling within the IC ₅₀ range of the 15H05 antibody line described here previously (FIG. 3). The significance of this activity on pSTAT3 signaling in feline FCFW4 cells has been demonstrated by previously positive in vivo efficacy results using chimeric and felinized antibodies in a feline pruritus model (FIG. 9 and FIG. 10).

1.23. Evaluation of the Efficacy of the Felinized Anti-IL-31 Antibody ZTS-5864 in Vivo in a Cat Pruritus Model

The in vivo efficacy of ZTS-5864 was evaluated in a feline IL-31 pruritus model as described previously in Section 1.14 of this Examples section. The results of this study are shown in Fig. 26. Prior to administration (day 7), it was shown that when challenged with IL-31 prior to administration of the drugs, animals from groups T01 and T02 developed the same itching response. During the study, up to 56 days, itching remained almost unchanged in the T01 group. In contrast, subcutaneous administration of ZTS-5864 (3.0 mg/kg) on day zero relieved pruritus after IL-31 challenge throughout the study, up to day 56 (FIG. 26, T02). The persistence of the reduction in the mean pruritus score in the T02 group at day 56 indicates a likely retention of antibody efficacy beyond this time point. These results highlight the robustness of the criteria used to screen for anti-feline IL-31 antibodies in this application. These results further support the positioning of these species-adapted antibodies as therapeutic agents for the treatment of IL-31 mediated disorders in cats.

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SEQUENCE LIST

<110> Zoetis Services LLC

Bammert, Gary F.

Dunham, Steven A.

<120> INTERLEUKIN-31 MONOCLONAL ANTIBODIES FOR VETERINARY USE

<130> ZP000226A

<150> 62/643940

<151> 2018-03-16

<160> 237

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15

<210> 55

<211> 5

<212> PRT

<213> Canis familiaris

<400> 55

Ser Tyr Val Met Thr

15

<210> 56

<211> 17

<212> PRT

<213> Canis familiaris

<400> 56

Gly Ile Asn Ser Glu Gly Ser Arg Thr Ala Tyr Ala Asp Ala Val Lys

1 5 10 15

gly

<210> 57

<211> 10

<212> PRT

<213> Canis familiaris

<400> 57

Gly Asp Ile Val Ala Thr Gly Thr Ser Tyr

1 5 10

<210> 58

<211> 11

<212> PRT

<213> Canis familiaris

<400> 58

Ser Gly Glu Thr Leu Asn Arg Phe Tyr Thr Gln

1 5 10

<210> 59

<211> 7

<212> PRT

<213> Canis familiaris

<400> 59

Lys Asp Thr Glu Arg Pro Ser

15

<210> 60

<211> 10

<212> PRT

<213> Canis familiaris

<400> 60

Lys Ser Ala Val Ser Ile Asp Val Gly Val

1 5 10

<210> 61

<211> 5

<212> PRT

<213> Canis familiaris

<400> 61

Thr Tyr Val Met Asn

15

<210> 62

<211> 17

<212> PRT

<213> Canis familiaris

<400> 62

Ser Ile Asn Gly Gly Gly Ser Ser Pro Thr Tyr Ala Asp Ala Val Arg

1 5 10 15

gly

<210> 63

<211> 8

<212> PRT

<213> Canis familiaris

<400> 63

Ser Met Val Gly Pro Phe Asp Tyr

15

<210> 64

<211> 11

<212> PRT

<213> Canis familiaris

<400> 64

Ser Gly Lys Ser Leu Ser Tyr Tyr Tyr Ala Gln

1 5 10

<210> 65

<211> 7

<212> PRT

<213> Canis familiaris

<400> 65

Lys Asp Thr Glu Arg Pro Ser

15

<210> 66

<211> 10

<212> PRT

<213> Canis familiaris

<400> 66

Glu Ser Ala Val Ser Ser Asp Thr Ile Val

1 5 10

<210> 67

<211> 122

<212> PRT

<213> Mus musculus

<400> 67

Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Thr Ser Gly Tyr Thr Glu Asn Asn Gln Arg Phe

50 55 60

Lys Asp Lys Thr Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Trp Gly Phe Lys Tyr Asp Gly Glu Trp Ser Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 68

<211> 366

<212> DNA

<213> Mus musculus

<400> 68

caggtccagc tgcagcagtc tgcagctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacatttact tcctacacga tacactggat aaaacagagg 120

cctggacagg gtctggaatg gattggaaac attaatccca ccagtggata cactgagaac 180

aatcagaggt tcaaggacaa gaccacattg actgtagaca gatcctccaa cacagcctat 240

ttgcaactgc acagcctgac atctgaggac tctgcggtct atttctgtgc aagatggggc 300

tttaaatatg acggagaatg gtccttcgat gtctggggcg cagggaccac ggtcaccgtc 360

tcctca 366

<210> 69

<211> 107

<212> PRT

<213> Mus musculus

<400> 69

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Thr Ile Thr Val Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Val Leu Ile

35 40 45

Asn Lys Ala Ser Asn Leu His Ile Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Asn

100 105

<210> 70

<211> 321

<212> DNA

<213> Mus musculus

<400> 70

gacatccaaa tgaaccagtc tccatccagt ctgtctgcat ccctcggaga cacaatcacc 60

gtcacttgcc gtgccagtca gggcatcagt atttggttaa gctggtacca gcagaaacca 120

ggaaatattc ctaaagtatt gatcaataag gcttccaact tgcacataggg agtcccacca 180

aggtttagtg gcagtggatc tggaacacat ttcacattaa ctatcaccag cctacagcct 240

gaagacattg ccacttacta ctgtctacag agtcaaactt atcctctcac gttcggaggg 300

gggaccaagc tggaaataaa c 321

<210> 71

<211> 121

<212> PRT

<213> Mus musculus

<400> 71

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Tyr Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Phe Pro Gly Asp Gly Gly Thr Lys Tyr Asn Glu Thr Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Thr Ser Val Ile Arg Asp Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 72

<211> 363

<212> DNA

<213> Mus musculus

<400> 72

caggttcagc tgcagcagtc tggagctgaa ctggtaaagc ctggggcttc agtgaagttg 60

tcctgcaagg cttctggcta caccttcaaa tactatgata taaactggggt gaggcagagg 120

cctgaacagg gacttgagtg gattggatgg atttttcctg gagatggtgg tactaagtac 180

aatgagacgt tcaagggcaa ggccacactg actacagaca aatcctccag cacagcctac 240

atgcagctca gcaggctgac atctgaggac tctgctgtct atttctgtgc aagagggggg 300

acttcggtga taagggatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 360

tca 363

<210> 73

<211> 111

<212> PRT

<213> Mus musculus

<400> 73

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Thr Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 74

<211> 333

<212> DNA

<213> Mus musculus

<400> 74

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat aattatggca ttagttttat gcactggtac 120

cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180

gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240

cctgtggaga ctgatgatgt tgcaacctat tactgtcagc aaagtaataa ggatccgctc 300

acgttcggtg ctgggaccaa gctggagctg aaa 333

<210> 75

<211> 124

<212> PRT

<213> Canis familiaris

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala His Ile Asn Ser Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Glu Val Tyr Thr Thr Leu Ala Ala Phe Trp Thr Asp Asn Phe Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 76

<211> 372

<212> DNA

<213> Canis familiaris

<400> 76

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctggggggtc cctgagactc 60

tcctgtgtgg cttctggatt caccttcagt agttatggca tgagctgggt ccgccaggct 120

ccagggaagg ggctgcagtg ggtcgcacac attaacagtg gtggaagtag cacatactac 180

gcagacgctg tgaagggacg attcaccatc tccagagaca acgccaagaa cacgctctat 240

ctgcagatga acagcctgag agctgaggac acggccgtct attactgtgt ggaggtttac 300

actacgttag ctgcattctg gacagacaat tttgactact ggggccaggg aaccctggtc 360

accgtctcct ca 372

<210> 77

<211> 110

<212> PRT

<213> Canis familiaris

<400> 77

Gln Ser Val Leu Thr Gln Pro Thr Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Asn Ile Gly Ile Leu

20 25 30

Ala Ala Thr Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Val Leu

35 40 45

Val Tyr Ser Asp Gly Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Asn Ser Ala Thr Leu Thr Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Thr Thr Leu

85 90 95

Asp Ala Tyr Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu

100 105 110

<210> 78

<211> 330

<212> DNA

<213> Canis familiaris

<400> 78

cagtctgtgc tgactcagcc gacctcagtg tcggggtccc ttggccagag ggtcaccatc 60

tcctgctctg gaagcacgaa caacatcggt attcttgctg cgacctggta ccaacaactc 120

cgggaaagg cccctaaagt cctcgtgtac agtgatggga atcgaccgtc aggggtccct 180

gaccggtttt ccggctccaa gtctggcaac tcagccaccc tgaccatcac tgggcttcag 240

gctgaggacg aggctgatta ttactgccag tcctttgata ccacgcttga tgcttacgtg 300

ttcggctcag gaacccaact gaccgtcctt 330

<210> 79

<211> 117

<212> PRT

<213> Canis familiaris

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Gln Trp Val

35 40 45

Ala Gly Ile Asp Ser Val Gly Ser Gly Thr Ser Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Phe Asn Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ser Gly Phe Pro Gly Ser Phe Glu His Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 80

<211> 351

<212> DNA

<213> Canis familiaris

<400> 80

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctgcagggtc cctgagactg 60

tcctgtgtgg cctctggatt caccttcagt gactatgcca tgagctgggt ccgccaggct 120

cctgggaggg gactgcagtg ggtcgcaggt attgacagtg ttggaagtgg cacaagctac 180

gcagacgctg tgaagggccg attcacaatc tccagagacg acgccaagaa cacactgtat 240

ctgcagatgt tcaacctgag agccgaggac acggccatat attactgtgc gagcgggttc 300

cctgggtcct ttgagcactg gggccagggc accctggtca ccgtctcctc a 351

<210> 81

<211> 104

<212> PRT

<213> Canis familiaris

<400> 81

Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Gly

20 25 30

Tyr Val Gly Trp Tyr Gln Gln Leu Pro Gly Thr Gly Pro Arg Thr Leu

35 40 45

Ile Tyr Tyr Asn Ser Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Arg Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Tyr Asp Arg Thr Phe

85 90 95

Asn Ala Val Phe Gly Gly Gly Thr

100

<210> 82

<211> 327

<212> DNA

<213> Canis familiaris

<400> 82

cagtctgtac tgactcagcc ggcctcagtg tctgggtccc tgggccagaa ggtcaccatc 60

120

cgggaacag gccccagaac cctcatctat tataacagtg accgaccttc gggggtcccc 180

gatcgattct ctggctccag gtcaggcacc acagcaaccc tgaccatctc tggactccag 240

gctgaggacg aggctgatta ttactgctca gtatatgaca ggactttcaa tgctgtgttc 300

ggcgggaggca cccacctgac cgtcctc 327

<210> 83

<211> 118

<212> PRT

<213> Canis familiaris

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Pro Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Asp Val Asn Ser Gly Gly Thr Gly Thr Ala Tyr Ala Val Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Leu Gly Val Arg Asp Gly Leu Ser Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 84

<211> 354

<212> DNA

<213> Canis familiaris

<400> 84

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctccagggtc cctgagactg 60

tcctgtgtgg cctctggatt caccttcagc agttatgaca tgacctgggt ccgccaggct 120

cctgggaagg gactgcagtg ggtcgcagat gttaacagtg gtggaactgg cacggcctac 180

gcagtcgctg tgaagggccg attcaccatc tccagagaca acgccaagaa aacactctat 240

ttacagatga acagcctgag agccgaagac acggccgttt attattgtgc gaaactaggt 300

gtgagagatg gtctttctgt ctggggccag ggcaccctgg tcaccgtctc ctcg 354

<210> 85

<211> 107

<212> PRT

<213> Canis familiaris

<400> 85

Ser Ser Val Leu Thr Gln Pro Ser Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Thr Ile Ser Cys Ser Gly Glu Ser Leu Asn Glu Tyr Tyr Thr

20 25 30

Gln Trp Phe Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Arg Asp Thr Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr His Thr Leu Thr Ile Ser Gly Ala Arg Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Glu Ser Ala Val Asp Thr Gly Thr Leu

85 90 95

Val Phe Gly Gly Gly Thr His Leu Ala Val Leu

100 105

<210> 86

<211> 321

<212> DNA

<213> Canis familiaris

<400> 86

tccagtgtgc tgactcagcc tccctcggta tcagtgtctc tgggacagac agcaaccatc 60

tcctgctctg gagagagtct gaatgaatat tatacacaat ggttccagca gaaggcaggc 120

caagcccctg tcttggtcat atatagggac actgagcggc cctctgggat ccctgaccga 180

ttctctggct ccagttcagg gaacacacac accctaacca tcagcggggc tcgggccgag 240

gacgaggctg actattactg cgagtcagcg gtcgacactg gaacccttgt ctttggcgga 300

ggcacccacc tggccgtcct c 321

<210> 87

<211> 117

<212> PRT

<213> Canis familiaris

<400> 87

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Arg Thr Tyr

20 25 30

Val Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Ser Ile Asn Gly Gly Gly Ser Ser Pro Thr Tyr Ala Asp Ala Val

50 55 60

Arg Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Gln Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Val Val Ser Met Val Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 88

<211> 351

<212> DNA

<213> Canis familiaris

<400> 88

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctgcagggtc cctgagactg 60

tcctgtgtgg ccctctggatt caccttcagg acctatgtca tgaactgggt ccgccaggct 120

cctgggaagg ggctgcaatg ggtcgcaagt attaacggtg gtggaagtag cccaacctac 180

gcagacgctg tgaggggccg attcaccgtc tccagggaca acgcccagaa ctcactgttt 240

ctgcagatga acagcctgag agccgaggac acagccgtgt atttttgtgt cgtgtcgatg 300

gttgggccct tcgactactg gggccaaggg accctggtca ccgtgtcctc a 351

<210> 89

<211> 102

<212> PRT

<213> Canis familiaris

<400> 89

Ser Ser Val Leu Thr Gln Pro Ser Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Thr Ile Ser Cys Ser Gly Glu Ser Leu Ser Asn Tyr Tyr Ala

20 25 30

Gln Trp Phe Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr His Thr Leu Thr Ile Ser Gly Ala Arg Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Glu Ser Ala Val Ser Ser Asp Thr Ile

85 90 95

Val Phe Gly Gly Gly Thr

100

<210> 90

<211> 321

<212> DNA

<213> Canis familiaris

<400> 90

tccagtgtgc tgactcagcc tccctcggta tcagtgtctc tggggcagac agcaaccatc 60

tcctgctctg gagagagtct gagtaactat tatgcacaat ggttccagca gaaggcaggc 120

caagcccctg tgttggtcat atataaggac actgagcggc cctctgggat ccctgaccga 180

ttctctggct ccagttcagg gaacacacac accctgacca tcagcggggc tcgggccgag 240

gacgaggctg actattactg tgagtcagca gtcagttctg atactattgt gttcggcgga 300

ggcacccacc tgaccgtcct c 321

<210> 91

<211> 124

<212> PRT

<213> Canis familiaris

<400> 91

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Thr Ile Asn Asn Asp Gly Thr Arg Thr Gly Tyr Ala Asp Ala Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Lys Gly Asn Ala Glu Ser Gly Cys Thr Gly Asp His Cys Pro Pro

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 92

<211> 372

<212> DNA

<213> Canis familiaris

<400> 92

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctgcggggtc cctgagactg 60

tcctgtgtgg cctctggatt caccttcagt agttatgcca tgaaatgggt ccgccaggct 120

cctgggaagg ggctgcagtg ggtcgcgact attaacaatg atggaaccag aacaggctac 180

gcagacgctg tgaggggccg attcaccatc tccaaagaca acgccaaaaa cacactgtat 240

ctgcagatgg acagcctgag agccgacgac acggccgtct attactgtac aaagggcaat 300

gccgaatccg gctgtactgg tgatcactgt cctccctact ggggccaggg aaccctggtc 360

accgtctcct ca 372

<210> 93

<211> 107

<212> PRT

<213> Canis familiaris

<400> 93

Ser Ser Val Leu Thr Gln Pro Ser Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Thr Ile Ser Cys Ser Gly Glu Ser Leu Asn Lys Tyr Tyr Ala

20 25 30

Gln Trp Phe Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ala Gly Asn Thr His Thr Leu Thr Ile Ser Gly Ala Arg Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Glu Ser Ala Val Ser Ser Glu Thr Asn

85 90 95

Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu

100 105

<210> 94

<211> 321

<212> DNA

<213> Canis familiaris

<400> 94

tccagtgtgc tgactcagcc tccctcggta tcagtgtctc tgggacagac agcaaccatc 60

tcctgctctg gagagagtct gaataaatat tatgcacaat ggttccaaca gaaggcaggc 120

caagcccctg tgttggtcat atataaggac actgagcggc cctctgggat ccctgaccga 180

ttctccggct ccagtgcagg caacacacac accctgacca tcagcggggc tcgggccgag 240

gacgaggctg actattactg cgagtcagca gtcagttctg aaactaacgt gttcggctca 300

ggaacccaac tgaccgtcct t 321

<210> 95

<211> 116

<212> PRT

<213> Canis familiaris

<400> 95

Glu Val Gln Leu Val Asp Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Phe Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Gln Trp Val

35 40 45

Ala Leu Ile Ser Ser Asp Gly Ser Gly Thr Tyr Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ile Phe Trp Arg Ala Phe Asn Asp Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 96

<211> 348

<212> DNA

<213> Canis familiaris

<400> 96

gaggtacaac tggtggactc tgggggagac ctggtgaagc ctggggggtc cctgagactc 60

tcctgtgtgg ccctctggatt caccttcagt acctacttca tgtcctgggt ccgccaggct 120

ccagggagggg ggcttcagtg ggtcgcactt attagcagtg atggaagtgg cacatactac 180

gcagacgctg tgaagggccg attcaccatc tccagagaca atgccaagaa cacgctgtat 240

ctgcagatga acagcctgag agccgaggac acggctatgt attactgtgc gatattctgg 300

cgggccttta acgactgggg cggggcacc ctggtcaccg tctcctca 348

<210> 97

<211> 110

<212> PRT

<213> Canis familiaris

<400> 97

Gln Thr Val Val Ile Gln Glu Pro Ser Leu Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr Ser

20 25 30

Asn Tyr Pro Gly Trp Tyr Gln Gln Thr Arg Gly Arg Thr Pro Arg Thr

35 40 45

Ile Ile Tyr Asp Thr Gly Ser Arg Pro Ser Gly Val Pro Asn Arg Phe

50 55 60

Ser Gly Ser Ile Ser Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Leu Tyr Thr Asp Ser

85 90 95

Asp Ile Leu Val Phe Gly Gly Gly Thr His Leu Thr Val Leu

100 105 110

<210> 98

<211> 330

<212> DNA

<213> Canis familiaris

<400> 98

cagactgtgg taatccagga gccatcactc tcagtgtctc caggagggac agtcacactc 60

acatgtggcc tcaactctgg gtcagtctcc acaagtaatt accctggctg gtaccagcag 120

acccgaggcc ggactcctcg cacgattatc tacgacacag gcagtcgccc ctctggggtc 180

cctaatcgct tctccggatc catctctgga aacaaagccg ccctcaccat cacaggagcc 240

cagcccgagg atgaggctga ctattactgt tccttatata cggatagtga cattcttgtt 300

ttcggcggag gcacccacct gaccgtcctc 330

<210> 99

<211> 117

<212> PRT

<213> Canis familiaris

<400> 99

Glu Val His Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Trp Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Arg

20 25 30

Gly Met Ser Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Tyr Ile Arg Tyr Asp Gly Ser Arg Thr Asp Tyr Ala Asp Ala Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Gly Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 100

<211> 351

<212> DNA

<213> Canis familiaris

<400> 100

gaggtgcatt tggtggagtc tgggggagac ctggtgaagc cttgggggtc cttgagactg 60

tcctgtgtgg cctctggatt cacctttagt gatcgtggca tgagctgggt ccgtcagtct 120

ccagggaagg ggctgcagtg ggtcgcatat attaggtatg atgggagtag gacagactac 180

gcagacgctg tggagggccg attcaccatc tccagagaca acgccaagaa cacgctctac 240

ctgcagatga acagcctgag agccgaggac acggccgtgt attactgtgc gagatgggac 300

ggtagttctt ttgactattg gggccaggggc accctggtca ccgtctcctc a 351

<210> 101

<211> 112

<212> PRT

<213> Canis familiaris

<400> 101

Asp Ile Val Val Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Thr Ala Ser Phe Ser Cys Lys Ala Ser Gln Ser Leu Leu His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Asp Trp Phe Arg Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Pro Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Gly Val Glu Ala Asp Asp Ala Gly Leu Tyr Tyr Cys Met Gln Ala

85 90 95

Ile His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Val Glu Leu Lys

100 105 110

<210> 102

<211> 336

<212> DNA

<213> Canis familiaris

<400> 102

gatattgtcg tgacacagac cccgctgtcc ctgtccgtca gccctggaga gactgcctcc 60

ttctcctgca aggccagtca gagcctcctg cacagtgatg gaaacacgta tttggattgg 120

ttccgacaga agccaggcca gtctccacag cgtttgatct acaaggtctc caacagagac 180

cctggggtcc cagacaggtt cagtggcagc gggtcaggga cagatttcac cctgagaatc 240

agcggagtgg aggctgacga tgctggactt tattactgca tgcaagcaat acactttcct 300

ctgacgttcg gagcaggaac caaggtggag ctcaaa 336

<210> 103

<211> 119

<212> PRT

<213> Canis familiaris

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Gly Ile Asn Ser Glu Gly Ser Arg Thr Ala Tyr Ala Asp Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Thr Gly Asp Ile Val Ala Thr Gly Thr Ser Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 104

<211> 357

<212> DNA

<213> Canis familiaris

<400> 104

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctgcagggtc cctgagactg 60

tcctgtgtgg cctctggatt caccttcagt agttatgtca tgacctgggt ccgccaggct 120

cctgggaagg gactgcagtg ggtcgcaggc attaatagtg aggggagtag gacagcctac 180

gcagacgctg tgaagggccg attcaccatc tccagagaca acgccaagaa tacactttat 240

ctacaaatag acagcctgag agccgaggac acggccatat attactgtgc gacaggcgat 300

atagtagcga ctggtacttc gtattggggc cagggcaccc tggtcaccgt ctcctca 357

<210> 105

<211> 107

<212> PRT

<213> Canis familiaris

<400> 105

Ser Asn Val Leu Thr Gln Pro Ser Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Thr Ile Ser Cys Ser Gly Glu Thr Leu Asn Arg Phe Tyr Thr

20 25 30

Gln Trp Phe Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Ile His Thr Leu Thr Ile Ser Gly Ala Arg Ala Glu

65 70 75 80

Asp Glu Ala Ala Tyr Tyr Cys Lys Ser Ala Val Ser Ile Asp Val Gly

85 90 95

Val Phe Gly Gly Gly Thr His Leu Thr Val Phe

100 105

<210> 106

<211> 321

<212> DNA

<213> Canis familiaris

<400> 106

tccaatgtac tgactcagcc tccctcggta tcagtgtctc tgggacagac agcaaccatc 60

tcctgctctg gagagactct gaatagattt tatacacaat ggttccagca gaaggcaggc 120

caagcccctg tgttggtcat atataaggac actgagcggc cctctgggat ccctgaccga 180

ttctccggct ccagttcagg gaacatacac accctgacca tcagcggggc tcgggccgag 240

gacgaggctg cctattactg caagtcagca gtcagtattg atgttggtgt gttcggcgga 300

ggcacccacc tgaccgtctt c 321

<210> 107

<211> 117

<212> PRT

<213> Canis familiaris

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Arg Thr Tyr

20 25 30

Val Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Ser Ile Asn Gly Gly Gly Ser Ser Pro Thr Tyr Ala Asp Ala Val

50 55 60

Arg Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Gln Asn Ser Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Phe Cys

85 90 95

Val Val Ser Met Val Gly Pro Phe Asp Tyr Trp Gly His Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 108

<211> 351

<212> DNA

<213> Canis familiaris

<400> 108

gaggtgcagc tggtggagtc tgggggagac ctggtgaagc ctgcagggtc cctgagactg 60

tcctgtgtgg ccctctggatt caccttcagg acctatgtca tgaactgggt ccgccaggct 120

cctgggaagg ggctgcaatg ggtcgcaagt attaacggtg gtggaagtag cccaacctac 180

gcagacgctg tgaggggccg attcaccgtc tccagggaca acgcccagaa ctcactgttt 240

ctgcagatga acagcctgag agccgaggac acagccatat atttttgtgt cgtgtcgatg 300

gttgggccct tcgactactg gggccatggg accctggtca ccgtgtcctc a 351

<210> 109

<211> 107

<212> PRT

<213> Canis familiaris

<400> 109

Ser Ser Val Leu Thr Gln Pro Ser Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Thr Ile Ser Cys Ser Gly Lys Ser Leu Ser Tyr Tyr Tyr Ala

20 25 30

Gln Trp Phe Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr His Thr Leu Thr Ile Ser Gly Ala Arg Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Glu Ser Ala Val Ser Ser Asp Thr Ile

85 90 95

Val Phe Gly Gly Gly Thr His Leu Thr Val Leu

100 105

<210> 110

<211> 321

<212> DNA

<213> Canis familiaris

<400> 110

tccagtgtgc tgactcagcc tccctcggta tcagtgtctc tggggcagac agcaaccatc 60

tcctgctctg gaaagagtct gagttactat tatgcacaat ggttccagca gaaggcaggc 120

caagcccctg tgttggtcat atataaggac actgagcggc cctctgggat ccctgaccga 180

ttctctggct ccagttcagg gaacacacac accctgacca tcagcggggc tcgggccgag 240

gacgaggctg actattactg tgagtcagca gtcagttctg atactattgt gttcggcgga 300

ggcacccacc tgaccgtcct c 321

<210> 111

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 111

Gln Val Leu Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Phe Cys Lys Ala Ser Gly Tyr Ser Phe Thr Tyr Tyr

20 25 30

Asp Ile Asn Trp Leu Arg Gln Ala Pro Glu Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Phe Pro Gly Asp Gly Gly Thr Lys Tyr Asn Glu Thr Phe

50 55 60

Lys Gly Arg Leu Thr Leu Thr Ala Asp Thr Ser Thr Asn Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Ala Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Thr Ser Val Ile Arg Asp Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Ala Leu Val Thr Val Ser Ser

115 120

<210> 112

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized mAb heavy chain variable region, from Mus

musculus and Felis catus

<400> 112

caggtgctgc tggtccagtc aggagcagag gtaaaaaagc ccggggcgag tgtcaagatt 60

ttctgtaagg cctccggata ctcttttacg tattacgata ttaactggct tcgccaggcc 120

cctgagcagg ggctcgaatg gatgggttgg atattccccg gagatggggg aaccaagtac 180

aacgaaacct tcaaggggag gctgaccctg actgcagata ccagcacgaa cacagtgtat 240

atggagttgt cctcactgcg atctgctgat actgccatgt actactgcgc tcgcggcggc 300

acttcagtta tcagggatgc catggactat tgggggcagg gcgcactcgt cactgtctcg 360

agc 363

<210> 113

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 113

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Gln Gly Thr

100 105

<210> 114

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 114

gaaatccaga tgacacaatc tcccagctcc ctcagcgcat ctcctggcga cagggtaacc 60

atcacctgcc gcgccagcga gtcagtagac aactatggca tatccttcat gcactggtat 120

caacaaaagc ccgggaaagt ccccaaactg ttgatttaca gagcaagcaa tctcgagtca 180

ggagtcccat ctcgcttctc tggttccggt tcgggaaccg acttcactct gacaatttct 240

tctctggagc ccgaggatgc cgctacatat tactgtcagc aaagcaataa agatccactg 300

accttcggac agggtaccaa gctggagatc aaa 333

<210> 115

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 115

Glu Val Val Leu Thr Gln Ser Ser Ala Phe Leu Ser Arg Thr Leu Lys

1 5 10 15

Glu Lys Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Asn Gln Ala Pro

35 40 45

Lys Leu Leu Val Lys Arg Ala Ser Asn Leu Glu Ser Gly Ala Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Pro Glu Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Gln Gly Thr

100 105

<210> 116

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 116

gaggtggtgc tgactcagag tagcgcgttt ctgtctcgga ccctgaaaga gaaagctacc 60

atcacgtgca gggcaagcga gagcgtggac aactatggta tcagcttcat gcattggtat 120

cagcagaaac ctaatcaggc gcctaagctg ctcgtgaaaa gagcctccaa ccttgagagc 180

ggcgcaccat caaggttttc aggaagtggc agcggggacag acttcaccct tacaatctct 240

agtccagagc cggaggacgc agctacctac tattgccagc aatccaataa agacccgttg 300

acattcggcc aaggtacc 318

<210> 117

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 117

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Gln Gly Thr

100 105

<210> 118

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 118

gagatccaga tgacccagtc tccatcctca ctgagtgcta gcccggggga tcgagtgact 60

ataacatgtc gggccagtga atcagtggac aactatggaa tcagttttat gcactggtat 120

cagcagaagc ccggccagcc accgaagctg ttgatttatc gcgcaagcaa tctggagtca 180

ggagtgccct ctagattttc tgggagcggt tctggcacag atttcacact cacaatatca 240

300

acttttggcc agggtacc 318

<210> 119

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 119

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Gln Gly Thr

100 105

<210> 120

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 120

gagatccaga tgacccagtc tccatcctca ctgagtgcta gcccggggga tcgagtgact 60

ataacatgtc gggccagtga atcagtggac aactatggaa tcagttttat gcactggtat 120

cagcagaagc ccggccaggt cccgaagctg ttgatttatc gcgcaagcaa tctggagtca 180

ggagtgccct ctagattttc tgggagcggt tctggcacag atttcacact cacaatatca 240

tccttggaac cggaagacgc agccacatac tattgccagc agagtaacaa ggaccctttg 300

acttttggcc agggtacc 318

<210> 121

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 121

Gln Val Leu Leu Val Gln Ser Gly Ala Glu Val Arg Thr Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Phe Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Thr Ile His Trp Leu Arg Gln Ala Pro Ala Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Asn Pro Thr Ser Gly Tyr Thr Glu Asn Asn Gln Arg Phe

50 55 60

Lys Asp Arg Leu Thr Leu Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Ala Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Trp Gly Phe Lys Tyr Asp Gly Glu Trp Ser Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 122

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized mAb heavy chain variable region, from Mus

musculus and Felis catus

<400> 122

caagtgcttc tggtgcaaag cggggcggaa gttaggaccc caggagcctc agtaaaaatt 60

ttttgtaagg catccggcta cagtttcacc agctacacta ttcactggct gaggcaggcc 120

ccggcccaag ggctggagtg gatgggaaat atcaatccca cgtctggcta tacagagaat 180

aaccaaaggt ttaaggatag gctgactctg acagctgaca catcaaccaa tacggcatac 240

atggagctct cctctctccg gagtgccgac accgccatgt actactgtgc tcggtggggg 300

tttaaatacg atggcgagtg gagcttcgac gtgtggggcg cgggcacaac cgtgaccgtc 360

tcgagc 366

<210> 123

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 123

Gln Val Leu Leu Val Gln Ser Gly Ala Glu Val Arg Thr Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Phe Cys Lys Ala Ser Gly Tyr Gly Phe Thr Ser Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ser Pro Ala Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Asn Pro Thr Ser Gly Tyr Thr Glu Asn Asn Gln Arg Phe

50 55 60

Lys Asp Arg Leu Thr Leu Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Ala Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Trp Gly Phe Lys Tyr Asp Gly Glu Trp Ser Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 124

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized mAb heavy chain variable region, from Mus

musculus and Felis catus

<400> 124

caggtgctgc tcgtgcagag cggagccgaa gtgaggacac ccggtgcgag tgtaaaaatt 60

ttttgcaagg caagcggcta cgggtttaca tcctatacca tccactgggt gaggcagtcc 120

ccagcgcagg gacttgaatg gatgggaaat attaatccaa caagcgggta tactgaaaac 180

aaccaaagat ttaaggacag actgacactc accgcagata catctacaaa tacagcctac 240

atggagttgt cttccctgcg gagtgccgac acggctatgt actactgtgc tcggtggggg 300

tttaagtatg atggcgaatg gtccttcgac gtctggggag ctggaaccac cgtgaccgtc 360

tcgagc 366

<210> 125

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 125

Gln Val Leu Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Phe Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Thr Ile His Trp Leu Arg Gln Ala Pro Glu Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Asn Pro Thr Ser Gly Tyr Thr Glu Asn Asn Gln Arg Phe

50 55 60

Lys Asp Arg Leu Thr Leu Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Ala Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Trp Gly Phe Lys Tyr Asp Gly Glu Trp Ser Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 126

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized mAb heavy chain variable region, from Mus

musculus and Felis catus

<400> 126

caggtcctct tggttcaaag cggagccgaa gtccgaaaac cgggtgcctc agtgaaaatc 60

ttctgtaagg cctccggcta tagtttcacg agttacacaa tccactggct gcgacaggca 120

ccagagcagg gactggagtg gatgggaaat ataaatccga cgtctgggta cacagaaaac 180

aaccagagat tcaaggatag attgacactg accgcggata ctagtacaaa tacggcttac 240

atggaactgt cctcactccg gtcagccgac accgccatgt attactgtgc tcgctggggg 300

ttcaagtatg atggagagtg gagcttcgac gtatggggag cgggaaccac tgtgaccgtc 360

tcgagc 366

<210> 127

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 127

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 128

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 128

gaaattcaga tgactcaatc accttcatct ttgagtgcat cacccggaga tagagttaca 60

atcacctgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccc 120

ggaaaggtcc cgaaactctt gatctacaag gcctctaacc tgcacattgg cgtgccaagc 180

cgattcagcg ggagcggaag tggcaccgac tttactctga cgatcagttc actggagccc 240

gaggacgctg caacatacta ttgtctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 129

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 129

Glu Ile Thr Met Thr Gln Ser Pro Gly Ser Leu Ala Gly Ser Pro Gly

1 5 10 15

Gln Gln Val Thr Met Asn Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln His Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Ser Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 130

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 130

gaaattacca tgacacaaag ccccggctcc ctggccggct cccccggaca gcaagtgacc 60

atgaattgtc gggccagcca gggaatttct atatggctct cttggtatca gcaaaaaccc 120

ggacagcacc ctaaacttct gatctacaaa gcaagtaact tgcacatcgg cgtccctgat 180

cgattcagtg gctcaggttc cggtacagat tttactctta ccatcagcaa tctgcaggct 240

gaggatgtgg caagctatta ctgtctccaa agtcagactt accctctgac atttgggggc 300

ggtaccaagc tggagatcaa a 321

<210> 131

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 131

Glu Val Val Leu Thr Gln Ser Ser Ala Phe Leu Ser Arg Thr Leu Lys

1 5 10 15

Glu Lys Ala Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Asn Gln Ala Pro Lys Leu Leu Val

35 40 45

Lys Lys Ala Ser Asn Leu His Ile Gly Ala Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Pro Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Gly Asp Gln

100 105

<210> 132

<211> 322

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 132

gaggtagtgc tgactcagtc ctccgccttc ttgtcaagaa ctctcaaaga gaaagcaaca 60

atcacttgtc gggcgtctca agggatatca atttggctga gctggtatca gcagaaacca 120

aatcaagcgc cgaaactgct ggtgaagaag gcctccaatc tccacattgg cgcaccagc 180

aggttttccg gcagtggctc tggcacagat ttcactctga ccatcagctc acccgagccc 240

gaagacgccg ctacatacta ttgcttgcaa tcccagacat accccctgac ttttggggga 300

ggtaccaagc tgggagatca aa 322

<210> 133

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 133

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Thr Ile Thr Val Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 134

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 134

gacattcaga tgaatcagtc tcctagctca ctgtcagcca gccttggaga caccattaca 60

gtcacttgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccc 120

ggaaaggtcc cgaaactctt gatctacaag gcctctaacc tgcacattgg cgtgccaagc 180

cgattcagcg ggagcggaag tggcaccgac tttactctga cgatcagttc actggagccc 240

gaggacgctg caacatacta ttgtctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 135

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 135

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Val Leu Ile

35 40 45

Asn Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 136

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 136

gaaattcaga tgactcaatc accttcatct ttgagtgcat cacccggaga tagagttaca 60

atcacctgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccg 120

ggcaatatcc caaaggtgct gattaacaag gcctctaacc tgcacattgg cgtgccaagc 180

cgattcagcg ggagcggaag tggcaccgac tttactctga cgatcagttc actggagccc 240

gaggacgctg caacatacta ttgtctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 137

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 137

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 138

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 138

gaaattcaga tgactcaatc accttcatct ttgagtgcat cacccggaga tagagttaca 60

atcacctgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccc 120

ggaaaggtcc cgaaactctt gatctacaag gcctctaacc tgcacattgg ggtcccccca 180

aggttcagcg gatctggatc cgggacccac tttactctga ccataacaag cctgcagcct 240

gaagacattg ctacctatta ctgcctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 139

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 139

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Thr Ile Thr Val Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Val Leu Ile

35 40 45

Asn Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 140

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 140

gacattcaga tgaatcagtc tcctagctca ctgtcagcca gccttggaga caccattaca 60

gtcacttgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccg 120

ggcaatatcc caaaggtgct gattaacaag gcctctaacc tgcacattgg cgtgccaagc 180

cgattcagcg ggagcggaag tggcaccgac tttactctga cgatcagttc actggagccc 240

gaggacgctg caacatacta ttgtctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 141

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 141

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Thr Ile Thr Val Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 142

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 142

gacattcaga tgaatcagtc tcctagctca ctgtcagcca gccttggaga caccattaca 60

gtcacttgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccc 120

ggaaaggtcc cgaaactctt gatctacaag gcctctaacc tgcacattgg ggtcccccca 180

aggttcagcg gatctggatc cgggacccac tttactctga ccataacaag cctgcagcct 240

gaagacattg ctacctatta ctgcctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 143

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 143

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Val Leu Ile

35 40 45

Asn Lys Ala Ser Asn Leu His Ile Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 144

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 144

gaaattcaga tgactcaatc accttcatct ttgagtgcat cacccggaga tagagttaca 60

atcacctgca gggcgagtca agggatctcc atatggttgt catggtatca gcagaaaccg 120

ggcaatatcc caaaggtgct gattaacaag gcctctaacc tgcacattgg ggtcccccca 180

aggttcagcg gatctggatc cgggacccac tttactctga ccataacaag cctgcagcct 240

gaagacattg ctacctatta ctgcctgcaa tctcagacct accctctcac ctttggagga 300

ggtaccaagc tggagatcaa a 321

<210> 145

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 145

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Val Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 146

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 146

gaaatccaga tgacacagtc ccccagtagc ctttccgctt caccggggcga tagagtcact 60

attacgtgca gggcctccca gggtatttct atctggctga gctggtatca gcagaagccc 120

ggtaatgtgc caaagctctt gatctacaag gcatctaacc ttcatatcgg agtgccctca 180

agatttagtg ggtcaggcag cggaaccgat ttcacattga ccattagttc tctggaacca 240

gaggacgctg ccacttacta ctgcctgcag tcccaaacat accctttgac ttttgggggg 300

ggtaccaagc tggagatcaa a 321

<210> 147

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 147

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 148

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 148

gagattcaga tgacccagag cccatcaagc ctctccgctt cccccggaga ccgggtgacc 60

atcacatgca gagcttcaca gggaatctca atctggctca gctggtatca gcagaagcca 120

ggcaagattc cgaagttgct tatctataag gccagtaacc tgcatatcgg agttccatca 180

agattcagtg gtagcggaag tgggacagat ttcactctca ccatcagctc cctcgaacca 240

gaggacgctg caacttacta ctgcctgcag tcccagacat atccacttac tttcggcggg 300

ggtaccaagc tggagatcaa a 321

<210> 149

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 149

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Val Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 150

<211> 322

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 150

gagattcaga tgactcagag cccatctagt ctctctgcat ctcccggaga cagagttacg 60

atcacctgca gggctagcca agggatatca atttggctgt cctggtatca gcaaaaacct 120

ggcaaagtgc caaaggtctt gatttacaaa gcatccaatt tgcacatcgg cgtccctagt 180

cgcttttccg ggtctggtag cggcaccgac ttcaccctca ccataagctc actcgagccg 240

gaagatgccg ctacttacta ttgcctgcag tctcagactt accccctgac tttcggcgga 300

ggtaccaagc tggagatcaa ac 322

<210> 151

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 151

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Asn Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 152

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 152

gagatccaga tgacgcagag ccctagcagc ctctctgcat ccccaggaga cagagtaaca 60

attacctgtc gcgccagcca gggaatatct atatggctgt catggtatca acagaaaccg 120

ggaaaggttc caaagctctt gatcaataag gctagcaatc tgcatattgg agtgccctcc 180

cgcttctctg gtagcggaag tggcacagat ttcaccctga ccattagtag tctggagcct 240

gaggatgcgg ccacctacta ctgcctccag tcccaaacct atcccctgac cttcggagga 300

ggtaccaagc tggagatcaa a 321

<210> 153

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 153

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Ile Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Ser Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 154

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 154

gaaattcaga tgactcagag tcctagcagc ctgtccgcaa gcccaggtga ccgagtcacc 60

ataacctgca gggccagtca ggggatctcc atatggctct cttggtatca acagaaaccc 120

ggcaatatcc ctaagctcct gatttataaa gcgtcaaatc tgcatatcgg ggtgccatca 180

agattctctg ggtccggctc aggaaccgac tttaccctga ccatttcttc tctcgaaccc 240

gaggatgccg ccacctatta ttgccttcaa agccagacat acccattgac cttcggcggc 300

ggtaccaagc tggagatcaa a 321

<210> 155

<211> 159

<212> PRT

<213> Canis familiaris

<400> 155

Met Leu Ser His Thr Gly Pro Ser Arg Phe Ala Leu Phe Leu Leu Cys

1 5 10 15

Ser Met Glu Thr Leu Leu Ser Ser His Met Ala Pro Thr His Gln Leu

20 25 30

Pro Pro Ser Asp Val Arg Lys Ile Ile Leu Glu Leu Gln Pro Leu Ser

35 40 45

Arg Gly Leu Leu Glu Asp Tyr Gln Lys Lys Glu Thr Gly Val Pro Glu

50 55 60

Ser Asn Arg Thr Leu Leu Leu Cys Leu Thr Ser Asp Ser Gln Pro Pro

65 70 75 80

Arg Leu Asn Ser Ser Ala Ile Leu Pro Tyr Phe Arg Ala Ile Arg Pro

85 90 95

Leu Ser Asp Lys Asn Ile Ile Asp Lys Ile Ile Glu Gln Leu Asp Lys

100 105 110

Leu Lys Phe Gln His Glu Pro Glu Thr Glu Ile Ser Val Pro Ala Asp

115 120 125

Thr Phe Glu Cys Lys Ser Phe Ile Leu Thr Ile Leu Gln Gln Phe Ser

130 135 140

Ala Cys Leu Glu Ser Val Phe Lys Ser Leu Asn Ser Gly Pro Gln

145 150 155

<210> 156

<211> 477

<212> DNA

<213> Canis familiaris

<400> 156

atgctctccc acacaggacc atccaggttt gccctgttcc tgctctgctc tatggaaacc 60

ttgctgtcct cccatatggc acccacccat cagctaccac caagtgatgt acgaaaaatc 120

atcttggaat tacagccctt gtcgagggga cttttggaag actatcagaa gaaagagaca 180

ggggtgccag aatccaaccg taccttgctg ctgtgtctca cctctgattc ccaaccacca 240

cgcctcaaca gctcagccat cttgccttat ttcagggcaa tcagaccatt atcagataag 300

aacattattg ataaaatcat agaacagctt gacaaactca aatttcaaca tgaaccagaa 360

acagaaattt ctgtgcctgc agatactttt gaatgtaaaa gcttcatctt gacgatttta 420

cagcagttct cggcgtgcct ggaaagtgtg tttaagtcac taaactctgg acctcag 477

<210> 157

<211> 165

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence corresponding to feline IL-31

wild-type with C-terminal His-tag

<400> 157

Met Leu Ser His Ala Gly Pro Ala Arg Phe Ala Leu Phe Leu Leu Cys

1 5 10 15

Cys Met Glu Thr Leu Leu Pro Ser His Met Ala Pro Ala His Arg Leu

20 25 30

Gln Pro Ser Asp Ile Arg Lys Ile Ile Leu Glu Leu Arg Pro Met Ser

35 40 45

Lys Gly Leu Leu Gln Asp Tyr Leu Lys Lys Glu Ile Gly Leu Pro Glu

50 55 60

Ser Asn His Ser Ser Leu Pro Cys Leu Ser Ser Asp Ser Gln Leu Pro

65 70 75 80

His Ile Asn Gly Ser Ala Ile Leu Pro Tyr Phe Arg Ala Ile Arg Pro

85 90 95

Leu Ser Asp Lys Asn Thr Ile Asp Lys Ile Ile Glu Gln Leu Asp Lys

100 105 110

Leu Lys Phe Gln Arg Glu Pro Glu Ala Lys Val Ser Met Pro Ala Asp

115 120 125

Asn Phe Glu Arg Lys Asn Phe Ile Leu Ala Val Leu Gln Gln Phe Ser

130 135 140

Ala Cys Leu Glu His Val Leu Gln Ser Leu Asn Ser Gly Pro Gln His

145 150 155 160

His His His His His His

165

<210> 158

<211> 495

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence corresponding to the feline gene

IL-31 wild-type encoded C-terminal His-tag

<400> 158

atgctttcac acgctggacc agcccgattc gccctcttcc tcctctgctg tatggagact 60

ctgttgccgt cccacatggc cccggcacat aggctgcagc cgtctgacat ccggaagatc 120

attctcgaac ttcgccccat gtcgaagggg ttgctgcaag actacctgaa gaaggagatc 180

ggcctgccg aaagcaacca ctcctcgctg ccttgcctgt caagcgattc ccagctgccc 240

cacattaacg gttccgccat cctcccgtac ttccgggcca tcagaccact gtcggacaag 300

aacaccatcg acaagatcat tgaacagctg gacaagctga agtttcagcg cgagcctgaa 360

gccaaagtgt ccatgcccgc cgataacttc gagcggaaga atttcattct cgcggtgctg 420

cagcagttct ccgcgtgcct ggagcacgtc ctgcaatccc tgaacagcgg acctcagcac 480

caccatcacc accat 495

<210> 159

<211> 148

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence corresponding to feline IL-31

N-terminal His-tag

<400> 159

Met Arg Gly Ser His His His His His His Gly Ser Ser His Met Ala

1 5 10 15

Pro Ala His Arg Leu Gln Pro Ser Asp Ile Arg Lys Ile Ile Leu Glu

20 25 30

Leu Arg Pro Met Ser Lys Gly Leu Leu Gln Asp Tyr Leu Lys Lys Glu

35 40 45

Ile Gly Leu Pro Glu Ser Asn His Ser Ser Leu Pro Cys Leu Ser Ser

50 55 60

Asp Ser Gln Leu Pro His Ile Asn Gly Ser Ala Ile Leu Pro Tyr Phe

65 70 75 80

Arg Ala Ile Arg Pro Leu Ser Asp Lys Asn Thr Ile Asp Lys Ile Ile

85 90 95

Glu Gln Leu Asp Lys Leu Lys Phe Gln Arg Glu Pro Glu Ala Lys Val

100 105 110

Ser Met Pro Ala Asp Asn Phe Glu Arg Lys Asn Phe Ile Leu Ala Val

115 120 125

Leu Gln Gln Phe Ser Ala Cys Leu Glu His Val Leu Gln Ser Leu Asn

130 135 140

Ser Gly Pro Gln

145

<210> 160

<211> 444

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence corresponding to the feline gene

IL-31 encoded N-terminal His-tag

<400> 160

atgagaggat cccatcacca tcaccaccac ggctcatctc atatggcccc cgcacatcgc 60

ctgcagccga gtgacattcg taaaattatc ttggagctgc gcccgatgtc caagggctta 120

ctgcaggatt atctgaagaa agagatcggg ctgcctgaaa gcaaccatag tagcctgccg 180

tgtttatcgt ctgatagcca gttaccacac atcaatggct ctgcgatttt gccctacttt 240

cgcgccatcc gtccgctgtc cgataaaaat accatcgaca aaattatcga acaactggat 300

360 aaattgaagt ttcagcgcga gcctgaagcg

cgcaaaaact ttattttagc ggtgttgcag cagttttctg cctgtctgga acacgtgctc 420

cagtcactca atagtgggcc acaa 444

<210> 161

<211> 165

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence corresponding to the mutant

11E12 feline IL-31 protein with a C-terminal His-tag

<400> 161

Met Leu Ser His Ala Gly Pro Ala Arg Phe Ala Leu Phe Leu Leu Cys

1 5 10 15

Cys Met Glu Thr Leu Leu Pro Ser His Met Ala Pro Ala His Arg Leu

20 25 30

Gln Pro Ser Asp Ile Arg Lys Ile Ile Leu Glu Leu Arg Pro Met Ser

35 40 45

Lys Gly Leu Leu Gln Asp Tyr Leu Lys Lys Glu Ile Gly Leu Pro Glu

50 55 60

Ser Asn His Ser Ser Leu Pro Cys Leu Ser Ser Asp Ser Gln Leu Pro

65 70 75 80

His Ile Asn Gly Ser Ala Ile Leu Pro Tyr Phe Arg Ala Ile Arg Pro

85 90 95

Leu Ser Asp Lys Asn Thr Ile Ala Lys Ile Ala Glu Gln Leu Asp Lys

100 105 110

Leu Lys Phe Gln Arg Glu Pro Glu Ala Lys Val Ser Met Pro Ala Asp

115 120 125

Asn Phe Glu Arg Lys Asn Phe Ile Leu Ala Val Leu Gln Gln Phe Ser

130 135 140

Ala Cys Leu Glu His Val Leu Gln Ser Leu Asn Ser Gly Pro Gln His

145 150 155 160

His His His His His His

165

<210> 162

<211> 495

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence corresponding to the gene of the mutant

feline IL-31 11E12 encoded C-terminal His-tag

<400> 162

atgctctctc acgccggtcc tgcccggttc gcactgttcc tcctctgttg catggagact 60

ctgcttccct cccacatggc accggcccat agactgcagc cgtccgacat cagaaagatc 120

atccttgaat tgcgccctat gagcaagggg ctgctgcagg attacctgaa aaaggagatc 180

ggcctgccgg aatcgaacca cagctcactg ccatgcctgt cctccgactc gcaactgccc 240

cacatcaatg gatccgccat tctgccgtac ttccgcgcta ttcggcctct ctccgacaag 300

aacaccatcg ccaagattgc cgagcagctg gataagctga agttccagag ggagccagaa 360

gccaaggtgt ccatgcccgc tgacaacttc gagcggaaga actttatcct cgcggtgctg 420

cagcagttct cagcgtgcct cgaacacgtc ttgcaaagcc tgaactcggg accccagcac 480

caccaccatc atcac 495

<210> 163

<211> 165

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence corresponding to the mutant

15H05 feline IL-31 protein with a C-terminal His-tag

<400> 163

Met Leu Ser His Ala Gly Pro Ala Arg Phe Ala Leu Phe Leu Leu Cys

1 5 10 15

Cys Met Glu Thr Leu Leu Pro Ser His Met Ala Pro Ala His Arg Leu

20 25 30

Gln Pro Ser Asp Ile Arg Lys Ile Ile Leu Glu Leu Arg Pro Met Ser

35 40 45

Lys Gly Leu Leu Gln Asp Tyr Leu Lys Lys Glu Ile Gly Leu Pro Glu

50 55 60

Ser Asn His Ser Ser Leu Pro Cys Leu Ser Ser Asp Ser Gln Leu Pro

65 70 75 80

His Ile Asn Gly Ser Ala Ile Leu Pro Tyr Phe Arg Ala Ile Arg Pro

85 90 95

Leu Ser Asp Lys Asn Thr Ile Asp Lys Ile Ile Glu Gln Leu Asp Lys

100 105 110

Leu Lys Phe Gln Arg Glu Pro Glu Ala Lys Val Ser Met Ala Ala Ala

115 120 125

Asn Phe Glu Arg Lys Asn Phe Ile Leu Ala Val Leu Gln Gln Phe Ser

130 135 140

Ala Cys Leu Glu His Val Leu Gln Ser Leu Asn Ser Gly Pro Gln His

145 150 155 160

His His His His His His

165

<210> 164

<211> 495

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence corresponding to the gene of the mutant

feline IL-31 15H05 encoded C-terminal His-tag

<400> 164

atgctctctc acgccggtcc tgcccggttc gcactgttcc tcctctgttg catggagact 60

ctgcttccct cccacatggc accggcccat agactgcagc cgtccgacat cagaaagatc 120

atccttgaat tgcgccctat gagcaagggg ctgctgcagg attacctgaa aaaggagatc 180

ggcctgccgg aatcgaacca cagctcactg ccatgcctgt cctccgactc gcaactgccc 240

cacatcaatg gatccgccat tctgccgtac ttccgcgcta ttcggcctct ctccgacaag 300

aacaccatcg acaagattat tgagcagctg gataagctga agttccagag ggagccagaa 360

gccaaggtgt ccatggccgc tgccaacttc gagcggaaga actttatcct cgcggtgctg 420

cagcagttct cagcgtgcct cgaacacgtc ttgcaaagcc tgaactcggg accccagcac 480

caccaccatc atcac 495

<210> 165

<211> 152

<212> PRT

<213> Artificial Sequence

<220>

<223> Equine IL-31 protein amino acid sequence c

C-terminal His-tag

<400> 165

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gly Pro Ile Tyr Gln Leu Gln Pro Lys Glu Ile Gln Ala

20 25 30

Ile Ile Val Glu Leu Gln Asn Leu Ser Lys Lys Leu Leu Asp Asp Tyr

35 40 45

Leu Asn Lys Glu Lys Gly Val Gln Lys Phe Asp Ser Asp Leu Pro Ser

50 55 60

Cys Phe Thr Ser Asp Ser Gln Ala Pro Gly Asn Ile Asn Ser Ser Ala

65 70 75 80

Ile Leu Pro Tyr Phe Lys Ala Ile Ser Pro Ser Leu Asn Asn Asp Lys

85 90 95

Ser Leu Tyr Ile Ile Glu Gln Leu Asp Lys Leu Asn Phe Gln Asn Ala

100 105 110

Pro Glu Thr Glu Val Ser Met Pro Thr Asp Asn Phe Glu Arg Lys Arg

115 120 125

Phe Ile Leu Thr Ile Leu Arg Trp Phe Ser Asn Cys Leu Glu His Arg

130 135 140

Ala Gln His His His His His His His

145 150

<210> 166

<211> 456

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of the equine IL-31 gene c

encoded by the C-terminal His-tag

<400> 166

atgggctggt cctgcatcat tctgtttctg gtggccacag ccaccggcgt gcactctgga 60

cctatctatc agctgcagcc caaagagatc caggccatca tcgtggaact gcagaacctg 120

agcaagaagc tgctggacga ctacctgaac aaagaaaagg gcgtgcagaa gttcgacagc 180

gacctgccta gctgcttcac cagcgattct caggcccctg gcaacatcaa cagcagcgcc 240

atcctgcctt acttcaaggc catctctccc agcctgaaca acgacaagag cctgtacatc 300

atcgagcagc tggacaagct gaacttccag aacgcccctg aaaccgaggt gtccatgcct 360

accgacaact tcgagcggaa gcggttcatc ctgaccatcc tgcggtggtt cagcaactgc 420

ctggaacaca gagcccagca ccaccaccat caccat 456

<210> 167

<211> 652

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of the feline extracellular domain

OSMR fused with human IgG1 Fc

<400> 167

Met Ala Leu Phe Ser Ala Phe Gln Thr Thr Phe Leu Leu Ala Leu Leu

1 5 10 15

Ser Leu Lys Thr Tyr Gln Ser Glu Val Leu Ser Glu Pro Leu Ser Leu

20 25 30

Ala Pro Glu Ser Leu Glu Val Ser Ile Asp Ser Ala Arg Gln Cys Leu

35 40 45

His Leu Lys Trp Ser Val His Asn Leu Ala Tyr His Gln Glu Leu Lys

50 55 60

Met Val Phe Gln Ile Glu Ile Ser Arg Ile Lys Thr Ser Asn Val Ile

65 70 75 80

Trp Val Glu Asn Tyr Ser Thr Thr Val Lys Arg Asn Gln Val Leu Arg

85 90 95

Trp Ser Trp Glu Ser Lys Leu Pro Leu Glu Cys Ala Lys His Ser Val

100 105 110

Arg Met Arg Gly Ala Val Asp Asp Ala Gln Val Pro Glu Leu Arg Phe

115 120 125

Trp Ser Asn Trp Thr Ser Trp Glu Glu Val Asp Val Gln Ser Ser Leu

130 135 140

Gly His Asp Pro Leu Phe Val Phe Pro Lys Asp Lys Leu Val Glu Glu

145 150 155 160

Gly Ser Asn Val Thr Ile Cys Tyr Val Ser Arg Ser His Gln Asn Asn

165 170 175

Ile Ser Cys Tyr Leu Glu Gly Val Arg Met His Gly Glu Gln Leu Asp

180 185 190

Pro Asn Val Cys Val Phe His Leu Lys Asn Val Pro Phe Ile Arg Glu

195 200 205

Thr Gly Thr Asn Ile Tyr Cys Lys Ala Asp Gln Gly Asp Val Ile Lys

210 215 220

Gly Ile Val Leu Phe Val Ser Lys Val Phe Glu Glu Pro Lys Asp Phe

225 230 235 240

Ser Cys Glu Thr Arg Asp Leu Lys Thr Leu Asn Cys Thr Trp Ala Pro

245 250 255

Gly Ser Asp Ala Gly Leu Leu Thr Gln Leu Ser Gln Ser Tyr Thr Leu

260 265 270

Phe Glu Ser Phe Ser Gly Lys Lys Thr Leu Cys Lys His Lys Ser Trp

275 280 285

Cys Asn Trp Gln Val Ser Pro Asp Ser Gln Glu Met Tyr Asn Phe Thr

290 295 300

Leu Thr Ala Glu Asn Tyr Leu Arg Lys Arg Ser Val His Leu Leu Phe

305 310 315 320

Asn Leu Thr His Arg Val His Pro Met Ala Pro Phe Asn Val Phe Val

325 330 335

Lys Asn Val Ser Ala Thr Asn Ala Thr Met Thr Trp Lys Val His Ser

340 345 350

Ile Gly Asn Tyr Ser Thr Leu Leu Cys Gln Ile Glu Leu Asp Gly Glu

355 360 365

Gly Lys Val Ile Gln Lys Gln Asn Val Ser Val Lys Val Asn Gly Lys

370 375 380

His Leu Met Lys Lys Leu Glu Pro Ser Thr Glu Tyr Ala Ala Gln Val

385 390 395 400

Arg Cys Ala Asn Ala Asn His Phe Trp Lys Trp Ser Glu Trp Thr Arg

405 410 415

Arg Asn Phe Thr Thr Ala Glu Ala Ala Asp Lys Thr His Thr Cys Pro

420 425 430

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

435 440 445

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

450 455 460

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

465 470 475 480

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

485 490 495

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

500 505 510

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

515 520 525

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

530 535 540

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

545 550 555 560

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

565 570 575

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

580 585 590

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

595 600 605

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

610 615 620

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

625 630 635 640

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

645 650

<210> 168

<211> 2076

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding an extracellular domain

feline OSMR fused with human IgG1 Fc

<400> 168

atggccctgt tcagcgcctt ccagaccacc ttcctgctgg ccctgctgag cctgaaaacc 60

taccagagcg aggtgctgag cgagcccctg tctctggccc ctgagagcct ggaagtgtcc 120

atcgacagcg ccagacagtg cctgcacctg aagtggagcg tgcacaacct ggcctaccac 180

caggaactga agatggtgtt ccagatcgag atcagccgga tcaagaccag caacgtgatc 240

tgggtggaaa actacagcac caccgtgaag cggaaccagg tgctgcggtg gtcctgggag 300

360

gcccaggtgc ccgagctgag attctggtcc aactggacct cctgggaaga ggtggacgtg 420

cagtctagcc tgggccacga ccccctgttc gtgttcccca aggacaagct ggtggaagag 480

ggctccaacg tgaccatctg ctacgtgtcc agaagccacc agaacaacat cagctgctac 540

ctggaaggcg tgcgcatgca cggcgagcag ctggacccta acgtgtgcgt gttccacctg 600

aagaacgtgc ccttcatcag agagacaggc accaacatct actgcaaggc cgaccagggc 660

gacgtgatca agggcatcgt gctgtttgtg tccaaggtgt tcgaggaacc caaggacttc 720

agctgcgaga cacgggatct gaaaaccctg aactgtacct gggcccctgg ctccgatgcc 780

ggactgctga ctcagctgtc ccagagctac accctgttcg agagcttcag cggcaaaaag 840

accctgtgca agcacaagag ctggtgcaac tggcaagtgt cccccgatag cggaaatg 900

tacaacttca ccctgaccgc cgagaactac ctgcggaaga gatccgtgca tctgctgttc 960

aacctgaccc acagagtgca ccccatggcc cccttcaacg tgttcgtgaa gaatgtgtcc 1020

gccaccaacg ccaccatgac atggaaggtg cacagcatcg gcaactactc caccctgctg 1080

tgtcagatcg agctggacgg cgagggcaaa gtgatccaga aacagaacgt gtcagtgaaa 1140

gtgaacggca agcacctgat gaagaagctg gaacccagca ccgagtacgc cgcccaggtg 1200

cgctgtgcca acgccaacca cttctggaag tggagtgaat ggacccggcg gaacttcacc 1260

acagccgaag ccgccgctga gaacgaggtg tccacaccta tgcaggccct gaccaccaac 1320

aaggacgacg acaacatcct gttccgggac tccgccaatg ccaccagcct gcctgtgcag 1380

gatagcagct ctgtgctgcc cgccaagccc gagaacatct cctgcgtgtt ctactacgag 1440

gaaaacttca cttgcacctg gtcccccgag aaagaggcca gctacacctg gtacaaagtg 1500

aagagaacct acagctacgg ctacaagagc gacatctgcc ccagcgacaa cagcaccaga 1560

ggcaaccaca ccttctgcag ctttctgccc cccaccatca ccaaccccga caactacacc 1620

atccaggtgg aagccgaa cgccgacggc atcatcaagt ccgacatcac ccactggtcc 1680

ctggacgcca tcacaaagat cgagcccccc gagatcttct ccgtgaagcc tgtgctgggc 1740

gtgaagagga tggtgcagat caagtggatc cggcccgtgc tggccccagt gtctagcacc 1800

ctgaagtaca ccctgcggtt caagaccgtg aacagcgcct actggatgga agtgaatttc 1860

accaaagagg acatcgaccg ggacgagaca tacaatctga ccggactgca ggccttcaca 1920

gagtacgtgc tggctctgag atgcgccacc aaagaatcca tgttttggag cggctggtcc 1980

caggaaaaga tgggcaccac cgaaggggt aagcctatcc ctaaccctct cctcggtctc 2040

gattctacgc gtaccggtca tcatcaccat caccat 2076

<210> 169

<211> 474

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of feline IL-31-Ra fused to Fc

human IgG1

<400> 169

Met Met Trp Pro Gln Val Trp Gly Leu Glu Ile Gln Phe Ser Pro Gln

1 5 10 15

Pro Ala Cys Ile Asp Leu Gly Met Met Trp Ala His Ala Leu Trp Thr

20 25 30

Leu Leu Leu Leu Cys Lys Phe Ser Leu Ala Val Leu Pro Ala Lys Pro

35 40 45

Glu Asn Ile Ser Cys Val Phe Tyr Tyr Glu Glu Asn Phe Thr Cys Thr

50 55 60

Trp Ser Pro Glu Lys Glu Ala Ser Tyr Thr Trp Tyr Lys Val Lys Arg

65 70 75 80

Thr Tyr Ser Tyr Gly Tyr Lys Ser Asp Ile Cys Pro Ser Asp Asn Ser

85 90 95

Thr Arg Gly Asn His Thr Phe Cys Ser Phe Leu Pro Pro Thr Ile Thr

100 105 110

Asn Pro Asp Asn Tyr Thr Ile Gln Val Glu Ala Gln Asn Ala Asp Gly

115 120 125

Ile Ile Lys Ser Asp Ile Thr His Trp Ser Leu Asp Ala Ile Thr Lys

130 135 140

Ile Glu Pro Pro Glu Ile Phe Ser Val Lys Pro Val Leu Gly Val Lys

145 150 155 160

Arg Met Val Gln Ile Lys Trp Ile Arg Pro Val Leu Ala Pro Val Ser

165 170 175

Ser Thr Leu Lys Tyr Thr Leu Arg Phe Lys Thr Val Asn Ser Ala Tyr

180 185 190

Trp Met Glu Val Asn Phe Thr Lys Glu Asp Ile Asp Arg Asp Glu Thr

195 200 205

Tyr Asn Leu Thr Gly Leu Gln Ala Phe Thr Glu Tyr Val Leu Ala Leu

210 215 220

Arg Cys Ala Thr Lys Glu Ser Met Phe Trp Ser Gly Trp Ser Gln Glu

225 230 235 240

Lys Met Gly Thr Thr Glu Glu Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

370 375 380

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 170

<211> 1422

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding feline IL-31 Ra,

fused with human IgG1 Fc

<400> 170

atgatgtggc cacaagtgtg gggcctggag atccagttca gcccccagcc tgcctgcatc 60

gatctgggca tgatgtgggc tcacgctctg tggaccctgc tgctgctgtg caagttttcc 120

ctggccgtgc tgcccgctaa gcctgagaac atcagctgcg tgttctacta tgaggagaac 180

ttcacctgta catggtcccc cgagaaggag gctagctata cctggtacaa ggtgaagaga 240

acatacagct atggctacaa gtctgatatc tgccccagcg acaactctac ccgcggcaat 300

cacacattct gttcttttct gccccctacc atcacaaacc ctgataatta taccatccag 360

gtggaggccc agaacgctga tggcatcatc aagtctgaca tcacccattg gtccctggac 420

gccatcacaa agatcgagcc acccgagatt ttctccgtga agcccgtgct gggcgtgaag 480

aggatggtgc agatcaagtg gatcaggcct gtgctggctc cagtgtccag caccctgaag 540

tatacactga gattcaagac cgtgaactcc gcttactgga tggaggtgaa cttcaccaag 600

gaggacatcg atagggacga gacctataat ctgacaggcc tgcaggcctt caccgagtac 660

gtgctggccc tgaggtgcgc tacaaaggag tccatgtttt ggtccggctg gagccaggag 720

aagatgggca ccacagagga ggataagacc cacacatgcc ctccatgtcc agctccagag 780

ctgctgggag gaccaagcgt gttcctgttt ccacctaagc ctaaggacac cctgatgatc 840

tctcgcaccc ctgaggtgac atgcgtggtg gtggacgtgt cccacgagga cccagaggtg 900

aagtttaact ggtatgtgga tggcgtggag gtgcataatg ccaagaccaa gcctagagag 960

gagcagtata acagcaccta ccgcgtggtg tctgtgctga cagtgctgca tcaggactgg 1020

ctgaacggca aggagtacaa gtgcaaggtg agcaataagg ccctgcctgc tccaatcgag 1080

aagaccatct ctaaggctaa gggacagcca agggagccac aggtgtatac actgccaccc 1140

agccgggagg agatgaccaa gaaccaggtg tctctgacat gtctggtgaa gggcttctac 1200

ccatctgata tcgctgtgga gtgggagtcc aatggccagc ccgagaacaa ttataagacc 1260

acacctccag tgctggattc tgacggctcc ttctttctgt actccaagct gaccgtggac 1320

aagagcaggt ggcagcaggg caacgtgttt tcttgctccg tgatgcatga ggctctgcac 1380

aatcattaca cacagaagag cctgtctctg tccccaggca ag 1422

<210> 171

<211> 335

<212> PRT

<213> Felis catus

<400> 171

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 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 Arg 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

<210> 172

<211> 1005

<212> DNA

<213> Felis catus

<400> 172

gcctccacca cggccccatc ggtgttccca ctggccccca gctgcgggac cacatctggc 60

gccaccgtgg ccctggcctg cctggtgtta ggctacttcc ctgagccggt gaccgtgtcc 120

tggaactccg gcgccctgac cagcggtgtg cacaccttcc cggccgtcct gcaggcctcg 180

gggctgtact ctctcagcag catggtgaca gtgccctcca gcaggtggct cagtgacacc 240

ttcacctgca acgtggccca cccgcccagc aacaccaagg tggacaagac cgtgcgcaaa 300

acagaccacc caccgggacc caaaccctgc gactgtccca aatgcccacc ccctgagatg 360

cttggaggac cgtccatctt catcttcccc ccaaaaccca aggacaccct ctcgatttcc 420

cggacgcccg aggtcacatg cttggtggtg gacttgggcc cagatgactc cgatgtccag 480

atcacatggt ttgtggataa cacccaggtg tacacagcca agacgagtcc gcgtgaggag 540

cagttcaaca gcacctaccg tgtggtcagt gtcctcccca tcctacacca ggactggctc 600

aaggggaagg agttcaagtg caaggtcaac agcaaatccc tcccctcccc catcgagagg 660

accatctcca aggccaaagg acagccccac gagccccagg tgtacgtcct gcctccagcc 720

caggagggagc tcagcaggaa caaagtcagt gtgacctgcc tgatcaaatc cttccacccg 780

cctgacattg ccgtcgagtg ggagatcacc ggacagccgg agccagagaa caactaccgg 840

acgaccccgc cccagctgga cagcgacggg acctacttcg tgtacagcaa gctctcggtg 900

gacaggtccc actggcagag gggaaacacc tacacctgct cggtgtcaca cgaagctctg 960

cacagccacc acacacagaa atccctcacc cagtctccgg gtaaa 1005

<210> 173

<211> 335

<212> PRT

<213> Artificial Sequence

<220>

<223> The amino acid sequence of the feline heavy chain,

modified to modulate antibody effector function

<400> 173

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 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 Ala Ala Gly Ala 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 Arg 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

<210> 174

<211> 1005

<212> DNA

<213> Artificial Sequence

<220>

<223> The nucleotide sequence encoding the feline heavy chain,

modified to modulate antibody effector function

<400> 174

gcctccacca cggccccatc ggtgttccca ctggccccca gctgcgggac cacatctggc 60

gccaccgtgg ccctggcctg cctggtgtta ggctacttcc ctgagccggt gaccgtgtcc 120

tggaactccg gcgccctgac cagcggtgtg cacaccttcc cggccgtcct gcaggcctcg 180

gggctgtact ctctcagcag catggtgaca gtgccctcca gcaggtggct cagtgacacc 240

ttcacctgca acgtggccca cccgcccagc aacaccaagg tggacaagac cgtgcgcaaa 300

acagaccacc caccgggacc caaaccctgc gactgtccca aatgcccacc ccctgaggcg 360

gctggagcac cgtccatctt catcttcccc ccaaaaccca aggacaccct ctcgatttcc 420

cggacgcccg aggtcacatg cttggtggtg gacttgggcc cagatgactc cgatgtccag 480

atcacatggt ttgtggataa cacccaggtg tacacagcca agacgagtcc gcgtgaggag 540

cagttcaaca gcacctaccg tgtggtcagt gtcctcccca tcctacacca ggactggctc 600

aaggggaagg agttcaagtg caaggtcaac agcaaatccc tcccctcccc catcgagagg 660

accatctcca aggccaaagg acagccccac gagccccagg tgtacgtcct gcctccagcc 720

caggagggagc tcagcaggaa caaagtcagt gtgacctgcc tgatcaaatc cttccacccg 780

cctgacattg ccgtcgagtg ggagatcacc ggacagccgg agccagagaa caactaccgg 840

acgaccccgc cccagctgga cagcgacggg acctacttcg tgtacagcaa gctctcggtg 900

gacaggtccc actggcagag gggaaacacc tacacctgct cggtgtcaca cgaagctctg 960

cacagccacc acacacagaa atccctcacc cagtctccgg gtaaa 1005

<210> 175

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of feline kappa light chain,

modified by the elimination of glycosylation (G-) at position 103

<400> 175

Arg Ser Asp Ala Gln Pro Ser Val Phe Leu Phe Gln Pro Ser Leu Asp

1 5 10 15

Glu Leu His Thr Gly Ser Ala Ser Ile Val Cys Ile Leu Asn Asp Phe

20 25 30

Tyr Pro Lys Glu Val Asn Val Lys Trp Lys Val Asp Gly Val Val Gln

35 40 45

Asn Lys Gly Ile Gln Glu Ser Thr Thr Glu Gln Asn Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Gln

65 70 75 80

Ser His Glu Lys Phe Ser Cys Glu Val Thr His Lys Ser Leu Ala Ser

85 90 95

Thr Leu Val Lys Ser Phe Gln Arg Ser Glu Cys Gln Arg Glu

100 105 110

<210> 176

<211> 330

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding a feline light chain

kappa modified by elimination of glycosylation (G-)

<400> 176

cggagtgatg ctcagccatc tgtctttctc ttccaaccat ctctggacga gttacataca 60

ggaagtgcct ctatcgtgtg catattgaat gacttctacc ccaaagaggt caatgtcaag 120

tggaaagtgg atggcgtagt ccaaaacaaa ggcatccagg agagcaccac agagcagaac 180

agcaaggaca gcacctacag cctcagcagc accctgacga tgtccagtac ggagtaccaa 240

agtcatgaaa agttctcctg cgaggtcact cacaagagcc tggcctccac cctcgtcaag 300

agcttccaga ggagcgagtg tcagagagag 330

<210> 177

<211> 335

<212> PRT

<213> Canis familiaris

<400> 177

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> 178

<211> 1005

<212> DNA

<213> Canis familiaris

<400> 178

gcctcaacaa ctgctcctag cgtgtttccc ctggccccta gctgcggaag tacctcaggc 60

agcacagtgg ccctggcttg tctggtgtct ggatatttcc ctgagccagt gaccgtgagt 120

tggaacagcg gctctctgac ctccggggtg cacacatttc catctgtgct gcagtctagt 180

ggcctgtact ccctgtcaag catggtgact gtgccttcct ctaggtggcc atcagaaact 240

ttcacctgca acgtggccca tcccgccagc aagaccaaag tggacaagcc cgtgcctaaa 300

agggagaatg gaagggtgcc aagaccacct gattgcccta agtgtccagc tccagaagcg 360

gcgggagcac caagcgtgtt catctttcca cccaagccca aagacacact gctgattgct 420

agaactcccg aggtgacctg cgtggtggtg gacctggatc cagaggaccc cgaagtgcag 480

atctcctggt tcgtggatgg gaagcagatg cagacagcca aaactcagcc tcgggaggaa 540

cagtttaacg gaacctatag agtggtgtct gtgctgccaa ttggacacca ggactggctg 600

aagggcaaac agtttacatg caaggtgaac aacaaggccc tgcctagtcc aatcgagagg 660

actatttcaa aagctagggg acaggctcat cagccttccg tgtatgtgct gcctccatcc 720

cgggaggaac tgtctaagaa cacagtgagt ctgacttgtc tgatcaaaga tttctttccc 780

cctgacattg atgtggagtg gcagagcaat gggcagcagg agccagaatc caagtacaga 840

accacaccac cccagctgga cgaagatggc tcctatttcc tgtacagtaa gctgtcagtg 900

gacaaatcta ggtggcagcg cggggatacc tttatctgcg ccgtgatgca cgaggctctg 960

cacaatcatt acacacaaga aagtctgtca catagccccg gcaag 1005

<210> 179

<211> 106

<212> PRT

<213> Canis familiaris

<400> 179

Arg Asn Asp Ala Gln Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp

1 5 10 15

Gln Leu His Thr Gly Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln

35 40 45

Asp Thr Gly Ile Gln Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser

65 70 75 80

His Glu Leu Tyr Ser Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr

85 90 95

Leu Ile Lys Ser Phe Gln Arg Ser Glu Cys

100 105

<210> 180

<211> 318

<212> DNA

<213> Canis familiaris

<400> 180

aggaacgacg cccagcctgc tgtgtatctg tttcagccct cccctgatca gctgcacact 60

ggctctgcta gtgtggtgtg tctgctgaac agcttctacc caaaggatat caatgtgaag 120

tggaaagtgg acggcgtgat ccaggatact gggattcagg agtccgtgac cgaacaggac 180

aaagattcaa catatagcct gagctccact ctgaccatgt ctagtaccga gtacctgagc 240

cacgaactgt attcctgcga gatcactcat aagtccctgc cctctaccct gatcaagagc 300

ttccagagat cagagtgt 318

<210> 181

<211> 164

<212> PRT

<213> Homo sapiens

<400> 181

Met Ala Ser His Ser Gly Pro Ser Thr Ser Val Leu Phe Leu Phe Cys

1 5 10 15

Cys Leu Gly Gly Trp Leu Ala Ser His Thr Leu Pro Val Arg Leu Leu

20 25 30

Arg Pro Ser Asp Asp Val Gln Lys Ile Val Glu Glu Leu Gln Ser Leu

35 40 45

Ser Lys Met Leu Leu Lys Asp Val Glu Glu Glu Lys Gly Val Leu Val

50 55 60

Ser Gln Asn Tyr Thr Leu Pro Cys Leu Ser Pro Asp Ala Gln Pro Pro

65 70 75 80

Asn Asn Ile His Ser Pro Ala Ile Arg Ala Tyr Leu Lys Thr Ile Arg

85 90 95

Gln Leu Asp Asn Lys Ser Val Ile Asp Glu Ile Ile Glu His Leu Asp

100 105 110

Lys Leu Ile Phe Gln Asp Ala Pro Glu Thr Asn Ile Ser Val Pro Thr

115 120 125

Asp Thr His Glu Cys Lys Arg Phe Ile Leu Thr Ile Ser Gln Gln Phe

130 135 140

Ser Glu Cys Met Asp Leu Ala Leu Lys Ser Leu Thr Ser Gly Ala Gln

145 150 155 160

Gln Ala Thr Thr

<210> 182

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Caninized lung variable region sequence

mAb chains, from Mus musculus and Canis

<400> 182

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Arg Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser

65 70 75 80

Arg Val Glu Ala Asp Asp Ala Gly Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 183

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

caninized light chain variable region mAb from Mus

muscle and canis

<400> 183

gacatcgtga tgacccagac ccccctgagc ctgagcgtgt cccctggcga gcctgccagc 60

atcagctgca gagccagcga gagcgtggac aactacggca tcagcttcat gcactggttc 120

cagcagaagc ccggccagag cccccagcgg ctgatctaca gagccagcaa cctggaaagc 180

ggcgtgcccg atcggtttag cggctctggc agcggcaccg acttcaccct gcggatctct 240

cgggtggaag ccgatgacgc cggagtgtac tactgccagc agagcaacaa ggaccccctg 300

acctttggcg ccggtaccaa gctggagatc aag 333

<210> 184

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Caninized lung variable region sequence

mAb chains, from Mus musculus and Canis

<400> 184

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser

65 70 75 80

Arg Val Glu Ala Asp Asp Ala Gly Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Lys Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 185

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

caninized light chain variable region mAb from Mus

muscle and canis

<400> 185

gatatagtga tgacacaaac tcctctcagt ctttccgtat caccgggaga accggcttcc 60

atttcctgtc gggcctcaga gtctgtggac aactacggga tatccttcat gcactggtat 120

cagcagaaac ccggccagcc ccctaaactc cttatttaca gggccagtaa tctggaaagc 180

ggtgtgcccg atcgatttag cggttccggg agcggcacag atttcaccct gcgaatctct 240

agagttgaag cggatgatgc aggagtatat tactgccagc aatccaataa ggatcccctt 300

acattcggcg cgggtaccaa gctggagatc aag 333

<210> 186

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of the feline kappa G- c light chain

modified C-terminus

<400> 186

Arg Ser Asp Ala Gln Pro Ser Val Phe Leu Phe Gln Pro Ser Leu Asp

1 5 10 15

Glu Leu His Thr Gly Ser Ala Ser Ile Val Cys Ile Leu Asn Asp Phe

20 25 30

Tyr Pro Lys Glu Val Asn Val Lys Trp Lys Val Asp Gly Val Val Gln

35 40 45

Asn Lys Gly Ile Gln Glu Ser Thr Thr Glu Gln Asn Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Gln

65 70 75 80

Ser His Glu Lys Phe Ser Cys Glu Val Thr His Lys Ser Leu Ala Ser

85 90 95

Thr Leu Val Lys Ser Phe Gln Arg Ser Glu Cys

100 105

<210> 187

<211> 324

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding the feline kappa chain G-

with modified C-terminus

<400> 187

cggagtgatg ctcagccatc tgtctttctc ttccaaccat ctctggacga gttacataca 60

ggaagtgcct ctatcgtgtg catattgaat gacttctacc ccaaagaggt caatgtcaag 120

tggaaagtgg atggcgtagt ccaaaacaaa ggcatccagg agagcaccac agagcagaac 180

agcaaggaca gcacctacag cctcagcagc accctgacga tgtccagtac ggagtaccaa 240

agtcatgaaa agttctcctg cgaggtcact cacaagagcc tggcctccac cctcgtcaag 300

agcttccaga ggagcgagtg ttag 324

<210> 188

<211> 323

<212> PRT

<213> Mus musculus

<400> 188

Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala

1 5 10 15

Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe

20 25 30

Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly

35 40 45

Val His Thr Phe Pro Ala Val Leu Glu Ser Asp Leu Tyr Thr Leu Ser

50 55 60

Ser Ser Val Thr Val Pro Ser Ser Pro Arg Pro Ser Glu Thr Val Thr

65 70 75 80

Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile

85 90 95

Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu

100 105 110

Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr

115 120 125

Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys

130 135 140

Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val

145 150 155 160

His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe

165 170 175

Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly

180 185 190

Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile

195 200 205

Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val

210 215 220

Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser

225 230 235 240

Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu

245 250 255

Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro

260 265 270

Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val

275 280 285

Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu

290 295 300

His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser

305 310 315 320

Pro Gly Lys

<210> 189

<211> 971

<212> DNA

<213> Mus musculus

<400> 189

ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact 60

ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct 120

ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg gagtctgacc 180

tctacactct gagcagctca gtgactgtcc cctccagccc tcggcccagc gagaccgtca 240

cctgcaacgt tgccccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg 300

attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc 360

ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg 420

tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg 480

tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca 540

gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca 600

acagtgcagc ttttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga 660

aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca 720

gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga 780

atgggcagcc agcggagaac tacaagaaca ctcagcccat catgaacacg aatggctctt 840

acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca 900

cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact 960

ctcctggtaa a 971

<210> 190

<211> 106

<212> PRT

<213> Mus musculus

<400> 190

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

1 5 10 15

Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

20 25 30

Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

35 40 45

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

65 70 75 80

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

85 90 95

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 191

<211> 321

<212> DNA

<213> Mus musculus

<400> 191

gctgatgctg caccaactgt atccatcttc ccaccatcca gtgagcagtt aacatctgga 60

ggtgcctcag tcgtgtgctt cttgaacaac ttctacccca aagacatcaa tgtcaagtgg 120

aagattgatg gcagtgaacg acaaaatggc gtcctgaaca gttggactga tcaggacagc 180

aaagacagca cctacagcat gagcagcacc ctcacgttga ccaaggacga gtatgaacga 240

cataacagct atacctgtga ggccactcac aagacatcaa cttcacccat tgtcaagagc 300

ttcaacagga atgagtgtta g 321

<210> 192

<211> 106

<212> PRT

<213> Homo sapiens

<400> 192

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 193

<211> 321

<212> DNA

<213> Homo sapiens

<400> 193

actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

240

cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300

ttcaacaggg gagagtgtta g 321

<210> 194

<211> 109

<212> PRT

<213> Canis familiaris

<400> 194

Asn Asp Ala Gln Pro Ala Val Tyr Leu Phe Gln Pro Ser Pro Asp Gln

1 5 10 15

Leu His Thr Gly Ser Ala Ser Val Val Cys Leu Leu Asn Ser Phe Tyr

20 25 30

Pro Lys Asp Ile Asn Val Lys Trp Lys Val Asp Gly Val Ile Gln Asp

35 40 45

Thr Gly Ile Gln Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr

50 55 60

Ser Leu Ser Ser Thr Leu Thr Met Ser Ser Thr Glu Tyr Leu Ser His

65 70 75 80

Glu Leu Tyr Ser Cys Glu Ile Thr His Lys Ser Leu Pro Ser Thr Leu

85 90 95

Ile Lys Ser Phe Gln Arg Ser Glu Cys Gln Arg Val Asp

100 105

<210> 195

<211> 330

<212> DNA

<213> Canis familiaris

<400> 195

aatgatgccc agccagccgt ctatttgttc caaccatctc cagaccagtt acacacagga 60

agtgcctctg ttgtgtgctt gctgaatagc ttctacccca aagacatcaa tgtcaagtgg 120

aaagtggatg gtgtcatcca agacacaggc atccaggaaa gtgtcacaga gcaggacaag 180

gacagtacct acagcctcag cagcaccctg acgatgtcca gtactgagta cctaagtcat 240

gagttgtact cctgtgagat cactcacaag agcctgccct ccaccctcat caagagcttc 300

caaaggagcg agtgtcagag agtggactaa 330

<210> 196

<211> 108

<212> PRT

<213> Sus scrofa

<400> 196

Ala Asp Ala Lys Pro Ser Val Phe Ile Phe Pro Pro Ser Lys Glu Gln

1 5 10 15

Leu Glu Thr Gln Thr Val Ser Val Val Cys Leu Leu Asn Ser Phe Phe

20 25 30

Pro Arg Glu Val Asn Val Lys Trp Lys Val Asp Gly Val Val Gln Ser

35 40 45

Ser Gly Ile Leu Asp Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Ser Leu Pro Thr Ser Gln Tyr Leu Ser

65 70 75 80

His Asn Leu Tyr Ser Cys Glu Val Thr His Lys Thr Leu Ala Ser Pro

85 90 95

Leu Val Lys Ser Phe Ser Arg Asn Glu Cys Glu Ala

100 105

<210> 197

<211> 327

<212> DNA

<213> Sus scrofa

<400> 197

gctgatgcca agccatccgt cttcatcttc ccgccatcga aggagcagtt agagacccaa 60

actgtctctg tggtgtgctt gctcaatagc ttcttcccca gagaagtcaa tgtcaagtgg 120

aaagtggatg gggtggtcca aagcagtggc atcctggata gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagcacc ctctcgctgc ccacgtcaca gtacctaagt 240

cataatttat attcctgtga ggtcacccac aagaccctgg cctcccctct ggtcaaaagc 300

ttcagcagga acgagtgtga ggcttag 327

<210> 198

<211> 108

<212> PRT

<213> Mustela vison

<400> 198

Arg Asn Asp Ala Gln Pro Ser Val Phe Leu Phe Gln Pro Ser Gln Asp

1 5 10 15

Gln Leu His Thr Gly Ser Ala Ser Val Val Cys Leu Leu Asn Gly Phe

20 25 30

Tyr Pro Lys Glu Val Thr Val Lys Trp Met Val Asp Gly Val Thr Lys

35 40 45

Asn Thr Gly Ile Leu Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Ile Pro Ser Thr Glu Tyr Leu

65 70 75 80

Ser His Glu Thr Tyr Ser Cys Glu Val Thr His Lys Ser Leu Ser Ser

85 90 95

Pro Leu Val Lys Ser Phe Gln Arg Ser Glu Cys Gln

100 105

<210> 199

<211> 327

<212> DNA

<213> Mustela vison

<400> 199

cggaatgatg cccagccatc cgtctttttg ttccaaccat ctcaggacca gttacatacc 60

ggcagtgcct ctgtcgtgtg cttgctgaat ggcttctacc ccaaagaagt cactgtcaaa 120

tggatggttg atggtgttac caaaaacaca ggcatcctag aaagtgtcac agaacaagac 180

agcaaggaca gcacctacag cctcagcagc accctgacga tccccagtac ggagtaccta 240

agtcatgaga cgtactcctg tgaggtcact cacaagagcc tgtcctcccc tcttgtcaag 300

agcttccaaa ggagcgagtg ccaatga 327

<210> 200

<211> 5

<212> PRT

<213> Mus musculus

<400> 200

Ser Tyr Trp Met Asn

15

<210> 201

<211> 17

<212> PRT

<213> Mus musculus

<400> 201

Met Ile Asp Pro Ser Asp Ser Glu Ile His Tyr Asn Gln Val Phe Lys

1 5 10 15

asp

<210> 202

<211> 9

<212> PRT

<213> Mus musculus

<400> 202

Gln Asp Ile Val Thr Thr Val Asp Tyr

15

<210> 203

<211> 17

<212> PRT

<213> Mus musculus

<400> 203

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ile Asn Gln Lys Asn His Leu

1 5 10 15

Ala

<210> 204

<211> 7

<212> PRT

<213> Mus musculus

<400> 204

Trp Ala Ser Thr Arg Glu Ser

15

<210> 205

<211> 9

<212> PRT

<213> Mus musculus

<400> 205

Gln Gln Gly Tyr Thr Tyr Pro Phe Thr

15

<210> 206

<211> 5

<212> PRT

<213> Mus musculus

<400> 206

Ser Tyr Trp Met Asn

15

<210> 207

<211> 17

<212> PRT

<213> Mus musculus

<400> 207

Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe Arg

1 5 10 15

asp

<210> 208

<211> 9

<212> PRT

<213> Mus musculus

<400> 208

Gln Asp Ile Val Thr Thr Val Asp Tyr

15

<210> 209

<211> 17

<212> PRT

<213> Mus musculus

<400> 209

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ile Asn Gln Lys Asn Phe Leu

1 5 10 15

Ala

<210> 210

<211> 7

<212> PRT

<213> Mus musculus

<400> 210

Trp Ala Ser Thr Arg Glu Ser

15

<210> 211

<211> 9

<212> PRT

<213> Mus musculus

<400> 211

Gln Gln His Tyr Gly Tyr Pro Phe Thr

15

<210> 212

<211> 118

<212> PRT

<213> Mus musculus

<400> 212

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Ile His Tyr Asn Gln Val Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Asp Ile Val Thr Thr Val Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 213

<211> 354

<212> DNA

<213> Mus musculus

<400> 213

caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgaactgggc gaagcagagg 120

cctggacaag gccttgaatg gattggtatg attgatcctt cagacagtga aattcactac 180

aatcaagtgt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagacaagat 300

atagtgacta cagttgacta ctggggccaa ggcaccactc tcacagtctc ctca 354

<210> 214

<211> 113

<212> PRT

<213> Mus musculus

<400> 214

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Asn Gln Lys Asn His Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ala Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Gly Tyr Thr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 215

<211> 339

<212> DNA

<213> Mus musculus

<400> 215

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttaca 60

atgagctgca agtccagtca gagcctttta tatagtatca atcaaaagaa ccacttggcc 120

tggttccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atcactagg 180

gaatctgggg tccctgctcg cttcacaggc agtggatctg gaacagattt cactctcacc 240

atcagcagtg tgaagactga agacctggca gtttattact gtcagcaagg ttatacctac 300

ccattcacgt tcggctcggg gacaaagttg gaaataaaa 339

<210> 216

<211> 118

<212> PRT

<213> Mus musculus

<400> 216

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Tyr Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Gln Ile Phe

50 55 60

Arg Asp Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gln Asp Ile Val Thr Thr Val Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 217

<211> 354

<212> DNA

<213> Mus musculus

<400> 217

caggtccaat tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60

tcctgcaagg cctatggcta caccttcacc agttactgga tgaactgggt gaaacagagg 120

cctggacaag gccttgaatg gattggtatg attgatcctt cagacagtga aactcactac 180

aatcaaatat tcagggacaa ggccacattg actatagaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct atttctgtgc aagacaagat 300

atagtgacta cagttgacta ctggggccag ggcaccactc tcacagtctc ctca 354

<210> 218

<211> 113

<212> PRT

<213> Mus musculus

<400> 218

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ser Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Gly Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 219

<211> 339

<212> DNA

<213> Mus musculus

<400> 219

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60

atgagctgca agtccagtca gagcctttta tatagtatca atcaaaagaa cttcttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atcactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaagtctga agacctggca gtttattact gtcagcaaca ttatggctat 300

ccattcacgt tcggctcggg gacaaagttg gaaataaaa 339

<210> 220

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 220

Asp Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Thr Cys Val Ala Ser Gly Phe Thr Tyr Ser Asn Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Arg Ile Asp Pro Tyr Gly Gly Gly Thr Lys His Asn Glu Lys Phe

50 55 60

Lys Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Val Arg Ser Gly Tyr Asp Tyr Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 221

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized mAb heavy chain variable region, from Mus

musculus and Felis catus

<400> 221

gacgtgcaac tggtggagag tggaggggac cttgtaaagc caggcgggtc cctgcgcctg 60

acctgtgtag ccagcggctt cacttactcc aactattgga tgcactgggt cagacaggcc 120

ccgggaaaag ggcttcagtg ggtggcaagg atcgatccct acggaggagg aacgaagcat 180

aacgagaagt tcaagcggag gtttactatc agtagagaca acgcgaaaaa tacactgtac 240

ctgcagatga atagtcttaa gacagaggat accgcgacct actattgcgt cagatccggc 300

tatgactatt actttgacgt ttggggacag ggcacactgg tcaccgtctc gagc 354

<210> 222

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 222

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His His Phe Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 223

<211> 321

<212> PRT

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 223

Gly Ala Ala Ala Thr Thr Cys Ala Ala Ala Thr Gly Ala Cys Cys Cys

1 5 10 15

Ala Gly Thr Cys Ala Cys Cys Cys Ala Gly Thr Thr Cys Cys Thr Thr

20 25 30

Gly Thr Cys Cys Gly Cys Cys Ala Gly Thr Cys Cys Cys Gly Gly Ala

35 40 45

Gly Ala Thr Cys Gly Cys Gly Thr Cys Ala Cys Cys Ala Thr Ala Ala

50 55 60

Cys Cys Thr Gly Cys Cys Gly Gly Gly Cys Cys Ala Gly Thr Gly Ala

65 70 75 80

Ala Ala Ala Thr Ala Thr Thr Thr Ala Thr Thr Cys Ala Thr Thr Cys

85 90 95

Cys Thr Cys Gly Cys Gly Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys

100 105 110

Ala Gly Ala Ala Gly Cys Cys Ala Gly Gly Cys Ala Ala Gly Gly Thr

115 120 125

Gly Cys Cys Gly Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala Thr Ala

130 135 140

Thr Ala Cys Ala Ala Cys Gly Cys Thr Ala Ala Cys Ala Cys Gly Thr

145 150 155 160

Thr Gly Gly Cys Cys Gly Ala Gly Gly Gly Gly Gly Thr Gly Cys Cys

165 170 175

Cys Ala Gly Thr Ala Gly Ala Thr Thr Cys Ala Gly Cys Gly Gly Gly

180 185 190

Thr Cys Cys Gly Gly Cys Ala Gly Thr Gly Gly Ala Ala Cys Cys Gly

195 200 205

Ala Thr Thr Thr Cys Ala Cys Cys Cys Thr Gly Ala Cys Ala Ala Thr

210 215 220

Thr Thr Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Cys Cys Ala

225 230 235 240

Gly Ala Ala Gly Ala Cys Gly Cys Cys Gly Cys Cys Ala Cys Ala Thr

245 250 255

Ala Cys Thr Ala Cys Thr Gly Cys Cys Ala Gly Cys Ala Thr Cys Ala

260 265 270

Cys Thr Thr Thr Gly Gly Gly Ala Cys Cys Cys Cys Thr Thr Thr Cys

275 280 285

Ala Cys Thr Thr Thr Cys Gly Gly Ala Thr Cys Cys Gly Gly Thr Ala

290 295 300

Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala Thr Cys Ala Ala

305 310 315 320

Ala

<210> 224

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 224

Asp Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Thr Cys Val Ala Ser Gly Phe Thr Tyr Ser Asn Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Arg Ile Asp Pro Tyr Gly Gly Gly Thr Lys His Asn Glu Lys Phe

50 55 60

Lys Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Val Arg Ser Gly Tyr Asp Tyr Tyr Phe Asp Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 225

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized mAb heavy chain variable region, from Mus

musculus and Felis catus

<400> 225

gacgtgcaac tggtggagag tggaggggac cttgtaaagc caggcgggtc cctgcgcctg 60

acctgtgtag ccagcggctt cacttactcc aactattgga tgcactgggt cagacaggcc 120

ccgggaaaag ggcttcagtg ggtggcaagg atcgatccct acggaggagg aacgaagcat 180

aacgagaagt tcaagcggag gtttactatc agtagagaca acgcgaaaaa tacactgtac 240

ctgcagatga atagtcttaa gacagaggat accgcgacct actattgcgt cagatccggc 300

tatgactatt actttgacgt ttggggacag ggcacactgg tcaccgtctc gagc 354

<210> 226

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Mus musculus and Felis catus

<400> 226

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Asn Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His His Phe Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 227

<211> 321

<212> PRT

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, from Mus

musculus and Felis catus

<400> 227

Gly Ala Ala Ala Thr Thr Cys Ala Ala Ala Thr Gly Ala Cys Cys Cys

1 5 10 15

Ala Gly Thr Cys Ala Cys Cys Cys Ala Gly Thr Thr Cys Cys Thr Thr

20 25 30

Gly Thr Cys Cys Gly Cys Cys Ala Gly Thr Cys Cys Cys Gly Gly Ala

35 40 45

Gly Ala Thr Cys Gly Cys Gly Thr Cys Ala Cys Cys Ala Thr Ala Ala

50 55 60

Cys Cys Thr Gly Cys Cys Gly Gly Gly Cys Cys Ala Gly Thr Gly Ala

65 70 75 80

Ala Ala Ala Thr Ala Thr Thr Thr Ala Thr Thr Cys Ala Thr Thr Cys

85 90 95

Cys Thr Cys Gly Cys Gly Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys

100 105 110

Ala Gly Ala Ala Gly Cys Cys Ala Gly Gly Cys Ala Ala Gly Gly Thr

115 120 125

Gly Cys Cys Gly Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala Thr Ala

130 135 140

Thr Ala Cys Ala Ala Cys Gly Cys Thr Ala Ala Cys Ala Cys Gly Thr

145 150 155 160

Thr Gly Gly Cys Cys Gly Ala Gly Gly Gly Gly Gly Thr Gly Cys Cys

165 170 175

Cys Ala Gly Thr Ala Gly Ala Thr Thr Cys Ala Gly Cys Gly Gly Gly

180 185 190

Thr Cys Cys Gly Gly Cys Ala Gly Thr Gly Gly Ala Ala Cys Cys Gly

195 200 205

Ala Thr Thr Thr Cys Ala Cys Cys Cys Thr Gly Ala Cys Ala Ala Thr

210 215 220

Thr Thr Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Cys Cys Ala

225 230 235 240

Gly Ala Ala Gly Ala Cys Gly Cys Cys Gly Cys Cys Ala Cys Ala Thr

245 250 255

Ala Cys Thr Ala Cys Thr Gly Cys Cys Ala Gly Cys Ala Thr Cys Ala

260 265 270

Cys Thr Thr Thr Gly Gly Gly Ala Cys Cys Cys Cys Thr Thr Thr Cys

275 280 285

Ala Cys Thr Thr Thr Cys Gly Gly Ala Thr Cys Cys Gly Gly Thr Ala

290 295 300

Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala Thr Cys Ala Ala

305 310 315 320

Ala

<210> 228

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Canis familiaris and Felis catus

<400> 228

Asp Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Thr Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Gly Ile Asp Ser Val Gly Ser Gly Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gly Phe Pro Gly Ser Phe Glu His Trp Gly Gln Gly Ala Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 229

<211> 351

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized heavy chain variable region mAb, Canis

familiaris and Felis catus

<400> 229

gatgtgcagc tggtggaaag cggcggcgat ctggtgaaac cgggcggcag cctgcgcctg 60

acctgcgtgg cgagcggctt tacctttagc gattatgcga tgagctgggt gcgccaggcg 120

ccgggcaaag gcctgcagtg ggtggcgggc attgatagcg tgggcagcgg caccagctat 180

gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa caccctgtat 240

ctgcagatga acagcctgaa aaccgaagat accgcgacct attattgcgc gagcggcttt 300

ccgggcagct ttgaacattg gggccagggc gcgctggtga ccgtgagcag c 351

<210> 230

<211> 132

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Canis familiaris and Felis catus

<400> 230

Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Gly Thr Leu Gly Gln

1 5 10 15

Arg Ile Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Gly

20 25 30

Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Met Gly Pro Lys Thr Val

35 40 45

Ile Tyr Tyr Asn Ser Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Ser Ser Gly Thr Leu Thr Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Tyr Asp Arg Thr Phe

85 90 95

Asn Ala Val Phe Gly Gly Gly Thr His Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ser Ala Pro Pro Arg Ser His Ser Ser Arg Pro Ile Ser Tyr Ala

115 120 125

Val Phe Cys Leu

130

<210> 231

<211> 327

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, Canis

familiaris and Felis catus

<400> 231

cagagcgtgc tgacccagcc gagcagcgtg agcggcaccc tgggccagcg cattaccatt 60

agctgcaccg gcagcagcag caacattggc agcggctatg tgggctggta tcagcaggtg 120

ccgggcatgg gcccgaaaac cgtgatttat tataacagcg atcgcccgag cggcgtgccg 180

gatcgcttta gcggcagcaa aagcggcagc agcggcaccc tgaccattac cggcctgcag 240

gcggaagatg aagcggatta ttattgcagc gtgtatgatc gcacctttaa cgcggtgttt 300

ggcggcggca cccatctgac cgtgctg 327

<210> 232

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Severe felinized variable region sequence

mAb chains, from Canis familiaris and Felis catus

<400> 232

Asp Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Thr Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

35 40 45

Ala Gly Ile Asp Ser Val Gly Ser Gly Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Gly Leu Lys Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gly Phe Pro Gly Ser Phe Glu His Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 233

<211> 351

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized heavy chain variable region mAb, Canis

familiaris and Felis catus

<400> 233

gatgtgcagc tggtggaaag cggcggcgat ctggtgaaac cgggcggcag cctgcgcctg 60

acctgcgtgg cgagcggctt tacctttagc gattatgcga tgaactgggt gcgccaggcg 120

ccgggcaaag gcctgcagtg ggtggcgggc attgatagcg tgggcagcgg caccagctat 180

gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa caccctgtat 240

ctgcagatga gcggcctgaa aaccgaagat accgcgacct attattgcgc gagcggcttt 300

ccgggcagct ttgaacattg gggccagggc accctggtga ccgtgagcag c 351

<210> 234

<211> 132

<212> PRT

<213> Artificial Sequence

<220>

<223> Felinized light variable region sequence

mAb chains, from Canis familiaris and Felis catus

<400> 234

Gln Ser Val Leu Thr Gln Pro Ser Ser Val Ser Gly Thr Leu Gly Gln

1 5 10 15

Arg Ile Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Gly

20 25 30

Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Met Gly Pro Lys Thr Val

35 40 45

Ile Tyr Tyr Asn Ser Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Ser Ser Gly Thr Leu Thr Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Val Tyr Asp Arg Thr Phe

85 90 95

Asn Ala Val Phe Gly Gly Gly Thr His Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ser Ala Pro Pro Arg Ser His Ser Ser Arg Pro Ile Ser Tyr Ala

115 120 125

Val Phe Cys Leu

130

<210> 235

<211> 327

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding sequence

felinized light chain variable region mAb, Canis

familiaris and Felis catus

<400> 235

cagagcgtgc tgacccagcc gagcagcgtg agcggcaccc tgggccagcg cattaccatt 60

agctgcaccg gcagcagcag caacattggc agcggctatg tgggctggta tcagcaggtg 120

ccgggcatgg gcccgaaaac cgtgatttat tataacagcg atcgcccgag cggcgtgccg 180

gatcgcttta gcggcagcaa aagcggcagc agcggcaccc tgaccattac cggcctgcag 240

gcggaagatg aagcggatta ttattgcagc gtgtatgatc gcacctttaa cgcggtgttt 300

ggcggcggca cccatctgac cgtgctg 327

<210> 236

<211> 106

<212> PRT

<213> Felis catus

<400> 236

Gly Gln Pro Lys Ser Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Asn

1 5 10 15

Glu Glu Leu Ser Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Ser Gly Leu Thr Val Ala Trp Lys Ala Asp Gly Thr Pro

35 40 45

Ile Thr Gln Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Ser Pro Asn Glu Trp Lys

65 70 75 80

Ser Arg Ser Arg Phe Thr Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Asn Val Val Pro Ala Glu Cys Ser

100 105

<210> 237

<211> 318

<212> DNA

<213> Felis catus

<400> 237

ggccagccca agagcgctcc ctccgtgacc ctgttccccc caagcaacga ggaactgagc 60

gccaacaagg ccaccctggt gtgcctgatc agcgacttct accccagcgg cctgaccgtg 120

gcctggaagg ccgatggcac ccctatcacc cagggcgtgg aaaccaccaa gccagcaag 180

cagagcaaca acaaatacgc cgccagcagc tacctgagcc tgagccccaa cgagtggaag 240

tcccggtccc ggttcacatg ccaggtgaca cacgagggca gcaccgtgga aaagaacgtg 300

gtgcccgccg agtgcagc 318

<---

Claims (83)

1. An anti-IL-31 antibody containing: VH-CDR1 (Heavy Chain Variable Region Hypervariable Region 1) containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 2 in SEQ ID NO: 3, VL-CDR1 (light chain variable region hypervariable region 1) containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL -CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. 2. An antibody according to claim 1 that binds to feline IL-31 and contains a VL chain (light chain variable region) containing changes in framework region 2 (FW2) selected from the group consisting of asparagine instead of lysine at position 42, isoleucine instead of valine at position 43, valine instead of leucine at position 46, asparagine instead of lysine at position 49, and combinations thereof. 3. An anti-IL-31 antibody containing: 1) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 15, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; 2) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 21, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 24; 3) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 25, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 26, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 27, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 28, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 30; 4) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 33, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 34, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 35, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 36; 5) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 37, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 38, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 39, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 42; 6) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 43, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 44, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 45, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 47, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 48; 7) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 49, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 50, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 51, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 52, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 53, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 54; 8) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 55, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 56, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 57, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 58, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 59, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 60; or 9) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 61, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 62, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 63, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 64, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 65, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 66. 4. An antibody according to any one of paragraphs. 1-3, which attenuates, inhibits, or neutralizes an IL-31-mediated pruritic or allergic condition in a mammal. 5. An antibody according to claim 4, wherein the mammal is selected from the group consisting of dog, cat and horse. 6. An antibody according to any one of paragraphs. 1-5, which is a chimeric antibody. 7. An antibody according to any one of paragraphs. 1-6, which is a caninized, felinized, equinized, fully canine, fully feline, or fully equine antibody. 8. An antibody according to claim 1, containing at least one of the group consisting of: VL chain containing the amino acid sequence shown in SEQ ID NO: 135 and VH chain containing the amino acid sequence shown in SEQ ID NO: 121. 9. An antibody according to claim 3, containing: 1) VL chain containing the amino acid sequence shown in SEQ ID NO: 77 and VH chain containing the amino acid sequence shown in SEQ ID NO: 75; 2) a VL chain containing the amino acid sequence shown in SEQ ID NO: 81 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 79; 3) a VL chain containing the amino acid sequence shown in SEQ ID NO: 85 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 83; 4) a VL chain containing the amino acid sequence shown in SEQ ID NO: 89 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 87; 5) a VL chain containing the amino acid sequence shown in SEQ ID NO: 93 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 91; 6) a VL chain containing the amino acid sequence shown in SEQ ID NO: 97 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 95; 7) a VL chain containing the amino acid sequence shown in SEQ ID NO: 101 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 99; 8) a VL chain containing the amino acid sequence shown in SEQ ID NO: 105 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 103; 9) a VL chain containing the amino acid sequence shown in SEQ ID NO: 109 and a VH chain containing the amino acid sequence shown in SEQ ID NO: 107; 10) VL chain containing the amino acid sequence shown in SEQ ID NO: 230 and VH chain containing the amino acid sequence shown in SEQ ID NO: 228; or 11) VL chain containing the amino acid sequence shown in SEQ ID NO: 234 and VH chain containing the amino acid sequence shown in SEQ ID NO: 232. 10. The use of antibodies according to any one of paragraphs. 1, 3, 8 and 9 for the treatment of an IL-31 mediated disorder in a subject. 11. The use of claim 10 wherein the IL-31 mediated disorder is an pruritic or allergic condition. 12. Use according to claim 11, wherein the pruritic or allergic condition is an pruritic condition selected from the group consisting of atopic dermatitis, eczema, psoriasis, scleroderma, and pruritus. 13. Use according to claim 12, wherein the pruritic or allergic condition is an allergic condition selected from the group consisting of allergic dermatitis, summer eczema, urticaria, equine fever, inflammatory airway disease, recurrent airway obstruction, hyperreactivity respiratory tract, chronic obstructive pulmonary disease and inflammatory processes resulting from autoimmunity. 14. The use of claim 10 wherein the IL-31 mediated disorder is tumor progression. 15. Use according to claim 14, wherein the IL-31 mediated disorder is eosinophilic disease or mastocytomas. 16. The use of antibodies according to any one of paragraphs. 1, 3, 8 and 9 to inhibit IL-31 activity in a mammal. 17. A veterinary composition for the treatment of an IL-31 mediated disorder in a mammal selected from the group consisting of dogs, cats and horses, comprising a therapeutically effective amount of an antibody according to any one of paragraphs. 1, 3, 8 and 9 and an acceptable carrier, diluent or excipient. 18. An antibody according to any one of paragraphs. 1, 3, 8 and 9 for use in the treatment of a mammal with an IL-31 mediated disorder. 19. The use of antibodies according to any one of paragraphs. 1, 3, 8 and 9 in the manufacture of a medicament for the treatment of a mammal with an IL-31 mediated disorder. 20. A method for detecting IL-31, including: (a) incubating a sample containing IL-31 in the presence of an antibody according to any one of paragraphs. 1, 3, 8 and 9; And (b) detection of an antibody associated with IL-31 in the sample. 21. The method of claim 20, further comprising quantifying IL-31 in the sample. 22. Selected nucleic acid to obtain a monoclonal antibody according to any one of paragraphs. 1-9 containing a nucleic acid sequence encoding: (1) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 15; (2) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 21; (3) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 25, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 27; (4) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 33; (5) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 37, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 38, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 39; (6) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 43, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 44, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 45; (7) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 49, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 50, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 51; (8) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 55, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 56, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 57; (9) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 61, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 62, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 63; or (10) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3. 23. Selected nucleic acid to obtain a monoclonal antibody according to any one of paragraphs. 1-9 containing a nucleic acid sequence encoding: (1) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 13, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 14, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 15 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (2) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 19, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 20, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 21 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 24; (3) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 25, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 26, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 27 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 28, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 30; (4) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 33 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 34, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 35, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 36; (5) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 37, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 38, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 39 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 42; (6) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 43, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 44, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 45 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 47, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 48; (7) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 49, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 50, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 51 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 52, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 53, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 54; (8) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 55, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 56, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 57 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 58, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 59, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 60; (9) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 61, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 62, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 63 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 64, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 65, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 66; or (10) VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 ; VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. 24. Selected nucleic acid to obtain a monoclonal antibody according to any one of paragraphs. 1-9 containing a nucleic acid sequence encoding: (1) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 18; (2) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 22, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 23, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 24; (3) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 28, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: thirty; (4) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 34, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 35, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 36; (5) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 40, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 41, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 42; (6) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 46, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 47, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 48; (7) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 52, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 53, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 54; (8) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 58, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 59, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 60; (9) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 64, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 65, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 66; or (10) VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. 25. An expression vector containing a nucleic acid according to claim 22. 26. An expression vector containing a nucleic acid according to claim 23. 27. An expression vector containing a nucleic acid according to claim 24. 28. A host cell that produces a monoclonal antibody according to any one of paragraphs. 1, 3, 8 and 9, containing the vector according to claim 25 and the vector according to claim 27 or the vector according to claim 26. 29. The method of obtaining antibodies according to any one of paragraphs. 1, 3, 8 and 9, comprising culturing the host cell according to claim 28 under conditions leading to the production of an antibody, and isolating the antibody from the host cell or host cell culture medium.

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